10.1007@978 3 030 25506 0

Fungal Biology
Ajar Nath Yadav

Sangram Singh
Shashank Mishra
Arti Gupta Editors
Recent
Advancement
in White
Biotechnology
Through Fungi
Volume 3: Perspective for Sustainable
Environments
Fungal Biology
Series Editors
Vijai Kumar Gupta
Department of Chemistry and Biotechnology
Tallinn University of Technology
Akadeemia tee, Tallinn, Estonia
Maria G. Tuohy
School of Natural Sciences
National University of Ireland Galway
Galway, Ireland
About the Series
Fungal biology has an integral role to play in the development of the biotechnology
and biomedical sectors. It has become a subject of increasing importance as new
fungi and their associated biomolecules are identified. The interaction between
fungi and their environment is central to many natural processes that occur in the
biosphere. The hosts and habitats of these eukaryotic microorganisms are very
diverse; fungi are present in every ecosystem on Earth. The fungal kingdom is
equally diverse, consisting of seven different known phyla. Yet detailed knowledge
is limited to relatively few species. The relationship between fungi and humans has
been characterized by the juxtaposed viewpoints of fungi as infectious agents of
much dread and their exploitation as highly versatile systems for a range of
economically important biotechnological applications. Understanding the biology
of different fungi in diverse ecosystems as well as their interactions with living and
non-living is essential to underpin effective and innovative technological
developments. This series will provide a detailed compendium of methods and
information used to investigate different aspects of mycology, including fungal
biology and biochemistry, genetics, phylogenetics, genomics, proteomics, molecular
enzymology, and biotechnological applications in a manner that reflects the many
recent developments of relevance to researchers and scientists investigating the
Kingdom Fungi. Rapid screening techniques based on screening specific regions in
the DNA of fungi have been used in species comparison and identification, and are
now being extended across fungal phyla. The majorities of fungi are multicellular
eukaryotic systems and therefore may be excellent model systems by which to
answer fundamental biological questions. A greater understanding of the cell
biology of these versatile eukaryotes will underpin efforts to engineer certain fungal
species to provide novel cell factories for production of proteins for pharmaceutical
applications. Renewed interest in all aspects of the biology and biotechnology of
fungi may also enable the development of “one pot” microbial cell factories to meet
consumer energy needs in the 21st century. To realize this potential and to truly
understand the diversity and biology of these eukaryotes, continued development of
scientific tools and techniques is essential. As a professional reference, this series
will be very helpful to all people who work with fungi and should be useful both to
academic institutions and research teams, as well as to teachers, and graduate and
postgraduate students with its information on the continuous developments in
fungal biology with the publication of each volume.
More information about this series at http://www.springer.com/series/11224

Ajar Nath Yadav • Sangram Singh
Shashank Mishra • Arti Gupta
Editors
Recent Advancement
in White Biotechnology
Through Fungi
Volume 3: Perspective for Sustainable
Environments
Editors
Ajar Nath Yadav Sangram Singh
Department of Biotechnology Dr. Ram Manohar Lohia Avadh University
Akal College of Agriculture Faizabad, Uttar Pradesh, India
Eternal University, Baru Sahib
Sirmour, Himachal Pradesh, India Arti Gupta
Department of Zoology
Shashank Mishra Dr. Ram Manohar Lohia Avadh University
Biotech Park Gonda, Uttar Pradesh, India
Lucknow, Uttar Pradesh, India
ISSN 2198-7777 ISSN 2198-7785 (electronic)

Fungal Biology
ISBN 978-3-030-25505-3 ISBN 978-3-030-25506-0 (eBook)
https://doi.org/10.1007/978-3-030-25506-0
© Springer Nature Switzerland AG 2019

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Foreword
White biotechnology is industrial biotechnology dealing with the potential applica-

tion of microbes and their enzymes for different processes in industry, agriculture,
pharmaceuticals, and environments. The main application of white biotechnology is
commercial production of various useful organic substances, such as acetic acid,
citric acid, acetone, and glycerin, and antibiotics like penicillin, streptomycin, and
mitomycin. The potential applications of fungi and fungal enzymes include being
environment-friendly solutions as an alternative to sustainable environments. One
of the first goals on white biotechnology’s agenda has been the production of biode-
gradable plastics and textiles. White biotechnology can provide an unlimited and
pure source of enzymes as an alternative to the harsh chemicals traditionally used in
industry for accelerating chemical reactions. Enzymes are found in naturally occur-
ring microorganisms, such as bacteria, fungi, and yeast, all of which may or may not
be genetically modified. In the twenty-first century, humans acquired skills to har-
ness fungi to protect human health (through antibiotics, antimicrobials, immuno-
suppressive agents, value-added products, etc.), which led to industrial-scale
production of enzymes, alkaloids, detergents, acids, and biosurfactants. Human
knowledge of white biotechnology has evolved to the point where today products
derived from white biotechnology often display better performance, higher sustain-
ability, and more commercially viable characteristics than products created from
traditional chemical procedures. Since then, white biotechnology has steadily
developed and now plays a key role in several industrial sectors, providing both
high-value nutraceuticals and pharmaceutical products. The fungal strains and bio-
active compounds also play an important role in environmental cleaning.
The present book volume on “Recent Advancement in White Biotechnology
Through Fungi” Volume 3: Perspective for Sustainable Environments is a very
timely publication, which provides state-of-the-art information in the area of white
biotechnology, broadly involving fungi and fungal-based products for sustainable
environments. The book volume comprises 20 chapters. Chapter 1 by Singh and
Singh describes fungal secretomes and their potential application for biodegrada-
tion of lignocelluloses and biopolymers for sustainable environments. Chapter 2
presented by Vishnoi and Dixit highlights eco-friendly techniques and fungal
v
vi Foreword
enzymes used for bioremediation for environmental cleaning. Chapter 3 by Kumar

et al. describes the genetic diversity of methylotrophic yeast and their impact on
environments. Chapter 4 by Periasamy et al. highlights the opportunities and chal-
lenges of white-rot fungi and their enzymes for the treatment of industrial dye efflu-
ents. Enshasy et al. describe the production of lignin-degrading enzymes by
Pleurotus ostreatus and their different industrial applications in Chap. 5. Chapter 6
details the functional attributes of fungal peroxidase and laccase enzymes for waste
treatments. Chandra and Enespa highlight the recent advancements in enzymes
from different fungal communities for bioremediation in Chap. 7. In Chap, 8,
Conejo-Saucedo and colleagues describe in detail the bioremediation of polycyclic
aromatic hydrocarbon-contaminated soil through fungal communities. Singh et al.
highlight the fungal production of novel enzymes and bioactive compounds for bio-
remediation of hazardous chemicals in Chap. 9. Mishra et al. explain β-glucosidases
produced by fungal communities and their potential biotechnological applications
in biomass degradation for the sustainable environments in Chap. 10. The roles of
fungi in climate change abatement through carbon sequestration have been described
by Malyan et al. in Chap. 11. Chapter 12 by Vyas and Yakubu describes microbial
enzymes and their applications in pulp the and paper industry. Kumar et al. high-
light the arbuscular mycorrhizal fungi-mediated mycoremediation of saline soil in
Chap. 13. Mondal et al. discuss the fungal enzymes for bioconversion of lignocel-
lulosic biomass in Chap. 14. Bioconversion of biomass to biofuel using fungal con-
sortium is discussed in Chap. 15 by Cherukuri and Akkina. Kumar et al. describe the
roles of fungi in the removal of heavy metals and dyes from wastewater by biosorp-
tion processes in Chap. 16. Kumar and Singh explain the impact of arbuscular
mycorrhizal fungi in global sustainable environments in Chap. 17. In Chap. 18
Kumar et al. describe the current scenario and future prospects of fungal phytore-
mediation of heavy metal-contaminated resources. Baker et al. highlight the biore-
mediation of xenobiotic compounds using fungal enzymes in Chap. 19. Finally, in
Chap. 20, the conclusion and future prospects on fungal white biotechnology are
described by Ajar Nath Yadav.
Overall, great efforts have been carried out by Dr. Ajar Nath Yadav, his editorial
team, and scientists from different countries to compile this book as a highly unique
and up-to-date source on fungal white biotechnology for the students, researchers,
scientists, and academicians. I hope that the readers will find this book highly useful
and interesting during their pursuit of fungal biotechnology.
Vice Chancellor Dr. H. S. Dhaliwal

Eternal University
Baru Sahib, Himachal Pradesh, India
Foreword vii
Dr. H. S. Dhaliwal is presently the Vice Chancellor of

Eternal University, Baru Sahib, Himachal Pradesh,
India. Dr. Dhaliwal holds a Ph.D. in Genetics from the
University of California, Riverside, USA (1975). He
has 40 years of research, teaching, and administrative
experience in various capacities. Dr. Dhaliwal is a
Professor of Biotechnology at Eternal University, Baru
Sahib, from 2011 to present. He had worked as
Professor of Biotechnology at IIT, Roorkee (2003–
2011); Founding Director of Biotechnology Centre,
Punjab Agricultural University, Ludhiana (1992–2003);
Senior Scientist and Wheat Breeder-cum-Director at
PAU’s Regional Research Station, Gurdaspur (1979–
1990); Research Fellow at FMI, Basel, Switzerland
(1976–1979); and D.F. Jones Postdoctoral Fellow at the
University of California, Riverside, USA (1975–1976).
Dr. Dhaliwal was elected as Fellow at the National
Academy of Agricultural Sciences, India, (1992);
worked as Visiting Professor at the Department of Plant
Pathology, Kansas State University, Kansas, USA
(1989); and was elected as Senior Research Fellow at
CIMMYT, Mexico (1987). He has many national and
international awards under his name such as the
Pesticide India Award from the Indian Society of
Mycology and Plant Pathology in 1988 and a cash
award from the Federation of Indian Chambers of
Commerce and Industry (FICCI) in 1985. He has to his
credit more than 400 publications including 250
research papers, 12 reviews, 15 chapters contributed to
books, 105 papers presented in meetings and confer-
ences and abstracted, 18 popular articles, and 2 books/
bulletins/manuals. His important research contributions
are identification of new species of wild diploid wheat
Triticum urartu and gathering of evidences to implicate
T. urartu as one of the parents of polyploid wheat; serv-
ing as team leader in the development of seven wheat
varieties, viz., PBW 54, PBW 120, PBW 138, PBW
175, PBW 222, PBW 226, and PBW 299, approved for
cultivation in Punjab and North Western Plains Zone of
India; molecular marker-assisted pyramiding of bacte-
rial blight resistance genes Xa21 and Xa13 and the
green revolution semi-dwarfing gene sd1 in Dehraduni
Basmati; and development of elite wheat lines bioforti-
fied for grain rich in iron and zinc through wide hybrid-
ization with related non-progenitor wild wheat species
viii Foreword
and molecular breeding. Dr. Dhaliwal made a signifi-

cant contribution to the development of life and epide-
miology life cycle of Tilletia indica fungus, the causal
organism of Karnal bunt disease of wheat, and devel-
opment of Karnal bunt-resistant wheat cultivar.
Dr. Dhaliwal had the membership of several task forces
and committees of the Department of Biotechnology,
Ministry of Science and Technology, Government of
India, New Delhi; served as Chairman of the Project
Monitoring Committee for Wheat Quality Breeding,
Department of Biotechnology, Ministry of Science and
Technology, Government of India (2007–2010), and
Chairman of the Project Monitoring Committee in
“Agri-biotechnology,” Department of Biotechnology,
Government of India, New Delhi (2014–2016); and
presently is a Member of the newly constituted Expert
Committee for DBT-UDSC Partnership Centre on
Genetic Manipulation of Crop Plants at UDSC, New
Delhi (2016 onward).
Foreword
Perspective for environmental sustainability is to main-

tain the persona of an environment for people and other
species. Some of the issues that create major environ-
mental sustainability problems include destruction of
the living environments (habitats) of native species,
release of polluting chemicals and other materials into
the environment, emission of greenhouse gases into the
atmosphere than can cause climate change, and deple-
tion of low-cost oil and other fossil fuels.
One promising alternative treatment strategy to
incineration is bioremediation which is to exploit the
ability of microorganisms to remove pollutants from
contaminated sites like water, air, and climate. Fungi are among the most potential
candidates of bioremediation as they are natural decomposers of waste matter and
secrete several extracellular enzymes capable of decomposing lignin and cellulose.
Fungi possess the biochemical and ecological capacity to degrade environmental
organic chemicals and to decrease the risk associated with metals, metalloids, and
radionuclides, either by chemical alteration or by influencing chemical
bioavailability.
CEO
Biotech Park, Lucknow, UP, India Prof. Pramod Tandon
ix
Preface
White biotechnology is drawing much attention as a solution to producing enzymes

and bioactive compounds for bioremediation for environmental cleaning through
eco-friendly techniques such as the use of fungi to synthesize hydrolytic enzymes
and compounds for plant growth promotion, biocontrol, and other processes for
agriculture, medicine, industry, pharmaceuticals, and allied sectors. White fungal
biotechnology is an emerging field in science that supports the revealing of novel
and vital biotechnological components. The fungi Aspergillus, Bipolaris, Cordyceps,
Fusarium, Gaeumannomyces, Myceliophthora, Penicillium, Phoma, Piriformospora,
Pleurotus, Trichoderma, and Xylaria are highly important fungal groups which can
be utilized for production of different antibiotics, enzymes, pigments, and peptides
useful in different processes of environmental cleaning.
The present book on “Recent Advancement in White Biotechnology Through
Fungi” Volume 3: Perspective for Sustainable Environments covers agriculturally
and industrially important fungi producing enzymes and bioactive compounds hav-
ing the potential application for cleaning polluted environments. The fungal com-
munity from different habitats such as those from extreme habitats and
plant-associated fungi having capability to produce extracellular enzymes, second-
ary metabolites, and bioactive compounds for diverse processes are targeted to be
applied in therapeutics, diagnostics, bioremediation, agriculture, industries, and
environments. This book volume will be immensely useful to those working in bio-
logical sciences, especially to microbiologists, microbial biotechnologists, bio-
chemists, researchers, and scientists of fungal biotechnology. We are honored that
the leading scientists who have extensive, in-depth experience and expertise in fun-
gal system and microbial biotechnology took the time and effort to develop these
outstanding chapters. Each chapter is written by internationally recognized research-
ers/scientists so the reader is given an up-to-date and detailed account of our knowl-
edge of white biotechnology and innumerable environmental applications of fungi.
We are grateful to the many people who helped to bring this book to light. Editors
wish to thank Dr. Eric Stannard, Senior Editor, Botany, Springer; Dr. Vijai Kumar
Gupta and Prof. Maria G. Tuohy, Series Editors, Fungal Biology, Springer; and
Ms. Saveetha Balasundaram and Mr. Rahul Kumar, Project Coordinators, Springer,
xi
xii Preface
for their generous assistance, constant support, and patience in initializing the vol-
ume. Dr. Ajar Nath Yadav gives special thanks to his exquisite wife Ms. Neelam
Yadav for her constant support and motivation in putting everything together. Dr.
Yadav also gives special thanks to his esteemed friends, well-wishers, colleagues,
and senior faculty members of Eternal University, Baru Sahib, India.
Baru Sahib, Himachal Pradesh, India Ajar Nath Yadav

Lucknow, Uttar Pradesh, India Shashank Mishra
Faizabad, Uttar Pradesh, India Sangram Singh
Gonda, Uttar Pradesh, India Arti Gupta
Contents
1 Secretomics of Wood-Degrading Fungi and Anaerobic

Rumen Fungi Associated with Biodegradation of Recalcitrant
Plant Biomass �� 1
Nasib Singh and Joginder Singh
2 Bioremediation: New Prospects for Environmental
Cleaning by Fungal Enzymes�� 17
Neha Vishnoi and Sonal Dixit
3 Genetic Diversity of Methylotrophic Yeast and Their Impact
on Environments�� 53
Manish Kumar, Raghvendra Saxena, Pankaj Kumar Rai,
Rajesh Singh Tomar, Neelam Yadav, Kusam Lata Rana,
Divjot Kour, and Ajar Nath Yadav
4 White Rot Fungi and Their Enzymes for the Treatment
of Industrial Dye Effluents�� 73
Dhevagi Periasamy, Sudhakarn Mani, and Ramya Ambikapathi
5 Pleurotus ostreatus: A Biofactory for Lignin-Degrading
Enzymes of Diverse Industrial Applications�� 101
Hesham El Enshasy, Farid Agouillal, Zarani Mat,
Roslinda Abd Malek, Siti Zulaiha Hanapi, Ong Mei Leng,
Daniel Joe Dailin, and Dalia Sukmawati
6 Extracellular Fungal Peroxidases and Laccases for Waste
Treatment: Recent Improvement �� 153
Shanmugapriya S., Manivannan G., Selvakumar Gopal,
and Sivakumar Natesan
7 Fungal Enzymes for Bioremediation of Contaminated Soil�� 189
Prem Chandra and Enespa
xiii
xiv Contents
8 Bioremediation of Polycyclic Aromatic Hydrocarbons (PAHs)

Contaminated Soil Through Fungal Communities�� 217
Ulises Conejo-Saucedo, Darío R. Olicón-Hernández,
Tatiana Robledo-Mahón, Haley P. Stein, Concepción Calvo,
and Elisabet Aranda
9 Role of Fungal Enzymes for Bioremediation of Hazardous
Chemicals�� 237
Nitika Singh, Abhishek Kumar, and Bechan Sharma
10 Biotechnological Applications of β-Glucosidases in Biomass
Degradation�� 257
Sushma Mishra, Deepika Goyal, Amit Kumar,
and Prem Kumar Dantu
11 Role of Fungi in Climate Change Abatement Through
Carbon Sequestration�� 283
Sandeep K. Malyan, Amit Kumar, Shahar Baram, Jagdeesh Kumar,
Swati Singh, Smita S. Kumar, and Ajar Nath Yadav
12 Microbial Enzymes and Their Application in Pulp
and Paper Industry�� 297
Abdulhadi Yakubu, Upasana Saikia, and Ashish Vyas
13 Arbuscular Mycorrhizal Fungi-Mediated Mycoremediation
of Saline Soil: Current Knowledge and Future Prospects �� 319
Dileep Kumar, Priyanka Priyanka, Pramendra Yadav, Anurag Yadav,
and Kusum Yadav
14 Fungal Enzymes for Bioconversion of Lignocellulosic Biomass�� 349
Subhadeep Mondal, Suman Kumar Halder,
and Keshab Chandra Mondal
15 Bioconversion of Biomass to Biofuel Using Fungal Consortium �� 381
Pavana Jyothi Cherukuri and Rajani Chowdary Akkina
16 Role of Fungi in the Removal of Heavy Metals and Dyes
from Wastewater by Biosorption Processes�� 397
Ajay Kumar, Vineet Kumar, and Joginder Singh
17 Impact of Arbuscular Mycorrhizal Fungi (AMF)
in Global Sustainable Environments�� 419
Sanjeev Kumar and Joginder Singh
18 Fungal Phytoremediation of Heavy Metal-Contaminated
Resources: Current Scenario and Future Prospects�� 437
Amit Kumar, Ashish K. Chaturvedi, Kritika Yadav, K. P. Arunkumar,
Sandeep K. Malyan, P. Raja, Ram Kumar, Shakeel Ahmad Khan,
Krishna Kumar Yadav, Kusam Lata Rana, Divjot Kour,
Neelam Yadav, and Ajar Nath Yadav
Contents xv
19 Fungal Enzymes for Bioremediation of Xenobiotic Compounds�� 463

Peter Baker, Araven Tiroumalechetty, and Rajinikanth Mohan
20 Fungal White Biotechnology: Conclusion and Future Prospects�� 491
Ajar Nath Yadav
Index�� 499
Contributors
Farid Agouillal Research Unit on Analysis and Technological Development in

Environment (URADTE), Centre de Recherche Scientifique et Technique en
Analyses Physico-Chimiques (CRAPC), Tipaza, Algeria
Rajani Chowdary Akkina Department of Microbiology & Food Science and
Technology, Institute of Science, GITAM (Deed to be University), Visakhapatnam,
Andhra Pradesh, India
Ramya Ambikapathi Department of Environmental Sciences, Tamil Nadu
Agricultural University, Coimbatore, India
Elisabet Aranda Department of Microbiology, Institute of Water Research,
University of Granada, Granada, Spain
K. P. Arunkumar Central Muga Eri Research and Training Institute, Central Silk
Board, Jorhat, Assam, India
Peter Baker Department of Biology, Colgate University, Hamilton, NY, USA
Shahar Baram Institute of Soil, Water and Environmental Sciences, The Volcani
Research Center, Agricultural Research Organization (ARO), Rishon LeZion, Israel
Concepción Calvo Department of Microbiology, Institute of Water Research,
Prem Chandra Department of Environmental Microbiology, School for
Environmental Sciences, Babasaheb Bhimrao Ambedkar (A Central) University,
Ashish K. Chaturvedi Water Management (Agriculture) Division, Centre for
Water Resources Development and Management, Kozhikode, Kerala, India
Pavana Jyothi Cherukuri Department of Microbiology & Food Science and
Technology, Institute of Science, GITAM (Deed to be University), Visakhapatnam,
Andhra Pradesh, India
xvii
xviii Contributors
Ulises Conejo-Saucedo Department of Microbiology, Institute of Water Research,

Daniel Joe Dailin Institute of Bioproduct Development (IBD), Universiti
Teknologi Malaysia (UTM), Johor Bahru, Malaysia
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti
Prem Kumar Dantu Department of Botany, Dayalbagh Educational Institute,
Deemed University, Dayalbagh, Agra, India
Sonal Dixit Department of Botany, University of Lucknow, Lucknow, India
Enespa Department of Plant Pathology, School of Agriculture, MPDC, University
of Lucknow, Lucknow, Uttar Pradesh, India
Hesham El Enshasy Institute of Bioproduct Development (IBD), Universiti
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti
City of Scientific Research and Technology Applications, New Burg Al Arab,
Alexandria, Egypt
Selvakumar Gopal Department of Microbiology, Alagappa University, Karaikudi,
Tamil Nadu, India
Deepika Goyal Department of Botany, Dayalbagh Educational Institute, Deemed
University, Dayalbagh, Agra, India
Suman Kumar Halder Department of Microbiology, Vidyasagar University,
Midnapore, West Bengal, India
Siti Zulaiha Hanapi Institute of Bioproduct Development (IBD), Universiti
Shakeel Ahmad Khan Centre for Environment Science and Climate Resilient
Agriculture, ICAR-Indian Agricultural Research Institute, New Delhi, India
Divjot Kour Department of Biotechnology, Akal College of Agriculture, Eternal
University, Baru Sahib, Sirmour, Himachal Pradesh, India
Abhishek Kumar Department of Biochemistry, Faculty of Science, University of
Allahabad, Allahabad, India
Ajay Kumar School of Bioengineering and Biosciences, Lovely Professional
University, Phagwara, Punjab, India
Amit Kumar Host Plant Section, Central Muga Eri Research & Training Institute,
Central Silk Board, Lahdoigarh, Jorhat, Assam, India
Dileep Kumar Department of Biochemistry, University of Lucknow, Lucknow,
Uttar Pradesh, India
Contributors xix
Jagdeesh Kumar Department of Hydrology, Indian Institute of Technology

Roorkee, Roorkee, Uttarakhand, India
Manish Kumar Amity Institute of Biotechnology, Amity University, Gwalior,
India
Ram Kumar Centre for Environment Science and Climate Resilient Agriculture,
ICAR-Indian Agricultural Research Institute, New Delhi, India
Sanjeev Kumar Department of Genetics and Plant Breeding, Lovely Professional
University, Jalandhar, India
Smita S. Kumar Center for Rural Development and Technology, Indian Institute
of Technology Delhi, New Delhi, India
Vineet Kumar School of Bioengineering and Biosciences, Lovely Professional
Ong Mei Leng Harita Go Green Sdn. Bhd., Johor Bahru, Johor, Malaysia
Roslinda Abd Malek Institute of Bioproduct Development (IBD), Universiti
Sandeep K. Malyan Institute of Soil, Water and Environmental Sciences, The
Volcani Research Center, Agricultural Research Organization (ARO), Rishon
LeZion, Israel
Sudhakarn Mani Department of Environmental Sciences, Tamil Nadu Agricultural
University, Coimbatore, India
Manivannan G. Department of Microbiology and Biotechnology, SVN College,
Madurai, Tamil Nadu, India
Zarani Mat Institute of Bioproduct Development (IBD), Universiti Teknologi
Malaysia (UTM), Johor Bahru, Malaysia
Sushma Mishra Department of Botany, Dayalbagh Educational Institute, Deemed
University, Dayalbagh, Agra, India
Rajinikanth Mohan Department of Biology, Colgate University, Hamilton, NY,
USA
Department of Biology, Mercyhurst University, Erie, PA, USA
Keshab Chandra Mondal Department of Microbiology, Vidyasagar University,
Subhadeep Mondal Department of Microbiology, Vidyasagar University,
Darío R. Olicón-Hernández Department of Microbiology, Institute of Water
Research, University of Granada, Granada, Spain
Dhevagi Periasamy Department of Environmental Sciences, Tamil Nadu
Agricultural University, Coimbatore, India
xx Contributors
Priyanka Priyanka Department of Biochemistry, University of Lucknow,

Pankaj Kumar Rai Department of Biotechnology, Invertis University, Bareilly,
P. Raja ICAR-IISWC, Regional Centre, Ooty, Tamil Nadu, India
Kusam Lata Rana Department of Biotechnology, Akal College of Agriculture,
Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India
Tatiana Robledo-Mahón Department of Microbiology, Institute of Water
Research, University of Granada, Granada, Spain
Upasana Saikia Department of Microbiology, School of Bioscience and
Bioengineering, Lovely Professional University, Phagwara, Punjab, India
Raghvendra Saxena Amity Institute of Biotechnology, Amity University, Gwalior,
India
Shanmugapriya S. Department of Molecular Microbiology, School of
Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
Bechan Sharma Department of Biochemistry, Faculty of Science, University of
Joginder Singh Department of Biotechnology, School of Bioengineering and
Biosciences, Lovely Professional University, Jalandhar, Phagwara, Punjab, India
Nasib Singh Department of Microbiology, Akal College of Basic Sciences, Eternal
University, Baru Sahib, Himachal Pradesh, India
Nitika Singh Department of Biochemistry, Faculty of Science, University of
Swati Singh Department of Environmental Science, Chaudhary Charan Singh
University, Meerut, Uttar Pradesh, India
Sivakumar Natesan Department of Molecular Microbiology, School of
Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
Haley P. Stein Department of Microbiology, Institute of Water Research, University
of Granada, Granada, Spain
Dalia Sukmawati Faculty of Mathematics and Natural Sciences, Universitas
Negeri Jakarta, Jakarta, Indonesia
Araven Tiroumalechetty Department of Biology, Colgate University, Hamilton,
NY, USA
Rajesh Singh Tomar Amity Institute of Biotechnology, Amity University,
Gwalior, India
Contributors xxi
Neha Vishnoi Department of Environmental Sciences, Babasaheb Bhimrao

Ambedkar University, Lucknow, India
Ashish Vyas Department of Microbiology, School of Bioscience and
Bioengineering, Lovely Professional University, Phagwara, Punjab, India
Ajar Nath Yadav Department of Biotechnology, Akal College of Agriculture,
Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India
Anurag Yadav Department of Microbiology, College of Basic Science and
Humanities, Sardarkrushinagar Dantiwada Agricultural University, Banaskantha,
Gujarat, India
Krishna Kumar Yadav Institute of Environment and Development Studies,
Bundelkhand University, Jhansi, Uttar Pradesh, India
Kritika Yadav Department of Botany, Dayalbagh Educational Institute, Agra,
Kusum Yadav Department of Biochemistry, University of Lucknow, Lucknow,
Neelam Yadav Gopi Nath P.G. College, Veer Bahadur Singh Purvanchal University,
Deoli-Salamatpur, Ghazipur, Uttar Pradesh, India
Pramendra Yadav Department of Biochemistry, University of Lucknow,
Abdulhadi Yakubu Department of Science Laboratory Technology, College of
Science and Technology, Jigawa State Polytechnic, Dutse, Nigeria
About the Editors
Ajar Nath Yadav is an Assistant Professor in the

Department of Biotechnology, Akal College of
Agriculture, Eternal University, Baru Sahib, Himachal
Pradesh, India. He has 4 years of teaching and 10 years
of research experiences in the field of Industrial
Biotechnology, Microbial Biotechnology, Microbial
Diversity, and Plant-Microbe Interactions. Dr. Yadav
obtained his doctorate degree in Microbial Biotechnology,
jointly from the Indian Agricultural Research Institute,
New Delhi, and Birla Institute of Technology, Mesra,
Ranchi, India; M.Sc. (Biotechnology) from Bundelkhand
University, India; and B.Sc. (CBZ) from the University
of Allahabad, India. Dr. Yadav has 111 publications,
which include 39 research papers, 15 review papers, 11
books, 1 Laboratory manual, 33 book chapters, 8 popu-
lar articles, 7 editorials, 2 technical reports, and 1 patent
with h-index of 25, i10-index of 57, and 1760 citations
(Google Scholar). Dr. Yadav has published 109 research
communications in different international and national
conferences. Dr. Yadav has got 12 Best Paper Presentation
Awards, one Young Scientist Award (NASI-Swarna
Jayanti Puraskar), and three certificates of excellence in
reviewing awards. Dr. Yadav received the “Outstanding
Teacher Award” in the 6th Annual Convocation in 2018
by Eternal University, Baru Sahib, Himachal Pradesh.
Dr. Yadav has a long-standing interest in teaching at the
UG, PG, and Ph.D. level and is involved in taking courses
in Agriculture Microbiology, Bacteriology, Bioprocess
Engineering and Technology, Environmental
Microbiology, Industrial Microbiology, and Microbial
xxiii
xxiv About the Editors
Biotechnology. Dr. Yadav is currently handling two proj-

ects, one funded by the Department of Environment,
Science and Technology (DEST), Shimla, entitled
“Development of Microbial Consortium as Bio-
inoculants for Drought and Low Temperature Growing
Crops for Organic Farming in Himachal Pradesh” as
Principal Investigator and another funded by HP Council
for Science, Technology and Environment (HIMCOSTE)
on “value-added products” as Co-PI. He also worked as
an Organizing Committee Member for seven interna-
tional conferences/symposia in the related field.
Presently he is guiding four scholars for Ph.D. degree
and one scholar for M.Sc. dissertation. To his credit
~6700 microbes (archaea, bacteria, and fungi) were iso-
lated from diverse sources and ~550 potential and effi-
cient microbes were deposited at culture collection of the
National Bureau of Agriculturally Important
Microorganisms (NBAIM), Mau, India. He has depos-
ited 2386 nucleotide sequences and 3 whole genome
sequences (Bacillus thuringiensis AKS47, Arthrobacter
agilis L77, and Halolamina pelagica CDK2) and 2 tran-
scriptomes to NCBI GenBank databases: in public
domain. Dr. Yadav and group have developed a method
for screening of archaea for phosphorus solubilization
for the first time. He has been serving as an Editor/
Editorial Board Member and Reviewer for more than 35
national and international peer-reviewed journals. He
has lifetime memberships of the Association of
Microbiologists of India; Indian Science Congress
Association, India; and National Academy of Sciences,
India. Please visit https://sites.google.com/site/ajarbio-
tech/ for more details.
Sangram Singh is an Associate Professor in the

Department of Biochemistry, Dr. Ram Manohar Lohia
Avadh University, Faizabad, India, and has 11 years of
teaching and 14 years of research experiences in the
field of Applied Biochemistry. Dr. Singh obtained his
Ph.D. in Biochemistry and M.Sc. in Biochemistry from
Dr. Ram Manohar Lohia Avadh University, Faizabad,
India. Dr. Singh has published 34 national and
international research papers, 2 books, and 2 book
chapters. He has presented nine papers in different
national and international symposia/seminars/
conferences/workshops.
About the Editors xxv
Shashank Mishra is presently working as Scientist

“C,” Biotech Park, Lucknow, Uttar Pradesh, India. He
obtained his doctorate degree in science (“Industrial
Biotechnology”) in 2015 from Birla Institute of
Technology, Mesra, Ranchi, India; M.Phil.
(Biotechnology) in 2008 from Alagappa University,
Tamil Nadu, India; M.Sc. (Botany) in 2005 from Dr.
R.M.L. Avadh University, Ayodhya, India; M.Sc.
(Biotechnology) in 2004 from Barkatullah University,
Bhopal, India; and B.Sc. (Botany and Chemistry) in
2001 from Dr. R.M.L. Avadh University, Ayodhya,
India. He has made pioneering contributions in the area
of Microbial Biotechnology, Natural Product Synthesis,
and Environmental Microbiology for Food,
Pharmaceutical, and Human Health. To his credit he
has 23 publications (7 research papers, 2 review articles,
3 books, and 11 book chapters) in different reputed
international and national journals and publishers with
138 citations, h-index of 5, and i10-index of 4 (Google
Scholar). He has reported for the first time high
concentration of phenolic compounds by optimizing
various parameters and published in peer-reviewed and
refereed international journals. He has published 16
abstracts in different conferences/symposia/workshops.
He has presented 16 papers (12 poster + 04 oral) in
conferences/symposia and got 1 Best Poster
Presentation Award. Dr. Mishra has contributed in
organizing seven conferences/workshops. He has
deposited three nucleotide sequences to NCBI GenBank
databases: in public domain. Dr. Mishra and group have
isolated and characterized three microbes (bacteria and
microalgae) from tulsi and paddy plantation site and
transformed ferulic acid into value-added phenolic
compounds, viz., vanillin, vanillic acid, and
4-vinylguaiacol. He has a long-standing interest in
teaching at the UG, PG, and Ph.D. level and is involved
in taking courses in Industrial Biotechnology,
Bioprocess Engineering and Technology, Environmental
Biotechnology, Environmental Microbiology, Industrial
Microbiology, Microbial Biotechnology, and
Techniques in Microbiology and Biotechnology. He is
a reviewer in six international journals including BMC
Microbiology, Indian Phytopathology, PLOS One,
xxvi About the Editors
Scientific Reports and Archive of Phytopathology and

Plant Protection, and 3 Biotech. He has lifetime
memberships of the Association of Microbiologists of
India (AMI) and Vigyan Bharati (VIBHA).
Arti Gupta is an Assistant Professor in the

Department of Zoology, Sri Avadh Raj Singh Smarak
Degree College, Bishunpur Bairiya, Gonda, India.
Dr. Arti Gupta received her B.Sc. in Botany, Zoology,
and Chemistry in 2001 and got her M.Sc. in
Biotechnology in 2003 from Chaudhary Charan Singh
University, Meerut, India. Dr. Arti Gupta obtained her
Ph.D. from Mahatma Jyotiba Phule Rohilkhand
University, Bareilly, India, in 2010 in Animal Science.
Dr. Gupta started her career in 2004 with teaching for
graduate and post-graduate students of Biotechnology
from D.A.V. (P.G.) College, Muzaffarnagar. In 2005,
she was appointed as a Research Intern at the Central
Drug Research Institute, Uttar Pradesh. In 2010, she
was appointed as Teaching Associate at the Govind
Ballabh Pant Engineering College, Pauri Garhwal,
Uttarakhand. In 2012, she worked as Scientist with Sun
Agrigenetics Pvt. Ltd., Vadodara, Gujarat, and has 9
years of teaching and 11 years of research experiences
in the field of Animal Biotechnology, Molecular Plant
Biotechnology, Molecular Animal Biotechnology,
Bioprocess Technology, and Microbiology. Dr. Gupta
has published one monograph, has edited three Springer
Nature Switzerland books and is currently editing other
Springer Nature books, has published 22 national and
international research papers, and has attended 36
national and international symposia/seminars/
conferences/workshops. Dr. Gupta has been awarded
University Topper (Gold Medal), M.Sc. (Biotech.), by
Ch. C. S. University, Meerut; Young Scientist Award
(Gold Medal) by the Zoological Society of India,
Lucknow; Best Poster Presenter by the Asian Journal
of Experimental Science, Jaipur; Best Poster Presenter
by the International Consortium of Contemporary
Biologists (ICCB) and Madhawi-Shyam Educational
Trust (MSET), Ranchi; Fellowship Award by the
International Consortium of Contemporary Biologists
(ICCB) and Madhawi-Shyam Educational Trust
About the Editors xxvii
(MSET); and Dr. V.P. Agarwal Gold Medal by D.A.V.

(P.G.) College, Muzaffarnagar. Dr. Gupta has lifetime
memberships of the Indian Science Congress
Association, Biotech Research Society of India,
Zoological Society of India, and International
Consortium of Contemporary Biologists.
Chapter 1
Secretomics of Wood-Degrading Fungi
and Anaerobic Rumen Fungi Associated
with Biodegradation of Recalcitrant Plant
Biomass
Nasib Singh and Joginder Singh
1.1 Introduction
Lignocellulose is a widely available recalcitrant plant biopolymer composed of

polymeric polysaccharides cellulose, hemicellulose, and heteropolymeric lignin
(Lewis and Yamamoto 1990; Eastwood et al. 2011; Bugg et al. 2011; Janusz et al.
2017; dos Santos et al. 2018; Bissaro et al. 2018; Brink et al. 2019; Ralph et al.
2019). According to an estimate, 550 billion tons of carbon are present in vegetation
in terrestrial ecosystems including forest ecosystems where dead wood is the major
form of the plant biomass (Siegenthaler and Sarmiento 1993; Krah et al. 2018).
This recalcitrant plant biomass is recognized as the most abundant carbon source in
terrestrial ecosystem. In woody plants (angiosperms and gymnosperms), cellulose
generally constitutes 40–50% of the dry weight, whereas the amount of hemicellu-
loses and lignin ranges from 15% to 30% (Krah et al. 2018; Adesogan et al. 2019).
Cellulose is a macropolymer of numerous glucose units attached linearly by β-1,4-
glycosidic linkages. It is responsible for rigidity and crystalline form of plant cell
walls (Baldrian and Valaskova 2008; McFarlane et al. 2014; Bissaro et al. 2018).
Hemicelluloses, on the other hand, are complex and heterogeneous plant polysaccha-
rides consisting of xylose, mannose, arabinose, glucose, galactose, and sugar acids.
Xyloglucans, xylans, mannans, and glucomannans are the main examples of hemicel-
luloses (Scheller and Ulvskov 2010). These are known to strengthen the plant cell wall
by filling the voids around cellulose fibrils and interacting with lignin.
N. Singh (*)
Department of Microbiology, Akal College of Basic Sciences, Eternal University,
Baru Sahib, Himachal Pradesh, India
J. Singh
Department of Biotechnology, School of Bioengineering and Biosciences,
Lovely Professional University, Jalandhar, Phagwara, Punjab, India
© Springer Nature Switzerland AG 2019 1

A. N. Yadav et al. (eds.), Recent Advancement in White Biotechnology Through
Fungi, Fungal Biology, https://doi.org/10.1007/978-3-030-25506-0_1
2 N. Singh and J. Singh
Hemicelluloses are considered as the second most abundant polysaccharide in

the nature (Saha 2003). Lignin is complex, aromatic, heteropolymeric, and most
indigestible parts of the plant cell wall exhibiting almost complete resistance to
hydrolytic degradation (Lewis and Yamamoto 1990; Janusz et al. 2017; Brink et al.
2019; Ralph et al. 2019). It contributes 15–30% of dry weight of vascular plant cell
walls (Lewis and Yamamoto 1990; Gall et al. 2017). It is a phenolic polymer com-
posed mainly of p-coumaryl, coniferyl, and sinapyl alcohols (Janusz et al. 2017; dos
Santos et al. 2018; Brink et al. 2019; Ralph et al. 2019). Lignin confers rigidity to
the plant cell walls and inhibits hydrolytic attacks on adjacent cellulose and hemi-
cellulose (Lewis and Yamamoto 1990; Gall et al. 2017; Ralph et al. 2019).
Lignocellulose-rich forest waste, dead woods, agro-food industry wastes, and left-
over crop residues offer a sustainable, eco-friendly, and abundant resource for
industrial-scale production of green energy and biofuels.
1.2 Degradation and Depolymerization of Plant Biomass
The aromatic nature of lignin is a major obstacle for biodegradation and mineraliza-
tion of lignocellulose (Lewis and Yamamoto 1990; Janusz et al. 2017; Bissaro et al.
2018; Brink et al. 2019). Lignin and its phenolic derivatives are known to inhibit
lignocellulolytic enzymes by adsorption or deactivation. In the biological world,
fungi are considered to be the most prolific producers of lignocellulolytic enzymes
(Blanchette 1991; Conesa et al. 2001; Bouws et al. 2008; Eastwood et al. 2011;
Girard et al. 2013; Edwards et al. 2017; Kameshwar et al. 2019; Yadav et al. 2019a, b).
These eukaryotic organisms play indispensable role in plant matter decomposition,
carbon cycle, and overall nutrients recycling. Most fungi are aerobic and facultative
anaerobic with the exception of strict anaerobic species present in the rumen of
herbivorous animals (Sirohi et al. 2012; Edwards et al. 2017; Hooker et al. 2019).
Filamentous fungi, in particular, secrete abundant enzymes to break down plant
matter and complex materials in the environment which is in turn absorbed through
hypha walls and utilized further for their growth and maintenance (Bouws et al.
2008; Eastwood et al. 2011; Girard et al. 2013; Edwards et al. 2017). Their involve-
ment in plant matter decomposition, association with plant roots as mycorrhiza,
association with photobionts in lichens, and presence in rumen and intestine of
wood-decaying insects are well established, widely reported, and extensively
reviewed earlier by several investigators.
Fungi and bacteria are indispensably involved in degradation of recalcitrant plant
biomass, thus contributing massively to carbon cycle in various ecosystems (Cragg
et al. 2015; Janusz et al. 2017). Surprisingly, animals such as ruminants, termites,
millipedes, and terrestrial isopods are dependent on their respective microbiomes
for lignocellulose degradation as none of them encode the entire enzymatic reper-
toire required for lignocellulose degradation (Sirohi et al. 2012; Edwards et al.
2017; Kameshwar et al. 2019). In this chapter, we will emphasize the roles and
secretomes of wood-degrading fungi and anaerobic rumen fungi associated with
1 Secretomics of Wood-Degrading Fungi and Anaerobic Rumen Fungi Associated… 3
plant biomass degradation. Fungi are among the most diversified and widely studied
Natural Biomass Utilization Systems (NBUS; Eastwood et al. 2011; Girard et al.
2013). Several fungi are equipped with enzymes which empower them to harvest
energy and nutrition from plant biomass, which otherwise indigestible for other liv-
ing organisms (Girard et al. 2013; Edwards et al. 2017; Janusz et al. 2017). Several
fungal species from class Agaricomycetes (Basidiomycota) are closely associated
with wood decay in different ecological niches (Krah et al. 2018). The saprotrophic
members of this class cause decay of dead woods as either white rot or brown rot.
The white rot fungi (WRF) and brown rot fungi (BRF) are the prominent colonizers
which degrade lignocellulose cell wall components of compact wood logs, branches,
and stumps (Martinez et al. 2004, 2009; Girard et al. 2013; Edwards et al. 2017;
Janusz et al. 2017; SistaKameshwar and Qin 2018; Reina et al. 2019) (Fig. 1.1). In
addition to white and brown rot, other wood-decaying manifestations, viz., soft rot
and gray rot, are also exhibited by some members of Basidiomycota and Ascomycota
(Riley et al. 2014).
Anaerobic rumen fungi (ARF) account for 8–20% of total microbial biomass of
rumen and alimentary tract of herbivorous mammals (Sirohi et al. 2012; Edwards
et al. 2017; Kameshwar et al. 2019; Hooker et al. 2019). First described in 1975 by
Colin Orpin (Youssef et al. 2013), these obligate anaerobic, filamentous, and motile
zoospore-forming fungi are assigned to phylum Neocallimastigomycota. Despite
their low numbers (106 per ml of rumen fluid), ARF are key players in lignocellulose
degradation in the rumen as these physically penetrate and disrupt the plant cell walls
and thus facilitate rapid growth of fibrolytic bacteria leading to optimal degradation
and utilization of lignocellulosic biomass (Sirohi et al. 2012; Youssef et al. 2013;
Fig. 1.1 Association of fungi with degradation and depolymerization of woods, fodder, and crop
residues
Solomon et al. 2016; Haitjema et al. 2017; Edwards et al. 2017; Hooker et al. 2019).
At present, only 11 genera have been cultured from rumen ecosystem of various
herbivorous animals. These genera are Neocallimastix, Anaeromyces, Caecomyces,
Cyllamyces, Orpinomyces, Piromyces, Buwchfawromyces, Feramyces, Oontomyces,
Pecoramyces, and Liebetanzomyces (Sirohi et al. 2012; Edwards et al. 2017; Hooker
et al. 2019; Li et al. 2019). In the next section, we have discussed the metabolic
capabilities of wood-degrading fungi and ARF involved in plant biomass degradation
and mineralization.
1.3 ecretomics and Mechanism of Lignocellulose

S
Biodegradation
Secretome, a term coined by Tjalsma et al. (2000), represents all the proteins and
cellular machineries which are secreted outside the plasma membrane into the
environment or extracellular matrix by a cell (McCotter et al. 2016). Plants, bacte-
ria, and fungi exhibit their unique and substrate-specific secretome under different
environmental conditions. Fungal secretome, therefore, essentially comprises
extracellular enzymes which are released exterior to the cell wall, usually in the
presence of lignocellulosic plant matter (Bouws et al. 2008; Eastwood et al. 2011;
Girard et al. 2013; Kameshwar et al. 2019). In the past decade, advances in protein
identification techniques and genome sequencing have enabled detailed investiga-
tion of the secretomes of many saprophytic, pathogenic, and symbiotic fungal
species revealing rich, diverse, and highly specific enzymatic profiles. The decon-
struction and mineralization of recalcitrant and indigestible plant biomass require
the synergistic and cooperative action of several hydrolytic, oxidative, and non-
hydrolytic enzymes (Blanchette 1991; Girard et al. 2013; Cragg et al. 2015;
Edwards et al. 2017; Janusz et al. 2017; Bissaro et al. 2018). An extensive knowl-
edge of fungal secretomes involved in recalcitrant plant biomass degradation is of
immense significance in the present scenario where increasing emphasis is devoted
toward sustainable bioeconomy. In the coming sections, we describe different
carbohydrate-active enzymes (CAZymes) involved in lignocellulose degradation
(Table 1.1 and Fig. 1.2). According to CAZy database, there are six types of
CAZymes, i.e., glycoside hydrolase (GH), carbohydrate esterase (CE), glycosyl-
transferase (GT), polysaccharide lyases (PL), auxiliary activity (AA), and carbohy-
drate-binding domains (Lombard et al. 2014). It is estimated that the proportion of
secreted proteins in fungal species ranges from 4 to 14% (Lowe and Howlett 2012).
Degradation of recalcitrant plant biomass, viz., dead wood, wheat straw, fodder,
etc., is accomplished by highly coordinated and synergistic actions of multiple
CAZymes exhibiting a combination of oxidative, hydrolytic, and non-hydrolytic
activities (Bugg et al. 2011; Lombard et al. 2014; Janusz et al. 2017; SistaKameshwar
and Qin 2018; Bissaro et al. 2018). The most difficult part of plant cell wall is
lignin and it needs to be degraded before enzyme can access cellulose and
Table 1.1 The enzymatic repertoire of wood-degrading fungi and anaerobic rumen fungi
associated with degradation of recalcitrant lignocellulosic plant biomass
Plant cell wall
component Enzymes involved CAZy families
Cellulose Endo-1,4-β-D-glucanase (EC 3.2.1.4) GH1, GH3, GH5_5, GH5_7,
Exo-1,4-β-D-glucanase (EC 3.2.1.91) GH5_9, GH5_12, GH5_45,
Cellobiohydrolase (EC 3.2.1.91) GH5_48 GH5_74, GH5_131,
β-Glucosidase (EC 3.2.1.21) GH5_148, GH6, GH7, AA3_2,
Cellobiose dehydrogenase AA3_4, AA7, AA8-AA3_1-
Glucose 1-oxidase (CBM1), AA8-AA12-CBM1,
Pyranose 2-oxidase AA9, AA10, AA11, AA13, AA14,
Gluco-oligosaccharide oxidases AA15
PQQ-dependent pyranose dehydrogenase
Lytic polysaccharide monooxygenases
(LPMOs)
Hemicellulose Endo-β-1,4-xylanases (EC 3.2.1.8) GH2, GH5_7, GH5_26, GH27,
β-D-xylosidase (EC 3.2.1.37) GH36, GH35, GH3, GH39, GH43,
1,4-β-D-endo-mannanases (EC 3.2.1.78) GH52, GH10, GH11, GH62,
1,4-β-D-mannosidases (EC 3.2.1.25) GH51, GH54, GH67, GH115,
β-1,4-Galactosidase GH12, GH16, GH74, GH31,
Galactomannan acetyl esterase AA5_2, AA14, CE1, CE1–CE7,
Arabinoxylan arabinofuranohydrolase CE12, CE16, GE/GCE, CE15
α-L-Arabinofuranosidases
α-Glucuronidases
Feruloyl esterases
Acetyl esterases
4-O-Methyl glucuronoyl methylesterases
Xyloglucan transferase/hydrolases
α-Xylosidases
Galactose 6-oxidase
Lytic xylan oxidase
Lignin Laccases (EC 1.10.3.2) AA1, AA2, AA3, AA5, AA6
Lignin peroxidases (EC 1.11.1.14)
Manganese peroxidases (EC 1.11.1.13)
Versatile peroxidases (EC 1.11.1.16)
Dye-decolorizing peroxidase (EC
1.11.1.19)
Glyoxal oxidase (EC 1.2.3.5)
Aryl alcohol oxidases (EC 1.1.3.7)
Pyranose 2-oxidase (EC 1.1.3.10)
Cellobiose dehydrogenase (EC 1.1.99.18)
Glucose oxidase (EC 1.1.3.4)
Chloroperoxidases (EC 1.11.1.10)
Glucose dehydrogenase (EC 1.1.99.10)
Aromatic peroxygenases (EC 1.11.2.1)
CAZy carbohydrate-active enzymes, CBM cellulose-binding domains, GHs glycoside hydrolases,
CE carbohydrate esterases, AA auxiliary activities, LPMOs lytic polysaccharide monooxygenases
Sources: Lewis and Yamamoto (1990), Eastwood et al. (2011), Bugg et al. (2011), Lombard et al.
(2014), Hori et al. (2014), Janusz et al. (2017), dos Santos et al. (2018), Bissaro et al. (2018), Brink
et al. (2019), Ralph et al. (2019)
Fig. 1.2 The carbohydrate-active enzymes involved in degradation and depolymerization of recal-
citrant plant biomass consisting primarily of lignin, cellulose, and hemicellulose. White rot fungi,
brown rot fungi, and anaerobic rumen fungi secrete an array either all or some of essentially
involved lignocellulolytic enzymes on variable substrates under in situ and in vitro conditions.
Lignocellulose degradation is achieved by concerted and simultaneous action of several hydrolytic
enzymes, oxidoreductases, peroxidases, free radicals, and other reaction mediators
hemicellulose components. This activity is achieved in white rot fungi by extracel-

lular enzymes, viz., oxidoreductases (AA2), lignin peroxidases, laccases, manga-
nese peroxidases, versatile peroxidases, copper radical oxidases (AA5),
dye-decolorizing peroxidases, and phenol-oxidizing multicopper oxidases (AA1),
and a number of mediators, e.g., reactive oxygen species, free radicals, and aro-
matic intermediates (Martinez et al. 2004; Kuuskeri et al. 2016; Janusz et al. 2017;
Bissaro et al. 2018; dos Santos et al. 2018). A detailed list of these enzymes is
provided in Table 1.1. Both lignin-modifying enzymes and lignin-degrading auxil-
iary enzymes are involved in lignin degradation process (Bugg et al. 2011; Janusz
et al. 2017; Bissaro et al. 2018). On the other hand, cellulose and hemicellulose
degradation into disaccharides and monosaccharides is accomplished with the help
of cellobiohydrolases (GH families GH6 and GH7), β-glucosidases (GH1 and

GH3), endoglucanases (GH5, GH9, GH12, GH44, and GH45), lytic polysaccha-
ride monooxygenases (AA9), polysaccharide lyases, and carbohydrate esterases
(Kuuskeri et al. 2016; Janusz et al. 2017; Bissaro et al. 2018). Exoglucanases from
GH family, GH6, GH7, and GH48, attack cellulose fibrils at the ends of the chain.
In case of hemicelluloses, it is endo-hemicellulases, exo-hemicellulases, and acces-
sory enzymes which cleave chains at various positions and locations (Table 1.1).
Compositional details of lignocellulosic plant biomass, microorganisms involved
in its biodegradation, and associated enzymatic repertoire have also been exten-
sively reviewed earlier by Blanchette (1991), Eastwood et al. (2011), Girard et al.
(2013), Lombard et al. (2014), Guerriero et al. (2015), Cragg et al. (2015), Janusz
et al. (2017), Gall et al. (2017), Edwards et al. (2017), Bissaro et al. (2018), dos
Santos et al. (2018), and Hooker et al. (2019). Fungal secretome studies are increas-
ingly facilitated by accelerated genome sequencing availability of advanced soft-
ware, databases, algorithms, analytical tools, prediction models, and improved
proteomic approaches (Table 1.2).
As depicted in Fig. 1.2, lignin is acted upon by oxidative enzymes (laccase, lig-
nin peroxidase, versatile peroxidase, manganese peroxidase) and auxillary activity
redox enzymes (glyoxal oxidase, pyranose oxidase, aryl alcohol oxidase, methanol
oxidase; Hori et al. 2014; Adesogan et al. 2019; Brink et al. 2019; Ralph et al. 2019).
During lignin depolymerization by fungi, laccases and manganese peroxidases
mainly act on phenols, and the nonphenolic lignin components are attacked by lig-
nin peroxidase, whereas versatile peroxidases act on both (Bugg et al. 2011; Janusz
et al. 2017; Brink et al. 2019; Ralph et al. 2019). In addition to numerous well-
characterized hydrolytic enzymes, lignocellulolytic fungi exhibit simultaneous
secretion of an array of lytic polysaccharide monooxygenases (LPMOs) and several
other oxidoreductases (Bissaro et al. 2018). LPMOs are a class of copper-dependent
enzymes classified as auxiliary activities (AA) and belong to families AA9, AA10,
AA11, AA13, AA14, and AA15 of CAZy (Bissaro et al. 2018). LPMOs are
known to hydroxylate carbons at scissile glycosidic bonds. Interestingly, H2O2
plays a crucial role in catalytic activity of LPMOs (Bugg et al. 2011; Janusz et al.
2017; Bissaro et al. 2018).
1.4 Wood-Degrading Fungi
The number of species of potent wood-degrading fungi is more than 1000; however,
the actual numbers are expected to be much higher. These fungi mainly exhibit
saprotrophic mode of nutrition but sometimes may be showing parasitic attributes
in forest ecosystems (Janusz et al. 2017). Ongoing research based on transcriptome
and secretome has offered considerable insights on enzymatic machinery, and
lignocellulose-degrading capabilities of several fungal species adapted to sapro-
phytic, plant pathogenic, symbiotic, anaerobic, and endosymbiotic life styles
(SistaKameshwar and Qin 2018). Secretomic studies offer deeper insights into
Table 1.2 List of some important fungal genome sequences, secretomes, transcriptome databases,
and software packages
Database/authority/Genome Description Weblink
CAZy database Carbohydrate-active http://www.cazy.org/
enzymes database
MycoCosm 1000 Fungal Genomes https://genome.jgi.doe.gov/mycocosm/
Project home
Ohm et al. (2014)
Genome sequence of First-ever genome https://genome.jgi.doe.gov/mycocosm/
Phanerochaete sequence of wood- home, http://genome.jgi-psf.org/
chrysosporium, a white rot decaying fungi Pchrysosporium2_2,
fungus Martinez et al. (2004)
Genome sequence of Postia First genome sequence Martinez et al. (2009)
placenta, a brown rot of brown rot fungi
fungus
Genome sequence of Genome sequence of Youssef et al. (2013)
Orpinomyces C1A anaerobic rumen fungus
FunSecKB Fungal Secretome http://bioinformatics.ysu.edu/
Knowledge Base secretomes/fungi.php
FunSecKB2 The Fungal Secretome http://bioinformatics.ysu.edu/
and Subcellular secretomes/fungi2/index.php
Proteome Knowledge
Base 2.1
SRA NCBI Sequence Read https://submit.ncbi.nlm.nih.gov/subs/sra
Archive
FungiDB – http://fungidb.org/fungidb/
eLignin eLignin Microbial www.elignindatabase.com
Database Brink et al. (2019)
FSD The Fungal Secretome http://fsd.snu.ac.kr/index.php?a=view
Database
Software packages used in transcriptome and secretome data analysis and prediction
1. SignalP v4.1
2. SecretomeP v1.027
3. TargetP v1.1
4. TMHMM v2.0
5. ProtComp v9.0
mechanistic details of lignocellulose degradation by various species of bacteria and

fungi in their respective natural habitats or under symbiotic associations with higher
organisms. These studies have the potential to elucidate the life style adaptation and
survival mechanisms of wood-degrading fungi on highly recalcitrant plant biomass
as sole carbon source. In addition, secretomic analyses can facilitate our search for
novel enzymes and exploitable secretary pathways essentially needed to establish
industrial-scale and sustainable biofuels production. Here, we specifically focus on
wood-decaying fungi and anaerobic rumen fungi well recognized for their excep-
tional lignocellulolytic potential. The challenging task of plant biomass depolymer-
ization in natural environments is primarily accomplished by filamentous fungi.
Wood-degrading fungi mostly belong to the class Agaricomycetes of phylum

Basidiomycota, although some members of Ascomycota are also of considerable
significance. Lignocellulolytic activity of wood-degrading fungi is manifested
through concerted action of oxidoreductases, peroxidases, glycoside hydrolases,
exoglucanases, endoglucanases, xylanases, and a number of other CAZymes (Gaskell
et al. 2016; Bissaro et al. 2018). In addition, the role of H2O2 and reactive oxygen
species is also crucial. The most extensively studied (in terms of transcriptome and
secretome) wood-degrading fungi are Phanerochaete chrysosporium, Phanerochaete
carnosa, Phlebia tremellosa, Trametes versicolor, Phlebia radiata, Bjerkandera
adusta, Irpex lacteus, Gloeophyllum trabeum, Agaricus bisporus, Stropharia coro-
nilla, Agrocybe praecox, Chondrostereum purpureum, Heterobasidium annosum,
Ceriporiopsis subvermispora, Phellinus pini, Lentinula edodes, Hericium clathroi-
des, Pleurotus ostreatus, Obba rivulosa, Postia placenta, Piptoporus betulinus,
Serpula lacrimans, Fomitopsis lilacinogilva, Ganoderma lucidum, Laetiporus por-
tentosus, Fomitiporia mediterranea, Pycnoporus cinnabarinus, Dichomitus squa-
lens, Punctularia strigosozonata, Botrytis cinerea, Stereum hirsutum, Pleurotus
eryngii, Fibroporia radiculosa, Wolfiporia cocos, Dacryopinax primogenitus,
Daedalea quercina, Laetiporus sulphurous, Neolentinus lepideus, Calocera cornea,
Fistulina hepatica, Hydnomerulius pinastri, and Coniophora puteana (Riley et al.
2014; Presley and Schilling 2017; SistaKameshwar and Qin 2018).
1.4.1 Secretomes of White Rot Fungi (WRF)
WRF have the unique distinction of being the only microorganism in the biological
world to completely degrade lignin, cellulose, and hemicellulose simultaneously
(Manavalan et al. 2015; Xie et al. 2016). WRF are members of Basidiomycota, the
largest phylum of kingdom Fungi (Blanchette 1991; Martinez et al. 2004; Krah
et al. 2018). During lignin degradation by WRF, the crystalline cellulose with a
bleached appearance is left behind which appears as white rot. This is why these are
called white rot fungi. WRF initiate lignin depolymerization by producing large
amount of free radicals mediated by oxidases and peroxidases (Martinez et al.
2004). With the help of a consortium of enzymes, WRF carry out mineralization of
lignocellulose plant matter to carbon dioxide and thereby ensure continuity of car-
bon cycle in the habitats characterized by forest litter, fallen trees, wooden stumps,
wood logs, etc. In return, WRF fulfil their energy and nutrition requirements from
the same substrate (Blanchette 1991; Martinez et al. 2004; Baldrian and Valaskova
2008; Eastwood et al. 2011; Krah et al. 2018; Bissaro et al. 2018).
WRF from phylum Basidiomycota are reported to efficiently degrade lignin,
cellulose, and hemicellulose present in the plant cell wall manifested through co-
secretory and synergistic action of hydrolytic, oxidative, and non-hydrolytic
enzymes, thus leaving a bleached fibrous residue (Krah et al. (2018). WRF can
uniquely attack lignin barrier first before gaining access to cellulose and hemi-
celluloses of plant cell wall. WRF also produce extracellular reactive oxygen
species mediated by peroxidases which facilitate physical disruption of crystalline

lignocellulose biomass leading to greater access for degradative enzymes (Martinez
et al. 2004; Bissaro et al. 2018). P. chrysosporium is a model organism for studying
lignin degradation. Its genome sequence is published and offers greater insights on
lignin-degradative enzymatic machinery and corresponding genomic organization
(Martinez et al. 2004; Ohm et al. 2014). It is considered as the most prolific pro-
ducer of CAZy, especially laccases, lignin peroxidases, manganese peroxidases,
and LPMOs among various WRF species (Singh and Chen 2008). P. chrysosporium
RP78 genome encodes >240 putative CAZymes belonging to 69 distinct families.
Among these, 166 were glycoside hydrolases, 57 glycosyltransferases, 40 putative
endoglucanases (GH5, GH9, GH12, GH61, and GH74), 14 carbohydrate esterases,
at least 9 β-glucosidases, and 7 exocellobiohydrolases (Martinez et al. 2004).
Further, ten lignin peroxidases, five manganese peroxidases, and several other
lignocellulolytic enzymes were encoded by its genome (Martinez et al. 2004).
P. chrysosporium is an excellent decomposer of soft- and hardwood, branches, logs,
leaves, etc. in forests.
The secretomes of a number of other WRF growing in situ or on various sub-
strates under in vitro conditions are now available in the literature. As compared
to P. chrysosporium, which carry out simultaneous degradation of cellulose,
hemicellulose, and lignin, Ceriporiopsis subvermispora exhibit unique and selec-
tive characteristic of lignin removal before initiating the cellulose degradation
(Hori et al. 2014). Chondrostereum purpureum, a basidiomycetous fungus, pro-
duces an extensive repertoire of lignocellulolytic enzymes (Reina et al. 2019).
Almost 50% of CAZy encoded by its genome belongs to GHs (GH5, GH6, GH7,
GH10, GH11, GH12, GH16, GH30), CEs, and cellulose-binding domains (CBMs)
which specifically target cellulose and hemicelluloses. In addition, 153 oxidore-
ductases, lignin-modifying enzymes, and auxillary activity (AA1, AA3) enzymes
are encoded by its genome under variable culture conditions (Reina et al. 2019).
Similarly, several families of CBMs and CEs were found to be encoded by C.
purpureum genome. Further, expression of 81 oxidoreductases was recorded
under substrate-specific culture conditions (Reina et al. 2019). Secretome analysis
of Pleurotus eryngii, an edible mushroom and white rot fungi, revealed the pro-
duction of seven glucanases, cellobiohydrolase, cellulose 1,4-beta-cellobiosidase,
glucosidases, 22 glycoside hydrolase (GH families GH1, GH6, GH12, GH16,
GH17, GH24, GH31, GH32, GH35, GH43, GH44, GH51, GH61, GH74, GH76,
GH78, GH79, GH88, GH92, GH95), and CBM of family 21. These findings con-
clusively established the rich cellulolytic enzymes repertoire in P. eryngii under
different substrate conditions (Xie et al. 2016). In another study, Kuuskeri et al.
(2016) studied the secretory enzyme profile of Phlebia radiata cultured in solid
state on spruce wood. The transcriptomic and secretomic analyses indicated
expression and secretion of oxidoreductase, glyoxal oxidases, alcohol oxidases,
cellobiohydrolases (GH6 and GH7), LPMO (AA9), lignin peroxidases, and acetyl
xylan esterase. Prominent upregulation of genes whose products are involved in
wood decay was observed at different growth periods.
1.4.2 Secretome of Brown Rot Fungi (BRF)
BRF are basidiomycetous fungi which occur as common pests of plants in conifer-
dominated woodlands (Presley and Schilling 2017). BRF decompose wood via
glycoside hydrolase-mediated saccharification and free radical oxidation (Baldrian
and Valaskova 2008). However, compared to WRF, BRF preferentially degrade cel-
lulose and hemicellulose whereas lignin is not depolymerized significantly thus
leaving behind a brownish residue with fragmented appearance (Krah et al. 2018).
Genomic analysis revealed evolution of BRF from WRF by gradual loss of genes
which encode for ligninolytic peroxidases (Januszet al. 2017). Absence of class II
peroxidases and cellobiohydrolases was reported in Wolfiporia cocos (Gaskell et al.
(2016). The important species of BRF are Gloeophyllum trabeum, Postia placenta,
Piptoporus betulinus, Serpula lacrimans, Fomitopsis lilacinogilva, Laetiporus por-
tentosus, Wolfiporia cocos, Fibroporia radiculosa, Dacryopinax primogenitus,
Daedalea quercina, Laetiporus sulphurous, Neolentinus lepideus, Calocera cornea,
Fistulina hepatica, Hydnomerulius pinastri, and Coniophora puteana (Krah et al.
2018; SistaKameshwar and Qin 2018).
Most of BRF are aerobic in nature and contribute a little inside digestive tracts of
herbivorous mammals. WRF vary significantly in terms of substrate specificity and
mechanisms of action. The genomic, phenotypic, and phylogenetic basis of these
variations is yet to be fully understood. Presley and Schilling (2017) studied in vitro
degradation of spruce wafers by two BRF, namely, Serpula lacrymans and
Gloeophyllum trabeum, using a proteomic approach. Upon initial colonization, oxi-
doreductase diversity was observed first followed by higher glycoside hydrolase
activity at later stages. Their findings suggest significant variations in their oxidore-
ductase profiles as indicated by presence of putative copper radical oxidase in S.
lacrymans but absence in G. trabeum. On the other hand, GMC oxidoreductase and
a xyloglucan-specific AA9 family protein were produced by G. trabeum but not by
S. lacrymans. S. lacrymans exhibited higher mannanase activity compared to G.
trabeum which showed elevated xylanase production. Interestingly, GH6 and cel-
lobiohydrolases (CBHs) were not detected in case of S. lacrymans. As compared to
93 proteins identified in S. lacrymans, the protein counts were 209 in G. trabeum.
Overall analysis of their secretomes indicates a two-step brown rot decay mecha-
nism manifested through entirely different biochemical routes. In another BRF spe-
cies Postia placenta from phylum Basidiomycota, 242 putative CAZY-encoding
genes were reported (Martinez et al. 2009). Among these, the number of GHs, CEs,
glycosyltransferases, and polysaccharide lyases were 144, 10, 75, and 6, respec-
tively. In addition, expansin-like proteins, laminarinases, chitinases, endoxylanases,
β-xylosidases, and L-α-arabinofuranosidases were identified. However, the
enzymes/proteins involved in lignin degradation, viz., lignin peroxidase, manga-
nese peroxidase, exocellobiohydrolases, versatile peroxidase, cellulose-binding
domains, and cellulose-binding endoglucanases, were entirely absent. This unique
secretome profile substantiated the non-action of P. placenta against lignin compo-
nents of plant biomass. Gaskell et al. (2016) determined 64 glycoside hydrolases
from Wolfiporia cocos growing on different media containing glucose, purified

crystalline cellulose, lodgepole pine, and aspen. More than fourfold upregulation of
hemicellulase-, endoxylanase-, and chitinase-encoding genes was observed.
Additionally, there was upregulation of genes involved in oxidative depolymeriza-
tion of cellulose.
1.5 Secretome of Anaerobic Rumen Fungi (ARF)
ARF have indispensable role in digestion of recalcitrant lignocellulosic feed materi-

als in digestive system of herbivorous animals. CAZymes in ARF exist either as free
enzymes or as cellulosome, a multiprotein complex (Haitjema et al. 2017). The
genome sequencing of four species of Neocallimastigomycota suggests that many of
these CAZymes have been acquired by horizontal gene transfer from rumen bacteria
(Youssef et al. 2013; Haitjema et al. 2017). The extensive CAZyme repertoire, cel-
lulosome, and extracellular proteases produced by Neocallimastigomycetes may
help these microbes compete with other rumen inhabitants for limited nutrients
(Youssef et al. 2013; Haitjema et al. 2017). As described earlier in Sect. 1.3, only 11
genera have been cultured and exhaustively investigated for secretome analyses.
Due to their strict anaerobic lifestyles, in vitro studies are limited on these fungi.
Still, a number of studies have offered insights in secretome profiles of ARF. Wang
et al. (2011) identified 25 families of glycosyl hydrolases (GHs) from anaerobic
rumen fungus Neocallimastix patriciarum W5 culture anaerobically on substrate
mixture comprising rice straw, napier grass, and sugarcane bagasse. Transcriptome
and secretome analysis revealed 25 putative GH families dominated by GH6 (15%),
GH10 (9.5%), GH5 (9.1%), and GH43 (9.1%). The main CAZymes were cello-
biohydrolase (EC 3.2.1.91), endoglucanase (EC 3.2.1.4), and xylanases. The num-
ber of cellulases and hemicellulases was found to be higher in N. patriciarum W5 as
compared to other plant matter-degrading fungi. The genome sequencing of
Orpinomyces sp. strain C1A by Youssef et al. (2013) revealed an efficient lignocel-
lulolytic enzymes repertoire comprising 357 glycoside hydrolase genes, 92 carbohy-
drate esterases genes, and 24 polysaccharide lyases genes. Interestingly, 220 genes
with fungal dockerin domain and 103 genes harboring carbohydrate-binding module
domains were also identified. Further, expansion of cellulolytic and hemicellulolytic
families, viz., GH6, GH9, Gh10, GH11, Gh43, GH45, and GH48, and reduction or
complete loss of families GH7, GH16, GH18, GH28, and GH61 were also observed
(Youssef et al. 2013). The genes attributing for an efficient and extensive glycoside
hydrolase machinery of this rumen fungus are believed to be acquired through hori-
zontal gene transfer from multiple ruminal bacteria present in rumen. A previous
study also reported presence of cellulase (GH family 48) containing two C-terminal
fungal dockerin domains from Piromyces equi (Steenbakkers et al. 2002).
Kameshwar and Qin (2018) compared the genome-wide annotations of five
ARF, namely, Neocallismatix californiae, Anaeromyces robustus, Orpinomyces sp.,
Piromyces sp. E2, and Piromyces finnis. Findings of this comprehensive analysis
revealed that ARF have the highest number of CAZyme-encoding genes compared
to other fungi. Moreover, the presence of genes for cellulosomes and carbohydrate
transport and metabolism strongly supported their remarkable polysaccharide-
degrading abilities. Surprisingly, the genes encoding for lignin-degrading auxiliary
activity enzymes, such as lignin peroxidase, laccase, manganese peroxidase, versa-
tile peroxidase, aryl alcohol oxidase, glyoxal oxidases, and glucose oxidases, were
completely lacking in their genomes. Among these five ARF species, Neocallimastix
californiae was found to possess the highest number of genes coding for CAZymes
involved in cellulolytic, hemicellulolytic, and pectinolytic activities (Kameshwar
and Qin 2018). A comparative analysis of transcriptome of four ARF, i.e.,
Anaeromyces mucronatus YE505, Neocallimastix frontalis 27, Orpinomyces joyonii
SG4, and Piromyces rhizinflata YM600 revealed that 8.1–11.2% of the entire tran-
scriptome were predicted CAZymes with highest in O. joyonii. About 40–44% of
the CAZymes-encoding contigs had one or more carbohydrate-binding modules
(Gruninger et al. 2018).
1.6 Conclusion
In spite of the availability of enormous amount of plant matter, woody substances,

crop residues, and agro-food industry by-products as an attractive renewable
resource, its industrial-scale utilization remains limited due to inherent structural
complexity and recalcitrance. The controlled decomposition of biomass in general
and of lignocellulose in particular involves a wide diversity of enzymatic activities
and chemical reactions, which are far from fully elucidated. Moreover, our knowl-
edge of the fungal secretion pathway is still at an early stage. Wood-degrading fungi
and anaerobic rumen fungi can accomplish this daunting task with remarkable effi-
ciency in natural environments using their specialized and sophisticated biomolecu-
lar machinery. The fungal secretomes have been explored to find enzymes and
enzyme combinations for paper, textile, and food manufacturing industries.
Similarly, low cost and sustainable processes for plant biomass conversion to biofu-
els can revolutionize industrial and environmental microbiology. The study of sec-
retomes of novel fungal genera/species is interestingly poised to elucidate novel
enzymes suitable for efficient plant cell wall degradation which can be exploited for
commercial biotechnological applications. White rot fungi in particular have tre-
mendous potential for biotechnological applications, bioremediation, pulp and
paper industries, and effluents treatment in different industrial settings. These fungi
possess remarkable potential for implementing eco-friendly, sustainable, and con-
solidated biological processing of lignocellulosic biomass for biofuel and biorefining
industries and bioremediation processes.
Acknowledgments NS is grateful to The Chancellor, Eternal University, for their financial support
and infrastructural facilities. The authors are thankful to Dr. Sumit Singh Dagar for his valuable
and expert suggestions.
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Chapter 2
Bioremediation: New Prospects
for Environmental Cleaning by Fungal
Enzymes
Neha Vishnoi and Sonal Dixit
2.1 Introduction
The planet ‘Earth’ is endowed with rich wealth of natural resources such as forests,
wildlife, land, soil, air, water, wind, plants and animals. One of the major problems
in present scenario faced by the earth is the contamination of soil, water, and air
with toxic chemicals released from numerous human activities. The battle between
human and natural resources started when humans began living a stable life rather
than a nomadic life. The use, overuse, and now the misuse of natural resources since
the beginning of the civilization have led to its depletion to an extent that today half
of our natural wealth are either depleted or at the edge of depletion (Gosavi et al.
2004). In early times, it was believed that our land and its resources are in abun-
dance and will remain available for decades. However, today, existing state of our
resources shows carelessness and negligence in using them. There are various
anthropogenic activities like use of chemical fertilizers in agriculture, release of
industrial waste, burning of fossil fuels, etc. which point toward the exploitation of
natural resources.
Rapid industrialization and the extensive use of pesticides and chemicals in agri-
culture and various fields have been proved to be the major cause of environmental
pollution. The continuous use of organic compounds has become a serious threat to
our eco system. Various industrial and anthropogenic activities have resulted in
increased polluted sites due to ignorance regarding production and proper use and
disposal of hazardous substances. There are various conventional techniques devel-
oped to decontaminate the contaminated sites. Conventional technique mainly
N. Vishnoi
Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University,
Lucknow, India
S. Dixit (*)
Department of Botany, University of Lucknow, Lucknow, India

18 N. Vishnoi and S. Dixit
removes the contaminated soil to a landfill or covers the contaminated sites but this
may create major risks in the excavation, handling, and transport of hazardous
material. At the same time, these methods are quite expensive and difficult to find
an alternative landfill sites for the final disposal of material. Other techniques like
usage of chemicals for decomposition, UV oxidation and dechlorination, and incin-
eration techniques have been employed and found to be effective in reducing the
level of contaminants chlorinated solvents, petroleum, ketones, polyaromatic hydro-
carbons (PAHs), trinitrotoluene (TNT), inorganic nitrogen, explosives, pesticides,
herbicides and heavy metals, but at the same time, they show several demerits such
as technical complexity, lack of public acceptance, and increased contaminants
exposure to site workers and nearby residents (Vidali 2001). Various physicochemi-
cal treatments such as coagulation with alum or lime followed by adsorption on
powdered activated carbon (PAC) are reported to yield high removal efficiency for
phenolics. But, on the other hand, these processes generate large volume of hazard-
ous sludge also and do not lead to the complete degradation of the contaminants
(Mehta and Chavan 2009). To overcome these problems, an alternative method
known as bioremediation has been used to completely remove or transform the
contaminants into some biodegradable forms. In this technique, natural biological
activity has been used to clean environment and its resources by degrading various
contaminants. It is proved to be a safer, cleaner, economical, and eco-friendly tech-
nology which received a high public acceptance and can often be carried out at any
site. According to Van Dillewijn et al. (2007), “bioremediation” is defined as the
process by which various biological agents primarily microorganisms used to
degrade or detoxify environmental contaminants. Bioremediation agents were
defined as microbiological cultures, enzyme, and nutrient additives that signifi-
cantly accelerate the rate of biodegradation to mitigate the effect of various con-
taminants. Bioremediation has been proved to be more advantageous than
conventional methods due to high efficiency, cost-effectiveness, minimal release of
chemical and biological sludge, selectivity to specific metals, regeneration of bio-
sorbent, and the possibility of metal recovery (Kratochvil and Volesky 1998).
A huge number of microbial enzymes and plant enzymes have been notified to
be implicated in the biodegradation of harmful organic contaminants. Several stud-
ies have proved that enzymes from white-rot fungi are effectual degraders of so
many organic pollutants like dyes, PAHs, phenols, polychlorinated biphenyls
(PCBs), and TNT (Yadav et al. 2019a, b). Numerous enzymes such as cellulase,
lipase, and protease prove to have the capability to lessen solid contents and number
of pathogens and enhance deflocculation in the sludge. Enzymes like hydrolases
and oxidoreductases are used widely in bioremediation. Extracellular enzymes are
those which are either secreted by the microorganisms (e.g., lignin peroxidase (LiP)
from white-rot fungi) or which go through the aqueous stage during an aerobic sub-
merged fermentation procedure (Bhargava et al. 2003). The extracellular enzymes
have been used in several industries for various applications, but currently they are
used widely for enhancing bioremediation practices. Each and every enzyme gener-
ally has only one particular function, for example, to lessen the activation energy for
the degradation of an intramolecular bond, but few enzymes can act on wide variety
2 Bioremediation: New Prospects for Environmental Cleaning by Fungal Enzymes 19
of varying substrates (Fullbrook 1996; Gianfreda and Rao 2004). These second
types of enzymes are mainly suitable for bioremediation purposes. These enzymes
are capable to react with xenobiotic and synthetic materials and so convert them
from a recalcitrant state to biodegradable form (Bollag 1992; Gianfreda and Rao
2004). Extracellular enzymes allow more competent treatment practices for biode-
gradable matter because they are capable of accelerating the rate of degradation of
these substances. The application of enzymes is gaining much attention because
enzymes can carry out the equivalent or even better function as compared to harsher
chemicals and they also do not produce any type of hazardous waste. While enzymes
are possibly little expensive because of the associated cost of extraction and purifi-
cation process, they can on the other hand tend to be very cost-effective as they
reduce waste disposal and heating requirements (Godfrey and Reichelt 1996;
Gianfreda and Rao 2004).
Fungus acts as an important agent in bioremediation because of their vigorous
morphology and varied metabolic ability. Bioremediation process by using fungi as
a tool for harmful organic remediation is an eco-friendly and sustainable way for
clean-up of contaminated sites. Profound investigation toward this area would
greatly assist in the development of advanced bioprocess technologies to decrease
the level of contamination as well as to find some novel valuable materials. This
chapter emphasizes on the importance of fungal enzymes for efficient bioremedia-
tion of a large variety of environmental pollutant. We will discuss in detail the mul-
tiple approach used by fungi for detoxification of various noxious and recalcitrant
compounds including prominent fungal enzymes such as oxidoreductase, laccases,
catalases, peroxidases, etc.
2.2 Bioremediation
Bioremediation is considered as one of the safer, cleaner, cost-effective, and eco-

friendly technologies for decontaminating sites which are contaminated with varied
range of pollutants. Various biological processes are involved for the removal/
reduction of hazardous chemicals present in the environment (Kour et al. 2019a, b;
Gianfreda and Rao 2004). The term “bioremediation” refers to all biochemical reac-
tions of natural attenuation which include all biotic and abiotic processes used to
minimize contaminant levels. This practice uses various biological agents such as
higher plants, bacteria, algae, yeast, fungi, and enzymes to degrade environmental
pollutants. Bioremediation can be further divided into phytoremediation and micro-
bial bioremediation (Fig. 2.1). Phytoremediation refers to the in situ utilization of
plants, their enzymes, roots, and associated microorganisms to degrade pollutants
present in soil, water, and air. Microbial bioremediation involves the use of micro-
organisms for conversion and removal of pollutants from various environmental
systems. The technique of bioremediation chiefly relies on microbes producing
enzymes utilized to detoxify the contaminants and changes them to harmless
products. Environmental conditions play a crucial role for microbial bioremediation
BIOREMEDIATION
(clean up of pollutants by living
organisms)
MICROBIAL BIOREMEDIATION PHYTOREMEDIATION

(remediation of pollutants by (remediation of pollutants by
using microbes) using plants)
ENZYMATIC BIOREMEDIATION
(remediation of pollutants by
enzymes secreted by organisms)
Oxidoreductase Lyases Peroxidase Hydrolase Dehalogenase
Fig. 2.1 An overview of types of bioremediation
as optimal conditions are necessary for the growth and vital activities of microor-
ganisms. Effective bioremediation often involves the alteration of environmental
conditions to permit growth of microbes and degradation to carry on at a faster rate.
Bioremediation techniques can be classified as in situ and ex situ on the basis of
removal and transportation of wastes (Fig. 2.2). In situ bioremediation technique
involves treatment of contaminated material at the same site, whereas ex situ
involves complete removal of contaminated material from one site to another site
where it has been treated using biological agents. When both the methods have been
compared, it was found that the rate of biodegradation and consistency of process
outcome differ between in situ and ex situ methods. The cost of ex situ bioremedia-
tion is relatively high as compared to in situ due to the cost of excavation of con-
taminated samples for treatment. Both the bioremediation methods depend
essentially on microbial metabolism, but in situ methods are favored over ex situ for
ecological restoration of contaminated site (Vidali 2001).
Microbes are ubiquitous on the biosphere due to their remarkable metabolic
activity and ability to grow in a diversified environmental condition (Yadav et al.
2015a, b, 2017b). The nutritional versatility of microbes can also be exploited for
breakdown of contaminants. Microorganisms act as an effective tool for pollutant
decontamination in soil, water, and sediments as they are advantageous over other
remediation procedures. They restore the original condition of natural surroundings
and prevent further pollution (Demnerova et al. 2005). Several strains of microbes
Fig. 2.2 An outline of

different methods of Intrinsic Engineered
bioremediation bioremediation bioremediation
In situ
(on site bioremediation)
Bioremediation
Ex situ
(off site bioremediation)
Solid Phase Slurry Phase
are known to act as agents of bioremediation under laboratory conditions. Microbes

can survive in extreme conditions of environment but effective bioremediation with
enzymatic processes usually prefer optimal conditions which are difficult to achieve
outside the laboratory (Bernhard-Reversat and Schwartz 1997; Vidali 2001; Dua
et al. 2002; Dana and Bauder 2011). Optimal conditions can be achieved easily in
ex situ technique than in situ technique. Therefore, in situ bioremediation by
enzymes may be considered as less effective and consumes more time and requires
a high concentration of enzyme when compared with ex situ bioremediation. The
growth of microbes gets affected by numerous factors like pH, temperature, oxygen,
soil structure, moisture, proper amount of nutrients, poor bioavailability of contami-
nants and presence of other toxic compounds. Bioremediation methods usually take
place under aerobic conditions, but anaerobic conditions may also allow degrada-
tion of recalcitrant compounds aided by microbes. Both intracellular and extracel-
lular enzymes produced by various groups of bacteria and fungi participate in the
degradation of xenobiotics (Hammel 1997; Vidali 2001).
The process of bioremediation depends on the metabolic potential of microor-
ganisms, accessibility, and bioavailability of pollutant for removal/detoxification of
pollutant (Antizar-Ladislao et al. 2008). The process of remediation can be acceler-
ated by the addition of various microorganisms (called seeding or inoculation) to a
contaminated site to enhance rate of biodegradation. The inoculums may be a blend
of non-indigenous microbes (specially selected and cultivated for their various
pollutant-degrading characteristics) from various contaminated environments, or it

may be a mixture of microbes selected from the site to be remediated or mass-cultured
in the laboratory. Addition of nutrients along with seeding process accelerates the
process of bioremediation (Boopathy 2000).
With the advances in biotechnology, bioremediation has become one of the sig-
nificant developing fields of ecological restoration that utilizes microorganisms to
minimize the concentration and toxicity of various chemical pollutants such as
heavy metals, dyes, pesticides, etc. Considerable efforts have been devoted for
developing low-cost removal technologies that can effectively immobilize dissolved
toxic metals. Different types of biomass have been tested to remove and/or recover
valuable metals for their possible reuse as reported by several authors (Say et al.
2001; Adhiya et al. 2002; Sheng et al. 2004). A strain of Pseudomonas putida was
registered in 1974 as the first patent agent for biological remediation to degrade
petroleum (Prescott et al. 2002). About 70 microbial genera were reported in 1991
(US Congress 1991) to degrade petroleum compounds and almost an equal number
has been added to the list in the successive two decades (Kumar et al. 2011).
2.2.1 Types of Bioremediation
Bioremediation can occur naturally on its own (natural attenuation or intrinsic bio-
remediation) or can be spurred via addition of fertilizers for the enhancement of
bioavailability within the medium (biostimulation). There are different types of
techniques under bioremediation processes such as biostimulation, bioattenuation,
bioaugmentation, bioventing, bioleaching, bioreactor and piles (Li and Li 2011).
2.2.1.1 Biostimulation
In this type of technique, specific nutrients at the polluted sites are injected to
stimulate the activity of indigenous microbes. It relies upon the stimulation of
indigenous or naturally existing microbial population by supplying fertilizers,
growth supplements and trace minerals and by providing other environmental
requirements like pH, temperature, and oxygen to accelerate their metabolism rate
and pathway (Kumar et al. 2011; Adams et al. 2015). The presence of minimal
amount of pollutant can also behave as stimulant by turning on the operons for
bioremediation enzymes. This type of strategic pathway is mostly continued with
the addition of nutrients and oxygen in order to help indigenous microorganisms.
These nutrients fulfil the nutritional demand of the microbes and are considered as
the basic building blocks of life. They permit microbial growth by providing the
basic requirement like energy, cell biomass, and enzymes to decontaminate the pol-
luted site. They also need some essential components like nitrogen, phosphorous and
carbon (Madhavi and Mohini 2012).
2.2.1.2 Bioattenuation
It is also known as natural attenuation which means elimination of varying concen-

trations of contaminants from surroundings. It is a physical phenomenon and takes
place through biological process via dispersion, dilution, diffusion, volatilization,
and sorption/desorption by chemical reactions like ion exchange, complexation and
abiotic transformation. Natural attenuation can also be termed as intrinsic remedia-
tion or biotransformation (Mulligana and Yong 2004). The nature can perform its
work in four different ways to decontaminate the environment with chemicals. First,
tiny bugs or microbes living in the soil and groundwater utilize some chemicals in
order to fulfil their food requirement. They transform these contaminants into water
and harmless gases on complete digestion. Second, chemicals are held by the soil
through sorption process which restricts their movement and ultimately does not
cause groundwater pollution. This process is not used in cleaning the chemicals but
creating obstruction in the flow of chemicals to other site. Third, when contaminants
move through soil and groundwater, their dilution occurs as it mixes with clean
water. This reduces the level of contamination. Fourth, the pollutants like oil and
various solvents easily convert their state from liquid to gas and are ultimately
destroyed by the sunlight. When the natural attenuation does not work quickly
or completely, then bioremediation can be accelerated by the process of either
biostimulation or bioaugmentation (Li et al. 2010).
2.2.1.3 Bioaugmentation
When natural or engineered population of microorganism responsible for degrada-

tion of contaminants is added to augment or enhance the biodegradative capability
of indigenous of microbes on the contaminated site, the process is known as bioaug-
mentation. The growth and degradation capacity of naturally present microbes can
be increased by their collection from remediation site, then culturing, making
genetic modification, and then returning to the same site. A study by Niu et al.
(2009) suggested that all vital microbes are present in their sites where soil and
groundwater are contaminated with chlorinated ethenes such as tetrachloroethylene
and trichloroethylene which are used to ensure that the in situ microorganisms can
completely convert these contaminants to non-toxic ethylene and chloride.
Bioaugmentation is the process in which engineered microbes are added as a
bioremediator in a system in such a manner so as to completely eradicate complex
pollutants in a fast manner. Genetically engineered microbes (GEMs) having diverse
metabolic profile of detoxification proved to be the suitable candidates for the deg-
radation of a wide range of environmental pollutant effectively (Malik and Ahmed
2012; Alwan et al. 2013; Gomez and Sartaj 2014). Natural species do not degrade
all the compounds quickly, so to facilitate their degradation, species must be modi-
fied at gene level through DNA manipulation. The capacity of degradation in geneti-
cally engineered microbes is more than natural species and they highly compete
with the indigenous species, predators and also with various abiotic factors. Due to
enhanced degradation properties, genetically engineered microbes have shown
potential for bioremediation of soil, groundwater and activated sludge contaminated
with a broad range of toxicants (Sayler and Ripp 2000; Thapa et al. 2012).
Researchers are continuously diverting their focal point toward the ways to augment
contaminated sites with various other non-native microbes especially genetically
modified microorganisms. The recombinant DNA and other molecular biological
techniques have shown certain advantages: (i) enabled amplification, disruption,
and modification of the targeted genes that encode the enzymes of metabolic path-
ways, (ii) minimized bottlenecks pathway, (iii) enhanced redox and energy genera-
tion, and (iv) played important role in recruiting heterologous genes to give new
characteristics (Liu et al. 2006). It can be said that the process of bioaugmentation
will open a new series of possibilities for future process of bioremediation.
Genetically engineered microorganisms (GEMs) are those whose genetic mate-
rial has been altered by using various techniques of genetic engineering inspired by
natural or artificial genetic exchange between microbes. This process is known as
recombinant DNA technology. Recombinant DNA techniques or natural genetic
material exchange between organisms resulted in the development of recombinant
living organisms. In the present scenario, scientists are successful in inserting the
suitable gene for the production of particular pollutant-degrading enzyme (Jain
et al. 2010). Recently, a number of opportunities come forward for the improvement
of degradation ability using genetic engineering strategies. The rate-limiting steps
in metabolic pathways can be increased and altered or new pathways incorporated
in microbial strains to enhance their degradation capacities for xenobiotics. Four
strategies are followed in genetic engineering microbes: (1) modification of enzyme
specificity and affinity, (2) pathway construction and regulation, (3) bioprocess
development, monitoring, and control, and (4) bioaffinity bioreporter sensor appli-
cations for chemical sensing, toxicity reduction, and end-point analysis. Vital genes
of bacteria are carried on a single chromosome but genes for enzymes which are
needed for the catabolism of some of the contaminants may be carried on plasmids.
Plasmids have been implicated in the catabolism. Therefore, GEMs can be used
efficiently for biodegradation process to optimize the eradication of hazardous
unwanted wastes, which symbolizes a research frontier with broad implications in
the coming future (Jain et al. 2011; Kulshreshtha 2013).
The major advantage of GEMs is that they accelerate the recovery of contami-
nated sites, increase substrate degradation, display a high catalytic or utilization
capacity with small amount of cell innoculum, and create eco-friendly environmen-
tal conditions by the mechanism of detoxification. The major drawbacks of using
genetic engineered microbes are that they are never used in traditional procedure
and the risk associated with the release of dead cellular mass. Biotic and abiotic
factors are directly correlated and also play significant role in the functioning of
microbes. Introduction of foreign modified strain to the system leads to deleterious
effects on the original structural composition, occurrence, and functional properties
of microbes.
2.2.1.4 Bioventing
Bioventing is involved in venting of oxygen through soil to stimulate growth of natu-

ral or introduced bacteria and fungus in the soil by providing oxygen to existing soil
microorganisms; indeed, it is functional in aerobically degradable compounds.
Bioventing uses low airflow rates to provide only enough oxygen to sustain microbial
activity. Oxygen is most commonly supplied through direct air injection into residual
contamination in soil by means of wells. Adsorbed fuel residuals and volatile
compounds are biodegraded which thereafter move slowly through biologically
active soil. Some researchers like Agarry and Latinwo (2015) proved bioventing to
be an effective technique in bioremediation of petroleum-contaminated soil.
2.2.1.5 Biopiles
In this technique, soil is excavated and then spread in the form of piles. Biopiles are
also known with other names such as biocells, bioheaps, biomounds, and compost
piles which are employed for reducing petroleum contaminants in excavated soils
during the course of biodegradation. In this technique, air is supplied to the biopile
system during a system of piping and pumps that either forces air into the pile under
positive pressure or draws air through the pile under negative pressure (Delille et al.
2008). The microbial activity is enhanced through microbial respiration which
results in the accelerated degradation of adsorbed petroleum contaminants (Emami
et al. 2012; Kumar et al. 2016).
2.2.2 Limitations of Bioremediation
Bioremediation is restricted to only those compounds that are degraded by biologi-

cal means and not to those which cannot be subjected to degradation. Important
concern should be taken to check the toxicity and persistent nature of products
synthesized during biodegradation. The biological methods are extremely specific
with culture requirements and at times are difficult to extrapolate the results from
lab to field. The time duration for these methods is comparatively more than for
excavation treatment. There are various factors affecting the process of bioremedia-
tion such as depletion of preferential substrates, lack of nutrients, toxicity and solu-
bility of contaminants, oxidation or reduction potential, and microbial interaction.
The outcome of each degradation process depends on microbes (biomass concentra-
tion, population diversity, and enzyme activities), substrate (physicochemical char-
acteristics, molecular structure, and concentration), and a range of environmental
factors (pH, temperature, moisture content, availability of electron acceptors, and
carbon and energy sources). These parameters affect the acclimation period of
microbes to the substrate. The molecular structure and contaminant concentration
have been shown to strongly affect the feasibility of bioremediation. The type of
microbial transformation depends on whether the compound serves as a primary,

secondary, or co-metabolic substrate (Boopathy 2000). All the contaminants are not
easily treated, accumulated, or degraded by bioremediation using microorganisms,
and the effects of microorganisms on the metal leaching associated with phytoreme-
diation have yet not received proper attention (Neagoe et al. 2009).
2.3 Fungi as Bioremediator
Mycoremediation or fungal remediation is a type of bioremediation which uses

fungi to remove the pollutants from the environment. Mycoremediation utilizes fun-
gal mycelium to expel the waste material from the contaminated site. Fungal colo-
nization can occur in a variety of habitats, i.e., fresh water and marine with complex
soil lattice. Fungi can flourish in varying extreme climatic conditions and germinate
through the dispersal of its spores in the atmosphere (Anastasi et al. 2013). There
are several reports which demonstrate that some fungi also grow well in industrial
effluent treatment plants (Zhang et al. 2013; Badia-Fabregat et al. 2015). Their
potential to survive in diverse habitats and secreting multiple enzymes enable them
to be the best candidate for bioremediation. The fungal mycelia secrete different
types of extracellular enzymes and acids that are utilized to convert the lignin and
cellulose into simpler forms.
Fungal species are specific for a particular pollutant which is an important
feature for fungal remediation. White-rot fungi like Phanaerochaete chrysosporium
and Polyporus sp. which are also known as ligninolytic fungi are employed for the
removal of numerous persistent or toxic environmental contaminants such as petro-
leum hydrocarbons, PAHs, explosives, PCBs, and organochlorine pesticides and
thus prove to be the best candidate for bioremediation (Wu and Yu 2007; Ayu et al.
2011). They are reported for the degradation of numerous organopollutants as
they secrete an enzyme which acts on lignin. White-rot fungi secrete extracellular
enzymes during the decaying process of lignin so they have been proved to be the
potential agents for oxidizing pollutants. A bird’s nest ligninolytic fungus known as
Cyathus bulleri which colonizes on dead herbaceous stems, wood chips, dung,
sticks, and other woody debris are found cost-effectively appropriate for the degra-
dation of lignin as it produces a single laccase, an internal peptide which shows
resemblance with enzyme laccase of white-rot fungi with differences in propor-
tions of some basic and hydrophobic amino acids.
Laccases are copper-containing enzymes using molecular oxygen as the electron
acceptor for catalyzing the oxidation of a varying range of phenolic and aromatic
amine compounds (Garg et al. 2008). Many workers have showed that the fungi
belonging to these groups were utilized for the oxidation of lignin which shows
structural similarity with PAH compounds by the secretion of extracellular enzymes
via mineralization with carbon dioxide as end product (Levin et al. 2003; Mai et al.
2004). The process of degradation is not yet decisively mapped but is expected that
intracellular and extracellular enzymes are responsible for oxidizing different
p ollutants under different environmental conditions. Fungal enzymes have also

been utilized for the degradation and decolorization of azo dyes (Vidali 2001;
Husain 2006).
Metals like uranium, cadmium, zinc, copper, and cobalt are biosorbed by
microbes through electrostatic interactions between the metal ions present in
solutions and cell walls (Bai and Abraham 2001). Chromium biosorption in fungi
like Ganoderma lucidum and Aspergillus niger takes place through ion exchange
mechanism (Muraleedharan and Venkobachar 1990; Ahluwalia and Goyal 2006).
Pleurotus ostreatus (tasty oyster mushroom) exhibit a broad range of applications in
bioremediation. A report by Favero et al. (1991) concluded that it is used in reme-
diation of soil contaminated with diesel oil and can be used in the conversion of
95% PAH to non-toxic components. Fungi which act as wood degraders are particu-
larly effective in degrading aromatic pollutants and chlorinated compounds (Arıca
et al. 2004). It is found that the indigenous microbe also participates with the fungi
for the mineralization of pollutants into carbon dioxide and water.
Yeasts possess the property to resist under unfavorable environmental conditions
and can accumulate metals and metalloids which are vital for structural and cata-
lytic functions (Chatterjee et al. 2011). Some examples of yeast species possessing
degrading capabilities are proved to be suitable representatives of remediation of
pollutants from environment: Candida, Clavispora, Debaryomyces, Leucosporidium,
Pichia, Rhodosporidium, Rhodotorula, Sporidiobolus, Sporobolomyces,
Stephanoascus, Trichosporon, and Yarrowia (Csutak et al. 2010). Dye decoloriza-
tion from industrial effluents by yeasts mainly occurs through three mechanisms:
biosorption, bioaccumulation, and biodegradation (Donmez 2002). Candida utilis
accumulates metal ions and radionuclides from the environment (Kujan et al. 2006).
Bioadsorption of lead and accumulation of free and complexed silver ions in yeast
Rhodotorula mucilaginosa has been achieved by metabolism-dependent and
metabolism-independent processes (Gomes et al. 2002). A comparative study was
conducted by Ksheminska et al. (2003) in which Pichia guilliermondii was used to
check uptake potential of chromium and it was concluded that chromium tolerance
increases on addition of riboflavin.
2.3.1 White-Rot Fungi
These are considered as important agents for biodegradation of lignin-containing

material in environment and therefore also contribute largely to the carbon recy-
cling. Previous studies showed the bioremediation prospective of white-rot fungi
like Bjerkandera adjustam, P. chysosporium, Pleurotus sp., and Trametes versicolor
in the degradation of recalcitrant compounds by secreting various ligninolytic
enzymes like laccases and peroxidases, which results in bioaccumulation, toxicity
to aquatic life, and deleterious effects to human beings (dos Santos Bazanella et al.
2013). White-rot fungi secrete ligninolytic enzymes that have been utilized for the
detoxification of a range of organic contaminants like pesticides from polluted
wastewaters (Rodríguez-Rodríguez et al. 2013). Extracellular ligninolytic enzymes

of fungi are also capable for the adsorption of various dyes. This property of white-
rot fungi established them as a governing power in the field of dye removal or decol-
orization as confirmed by the decolorization of Direct Blue 14 by different species
of Pleurotus (Singh et al. 2013a) and Remazol Brilliant Blue-R by Agaricomycete
(a white-rot fungus from Amazon forest) (dos Santos et al. 2015). Various fungal
species like Coriolus versicolor, Hirschioporus larincinus, Inonotus hispidus, P.
chrysosporium, and Phlebia tremellosa have been previously examined for the
decolorization of different dye effluents (Jebapriya and Gnanadoss 2013).
White-rot fungi like Lentinus tigrinus and T. versicolor were reported for the
decontamination of soil contaminated with cresolate via. bioaugmentation process
(Lladó et al. 2013). Residues of petroleum hydrocarbons and PAHs with high
molecular weight are mainly responsible for the pollution of cresolate soil. This
problem of petroleum residue contamination can be solved by significant degrada-
tion of these residues through biostimulation by means of lignocellulosic substrate
together with bioaugmentation of fungi. However, there is a usual chance with this
treatment that it may support the growth and intensification of local microorganisms
which can rule over the augmented microbes. This can pressurize the requirement
to authenticate such kind of study at a small level prior to the field applications.
Besides these applications of ligninolytic enzymes for bioremediation of a range of
substances, laccases have also been utilized for the degradation of substituted
organic compounds with improved removal efficiencies (Cutright and Erdem 2012;
Fan et al. 2013; Purnomo et al. 2013). Considering the significance of such features
in bioremediation, laccase production was maximized in T. versicolor and P. ostrea-
tus by solid-state fermentation on orange peels. Further examination of bioremedia-
tion capabilities of these white-rot fungi revealed enhanced potential for PAHs such
as phenanthrene and pyrene removal (Rosales et al. 2013). For an improved percep-
tive and utilization of bioremediation potential of fungi completely, genomic-level
research of these fungi is needed.
2.3.2 Marine Fungi
The application of marine fungi in bioremediation of heavy metals and hydrocar-

bons along with their capability to produce biosurfactants, secondary metabolites,
polyunsaturated fatty acids, polysaccharides, and novel enzymes are well acknowl-
edged a long time ago (Damare et al. 2012). The adaptation ability of marine fungi
toward extremophilic conditions like high salinity and pH variations proved an
advantage in comparison to terrestrial fungal species. The effectiveness of marine
microorganism in metal removal provides an additional opportunity of using these
extremophilic microbes for bioremediation purposes. Thatoi et al. (2013) examined
the application of mangroves marine fungus for their enormous ecological and bio-
technological functions like production of novel enzymes, drugs, biopesticides, bio-
diesel and their potential use in bioremediation. Numerous aspects related to fungi
have been examined to increase the bioremediation potential of fungi for removal of
hazardous and persistent organic contaminants.
The noteworthy role of enzymes obtained from marine fungi and its biotechno-
logical significance were demonstrated by Bonugli-Santos et al. (2015).
Bioremediation properties of enzymes of marine basidiomycetes obtained from
marine sponges and Cerrena unicolor (marine white-rot basidiomycete) were suc-
cessfully examined for decolorization of dyes Remazol Brilliant Blue-R and anthra-
quinone Reactive Blue 4, respectively (Bonugli-Santos et al. 2012). Divya et al.
(2013) documented distinctive feature of marine fungi Trichoderma viride Pers
NFCCI-2745, which was isolated from a phenolic-polluted estuary, for production
of salinity- and phenol-tolerant laccase enzyme. Biotransformation of persistent
organic contaminants like PCB 118 by marine fungi Penicillium sp. and Trichoderma
harzianum can be affected by biostimulation and bioaugmentation process (Verma
et al. 2012; Gao et al. 2013; Vacondio et al. 2015). Several other marine-originated
fungi like Aspergillus, Penicillium, and Mucor and slime mold also showed effec-
tive bioremediation properties in removal of crude oil; however, higher concentra-
tion of crude oil was observed to be toxic for fungi (Hickey 2013).
2.3.3 Extremophilic Fungi
Microbes colonizing extreme environments are very crucial for various industrial
applications as they produce extremophilic enzymes which depict several unique
properties such as tolerance against temperature, pH and other extreme environ-
mental conditions (Yadav 2018, 2019; Yadav et al. 2016, 2017b; Neifar et al. 2015).
Fungi showed tolerance against high level of toxicants present in the effluent treat-
ment plant and utilized for numerous bioremediation applications. These properties
make them potent candidates for economical, eco-friendly bioconversions of raw
materials into non-toxic forms used in food industries, leather processing, textiles
manufacture, and animal feed preparation (Nigam 2013). Microbes have been
employed for the removal of heavy metal contaminants from environment.
Biosynthesis of nanoparticles provides a safer pathway for metal removal (Sinha
et al. 2014). A psychrophilic fungus named Cryptococcus sp. isolated from deep-
sea sediments showed tolerance and growth in presence of elevated levels of heavy
metals concentration up to 100 mg/L ZnSO4, CuSO4, Pb(CH3COO)2, and CdCl2
(Singh et al. 2013b).
Many hydrolytic enzymes were reported which showed activity under extremo-
philic conditions such as high salinity and crude oil concentration which is a result
of drilling waste from oil belts (Yadav et al. 2017a, 2018). Laccases were observed
to be responsible for bioremediation activity in Pestalotiopsis palmarum in the pres-
ence of wheat bran and production of lignin peroxidases occurred when heavy crude
oil was present in high concentration and it was utilized by fungi as the sole carbon
and energy source (Betancor et al. 2013; Naranjo-Briceno et al. 2013). On the other
hand, enzymes like chitinases synthesized by a psychrophilic fungus named
Lecanicillium muscarium was employed for increasing the activity of insecticides

which showed capability to act on insect chitin exoskeleton (Li et al. 2013;
Narayanan et al. 2013). Not only literature about the bioremediation studies by
extremophilic fungi is important but their isolation from extreme habitats such as a
deep biosphere represented by fumarolic ice caves on Antartica’s Mt. Erebus can
also be used for identifying unique properties of fungi capable of utilizing energy
sources other than photosynthesis and also provides information about possible
anthropogenic sources of contamination of such extreme places (Connel and
Staudigel 2013).
2.3.4 Symbiotic Fungi with Plants
Fungi are known to build mutual and close association with plants in order to rise
above the barrier of limited growth under varied environmental conditions. A sym-
biotic relationship between plants and fungi is represented by arbuscular mycor-
rhizal fungi (AMF) (Yadav et al. 2019a). In this association, fungus accelerates the
degradation of pollutants by providing higher surface area for absorption through its
hyphae and spores and thereby converts the pollutants into mobile form and binds
to the root. Fester (2013) observed AMF colonization in the root sample of plants
which are utilized for phytoremediation of polluted groundwater in a constructed
wetland. Numerous arbuscular mycorrhizal fungi such as Aspergillus nidulans,
Bjerkandera adusta, Trametes hirsuta, T. viride, Funalia trogii, Irpex lacteus, and P.
ostreatus could thrive in the toxic environment of dyes and were employed for
decolorization of textile industry effluents (Tegli et al. 2013). Arbuscular mycor-
rhizal fungus named Rhizophagus custos showed elevated level of tolerance against
anthracene, a polyaromatic hydrocarbon, and converts it to the less toxic anthraqui-
none by-product (Aranda et al. 2013). Quinoa plants showed enhanced uptake of
137 Cesium on loamy soil after being inoculated with a commercial AM product
which also represents mycorrhizal association due to root colonization (Vinichuk
et al. 2013).
Ectomycorrhizal fungi such as Suillus bovinus and Rhizopogon roseolus repre-
senting association with Pinus have been reported for the removal of calcium from
the surroundings. Environmental factors such as pH, type of nutrient and tempera-
ture also play a crucial role in the decontamination process (Sousa et al. 2014).
Other uses of such fungi have been targeted at overcoming technical barriers of
algal bio-fuels and photosynthetic biorefineries by cocultivation of microalgae and
fungi for the complete removal of single algal cells from fermentation medium. This
allowed their extraction and harvesting in a simple manner by simple filtration with
result of increased biomass, lipids, and bio-product yields (Xie et al. 2013). There
are several advantages of coculture studies for bioremediation, but their applications
are complex and require immense understanding about the interaction between
diversified metabolic pathways from different organisms.
2.4 Fungal Enzymes in Bioremediation
Common enzymes of fungal origin used in bioremediation include amylases, cata-

lases, cellulases, laccases, lipases, peroxidases, proteases, xylanases, etc. which
have potential industrial applications and are used in managing organic waste
(Marco et al. 2013). These fungal enzymes may be utilized for hydrolysis of poly-
meric materials, for example, cellulose, protein, lipid, starch, and xylan from
organic wastes such as kitchen waste, vegetable market waste, leaf, litter, etc. The
hydrolyzed substances can be further used for composting or for manufacture of
significant products such as volatile fatty acids and biogas (Khardenavis et al. 2013;
Hattori et al. 2015). The group of white-rot fungi synthesizes various types of ligni-
nolytic enzymes which rely on the fungal species and environmental conditions,
which cannot only be used for lignin degradation of ligno-cellulosic materials but
also in the remediation of different recalcitrant pollutants including dyes. These
enzymes alter azo dye configuration by damaging the chromophoric linkages which
lead to the synthesis of phenoxyl radicals in the reactions (Gül and Dönmez 2013).
Ligninolytic enzymes produced by the group of white-rot fungi have been divided
into two groups: peroxidases (lignin peroxidases and manganese peroxidases) and
laccases (Jebapriya and Gnanadoss 2013; He et al. 2015).
Several enzymes secreted by white-rot basidiomycetes such as laccases and
some peroxidases are very well known for the degradation of persistent organic
xenobiotics (Ikehata 2015). Such enzymes are also secreted by extremophilic fungi
which can be used for remediation of pollutants under extreme conditions like high
salinity, extra-heavy crude oil contamination, etc. Currently, much attention has
been given on the modification of enzymes from white-rot fungi through recombi-
nant gene expression techniques and protein engineering process, which can be
used effectively and eco-friendly for remediation of toxic pollutants (Fonseca et al.
2013; Sakaki et al. 2013; Syed et al. 2013; Wong et al. 2013). An overview of com-
mon fungal enzymes used for bioremediation of different pollutants is given in
Table 2.1.
2.4.1 Enzymological Background
Enzymes are biological moieties which act as a catalyst and modify a reaction’s rate
by providing suitable conditions which reduce the activation energy of the reaction,
without being used with substrates of the reaction. Enzymes are formed naturally by
almost every known organism to help in the processes such as cell synthesis, diges-
tion, and metabolism (Madigan et al. 2003). An enzyme might comprise a protein
or a glycoprotein and a polypeptide moiety. The segments of an enzyme which are
directly involved with the catalytic process are known as active sites (Fig. 2.3).
An enzyme may have other groups associated with the active sites through either
covalent or noncovalent bonds which are crucial for catalytic activity. The protein
Table 2.1 An overview of the potential of common fungal enzymes for bioremediation
SN Enzyme Compound Fungi References
1. Lignin Pyrene Phanerochaete Hammel (1997)
Peroxidase chrysosporium
(LiP)
Aminodinitrotoluenes P. chrysosporium Cameron et al.
(2000)
Bentazon (herbicide) P. chrysosporium Castillo et al.
(2000)
Trichlorophenol Cotiolos versicolor, Leontievsky
Panus tigrinus et al. (2000)
Poly aromatic hydrocarbon Pleurotus pulmonarius Lau et al. (2003)
(PAH)
Delor 106 [Polychlorinated Trametes versicolor Novotny et al.
biphenyl (PCB)] (2004)
Phencyclidine (PCP) T. versicolor, Trametes Ford et al.
sp. (2007)
Remazol Brilliant Blue R Irpex lacteus Novotny et al.
(2004)
PAH P. chrysosporium Wang et al.
(2009)
2. Manganese Trinitrotoluene (TNT) Nematolloma frowardii Scheibner and
Peroxidase Stropharia Hofrichler
(MnP) rugosoannulata (1998)
Trichlorophenol C. versicolor, Leontievsky
P. tigrinus et al. (2000)
TNT P. chrysosporium Cameron et al.
(2000)
PAH P. pulmonarius Lau et al. (2003)
Delor 106 (PCB) T. versicolor Novotny et al.
(2004)
Remazol Brilliant Blue R I. lacteus Novotny et al.
(2004)
PCP T. versicolor, Ford et al.
Trametes sp. (2007)
PAH Bjercandera adusta Valentin et al.
(2007)
Di(2-ethylhexyl) phthalate, P. pulmonarius Chiu et al.
heavy metals, total petroleum (2009)
hydrocarbons
Bentazon P. chrysosporium Castillo et al.
(2000)
PAH P. chrysosporium Wang et al.
(2009)
Reactive black 5, Veratryl Ceriporiopsis Fernández-
alcohol subvermispora Fueyo et al.
(2014)
(continued)
Table 2.1 (continued)

3. Laccase 2,6 dimethoxy phenol Pleurotus ostriatus Hublik and
Schinner (2000)
Phenolic compounds C. versicolor Davis and Burns
(1992)
Trichlorophenol C. versicolor, Leontievsky
P. tigrinus et al. (2000)
2,4-dichlorophenol, C. versicolor Ullah et al.
pentachlorophenol (2000)
Bromophenol blue, Pycnoporus sanguineus Mayer and
Malachite green, Staples (2002)
Orange G,
Amalanth
TNT Trametes villosa Wang et al.
(2002)
PAH P. pulmonarius Lau et al. (2003)
Anthracene and benzo pyrene T. versicolor Dodor et al.
(2004)
4- hydroxybiphenyl T. versicolor, Keum and Li
Pleurotus ostriatus (2004)
Delor 106 (PCB) T. versicolor Novotny et al.
(2004)
Remazol brilliant blue R I. lacteus Novotny et al.
(2004)
PCP T. versicolor, Ford et al.
Trametes sp. (2007)
Atrazine (herbicide) T. versicolor Bastos and
Magan (2009)
Di(2-ethylhexyl) phthalate, P. pulmonarius Chiu et al.
heavy metals, total petroleum (2009)
hydrocarbons
Flexographic inks T. villosa, Fillat et al.
Coriolopsis rigida, (2012)
Pycnoporus coccineus
Myceliopthora
thermophila
Pesticides P. chrysosporium, dos Santos
T. versicolor, Bazanella et al.
Pleurotus sp., (2013)
B. adjusta
Phenols T. versicolor Margot et al.
(2013)
PCB Doratomyces nanus, Mouhamadou
D. purpureofuscus, et al. (2013)
D. verrucisporus,
Myceliophthora
thermophila, Phoma
eupyrena,
Thermoascus crustaceus
(continued)

Phenylurea herbicide diuron Mortierella Ellegaard-Jensen
et al. (2013)
Gasoline Exophiala xenobiotica Isola et al.
(2013)
Anthracene Armillaria sp. Hadibarata et al.
(2013)
Naphthalene Pleurotus eryngii Hadibarata et al.
(2013)
Organic pollutants White-rot Ikehata (2015)
basidiomycetes
4. Lipase PAHs Aspergillus sp., Lladó et al.
Curvularia sp., (2013)
Drechslera sp., Chang et al.
Fusarium sp., (2015)
Lasiodiplodia sp., Balaji et al.
Mucor sp., (2014)
Rhizopus sp.,
Tricoderma sp.
5. Catalase Heavy metals Aspergillus sp., Akhtar et al.
Curvularia sp., (2013)
Acrimonium sp.,
Pythyme sp.
A. flavus Kurniati et al.
(2014)
Malondialdehyde A. foetidus Chakraborty
et al. 2013
Heavy metals Penicillium sp. Deshmukh et al.
Rhizopus sp. (2016)
6. Peroxidase Textile dye decolorization A. niger, Jebapriya and
A. foetidus, Gnanadoss
T. viride, (2013)
A. sojae,
Geotrichum candidium,
Penicillium sp.,
Pycnoporus
cinnabarinus
Trichoderma sp.
Dye decolorization White-rot fungi Ma et al. (2014)
B. adusta,
Ceriporia
metamorphosa,
Ganoderma sp.
Pesticides P. chrysosporium, dos Santos
T. versicolor, Bazanella et al.
Pleurotus sp., (2013)
B. adjusta
Organic pollutants White-rot Ikehata (2015)
basidiomycetes
Complex of
enzyme &
substrate
Substrate
Active sites
Reaction
products
Enzyme
Enzyme
Fig. 2.3 A schematic diagram showing mechanism of enzymatic reactions
or glycoprotein part in an enzyme is known as the apoenzyme, and the nonprotein

moiety is called the prosthetic group. The combination of both apoenzyme and
prosthetic group forms the holoenzyme.
Enzyme names are given according to the role of the enzyme and type of the
reaction catalyzed (Lehninger et al. 2004). The identification of a particular enzyme
is possible through its enzyme commission (E.C.) number. The assignment of E.C.
numbers is described in guidelines set out by the International Union of Biochemistry.
All known enzymes fall into one of these six categories. The six main divisions are
(1) the oxidoreductases, (2) the transferases, (3) the hydrolases, (4) the lyases, (5)
the isomerases, and (6) the ligases (synthetases). Oxidoreductases catalyze the
transfer of electrons and protons from a donor to an acceptor. Transferases catalyze
the transfer of a functional group from a donor to an acceptor. Hydrolases facilitate
the cleavage of C–C, C–O, C–N and other bonds by water. Lyases catalyze the
cleavage of these same bonds by elimination, leaving double bonds (or, in the
reverse mode, catalyze the addition of groups across double bonds). Isomerases
facilitate geometric or structural rearrangements or isomerizations. Finally, ligases
catalyze the joining of two molecules (Lehninger et al. 2004).
2.4.1.1 Oxidoreductases
The oxidoreductase enzyme facilitates detoxification of organic pollutants by dif-

ferent fungi via oxidative coupling mechanism (Gianfreda et al. 1999). Fungal
enzymes cleave the chemical bonds of substrates through biochemical reactions
releasing energy and assist the transfer of electrons from a reduced organic substrate
to another chemical compound which acts as an acceptor. The contaminants are
finally oxidized to non-toxic compounds during the process of oxidation coupled
with reduction. Oxidoreductases through the process of detoxification convert toxic
xenobiotics into harmless compounds. Xenobiotics like phenolic or anilinic com-
pounds get transformed into non-toxic form through the process of polymerization,
copolymerization, or binding to humic entities (Park et al. 2006). Oxygenases rep-
resent the oxidoreductase group of enzymes. Organic compounds mainly of haloge-
nated nature like herbicides, insecticides, fungicides, plasticizers and intermediates
used for the synthesis of chemicals consist the largest groups of environmental pol-
lutants ubiquitous in nature. These pollutants are degraded by specific oxygenases.
The dehalogenation reactions of halogenated methanes, ethanes, and ethylenes are
mediated by oxygenases in combination with multifunctional enzymes (Fetzner and
Lingens 1994).
Chlorinated phenolic compounds are considered to be the most abundant recal-
citrant wastes found in the effluents generated by the paper and pulp industry. It
generates mainly recalcitrant wastes in their effluent. The partial degradation of
lignin during pulp bleaching process results in the formation of these recalcitrant
compounds. Fungal populations are considered to be appropriate candidate for the
decontamination of sites contaminated with chlorinated phenolic compounds. The
production of extracellular oxidoreductase enzymes like laccase, manganese per-
oxidase and lignin peroxidase by the action of fungi is useful for bioremediation.
Filamentous fungi penetrate the soil pollutants more efficiently than bacteria
(Rubilar et al. 2008).
2.4.1.2 Laccases
Laccases represent a group of copper-based enzymes synthesized by fungi. They

utilize copper as a cofactor and molecular oxygen as cosubstrate which further gets
reduced to water during oxidation process of xenobiotics (Gianfreda et al. 1999;
Mai et al. 2000). Laccases are known to occur in multiple. Various isoenzyme forms
of laccases have been reported. Each enzyme is coded by an individual gene. The
expression of gene depends on the nature of inducer (Giardina et al. 1995; Rezende
et al. 2005). Most of the phenolic, non-phenolic, and aromatic compounds are oxi-
dized by laccases and an increase in their activity about 20 times was observed in
the fungi like T. versicolor (Margot et al. 2013). Both intracellular and extracellular
laccases were produced by many genera of fungi which catalyze the oxidation of
ortho and paradiphenols, aminophenols, polyphenols, polyamines, lignins, aryl
diamines, and some inorganic ions (Mai et al. 2000; Ullah et al. 2000; Rodríguez
Couto and Toca Herrera 2006). In spite of oxidation of phenolic and methoxyphe-
nolic acids, laccase also performs the function of decarboxylation and demethyl-
ation. These enzymes participate in the depolymerization of many aromatic
compounds. This enables them to be potent candidates to function as a biocatalyst
in the production of numerous dyes and colorants, thereby reducing the cost and
energy expenditure in their synthesis with no residual material (Polak and Jarosz-
Wilkolazka 2012). These compounds are also utilized by microbes as a nutrient
source or repolymerized to humic materials by laccase (Kim et al. 2002). Laccases
signify an appealing group of ubiquitous, oxidoreductase enzymes among biologi-
cal agents contributing great prospective in biotechnological and bioremediation
applications (Gianfreda et al. 1999).
Their non-specific property for varying range of substrates ultimately makes
them ideal catalyst for numerous industrial applications. Enzymes have been
explored to be used extensively for their efficient bioremediation potential
(Vishwanath et al. 2014). Fillat et al. (2012) demonstrated deinking process in
printed paper on recycling by the enzyme laccases produced by the three fungi
belonging to group basidiomycetes (Trametes villosa, Coriolopsis rigida,
Pycnoporus coccineus) and other to the ascomycetes (Myceliopthora thermophila)
in the presence of synthetic mediators. Due to the synthetic origin of the textile
dyes, they remain unaltered in the environment on exposure to sunlight, water, and
other external factors and thus subsequently impart toxicity to the ecosystem.
Fungal marine laccases were used for the color removal, detoxification, and miner-
alization of Reactive Blue 4 dye in the studies of Verma et al. (2012) and Vishwanath
et al. (2014) for the first time. An endocrine disrupting chemical named Bisphenol
A is degraded by laccase isolated from Fusarium incarnatum as reported by Chhaya
and Gupte (2013). Laccases were found to be produced at low temperature in
Himalayan region by some extremophilic fungi like Penicillium pinophilum (Dhakar
et al. 2014). Less literature is available about the mechanism of laccase under
extreme conditions. However, only few laccases from fungus belonging to the group
ascomycetes like Melanocarpus albomyces and Thielavia arenaria were explored
with crystal nature. They differ from other fungus by having conserved ‘C-terminal
plug’ most likely used in proton transfer processes (Kallio et al. 2011). Changes in
pH can be directly correlated with the substrate specificity and enzyme affinity. An
inhibition in the production of laccase was observed by the presence of various
chemicals like halides, azides, cyanides, and hydroxide (Xu 1996). The synthesis of
laccase in fungi is sensitive to the concentration of nitrogen. High levels of nitrogen
are directly proportional to the production of recombinant. It can be produced by
either homologous or heterologous means (Gianfreda et al. 1999). Laccases show
remarkable potential in bioremediation, but their utilization is limited due to their
low shelf-life. Immobilization or tailoring of enzymes is considered as an appropri-
ate method in order to overcome this problem and enhancing their efficiency (Mate
et al. 2011; Torres-Salas et al. 2013; Patel et al. 2014).
2.4.1.3 Catalase
The accumulation of reactive oxygen species (ROS) in biological systems results in

the damage at cellular level. These species cause deleterious effects to cellular mac-
romolecules. Fungi produce monofunctional catalases and bifunctional peroxidase/
catalase enzymes for defense mechanism at primary level in order to cope up with
the toxicity of reactive oxygen species. Lindane inhibits the production of catalase
and thus accelerates the generation of reactive oxygen species which ultimately
declines the growth of Saccharomyces cerevisiae (Pita et al. 2013). The production
of reactive oxygen species in microbes has been induced by the presence of several
heavy metals like lead, copper, cadmium, zinc, etc. Several studies have shown that
presence of heavy metals on the generation of reactive oxygen species is directly
correlated, and it leads to the production of anti-oxidative enzymes. Studies of
Chakraborty et al. (2013) demonstrated that fungus Aspergillus foetidus showed
tolerance against 200 mg/L concentration of Pb (II) by the production of anti-
oxidative enzymes including laccase for the detoxification of malondialdehyde and
hydrogen peroxide. A study conducted by Mitra et al. (2014) showed that an increase
in tolerance level of Aspergillus sp. against oxidative stress induced by the presence
of varying concentrations of heavy metals such as 100 mg/L Cu(II) and 750 mg/L
Zn(II) is achieved by the production of catalase. Complete literature about the effect
of heavy metals on fungal physiology is not yet known. An inhibition in catalase
and peroxidase activity and increase in cytochrome P450 (CYP450) activity were
observed in the case of P. chrysosporium when exposed to 50–100 μM concentra-
tion of cadmium or lead (Zhang et al. 2015). An increase in the catalase activity was
observed in the case of fungal consortia A. niger, Penicillium sp., and Rhizopus sp.
when exposed to heavy metals like lead and copper at 50 mg/L (Thippeswamy et al.
2014). In a study by Lin et al. (2009), it was demonstrated that catalase activity
could be employed effectively for monitoring bioremediation potential of fungus,
and it decreases with increase in the oil concentration in sites contaminated with oil
residues. Catalase proved to be an effective tool for providing tolerance capacity to
fungi for decontamination of contaminated sites. Thus fungi producing enzymes
play a significant role in the bioremediation of contaminated sites with metals.
2.4.1.4 Peroxidases
Peroxidases catalyze the oxidation of lignin and other phenolic compounds at the
expense of hydrogen peroxide in the presence of a mediator. These peroxidases
belong to proteins of heme and nonheme nature. The heme peroxidases are divided
into two broad groups. First group is found in animals, plants, fungi, and prokary-
otes while the second group peroxidases have been subdivided into three distinct
classes on the basis of sequence comparison. Class I represents enzymes of intracel-
lular nature including yeast cytochrome c peroxidase, ascorbate peroxidase from
plant origin, and gene-duplicated catalase peroxidases from bacteria. Class II repre-
sents fungal-secreted heme peroxidases from P. chrysosporium like lignin
peroxidases and manganese peroxidases. Lignin peroxidases and manganese per-

oxidases were reported for the utilization of hydrogen peroxidase and manganese in
carrying out their vital activities. These enzymes were reported for the degradation
of recalcitrant toxic compounds by the group of white-rot and basidiomycetes fungi.
Versatile peroxidase enzymes are specific for a broad range of substrates. They oxi-
dize both phenolic and non-phenolic compounds and thus they are highly efficient
for bioremediation (Karigar and Rao 2011). Degradation of lignin in wood was
reported by the class II peroxidases. Class III represents the secretory plant peroxi-
dases such as those from horseradish, barley, or soybean. These peroxidases behave
as a biosynthetic enzyme involved in the plant cell wall formation and lignification
processes (Hiner et al. 2002; Koua et al. 2009). Non-heme peroxidases are not evo-
lutionarily linked and form five independent families. They include thiol peroxi-
dases, alkyl hydroperoxidases, non-heme haloperoxidase, manganese catalase, and
NADH peroxidases. Thiol peroxidases represent the largest family when compared
with others. They also include two subfamilies such as glutathione peroxidases and
peroxy redoxins (Koua et al. 2009).
Another group of heme peroxidases were reported as dye-decolorizing peroxi-
dases (DyPs) and unspecific peroxygenases (UPO) which utilize hydrogen peroxide
for catalyzing oxidations of varying non-phenolic lignin compounds and other
organic compounds. This group does not fit in the above classification system (Liers
et al. 2013; Strittmatter et al. 2013; Hofrichter and Ullrich 2014). One of such per-
oxidases produced by B. adusta was declared for the disruption of the phthalocya-
nine ring in phthalocyanine dyes. This has been achieved by the cleavage of azo
bond and ultimately leading to the decolorization of azo and phthalocyanine dyes.
Five fungal dye-decolorizing peroxidases of high redox nature were demonstrated
by Liers et al. (2013) which possess catalytic properties of both lignin peroxidases
and versatile peroxidases as confirmed from their capability to oxidize non-phenolic
aromatic compounds and Reactive Black B. The study highlighted the need to fur-
ther distinguish peroxidase activities in crude enzyme mixtures of fungi posing dif-
ficulty in classifying the dye-decolorizing peroxidases from lignin peroxidases and
versatile peroxidases. Such type of classification based on catalytic specificity was
suggested only after the refinement of the different enzymes.
2.4.1.4.1 Lignin Peroxidases
They belong to the group of heme proteins secreted mainly by the members of
white-rot fungi during their secondary metabolism. These enzymes degrade lignin
and other phenolic compounds in the presence of cosubstrate H2O2 and veratryl
alcohol which acts as a mediator. During the course of reaction, H2O2 gains elec-
trons from lignin peroxidases and thus reduced to H2O. The oxidized form of lignin
peroxidases gained electron from veratryl alcohol and thereafter returns to its origi-
nal reduced state with the formation of veratryl aldehyde. It gets reduced back to
veratryl alcohol by gaining an electron from substrate. This ultimately results in the
oxidation of halogenated phenolic compounds, polycyclic aromatic compounds and
other aromatic compounds followed by a series of nonenzymatic reactions (Yoshida

1998; Ten Have and Teunissen 2001). Lignin peroxidases play a crucial role in the
biodegradation of lignin which is a constituent of the plant cell. It also oxidizes
aromatic compounds with redox potentials more than 1.4 V (NHE) by the single-
electron abstraction mechanism. The exact redox mechanism is still not properly
understood (Piontek et al. 2001).
2.4.1.4.2 Manganese Peroxidases
They are an heme group of enzymes produced extracellularly from the basidiomy-
cetes group of fungus responsible for the degradation of lignin which oxidizes Mn2+
to Mn3+ in a multistep manner. Mn2+ initiates the production of manganese peroxi-
dases and also acts as a substrate for it. Manganese peroxidases generate Mn3+
which plays an important role in the oxidation of varying phenolic compounds by
acting as a mediator (Ten Have and Teunissen 2001). Recently, Ceriporiopsis sub-
vermispora was used for the production of enzyme manganese peroxidases which
were modified through genetic manipulation to increase the stability even at acidic
pH 2. The acid-stable nature of Mn2+ and its high oxidizing activity were confirmed
by analyzing its crystal structure as a scaffold. An engineered enzyme of stable
nature was found for oxidizing Reactive Black 5 and veratryl alcohol (Fernández-
Fueyo et al. 2014).
2.4.1.4.3 Versatile Peroxidases
Versatile peroxidase enzymes are found capable of oxidizing Mn2+, methoxyben-

zenes, and phenolic aromatic substrates. Versatile peroxidases are specific for a
broad range of substrates. They oxidize substrates in the absence of manganese
when compared with other group of peroxidases. Both phenolic and non-phenolic
lignin model dimmers were found to be oxidized by VP (Ruiz-Dueñas et al. 2007).
A highly well-organized versatile peroxidase production system in excess was for-
mulated for various biotechnological applications in industries and removal of
xenobiotic compounds of recalcitrant nature from the environment (Mai et al. 2000;
Tsukihara et al. 2006).
2.4.2 Advantages of Enzymatic Bioremediation
Enzymes are adapted to act in a varied range of environmental conditions with-

out alteration in their active nature (Ahuja et al. 2004; Gianfreda and Rao 2004).
An example depicts that protease enzyme is capable of functioning in the pH
range 4–11 but the experiment retards at pH 11. The enzyme was found still to be
active at this moment with temperature lesser than 20 °C and greater than 70 °C
(Whiteley et al. 2002). Enzymes are found to work effectively in diverse environ-
ments especially when they present in immobilized form. This property makes the
enzymes more opposed to hard environmental conditions and can be recycled and
recovered when they are not needed (Gianfreda and Rao 2004). Several workers
found that xenobiotics like phenol, PCBs, herbicides and dyes were degraded by the
laccase (Ullah et al. 2000; Dodor et al. 2004; Novotný et al. 1997; Mougin et al.
2000; Mayer and Staples 2002). Enzymatic degradation of several xenobiotic com-
pounds was found to be a feasible method, but at the same time, the rate of reaction
for each compound should be minimized for a system (Alvarez-Cohen and Speitel
Jr 2001). Enzymatic treatment of contaminated site is an eco-friendly process over
conventional methods. These procedures are relatively time-consuming in compari-
son to conventional methods without imparting deleterious effect on the environ-
ment. Enzymes are proteins with degradable nature, i.e., when they are not recovered,
they are degraded in the nature without leaving any harmful residues. So, enzymatic
processes are found to be an attractive alternative for the decontamination of
contaminated sites.
2.4.3 Disadvantages of Enzymes
Although enzymatic technology is very promising, it has limitations. Microbes can

reproduce and increase their population in order to consume a large amount of sub-
strate, but extracellular enzymes cannot. Enzymes cannot reproduce themselves,
meaning that any increase in enzyme population must come from outside of the
system (i.e., humans adding more enzymes to the system). It has been noticed that
enzymes lose their reactive property after their interaction with the pollutants and
thus get converted into inactive entities. (Gianfreda and Rao 2004). The concentra-
tion of enzyme is dependent on the reaction rate so their concentrations must be
checked and controlled properly so as to optimize enzyme kinetics for site-specific
situations. Enzymes do not possess the reproductive nature so they are not able to
adapt themselves as microbes during mutation. Mutations allow microorganisms to
withstand harsh conditions and able to metabolize new substrates (Madigan et al.
2003). Enzymes can withstand varying range of environmental conditions only
within their limits of adaptation. The main drawback of utilizing extracellular
enzymes for bioremediation is their high costs (Duran and Esposito 2000). The cost
of producing enzymes employed for bioremediation in pure state is relatively high
as purity directly correlates with their specific functions and showing no adverse
effects. The cost of synthesis of enzyme solutions in crude form is comparatively
cheaper, but it tends to show side effects (Fullbrook 1996). The reduction of produc-
tion expenditure can be achieved by the selection of cheaper substrates for the
growth of microbes in the production of enzymes. The focus of advancement in
technology is in the process for the replication of bacteria and fungi used for the
synthesis of enzymes (Ahuja et al. 2004; Gianfreda and Rao 2004).
2.4.4 Scope of Enzymatic Bioremediation
For bioremediation of various pollutants, fungi have now been proved to play a
noteworthy role. Variety of toxic pollutants, for instance, dyes, pesticides, pulp and
paper industry effluents, persistent organic pollutants (POPs), PAHs, petroleum
hydrocarbons, leather tanning effluents, etc., can be degraded by using different
fungal enzymes. Various fungi like Acrimonium, Aspergillus, and Curvularia were
reported for metal tolerance and metal removal ability (Akhtar et al. 2013). Few
basidiomycetes such as T. versicolor and white-rot fungi P. ostreatus have been
studied for degradation of PAHs in solid-state fermentation during growth phase on
agro-industrial wastes (Rosales et al. 2013). Pollutant from coffee industries like
coffee pulp can also be remediated by using fungi such as Aspergillus restrictus,
Chrysosporium keratinophilum, Fusarium solani, Gliocladium roseum, Penicillium
and Stemphylium under controlled environmental conditions with additional nutri-
ents input (Nayak et al. 2013). A variety of fungi of different groups including
white-rot fungi, Aspergillus, Penicillium, etc. have been reported to degrade and
decolorize diversified pollutants from various industries like sugar mill, textile dye,
paper and pulp industry, and leather tanning industry, which indicate that fungi can
be used for a range of substrates (Bennett et al. 2013; Buvaneswari et al. 2013;
Duarte et al. 2013; Jebapriya and Gnanadoss 2013; Reya et al. 2013; Huang et al.
2014). Bioremediation of PAHs in presence of A. niger and P. chrysosporium
revealed considerable removal of petroleum hydrocarbons from petrol- and diesel-
polluted soil (Maruthi et al. 2013). Some studies showed the complete remediation
of pesticides such as chlorpyrifos and also its metabolite 3,5,6-trichloro-2-pyridi-
nol (TCP) from contaminated soils by using fungi A. niger JAS1 without any sup-
plementary nutrient input (Silambarasan and Abraham 2013). The degradation of
metabolite TCP by chlorpyrifos-degrading fungal strain was a considerable finding
in view of the antimicrobial property and catabolite repression character showed
by TCP.
2.5 Conclusion and Future Prospects
Though there have been several reports on the bioremediation potential of fungal
species, profound evaluation of the versatile function of fungi in remediation of
xenobiotic compounds suggesting the descriptive mechanism of fungi for attaining
this task is missing. In the present chapter, we have tried to elaborate the role of
fungal enzymes in bioremediation of synthetic compounds with special prominence
to organic pollutant. Besides enzymes like peroxidases and laccases, some stress
response proteins like ABC transporters also play an active role in the removal of
toxic contaminants in fungi, and there is a demand for exploring these genes for
further applications. Thus, a necessity arises to search new techniques such as
genetically modified microorganisms or making consortia by combining plants,
bacteria and fungi for providing appealing opportunities in bioremediation technique.

A continuous investigation for the new biological form is required for proper man-
agement of increasing pollution and contamination. Therefore, bioremediation is still
considered as a developing technology to regulate the day-to-day environmental
problems faced by humans residing in an area.
Acknowledgments The author Sonal Dixit acknowledges DSKPDF Cell, Pune, India, and
University Grant Commission, New Delhi, India, for the financial assistance in the form of
D.S. Kothari Postdoctoral Fellowship (F4-2/2006 (BSR)/BL/15-16/0156). There are no conflicts
of interest among authors.
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Chapter 3
Genetic Diversity of Methylotrophic Yeast
and Their Impact on Environments
Manish Kumar, Raghvendra Saxena, Pankaj Kumar Rai,

Rajesh Singh Tomar, Neelam Yadav, Kusam Lata Rana,
Divjot Kour, and Ajar Nath Yadav
3.1 Introduction
In the environment, abundant reduced C1-compounds are available. Reduced car-

bon compounds, such as methane and methanol, utilised by methylotrophs, which
have the ability to utilise C1-compounds as the sole source of carbon and energy,
also appear to be cosmopolitan in nature. There is a remarkable difference between
prokaryotic methylotrophs and eukaryotic methylotrophs. Pokaryotic methylo-
trophs utilize carbon substrate like methanol, methane and methylamine while
methanol is used as carbon substrate and methylamine as nitrogen source by eukary-
otic methylotrophs. Methylotrophic yeast comprises genera like Pichia, candida and
some other related genera close to Pichia i.e. Kuraishia, Ogataea and Komagataella
(Yurimoto et al. 2011).
Different lineages of methylotrophic yeast utilising methanol as sole source of
carbon and energy were documented and described (de Koning and Harder 1992).
The similar metabolic pathway for methanol utilisation was followed by all methy-
lotrophic yeasts, and they composed of several enzymes localised in peroxisomes
which proliferate during growth of yeast in methanol (Veenhuis et al. 1992; Yurimoto
M. Kumar · R. Saxena · R. S. Tomar

Amity Institute of Biotechnology, Amity University, Gwalior, India
P. K. Rai
Department of Biotechnology, Invertis University, Bareilly, Uttar Pradesh, India
N. Yadav
Gopi Nath P.G. College, Veer Bahadur Singh Purvanchal University,
Deoli-Salamatpur, Ghazipur, Uttar Pradesh, India
K. L. Rana · D. Kour · A. N. Yadav (*)
Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib,
Sirmour, Himachal Pradesh, India
e-mail: ajar@eternaluniversity.edu.in

54 M. Kumar et al.
et al. 2002). The methylotrophic yeast with regulated promoters of methanol

oxidising genes is reported to be used in the study of production of recombinant
proteins as well as industrial proteins (Ravin et al. 2013). The diversity of methylo-
trophic yeast involved in glycerol metabolism was also discussed in one of the
earlier reports in which methylotrophic strains such as Candida boidinii no. 2201,
Hansenula ofunaensis and Hansenula polymorpha DL-1 were identified with
specific enzyme activity of glycerol kinase (GK), glycerol dehydrogenase (GDH)
and both GK and GDH, respectively (Tani and Yamada 1987).
Methanol is considered as a very recent alternative carbon source that replaces the
petroleum and coal (Olah 2005). It is considered that methanol is formed with the
combination of CO and H2 or from CO2 with the help of H2 gas. Methane, an abun-
dant natural carbon substrate produces CO and H2. Since methanol is a cheaper
substrate, it can be used as feedstock for different biochemical and biotechnological
processes. Methane oxidizing bacterial communities are responsible for release of
methanol from methane and further triggers the decomposition of lignins and pectins
containg methylester and methoxyl groups respectively (Mitsui et al. 2003; Nakagawa
et al. 2000). Methylotrophs and methylotrophic yeasts oxodise CO2.
Thus, methylotrophs play indispensable roles in the global carbon cycle between
methane and CO2 called “the methane cycle”. A thorough understanding of the
molecular basis of methylotrophy is needed not only to better understand the global
methane cycle but also to permit more efficient use of methanol as a renewable
carbon source. In the recycling of carbon in the environment, methylotrophic yeast
plays a very crucial role. The ability of C. Boidinii to grow over pectin as a substrate
shows its methylotrophic metabolism (Nakagawa et al. 2000). The intensive
researches explain the beneficial relationship between plants and methylotrophic
microbial communities (Kumar et al. 2019; Meena et al. 2012; Verma et al. 2013,
2014, 2015a, b, 2016a, b; Yadav 2009). Moreover, the interaction between plants
and methylotrophic yeasts has not been characterised and very less report or docu-
mentation is available (Limtong et al. 2005; van der Klei et al. 2006). Therefore,
keeping in view the importance of the methylotrophic yeast in the agriculture,
industry and environments, the present chapter deals with the impact of methylotro-
phic yeast in environments and describes recent insights into the molecular basis of
yeast methylotrophy.
3.2 Genetic Diversity of Methylotrophic Yeast
Yeasts are well known for their beneficial activity for humankind by their exploita-
tion and application in the food, beverages and in the production of various types of
biochemicals. Since they contain a significant content of vitamin B, amino acids and
minerals, they can be used as a food supplement also. Moreover, methylotrophic
yeast can be used in the gene regulation study in eukaryotes and as biofactories for
heterologous and homologous proteins (Cremata and Díaz 1999; Negruţă et al.
2010) (Table 3.1). This group of yeast is able to survive by metabolising
3 Genetic Diversity of Methylotrophic Yeast and Their Impact on Environments 55
Table 3.1 Details of various strains of methylotrophic yeast and their genetic diversity
Gene for identification/
Methylotrophic yeast functionality/phylogeny Isolation source Reference
Candida D1/D2 region of LSU rRNA YM agar, 2% Suh and Zhou (2010)
parapolymorpha gene malt agar,
Candida rishirensis D1/D2 domain of LSU rRNA Soil of Rishiri Nakase et al. (2010)
gene island
Hansenula Formate dehydrogenase Methanol-grown Ravin et al. (2013)
polymorpha yeast cells
Hansenula Dihydroxyacetone synthase Methanol-grown Ravin et al. (2013)
Hansenula Formaldehyde dehydrogenase Methanol-grown Ravin et al. (2013)
Hansenula Whole genome sequence Methanol- and Ravin et al. (2013)
polymorpha DL1 analysis Glucose-grown
yeast cells
Hansenula HpELO1, a fatty acid elongase – Prasitchoke et al.
polymorpha gene (2007)
Kluyveromyces D1/D2 domain of LSU rRNA – Negruţă et al. (2010)
gene
Komagataella phaffii Methanol-inducible gene Culture media Ohsawa et al. (2018)
expression with methanol
Komagataella phaffii KpMit1 transcription factor Culture media Leão-Helder et al.
gene expression with methanol (2003)
Meyerozyma candida D1/D2 domain of LSU rRNA – Negruţă et al. (2010)
gene
Ogataea angusta SSU rRNA gene, internal YM agar, 2% Suh and Zhou (2010)
transcribed spacers (ITS) malt agar,
including 5.8S rRNA gene, cornmeal agar,
and the D1/D2 region of LSU and V8 juice
rRNA gene agar
Ogataea D1/D2 domains of the Soil and tree Limtong et al. (2008)
chonburiensis large-subunit rDNA sequence exudates
Ogataea D1/D2 large-subunit ribosomal From tree Morais et al. (2004)
falcaomoraisii DNA exudates
Ogataea glucozyma, SSU and LLU rRNA – Naumov et al. (2018)
Ogataea haglerorum D1/D2 LSU rRNA gene, Naumov et al. (2017)
sp. ITS1-5.8S-ITS2, and
translation elongation
factor-1α (EF-1 α)
Ogataea henricii, SSU and LLU rRNA – Naumov et al. (2018)
Ogataea minuta SSU and LLU rRNA – Naumov et al. (2018)
Ogataea minuta, SSU and LLU rRNA – Naumov et al. (2018)
Ogataea D1/D2 domains of the Soil and tree Limtong et al. (2008)
nakhonphanomensis large-subunit rDNA sequence exudates
Ogataea D1/D2 large-subunit ribosomal Leaf and rotten Péter et al. (2008)
nitratoaversa DNA and ITS sequence wood
(continued)
56 M. Kumar et al.

Gene for identification/
Methylotrophic yeast functionality/phylogeny Isolation source Reference
Ogataea SSU and LLU rRNA – Naumov et al. (2018)
philodendra,
Ogataea polymorpha, SSU and LLU rRNA – Naumov et al. (2018)
Ogataea Gene encoding Hac1 Grown in Phithakrotchanakoon
thermomethanolica transcription factor culture media et al. (2018)
TBRC656
Ogataea Novel gene expression based – Puseenam et al.
thermomethanolica on maltase (mal) gene (2018)
TBRC656
Ogataea, D1/D2 domain of LSU rRNA – Negruţă et al. (2010)
Millerozyma gene
Pichia pastoris DAS (Dihydroxyacetone – Ahmad et al. (2014)
synthase)
Pichia pastoris FLD1 (Formaldehyde – Ahmad et al. (2014)
dehydrogenase)
Pichia pastoris THL1 (Thiamine biosynthesis – Ahmad et al. (2014)
gene)
Pichia pastoris ADH1 (Alcohol – Ahmad et al. (2014)
dehydrogenase)
Pichia pastoris AOX1 gene (Alcohol – Ahmad et al. (2014)
dehydrogenase)
Pichia pastoris ICL1 (isocitrate lyase) – Ahmad et al. (2014)
Pichia pastoris Used as recombinant gene Methanol-grown Young et al. (2012)
expression system yeast cells
Pichia pastoris AOX1 and AOX2 genes – Cereghino and Cregg
encoding alcohol oxidase (2000)
Pichia sp. D1/D2 domain of LSU rRNA – Negruţă et al. (2010)
gene
Pichia sp. N002 D1/D2 domain of LSU rRNA Soil Limtong et al. (2005)
gene
Pichia sp. N069 D1/D2 domain of LSU rRNA Soil Limtong et al. (2005)
gene
Pichia sp. PT31T D1/D2 domain of LSU rRNA Soil Limtong et al. (2005)
gene
Sacharomyces sp. D1/D2 domain of LSU rRNA – Negruţă et al. (2010)
gene
Trichosporon Ribosomal DNA-based Oil- Kaszycki et al. (2006)
cutaneum characterisation contaminated
soil
monocarbonic compounds such as formaldehyde and methanol (Kaszycki et al.

2001). Methylotrophic yeasts are able to grow on extract of woods and other pectic
material especially in fruits and vegetable products (Craveri et al. 1976). The woody
materials are the source of methanol because of the presence of metoxi chain in
Fig. 3.1 Illustration of methylotrophic yeast diversity based on genes involved in their metabo-
lism and physiology
lignin. Figure 3.1 illustrates the diversity of methylotrophic yeast based on genes
involved in their metabolism and physiology.
Two methylotrophic yeast strains H. polymorpha and P. pastoris were utilised in
the production of heterologous proteins. Apart from this, H. polymorpha is used for
studying gene regulation of enzymes associated with abiotic stress tolerance, meth-
anol metabolism, heavy metal resistance and nitrate assimilation. They are widely
used in the methanol-contaminated waste water treatment also (Kaszycki and
Koloczek 2002; Kaszycki et al. 2001). The methylotrophic yeast has the ability to
grow in extreme environment also. In one of the investigations, three novel strains
of thermotolerant methylotrophic yeast, which belong to genus Pichia, were
reported. The methylotrophic strains were designated as N002, N069 and
PT31T. The Pichia strains were isolated from soil (taken from Thailand) enriched
with methanol. Thermotolerant yeast is found to grow at 20 °C (minimum temp.)
but no limit for the maximum temperature for growth (Arthur and Watson 1976).
According to this definition, methylotrophic yeast growing at 20 °C up to a temp. of
37 °C will be called as a thermotolerant methylotrophic yeast (Limtong et al. 2005).
The presence of helmet-/hat-shaped ascospores, multilateral budding, ubiquinon
58 M. Kumar et al.
Q-7 and negative for Diazonium blue B and urease reactions are the basic character-
istics of genus Pichia. They do not have ballistospore and arthrospores also. The
phylogenetic analysis based on D1/D2 domain of large subunit rDNA revealed the
closeness with Pichia dorogensis. Because of the differences in phenotypic appear-
ance, the above three strains were designated as novel species of Pichia and the
name proposed was Pichia thermomethanolica sp. nov. The type strain is PT31T
(Limtong et al. 2005).
In a report, whole genome sequencing of a methylotrophic yeast H. polymorpha
was performed and total transcripts were analysed from the yeast culture grown in
methanol and glucose as well. A total of 9 Mb size of genome was sequenced for
Hansenula polymorpha DL1.
In a transcriptome analysis of H. polymorpha under methanol growth condition,
40% genome expression has shown the identified unregulated and abundant gene
expression through RNA-seq analysis along with alternate splicing events. From
seven chromosomes of H. polymorpha, different proteins of subtelomeric region
were identified and the evolutionary relationship established revealed the closeness
of H. polymorpha with both non-methylotrophic and methylotrophic yeast Dekkera
bruxellensis and Pichia pastoris respectively. In the investigation, phylogenetic
analysis indicated the methylotrophic evolutionary pattern in filamentous fungi and
yeasts (Ravin et al. 2013).
Evolutionary analysis based on methanol-utilising pathway genes evaluated the
relatedness of methylotrophic yeasts using NCBI nucleotide database and associ-
ated tools. Different genes involved in methanol utilisation pathway such as AOX
(alcohol oxidase), DAS (dihydroxyacetone synthase), FDH (format dehydrogenase)
and DAK (dihydroxyacetone kinase) were identified against BLAST searches.
Using fast minimum evolution algorithm, phylogenetic tree was prepared to see the
evolutionary relatedness. The phylogenetic analysis based on these genes and
MEGA software revealed the position of methylotrophic yeasts (Okonechnikov
et al. 2012).
Methylotrophic yeast was reported to be a suitable expression system also. To
enhance the yields of complex proteins having unnatural amino acids, a recombi-
nant gene expression system was developed in methylotrophic yeast Pichia pasto-
ris. In the investigation, it was emphasised that by modulating aaRS level, the
optimization of expression of unnatural amino acids in the methylotrophic host cell
was done and better than as reported earlier in Saccharomyces cerevisiae (Young
et al. 2012). Earlier, S. cerevisiae was considered to be specific for unnatural amino
acids, but in this investigation, it was explained that a mutant of recombinant human
serum albumin with p-phenylalanine is expressed efficiently in the methylotrophic
yeast system and therefore allows the higher production of complex proteins whose
gene expression is difficult in the existing systems (Young et al. 2012).
In a study, the alcohol oxidase activity was analysed in Pichia pastoris on the
basis of two genes AOX1 and AOX2 and their expression analysis in the cell
(Tschopp et al. 1987). The AOX1 gene expression was observed undetectable when
cells are grown in the media with carbon sources other than methanol (Cregg et al.
1989). In a study, the genes encoding polyunsaturated fatty acids are targeted for the
identification and phylogenetic analysis of methylotrophic yeast Hansenula poly-

morpha. In the investigation, gene encoding fatty acid elongase, HpELO1, was iden-
tified and characterised. The HpELO1 gene encoding a protein has 319 long amino
acids, and it contains 5 different conserved membrane-spanning regions in yeast Elo
protein family. The phylogenetic analysis based on amino acid sequences revealed
that HpELO1 gene is an orthologue of S. cerevisiae ELO3. ELO3 gene is responsible
for the elongation of VLCFAs (very long chain fatty acids) (Fang et al. 2017; Hong
et al. 2019; Prasitchoke et al. 2007; Řezanka et al. 2018).
To see the clear classification and taxonomy of Hansenula (Ogataea) polymor-
pha and other related species, the phylogenetic relatedness was observed based on
conserved gene sequences (Suh and Zhou 2010; Yoo et al. 2019). The phylogenetic
analysis was done based on ribosomal gene sequences of ITS and D1/D2 region of
LSU rRNA gene, and this molecular analysis revealed that most of the O. Polymorpha
strains were different phylogenetically from type strain of Pichia angusta (ATCC
14755), Ogataea thermophila was found to be evolutionarily related to
O. Polymorpha and two novel strains of Candida (ATCC 26012 and ATCC 58401)
were close to O. polymorpha. The character-based method was applied to construct
the phylogenetic tree. The maximum likelihood-based and parsimonious trees were
constructed by taking sequences of ITS and D1/D2 region of LSU rRNA gene (>1
Kb). The result showed the close evolutionary relatedness of O. angusta and C.
parapolymorpha with O. polymorpha along with some significant evolutionary dis-
tances in the tree constructed. A very closest matching and relatedness was observed
in case of O. thermophila and O. polymorpha with 100% bootstrap value and there-
fore were grouped in the same clade (Suh and Zhou 2010).
The SSU (small subunit) and LSU (large subunit) ribosomal gene sequence-based
study revealed that five different methylotrophic yeast species of Ogataea genus,
that is, O. henricii, O. philodendra, O. glucozyma, O. minuta and O. polymorpha
were reported earlier but a variety of Ogataea minuta var. nonfermentas was dis-
tantly related with genus Pichia (Kurtzman et al. 2008; Naumov et al. 2018; Yamada
et al. 1994). Thereafter, O. minuta var. nonfermentas was reclassified separately as
O. nonfermentas (Kurtzman and Robnett 2010). The multigene-based identifica-
tion and phylogenetic analysis illustrated more than 37 species of methylotrophic
yeast and recently 67 species of Ogataea genus were reported (Kurtzman 2009; Lu
et al. 2017). The multigene analysis involved different types of genes for the study
such as LSU rRNA, SSU rRNA, elongation factor EF-1α and mitochondrial SSU
rRNA gene. The multigene analysis revealed differentiation among genera such as
Pachysolen, Nakazawaea, Ambrosiozyma, Komagataella, Phaffomyces and Ogataea
(Kurtzman and Robnett 2010). The pioneer of yeast molecular phylogeny,
K.P. Kurtzman, described the phylogenetic classification of yeasts based on multi-
ple genes and evolutionary relationship was observed.
In one of the investigations, a total of thirteen methylotrophic yeast strains utilis-
ing methanol as a carbon substrate (forming ascospore) were isolated and identified
from the sap exudates of a tree Sclerobium sp. from Costa Rica and Brazil. Their
characterisation for the identification and phylogenetic study was based on the
sequence analysis of D1/D2 large subunit rDNA. The ribosomal gene sequence
60 M. Kumar et al.
analysis and neighbour joining method of phylogenetic tree construction revealed

their identification as predominant species of Ogataea genus (syn. Pichia) and
Candida sp. Later, few new isolates were identified as Ogataea falcaomoraisii
(Morais et al. 2004).
Three methylotrophic yeast strains were isolated from leaf and rotten wood sam-
ples of temperate forest in Hungary. The strains were found to be nitrate negative
and assimilating methanol as a source of carbon. The D1/D2 large subunit rRNA
gene sequence analysis grouped these strains in a clade of Ogataea sp. The strains
have similar sequences and differ from genetically related and close species Pichia
pilisensis. A novel methylotrophic yeast species Ogataea nitratoaversa was pro-
posed to accommodate these nitrate-negative yeast strains. The variation in the D1/
D2 and ITS sequences was observed due to several substitutions. Since the investi-
gation does not allow the inclusion of nitrate-negative strains, they were named as
Ogataea yamada, maeda and mikata (Péter et al. 2008).
In one of the earlier studies, two novel thermotolerant methylotrophic yeast
strains were reported from soil and tree exudates from Thailand. The biochemical
and phenotypic characterisation included the nitrate assimilation, hat-shaped asco-
spore formation, ubiquinone study, multilateral budding, urease reaction and other
observations to identify the strains. The sequence analysis of D1/D2 rRNA gene
revealed the phylogenetic relatedness between the species. The sequence analysis
justifies two different strains PT44T and S051T. The PT44T strain was close to Pichia
(Ogataea) dorogensis, whereas S051T strain was closely related to Pichia thermo-
methanolica with some nucleotide substitutions in the phylogenetic tree constructed.
The biochemical, molecular, physiological and phenotypic characterisation of the
strains proved them novel strains of genus Ogataea and further proposed with the
name of Ogataea chonburiensis sp. nov. and Ogataea nakhonphanomensis sp. nov.,
respectively. Moreover, thermotolerant Pichia siamensis was renamed as Ogataea
siamensis and Pichia thermomethanolica was renamed as Ogataea thermometha-
nolica in this study (Limtong et al. 2005).
3.3 Genetic Regulation in Yeast Methylotrophy
A number of methylotrophic yeast strains are reported and characterised by the

identification of C1 carbon substrate-inducible gene expression analysis. For the
yeast methylotrophy, different essential enzymes such as AOX and DAS and others
are required to carry out formaldehyde oxidation metabolic pathway (Nakagawa
et al. 1999; Sakai et al. 1998; Yurimoto et al. 2011). Molecular mechanism of
methanol-inducible gene expression is represented during growth on various carbon
sources (Fig. 3.2). Genes responsible for carbon substrate metabolism in methylo-
trophic yeast were investigated in C. boidinii by cloning the genes coding methanol-
metabolising enzymes (Yurimoto et al. 2002). Figure 3.2 represents the methanol
metabolism in methylotrophic yeasts (Fig. 3.3).
Fig. 3.2 Molecular mechanism of methanol-inducible gene expression. (a) Relative expression
levels of H. polymorpha MOX (encoding AOD), C. boidinii AOD1, and C. boidinii DAS1 during
growth on various carbon sources. On glucose-containing media, expression is completely
repressed. When glucose is completely consumed or cells are shifted to glycerol medium, a dere-
pressed level of expression of the AOD genes is induced (derepression) and the extent of derepres-
sion of the AOD genes differs between H. polymorpha and C. boidinii. When cells are grown on
methanol, the maximum level of expression of AOD genes is achieved not only by derepression but
also by methanol-specific gene activation. The induction of DAS1 on methanol medium is achieved
only by methanol-specific gene activation. (b) During growth on glucose, expression of methanol-
inducible genes is repressed. When cells are shifted to methanol, initially, a Trm2p-related dere-
pression event occurs followed by a Trm1p-related methanol-specific gene activation. (Adapted
from Yurimoto et al. (2011))
For the heterologous protein expression and production, Pichia pastoris strain is
generally preferred. In a study, the gene copy number was determined for P. pastoris
along with real-time PCR assay for the quantification of integrated expression cas-
settes (Abad et al. 2010). In yeast methylotrophy, the expression of genes involved
in methanol metabolism is regulated by the presence of carbon source. The expres-
sion and repression of genes were studied by Ohsawa et al. (2018) in which it was
62 M. Kumar et al.
CH3OH
Cytosol
NAD+ NADH
Peroxisome
CH OH CH2(OH)OCH3 HCOOCH3
O2 3
ADH (MFS)
AOD
1/2 O2 + H2O H2O2 NAD+ NADH
CTA
GS-CH2OH GS-CHO FGH
HCHO GS-CH2OH HCOOH
FLD
RCOOH RCOOOH GSH GSH
DAS Xu5P Xu5P NAD+

Pmp20 FDH
GSSG GSH DHA GAP 1/3 GAP NADH
CO2
Rearrangements
DHA GAP
FBP F6P Cell
GLR DAK constituents
DHAP
Fig. 3.3 Outline of methanol metabolism in methylotrophic yeasts. Enzymes: ADH (MFS), alco-
hol dehydrogenase (methyl formate-synthesising enzyme); AOD, alcohol oxidase; CTA, catalase;
DAK, dihydroxyacetone kinase; DAS, dihydroxyacetone synthase; FDH, formate dehydrogenase;
FGH, S-formylglutathione hydrolase; FLD, formaldehyde dehydrogenase; GLR, glutathione
reductase; Pmp20, peroxisome membrane protein which has glutathione peroxidase activity.
Abbreviations: DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; F6P, fructose
6-phosphate; FBP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; GS-CH2OH,
S-hydroxymethyl glutathione; GS-CHO, S-formylglutathione; GSH, reduced form of glutathione;
GSSG, oxidised form of glutathione; RCOOOH, alkyl hydroperoxide; Xu5P, xylulose 5-phosphate.
(Adapted from Yurimoto et al. (2011))
discussed that a significant and maximum gene expression was observed in the
presence of methanol, whereas a low level of gene expression was observed in the
absence of methanol (derepression). The characterisation and identification of vari-
ous transcription factors involved in the expression and regulation of methanol-
inducible gene expression was done by Ohsawa et al. (2018). Transcription factors
KpMit1, leads to the repression of methanol-inducible gene expression or the pres-
ence of glucose leads to the repression of methanol-inducible gene expression
(Hartner and Glieder 2006; Yamashita et al. 2009). Transcription factors such as
KpMit1 in Komagataella phafi (Wang et al. 2016) and KpPrm1 in Candida boi-
dinii (Sahu et al. 2014) along with Hap complex are involved in methanol induc-
tion, whereas transcription factors like KpMxr1 and CbTrm2 are involved in
derepression (Lin-Cereghino et al. 2006). KpMxr1 and CbTrm2 are homologues to
S. cerevisiae Adr1.
In a recent investigation, the function of a gene encoding Hac1 transcription
factor was characterised in thermotolerant methylotrophic yeast Ogataea thermo-
methanolica TBRC656 (OtHAC1). Hac1 generally triggers the unfolded protein
response pathway in yeasts. Under the characterisation study of this gene, the
comparative proteomic analysis was done between OtHAC1 mutant and wild-type
Ogataea strain. About 400–500 proteins were detected and gene encoding Hac1
annotated different functions involved in transcription, translation, oxidative stress
and secretary pathway. Subsequently, two different novel OtHAC1-dependent pro-

teins, viz. Iml1 and Npr2, were also identified which are responsible for the regulation
of autophagy. This research on methylotrophic yeast therefore revealed the regulation
of different metabolic pathways or processes through OtHAC1 gene expression in
thermotolerant Ogataea thermomethanolica TBRC656 (Phithakrotchanakoon
et al. 2018).
In Pichia pastoris, the regulation of AOX gene expression is done through repres-
sion/derepression mechanism along with induction mechanism and mostly this
gene regulation resembles the regulation of GAL1 gene expression in Saccharomyces
cerevisiae. The rich level of methanol in the media facilitates the high rate of tran-
scription in case of methylotrophic yeast and the repression of gene regulation in the
presence of glucose (a repressing carbon substrate) was not seen unlike GAL1 gene
in case of Saccharomyces cerevisiae. The rate of gene expression was found directly
proportional to the presence of methanol in the medium (Tschopp et al. 1987).
The methylotrophic yeast Ogataea thermomethanolica TBRC656 is a well-known
host cell for the heterologous protein expression. In this thermotolerant methylotro-
phic yeast, maltase gene (mal) promoter-based new gene expression system was
developed. The gene expression of xylanase and phytase was found to be enhanced
many fold when Ogataea thermomethanolica TBRC656 was supplemented with
sucrose in media. The increase in fold expression was due to activation of OtMal
promoter gene as compared to constitutive OtGAP promoter gene. The presence of
sucrose in the media also activates the more expression of OtMal promoter gene as
compared to methanol-inducible OtAOX promoter. This enhances the enzyme
activity by increasing higher gene expression of reporter genes coding xylanase and
phytase. Therefore, it was suggested that methylotrophic yeast in the presence of
sucrose as source of carbon substrate can be utilised for the production of heterolo-
gous proteins at large scale (Puseenam et al. 2018).
3.4 Methylotrophic Yeast and Impact to the Environments
The methylotrophic communities have been found to be applicable in diverse poten-

tial biotechnological applications (Fig. 3.4). The methylotrophic communities have
been reported from diverse habitats including plant microbiomes as rhizospheric
(Verma et al. 2013, 2014; Yadav 2017), endophytic (Rana et al. 2018; Verma et al.
2015a, 2016b) and epiphytic (Verma et al. 2015b, 2016a) and natural habitats as
well as from different extreme environments of acidic, alkaline, drought, low tem-
perature (Yadav 2015; Yadav et al. 2015a, b, 2016, 2019c), salinities and radiations
(Yadav and Saxena 2018; Yadav et al. 2015c; Yadav and Yadav 2018). The methylo-
trophic communities having potential and efficient multifarious plant growth-
promoting attributes have been used for plant growth promotion, crop yield, and soil
health for sustainable agriculture (Biswas et al. 2018; Yadav et al. 2017; Yadav
2009). Along with agricultural application, the methylotrophic communities have
been reported to use in different processes in medical, industrial and pharmaceutical
64 M. Kumar et al.
Fig. 3.4 Biotechnological application of methylotrophic yeast. (Adapted with permission from
Kuroda and Ueda (2011))
sectors as well as in environment for sustainable future (Rastegari et al. 2019; Yadav
et al. 2019a, b).
The genetically engineered thermotolerant methylotrophic yeast strains are
reported to have properties of bioremediation (Kour et al. 2019; Suman et al. 2016;
Yadav et al. 2018). Particularly chromate bioremediation was observed in Hansenula
polymorpha which triggers the many fold enhanced gene expression of FCb2 gene
coding flavocytochrome b2 enzyme as compared to parental strain (Smutok et al.
2011). The flavocytochrome b2 enzyme is known to be specific for L-lactate. In the
presence of L-lactate, the enzyme flavocytochrome b2 leads to the reduction of
chromate by living cells. Pichia pastoris is reported to be involved in the degrada-
tion of azo dyes and anthraquinone dyes and bioremediation of different xenobiotic
compounds (Colao et al. 2006). In the production of fungal laccase, the expression
of lccI gene was done in Pichia pastoris from cDNA synthesised from the white rot
fungus Trametes togi.
Methylotrophic yeast isolated from oil-contaminated soil has distinct enzymatic
activity and identified as Trichosporon cutaneum. Later, the identification was con-
firmed by ribosomal DNA-based molecular characterisation. In the isolate, methanol
assimilation was found and can use formaldehyde also as a source of carbon sub-
strate along with other carbon substrates like ethanol, glycerol, glucose and other
petroleum derivatives. The microorganism was proved as an extremophile. In the
isolate, different enzymatic activities were observed such as formaldehyde reduc-
tase, unspecific aldehyde dehydrogenase and formaldehyde dehydrogenase activity.
Therefore, the biochemical, metabolic and physiological characteristics of methylo-
trophic isolate explore the new possibilities in the field of environmental biotech-
nology (Kaszycki et al. 2006).
Pichia pastoris is well-known yeast used in the production of animal contaminant-
free hydroxylated collagen. The enzyme-based and molecular methods are utilised
in the production and selection of triple-transformed Pichia pastoris strain, useful
in the expression of P4H (prolyl 4-hydroxylase) tetramer obtained from marine
sponge Chondrosia reniformis along with a hydroxylated collagen from the same
animal (Pozzolini et al. 2015).
The environmental pollutants like household, industrial wastes and oil spills cost
a lot to the sustainability of environment, and therefore some strains of methylotro-
phic yeast were found to be a better alternative for the bioremediation of these
potent pollutants. The restoration of ecological balance is achieved by diminishing
the level of environmental pollution. Methylotrophic yeast are able to degrade a
number of xenobiotic compounds as a source of carbon. Methylotrophic strains
such as Rhodosporidium, Pichia, Trichosporon, Rhodotorula and Yarrowia are able
to degrade xenobiotic compounds like phenol, aromatic compounds and polar com-
pounds. The degradation intensity decreases from n-alkanes to polar and aromatic
carbon substrates (Csutak et al. 2010).
Genetically engineered methylotrophic yeast Hansenula polymorpha is known
for the development of lactate-selective microbial biosensor. This thermotolerant
yeast was utilised for the overproduction of lactate:cytochrome c-oxidoreductase
enzyme system [FC b(2)] by overexpression of the CYB2 gene encoding FC b(2).
The strong alcohol oxidase promoter of H. polymorpha controls the gene expression
of HpCYB2 gene, and it was transformed into the host methylotrophic yeast strain
H. polymorpha C-105 (gcr1 catX) in the absence of catalase activity and with glu-
cose repression. Using a cathodic electrodeposition polymer, the cells are immobil-
ised over graphite base. The redox mediator phenazine methosulphate used with
this reacts with FC b (2) inside the cell in the presence of L-lactate. A higher Km
value is observed in a biosensor based on recombinant methylotrophic yeast with a
higher linear range towards lactate (Smutok et al. 2011).
Now it is very clear that methylotrophic yeast can be exploited as a suitable
microbial culture used in the heavy-load wastewater treatment process. The pure
monoculture of yeast can be utilised as a biofilter for the treatment of concentrated
wastewater. The dilution of contaminants is done through degradation of wastes
generated as a result of several technological processes. In earlier reports, it was
66 M. Kumar et al.
illustrated that up to 10 g/l of formaldehyde can be diluted with the help of these
biofilters. Moreover, methylotrophic yeast can be useful in the bioremediation of
soil contaminated with oil (Kaszycki et al. 2006). Since methylotrophic yeast is
well known for the production of heterologous proteins, they can change the prod-
uct or compounds after processing due to genetic changes in their genome. Yeast
cells do not contain pyogenes, pathogens, and viral inclusions and therefore can be
used in the production of therapeutic administration. Earlier it was assumed that
mainly S. cerevisiae was utilised for the production of pharmaceutical proteins but
later methylotrophic yeast Hansenula polymorpha was used widely in modern
genetics for the production of pharmaceuticals. Hansenula polymorpha possess
especial advantageous characteristics as a host cell for the production of pharma-
ceuticals proteins. The pharmaceutical protein production system based on methy-
lotrophic yeast Hansenula polymorpha has been established for different vaccines,
serum proteins and other important therapeutics. In future, different products based
on H. Polymorpha will be introduced to market after preclinical and clinical trials
(Gellissen and Melber 1996).
The molecular-level analysis enhances our understanding of methylotrophic yeast

structure and function. The gene-level diversity and their study for the phylogenetic
relatedness helped us to understand the establishment of methylotrophic yeast with
the natural ecosystems. The methylotrophic yeasts are used in the research area and
in biotechnological applications, one of the most important being the production of
heterologous proteins of a large industrial and medical importance. The biotechno-
logical production of heterologous proteins is reported from methylotrophic yeast
Pichia pastoris which is renamed as Komagataella pastoris after recent taxonomy.
The increase of SCP (Single Cell Protein) requirements or the remediations of the
polluted systems by making use of natural alternatives represent important reasons
for the necessity of characterising the methylotrophic yeasts. Moreover, further
sophisticated and intensive research in the field of yeast methylotrophy and its
molecular basis will explore and reveal new insights of physiological functions
along with its importance in the natural ecosystem.
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Chapter 4
White Rot Fungi and Their Enzymes
for the Treatment of Industrial Dye
Effluents
Dhevagi Periasamy, Sudhakarn Mani, and Ramya Ambikapathi
4.1 Introduction
Dyes are organic colourants, which are indispensable, as they are widely used in all
fields of industry to envisage a colourful world. Among the different classes of dyes,
azo dyes are most commonly used in textile industry, and nearly 10–15% of the dye
stuffs used do not bind to the fabrics. The unabsorbed dyes were released into sew-
age treatment systems and in other water resources of the environment (Anliker
1979; Chudgar 1985; Zollinger 1961). It was shown that in environment, an
unchanged azo dye was subjected to a reductive transformation of the azo bond. The
reductive ring cleavage of the azo linkage is due to unspecific cytoplasmic reduc-
tases, resulting in the formation and accumulation of colourless aromatic amines
(Chung 2000). The resultant products formed may be toxic, mutagenic and carcino-
genic to animals and humans. Biological degradation is a popular, viable and attrac-
tive technology that uses the metabolic potential of microorganisms. In recent days,
the potential of white rot fungi for the degradation of recalcitrants is gaining
importance.
White rot fungi (WRF) that come under the division Eumycota are heteroge-
neous group of fungi having capacity to degrade a wide variety of recalcitrant com-
pounds. It contains the mushrooms, puffballs, conks and crust-like fungi which are
used as food source. The xenobiotic degradation capacity of the WRF fungi may be
due to extracellular, non-specific enzymes. Recently, white-rot fungi and their
ligninolytic enzymes, including laccase, manganese peroxidase (MnP) and lignin
peroxidase (LiP), as well as H2O2-producing oxidases, were explored intensively for
the degradation of a wide range of xenobiotics. These fungi are having the capacity
to break down the lignin in wood without degrading cellulose, and sometimes
D. Periasamy (*) · S. Mani · R. Ambikapathi

Department of Environmental Sciences, Tamil Nadu Agricultural University,
Coimbatore, India

74 D. Periasamy et al.
both lignin and cellulose will be degraded. The non-specific enzymes enable a number
of advantages which are not been found in other bioremediation systems.
The white rot fungus oyster mushroom (Pleurotus ostreatus) prefers lignin instead
of polysaccharides during the degradation. Some of the commonly cultivated
Pleurotus ostreatus and other oyster mushrooms will not grow on living trees. They
are not parasitic and attack only already dying trees from other causes. During the
degradation, the phenyl propane alkyl side chains found abundantly in the lignin was
decayed by WRF. One other white rot fungus, Phanerochaete chrysosporium, shows
no preference to lignocellulosics. Armillaria spp. is a white rot fungus called as honey
mushroom which is notorious for attacking living trees. Turkey tail, artist’s conk and
tinder fungus are some of the other white rot fungi involved in degradation.
4.2 Textile Dyes
Colour has become an important aesthetic factor in the textile world. Different dyes
and pigments are used as colourants, which are based on two major chemistries: azo
and anthraquinone. Among these two, azo dyes are largely used in textile industries,
and many of these dyes enter wastewater treatment facilities (Chung et al. 1978;
Sudhakar et al. 2002). These dyes exhibit high resistance to microbial degradation
in wastewater treatment systems and retain their colour, structural integrity upon
exposure to sunlight, soil, bacteria and human sweating. Synthetic fabrics like
nylon, rayon and polyester necessitate the production of new dyes that can bind with
these materials strongly. In addition, updating of azodyes to match the changing
social scenario, ideas and styles is also a must. Nearly 1,00,000 commercially avail-
able dyes (7 × 105 metric tons) are produced annually (Zollinger 2003). More than
8000 chemicals associated with textile dyeing process are listed in the Colour Index.
Brighter, longer-lasting colours with better binding ability are often necessary to
satisfy the emerging demand.
A multitude of dyes were used in textile industry and different classes of these
dyes include azo, acid, reactive, metal complex, disperse, vat, mortant, direct, basic,
suphur, etc. (Vijaya et al. 2003). These dyes are usually aromatic and heterocyclic
compounds (Vyas and Molitoris 1995) and some are toxic and carcinogenic.
Azo dyes contain one to many N=N double bonds, hence many different struc-
tures are possible (Cripps et al. 1990; Zollinger 2003). For example, monoazo dyes
have only one N=N double bond, while diazo and triazo dyes containing two and
three N=N double bonds, respectively. The azo groups are linked to aromatic het-
erocyclic or enolizable aliphatic groups, but also attached with benzene and naph-
thalene rings (Zollinger 2003). Aromatic heterocyclic, enolizable aliphatic groups
side chains are necessary for imparting the dye colour, with different shades and
brightness. Dye molecules are generally described as chromogen, which contains
nucleophiles often referred to as auxochromes and the aromatic groups are called as
chromophores. Synthesis of most azo dyes involves diazotization of a primary
aromatic amine, followed by coupling with one or more nucleophiles. Amino- and
hydroxyl- groups are the commonly used coupling components. Because of the
4 White Rot Fungi and Their Enzymes for the Treatment of Industrial Dye Effluents 75
possibilities of the synthesis of diversity of dye components, a large number of

structurally different azo dyes exist and are used in industry (McCurdy 1991).
Worldwide production of organic dyes is currently estimated at nearly 4,50,000
tons, with 50,000 tons being lost in effluents during application and manufacture
(Lewis 1999).
Reactive dyes are coloured compounds that contain one or two functional groups
capable of forming covalent bonds with the active sites in fibres. Almost 80–95% of
the reactive dyes are based on azochromogen (Edwards 2000; Zollinger 2003). The
carbon and phosphorus atom found in the dye molecule will bind with hydroxyl
groups in cellulose, amino, thiol, and hydroxyl groups in wool, or amino groups in
polyamides. Most fibre-reactive azo dyes are used for dyeing cellulosic materials,
such as cotton, and are a major source of dye waste in textile effluents. Fibre-reactive
azo dyes exhibit a high wet-fastness, due to their ability to covalently bond to sub-
strates. However, dyes that hydrolyse in solution prior to bonding to a substrate are
often lost in the washing (Loyd 1992). Schematic diagram of fibre-reactive azo dyes
is given in Fig. 4.1.
Amino and alkylamino groups are generally used for bridging chromogen and
reactive group. The bridging group that binds chromogen and the reactive group
must be stable, must be water-soluble and should have flexibility. Mono-, di-, and
trichlorotriazinyl are all examples of reactive functional groups, which help to bond
the dye molecule with a substrate through nucleophilic substitution, sometimes
addition also. Nearly 200 different reactive dye groups are patented with an addition
of 25 reactive azo dyes in the list per year from 1998, which was nearly five times
as many as from other classes of azo dyes (Freeman and Sokolowska 1999). With
such a disproportionate production rate, it is not surprising that a large percentage
of dye pollution problems are related to fibre-reactive azo dyes.
India, the former USSR, Eastern Europe, China, South Korea and Taiwan con-
sume approximately 600 thousand tons (kt) of dyes per annum (Ishikawa and Leder
2000). The total annual world textile dye production is estimated at about 800 kt
(Will et al. 2000; Zollinger 2003). Now Asia is being the largest dyestuff market
(about 42%). Classification of dyestuffs into native and synthetic dyes may not be
sufficient nowadays, since many natural substances are synthesized easily.
According to the chemical structure, the dyes are classified systematically and given
as colour index (Table 4.1). This indexing also provides information about the
biodegradability of the dyes. A listing of synthetic dyes according to their most
predominant chemical structures is given in Table 4.2.
Fig. 4.1 Schematic diagram of fibre-reactive azo dye

Table 4.1 Classes of synthetic dyes according to colour index

Chemical class Code Chemical class Code
Nitroso 10,000 Thiazole 49,000
Nitro 10,300 Indamine/Indophenol 49,400
Monoazo 11,000 Azine 50,000
Disazo 20,000 Oxazine 51,000
Trisazo 30,000 Thiazine 52,000
Polyazo 35,000 Sulphur 53,000
Azoic 37,000 Lactone 55,000
Stilbene 40,000 Aminoketone 56,000
Carotenoid 40,800 Hydroxyketone 57,000
Diphenylmethane 41,000 Anthraquinone 58,000
Triarylmethane 42,000 Indigoid 73,000
Xanthene 45,000 Phthalocyanine 74,000
Acridine 46,000 Natural 75,000
Quinoline 47,000 Oxidation base 76,000
Methine 48,000 Inorganic 77,000
(Source: Wesenberg et al. 2003)
4.3 Dyes Used in India
Indian dyestuff industry produces around 60,000 metric tons of dyes, which is 6.6%
of total colourants used globally (Teli 2008). Today India exports dyes to USA,
Turkey, Bangladesh, China and Germany on which once dependent for imports. The
various dyes and dyestuffs used in India are azodyes, disperse dyes, ingrain dyes,
naphthols, vat dyes, reactive dyes, pigment emulsion, sulphur dyes and other dyes.
The type of dyes and chemicals used in the textile industry are found to differ
depending on the fabrics manufactured (Table 4.3). The quantity of wastewater pro-
duced from composite industries (cotton and synthetics) was estimated as 840 m3/
day, with synthetic industry alone 180 m3/day. The textile processing and blending
industry produces about 150 m3/day and 1500 m3/day, respectively, whereas wool-
len industry uses 2700 m3/day (Sarayu and Sandhya 2012).
4.4 escription of Dyeing Process and Sources of Effluent

D
Generation
Among high water-consuming industries, dye industry is also one, which dominates
in Coimbatore, Tiruppur and Erode districts of Tamil Nadu. Substantial volume of
water and numerous chemicals are used to each kilogram of hosiery. Nearly 200–
300 litres of water is consumed for a kilogram of yarn and 75–90% of which is
discharged as effluent containing organic and inorganic pollutants (Banat et al.
1997). Dyes have strong staining properties and they are visible even at a concentra-
tion of 1 ppm. The volume of waste water generated from textile industry is very
Table 4.2 Structure of synthetic dyes used in textile industries
Name of dyes Chemical structure
Reactive Orange 96 (N=N)
Remazol Brilliant Blue
Bromothymol Blue
Reactive Orange 16
Disperse Violet 93
Amaranth dye
Congo Red
Remazol Black 5
Reactive Orange 16 s
Table 4.3 Type of dyes and chemicals used by the Indian textile industries
Material/
S. No. Dyes Fabrics References
1. Acid, metal–complex and reactive Wool, Chavan
Silk (2001),
2. Reactive, direct, vat, sulphur, indigo, etc. Cotton Ghaly et al.
3. Cellulosics and pigments All kinds (2014),
of Teli (2008)
materials
4. Cationic Acrylics
5. Reactive dyes (Remazol, Procion MX and Cibacron F), direct Cellulosic
dyes (Congo Red, Direct Yellow 50 and Direct Brown 116), fibres
Naphthol dyes (Fast Yellow GC, Fast Scarlet R and Fast Blue
B) and indigo dyes (Indigo White, Tyrian Purple and Indigo
Carmine)
6. Acid dyes (azo dyes, triarylmethane dyes and anthraquinone Protein
dyes) and Lanaset dyes (Blue 5G and Bordeaux B) fibres
7. Dispersed dyes (Disperse Yellow 218 and Disperse Navy 35), Synthetic
basic dyes (Basic Orange 37 and Basic Red 1) and direct dyes fibres
high, and the average concentration of 300 mg/L was also reported in many indus-
tries. Until recently, the waste water generated from these industries were dis-
charged into sewage, running or stagnant water bodies and adjoining waste lands
without proper treatment. Due to high composition, variety and colour intensity,
wastewater from textile industry cannot be treated satisfactorily.
However, due to stringent environmental regulations and awareness, the dye
industries waste water is treated at Common Effluent Treatment Plants (CETP) or at
individual factory premises before recycling. Perhaps, before the introduction of
CETP or individual treatment plants, sizeable land area was contaminated already
by this industrial waste water. The physicochemical characteristics of untreated textile
and dye industries effluent indicate that it will have deleterious effect on soil and soil
biological properties. The concentrations of most of the chemical parameters of the
waste water were well above the critical limits fixed by Central and State Pollution
Control Board of India. Characterizing the untreated textile and dye industry waste
water, Gupta (1992) reported a pH range of 10–11.5, electrical conductivity in the
range of 8.5–13.9 dS/m and BOD in the range of 400–800 mg/L. Besides high-sol-
uble salts, the effluent also contains toxic trace metals such as lead (1.3 mg/L) and
chromium (5–20 mg/L) beyond the critical limits (Kothandaraman et al. 1976). Dye
effluent with a pH of 4–12, colour to the level of 500–2000 Pt-Co units, Chromium
(VI) 1–4 mg/L and sulphide 0–50 mg/L was reported by Puscas et al. (2003)
(Fig. 4.2). The following steps are involved in the dyeing process:
(a) Scouring – The raw material, mainly the hosiery cloth, is subjected to the
scouring operation for eliminating the cotton impurities by boiling the cloth in
a vessel at about 80 °C for about three hours.
(b) Bleaching – H2O2 and caustic soda of 3% strength is mixed in the vessel thor-
oughly prior to carrying out the scouring operation.
Fig. 4.2 Dyeing process in textile industries
(c) Neutralizing with acid and washing – The contents are washed using the
alkaline water followed by the acid-wash for neutralizing and again the wash-
ing operation is repeated. After the final washing of the bleached cloth, the
dyeing operation is carried out by transferring the material into the dyeing vat.
(d) Dyeing – The dyeing solution is prepared by dissolving the dyes as per the
recipe for a particular shade in the winch along with the salts such as sodium
chloride (40–60 g/lit strength) and soda ash for exhaustion and fixing the dyes,
respectively. The dyeing operation is done at 70 °C and for about three hours to
achieve the desired shade.
4.4.1 Characterization of the Raw Effluents
Textile industry wastewater contains dyes to the level of 20–200 mg/L, and about
10–20% of the dyes are present in effluents along with other organic and inorganic
accessory chemicals. Reactive dyes, sodium chloride, soda ash, caustic soda, wet-
ting oil, industrial soap powder, hydrochloric acid, acetic acid, softening agent and
fixing agents are some of the accessory chemicals used in textile industries. Tiruppur
has large sources of bleaching and dyeing industries which generate between 100
and 120 MLD of effluents. Due to poor dyeing process in the textile industry,
10–15% of the dyes are lost in the effluents of textile units, rendering them highly
coloured (Boer et al. 2004; Vaidya 1982). It is estimated that 280,000 tons of textile
dyes are discharged in such industrial effluents every year worldwide (Maas and
Chaudhari 2005). The raw effluent samples were collected based on composite
sampling method from Tirupur. The samples were collected on hourly basis and
homogenized, and then the representative samples were analysed (Table 4.4.)
4.5 Enzymes of White Rot Fungi
With the advancement of biotechnological tools, the use of fungi or bacteria, often
in combination with physico-chemical processes were used eco-efficiently for com-
bating this pollution source (Yadav et al. 2016, 2017, 2018, 2019a, b). Among those
microorganisms, WRF are most efficient in breaking down synthetic dyes. These
established a diverse eco-physiological group comprising mostly basidiomycetous
(and, to a lesser extent, litter-decomposing) fungi capable of extensive aerobic lignin
mineralization and depolymerization. This property is based on one or more extra-
cellular lignin-modifying enzymes (LMEs) produced by white rot fungi, and these
enzymes are also capable of degrading a wide range of xenobiotics (Beydilli et al.
1998; Borchert and Libra 2001; McMullan et al. 2001; Robinson et al. 2001;
Willmott et al. 1998; Zissi and Lyberatos 2001).
In 1896, laccase was identified first time in fungi by both Bertrand and Laborde.
Most of the laccases have been widely isolated from fungal origin specifically from
white rot fungi belonging to Ascomycetes, Deuteromycetes and Basidiomycete
(Gochev and Krastanov 2007). Many other earlier studies also reported laccase
production from white rot fungi (Kiiskinen et al. 2004) such as Phlebia radiata
(Niku-Paavola et al. 1988), Trametes versicolor (Bourbonnais et al. 1995) and
Pleurotus ostreatus (Palmieri et al. 2000). Some of Trichoderma sp. are also studied
as laccase source. For example, T. harzianum (Hölker et al. 2002), T. atroviride
(Hölker et al. 2002) and T. longibrachiatum produced higher amount of laccase.
Velázquez-Cedeño et al. (2004) observed that the laccase synthesis was found to be
higher in mixed cultures than the pure cultures. Liophora terristrus, Phanerochaete
chrysosporium, Lenzitis betulina and Stereum ostrea (Viswanath et al. 2008) are
some of the important Basidiomycetes which have been reported as the sources of
laccases.
WRF possess not only laccases, it also possess a wide range of various enzymes
such as extracellular ligninolytic enzymes (manganese peroxidase, lignin perox-
ides) and hydrolytic enzymes (cellulase, xylanase, pectinase) (Teerapatsakul et al.
2007). The expression pattern of enzymes mainly depends on the organism itself.
Certain WRF produce manganese peroxidase and lignin peroxidase, but not laccase;
meanwhile, others produce laccase and manganese peroxidase, but not lignin per-
oxidase (Hatakka 1994). Therefore, among various types of WRF, some can decom-
pose all of the lignocellulose components in wooden material, while some can
degrade hemicellulose and lignin (Fang et al. 2008). These enzymes are mostly
important in industrial purpose and have a great potential in the processes of degra-
dation of recalcitrant substances (Tortella et al. 2008).
4.5.1 Lignin Peroxidase (LiP)
WRF can degrade lignin and a range of diverse environmental pollutants by means
of their extracellular ligninolytic systems. Purified forms of LiP have been found to
directly oxidize recalcitrant xenobiotic compounds such as polycyclic aromatic
hydrocarbons, chlorophenols and azo dyes (Collins et al. 1997). LiPs are proposed
to oxidize lignin with free radicals generated through oxidation of various secreted
metabolites (e.g. veratryl alcohol). The most effective stimulant was found to be
veratryl alcohol, a secondary metabolite produced by ligninolytic cultures of WRF
(Sugiura et al. 2003). Veratryl alcohol plays an important role in LiP catalysis. LiP
is oxidized by H2O2 to form a two electron intermediates, compound I, which oxi-
dizes substrates by one electron, forming the more reduced enzyme intermediate,
compound II. Compound II can then oxidize substrates by one electron, returning
the enzyme to the ferric state. However, compound II has a very high reactivity with
H2O2; therefore, in the presence of a poor substrate and excess H2O2, it is instead
converted to an inactive form of the enzyme, compound III. Veratryl alcohol, when
present, is a more favourable substrate for compound II and functions to convert it
to the resting enzyme, completing the catalytic cycle (Vasina et al. 2017) (Fig. 4.3).
4.5.2 Manganese Peroxidase (MnP)
MnP plays a vital role in lignin degradation, as it is found in all lignin-degrading

WRF. This haem protein belongs to the commonly occurring class II peroxidase
group in basidiomycetous fungi and has a highly specific Mn2+ binding site. In the
binding site of classical long MnPs, there are three amino acid residues while several
fungal Mn2+-oxidizing enzymes with an additional tryptophan residue on the enzyme
surface have been found (Hofrichter et al. 2001). These enzymes are called VPs or
hybrid MnPs, bearing resemblance to LiPs and able to perform oxidation though
Table 4.4 Characteristics of combined raw effluent from Tirupur

S. No. Parameters Value
1 pH 7.5–10
2 EC (dS m−1) 10–16
3 Colour (NTU) 300–324
4 Turbidity (NTU) 100–770
5 Total dissolved solids (mg L−1) 6250–8205
6 Total suspended solids (mg L−1) 60–120
7 Biochemical oxygen demand (mg L−1) 75–500
8 Chemical oxygen demand (mg L−1) 288–1200
9 Total organic carbon (mg L−1) 100–111
10 Total hardness (mg L−1) 500–900
11 M-Alkalinity (mg L−1) 570–1000
12 P-Alkalinity (mg L−1) 20–80
13 Calcium (mg L−1) 37–400
14 Magnesium (mg L−1) 250–847
15 Oil and grease (mg L−1) 10–20
16 Chloride (mg L−1) 200–3900
17 Sulphate (mg L−1) 460–3000
18 Sodium (mg L−1) 2000–3000
19 Potassium (mg L−1) 40–100
20 Carbonate (mg L−1) 30–100
21 Bicarbonate (mg L−1) 700–1200
22 Total phosphate (mg L−1) 0.9–4.0
23 Fluorides as F (mg L−1) 1.1–4.0
24 Nitrate as N (mg L−1) 0.6–6.0
25 Ammonical nitrogen (mg L−1) 2.0–6.0
26 Total Kjeldal nitrogen (mg L−1) 10.0–40.0
27 Total iron (mg L−1) 0.195–2.00
28 Barium (mg L−1) 0.15–0.25
29 Boron (mg L−1) 0.50–1.00
30 Aluminium (mg L−1) 0.10–1.20
31 Zinc (mg L−1) 0.017–0.50
32 Lead (mg L−1) 0.046–0.25
33 Manganese (mg L−1) 0.05–0.30
34 Copper (mg L–1) 0.041–0.20
35 Chromium (mg L−1) 0.00–0.05
36 Cobalt (mg L−1) 0.05–0.10
37 Nickel (mg L−1) 0.031–0.20
38 Cadmium (mg L−1) 0.04–0.06
39 Arsenic (mg L−1) BDL
40 Total silica (mg L−1) 20.0–40.0
41 Strontium (mg L−1) 1.00–2.00
Sources: Sivasamy (2008)
Fig. 4.3 Lignin peroxidase (LiP) catalytic cycle
long-range electron transfer as well. The evolution of these class II peroxidases

appears to be closely related to each other and even consistent with the sharp decline
in coal accumulation rate during the Permo-Carboniferous period (Maijala et al.
2008). Lignin is the main precursor for coal. MnP catalyses the oxidation of Mn2+
ions to highly reactive Mn3+ ions. Chelated Mn3+ in turn acts as low molecular weight
mediators that are able to attack phenolic structures. MnP is able to cause substantial
depolymerization in in vitro biomass treatments (Floudas et al. 2012). Potential appli-
cations for MnP include pulp bleaching, biomechanical pulping, dye decolourization,
bioremediation and production of highly valuable chemicals from residual lignin
from biorefineries, pulp and paper side-streams (Järvinen et al. 2012) (Fig. 4.4).
4.5.3 Laccase
Laccase is a part of broad group of enzymes called polyphenol oxidases containing

copper atoms in the catalytic centre and are usually called multicopper oxidases.
Laccases contain three types of copper atoms, one of which is responsible for their
Fig. 4.4 Manganese peroxidase (MnP) catalytic cycle
characteristic blue colour. The enzymes lacking a blue copper atom are called yellow
or white laccases (Diamantidis et al. 2000). Typically laccase-mediated catalysis
occurs with reduction of oxygen to water accompanied by the oxidation of substrate.
Laccases catalyse the oxidation of a broad range of substrates such as ortho and para-
diphenols, methoxy-substituted phenols, aromatic amines, phenolic acids and sev-
eral other compounds coupled to the reduction of molecular oxygen to water with
one-electron oxidation mechanism (Morozova et al. 2007). Laccase is most widely
distributed in a wide range of higher plants, fungi and bacteria. Laccases are secreted
out in the medium extracellulary by several fungi during the secondary metabolism
but not all fungal species produce laccase such as Zygomycetes and Chytridiomycetes.
Fungi belonging to Deuteromycetes, Ascomycetes as well as Basidiomycetes are
known producers of laccase (Sadhasivam et al. 2008). Laccase is currently the focus
of much attention because of its diverse applications such as dye decolourization,
waste detoxifications and bioremediation applications (Fig. 4.5).
4.6 actors Influencing Enzyme Production and Dye

F
Degradation
Secretion and synthesis of these enzymes are often stimulated by limited levels of
nutrients such as nitrogen or carbon sources. Production of manganese peroxide and
lignin peroxide is generally induced by agitation in submerged white rot fungi liq-
uid culture, while laccase production is frequently enhanced by agitation. These
features are considered as important roles in the process design and optimization of
fungal treatment of colour-containing effluents (Wesenberg et al. 2003). While sev-
eral studies were devoted to bio-decolourization of the textile azo dyes, far less
Fig. 4.5 Laccase catalytic cycle
attention has been paid to synthetic dye bath effluent in which the presence of salts
and high dye concentration may be inhibitory to biological agents (Faraco et al.
2009; Gomaa et al. 2008). Different WRF and their enzymes involving in dye deg-
radation are given in Table 4.5.
4.6.1 Effect of Inducers on Enzyme Production
Laccase production of white rot fungi mainly depended on the condition of fungal
cultivation and media supporting. Lignolytic systems of WRF were mainly stimu-
lated during the secondary metabolic phase and were prompted by nitrogen concen-
tration or when sulphur or carbon became limiting. Fungi produced lower
concentration of laccase, but higher concentrations can be attained with the addition
of supplements to media like veratryl alcohol, 2,3xylidine and lignin. Response
surface methodology (RSM) was applied to optimize the decolouration of the diazo
dye Reactive Black 5 (RB5) by crude laccase from the white rot fungus Trametes
pubescens (Roriz et al. 2009). Laccase production of white rot fungus (Pycnoporus
sanguineus) and its growth in a bubble column reactor were studied. Different
inducers like copper sulphate, ethanol, saw dust and superficial gas velocities are
used to increase the growth and laccase production (Karim and Annuar 2009).
4.6.2 Effect of Nitrogen Source
The laccase production was influenced by the sources of nitrogen used in the media.
Peralta-Zamora et al. (2003) isolated four WRF, and Lentinus edodes displayed the
greatest decolourization ability both in terms of extent and rapidity of decolouriza-
tion. The dyes used were Reactive Red 195 (0.025%), Reactive Blue 19 (0.05%),
Table 4.5 White rot fugal species and their enzymes involving in dye degradation
Organisms Enzymes Dye Reference
Dichomitus Manganese- Cresol RedTPM, Brilliant Green Périé and Gold
squalens peroxidase (TPM), Crystal Violet (TPM), OrangeII (1991)
(N=N), Congo Red (N=N)
Bjerkandera adusta Lignin Reactive Violet 5 (N=N), Kaal et al.
peroxidase Reactive Orange 96 (N=N), (1995)
Reactive Black 5 (N=N), Reactive Blue
38 (PC)
Reactive Blue 15 (PC) and Remazol
Brilliant Blue RPAQ, Poly R-478
(PAQ)
Pleurotus eryngii Lignin Reactive Violet 5 (N=N), Reactive Blue Heinfling et al.
peroxidase 38PC by MnP Reactive Black 5 (N=N) (1998)
Bjerkandera adusta Manganese- Reactive Black 5 (N=N), Reactive Heinfling et al.
peroxidase Violet 5 (N=N), Reactive, Blue 38PC (1998)
Pleurotus and versatile Dyes Mester and
Bjerkandera peroxidases Field (1998)
(VP)
Irpex lacteus Laccase Naphtol Blue Black (N=N), Methyl Novotný et al.
Red (N=N), Congo Red (N=N), (2000)
Remazol Brilliant Blue R (PAQ),
Copper (II)
phthalocyaninetetrasulphonic acid
Tetrasodium salt (MC), Bromophenol
Blue (TPM),
Poly R-478 (PAQ)
Phanerochaete Manganese- Azure Blue, Azo dyes, Cresol Red Cameron et al.
chrysosporium peroxidase (TPM), Bromophenol Blue (TPM), (2000)
Crystal Violet (TPM)
Irpex lacteus Lignin Methyl Red (N=N), Naphtol Blue Novotný et al.
peroxidase Black (N=N), Congo Red (N=N), (2000)
Bromophenol
Blue (TPM), Remazol Brilliant Blue R
(PAQ), Copper (II) phthalocyaninetetra
sulphonic acid tetrasodium salt(MC)
and Poly R-478(PAQ)
Phanerochaete Lignin Azo dyes, Azure Blue, Cresol Red Cameron et al.
Chrysosporium peroxidase (TPM), Crystal Violet (TPM), (2000)
Bromophenol Blue (TPM)
Phlebia (Merulius) Lignin Cibacron Red, Remazol Red, Remazol Kirby et al.
Tremellosa peroxidase Navy Blue Cibacron Orange, Remazol (2000)
Golden Yellow, Remazol Blue,
Remazol Black B,
Remazol Turquoise Blue
(continued)

Irpex lacteus Manganese Congo Red (N=N), Methyl red (N=N), Novotný et al.
peroxidase Naphtol Blue Black (N=N), (2000)
Bromophenol
Blue (TPM), Remazol Brilliant Blue R
(PAQ),
Copper (II)
phthalocyaninetetrasulphonic acid
Tetrasodium salt (MC), Poly R-478
(PAQ)
Trametes(Coriolus) Lignin Remazol Brilliant Blue Everzol Novotný et al.
Versicolor peroxidase Turquoise Blue GPC, R(PAQ), Poly (2000)
R-478(PAQ), Everzol Red RBN,
Everzol Yellow 4GL, Everdirect Supra
Yellow (PG), Orange K-GL
Pleurotus ostreatus, LiP and/or Triarylmethane, Anthraquinonic, and Abadulla et al.
Schizophyllum MnP in Indigoid textile dyes (2000)
commune, addition to
Sclerotium rolfsii, Lac
Neurospora crassa
Phellinus gilus, Lignin Vat textile dyes Balan and
Pleurotus sajor- peroxidase Monteiro
caju, Pycnoporus (2001)
sanguineus,
Phanerochaete
chrysosporium
Phanerochaete Manganese- Amaranth dye, Orange G dye, New Chagas and
chrysosporium peroxidase coccine dye, Tartrazine dye Durrant (2001)
Lentinula (Lentinus) Manganese- Remazole Brilliant Blue R Boer et al.
edodes peroxidase (2004)
Trametes versicolor Lignin Remazol Brilliant Blue R Christian et al.
peroxidase (2005)
T. trogii Laccase Azo and triarylmethane dyes Zouari-
Mechichi et al.
(2006)
Perenniporia Laccase Neolane pink, neolane blue, and Ben Younes
tephropora remazol brilliant blue R (RBBR) et al. (2007)
Phanerochaete Manganese- Direct Green 6, Direct Blue 15, Congo Urek and
chrysosporium peroxidase Red Pazarlioglu
(2007)
Scyzophyllum Manganese- Solar Golden Yellow R Asgher et al.
commune peroxidase (2008)
Cerrena unicolor Laccase Acid Blue 62, Acid Blue 40, Reactive Michniewicz
Blue 81, Direct Black 22, Acid Red 27 et al. (2008)
Bjerkandera sp. Manganese- Reactive Blue 38PC, Orange IIN = N, Moreira-Neto
peroxidase Poly R-478PAQ et al. (2013)
Trametes versicolor Laccase Indigo dye Rita de Cássia
et al. (2013)
(continued)

Lentinus tigrinus Manganese- Reactive Blue 38(PC), Orange II Moreira-Neto
peroxidase (N=N), Poly R-478(PAQ) et al. (2013)
Lentinus tigrinus Laccase Reactive Blue 38(PC), Orange II Moreira-Neto
(N=N), Poly R-478(PAQ) et al. (2013)
Phanerochaete Laccase Red 2BAB Jain et al.
chrysosporium (2000)
New coccine, Amaranth, Orang G, Chagas and
Tartrazine Durrant (2001)
Congo Red (N=N) Gill et al.
(2002)
Pleurotus ostreatus, Laccase Coralene Golden Yellow, Coralene Kunjadia et al.
Pleurotus sapidus, Navy Blue and Coralene Dark Red azo (2016)
Pleurotus florida dyes
Reactive Black 5 (0.05%) and Reactive Yellow 145 (0.05%). The colour removal by
fungal hyphae is mainly by the mechanism of degradation by extracellular and
intracellular enzymes (Chagas and Durrant 2001). In a nitrogen-deficient mineral
salts medium, Robinson et al. (2001) studied four WRF, Bjerkandera adusta,
Phlebia tremellosa, Pleurotus ostreatus and Coriolus versicolor, for testing their
ability to produce manganese peroxidase (MnP), lignin peroxidase (LiP) and lac-
case. B. adusta and P. tremellosa were selected, on the basis of their high enzyme
production potential, for the degradation of five dyes in an artificial textile effluent.
In N-rich (C:N ratio, 11.6:1 and N-limited, 116:1) conditions, degradation experi-
ments were carried out to examine degradation potential. At a dye concentration of
100 mg/litre, in N-rich media, P. tremellosa degraded 79% in 9 days, B. adusta
degraded 85% of the dyes in 7 days, and in N-limited conditions, 86% of the
effluent was degraded in 9 days by B. adusta and 74% by P. tremellosa in 11 days.
The results revealed that addition of nitrogen had no significant effect on dye degra-
dation percentage by B. adusta, with a slight increase for P. tremellosa and nitrogen
supplementation reducing the decolourization time.
In nitrogen-limited glucose ammonium media, Phanerochaete sordida deco-
lourized dye mixtures (reactive textile dyes, including azo and anthraquinone dyes
200 mg/L each) within 48 h by 90%. Manganese peroxidase (MnP) was engaged in
dye decolourization by P. sordida. Wastewaters from textile industries often contain
polyvinyl alcohol (PVA) which inhibited MnP reaction system and decolourization
(Reactive Red 120) potential of Phanerochaete sordida. Addition of Tween 80 to
the mixtures in the occurrence of PVA improved the decolourization of RR120
which showed that PVA could hinder with lipid peroxidation or consequent attack
to the dye (Harazono and Nakamura 2005). D’Souza-Ticlo et al. (2006) reported the
effect of KNO3, glycine, glutamic acid, corn steep liquor and beef extract under
stationary conditions. The cultures were oxygenated every third day with pure oxy-
gen for 1 min using Pasture pipettes and Tygon tubing under sterile conditions. They
obtained when glutamic acid was used as nitrogen source.
In majority of the cases, nitrogen-limited medium increased the production of

lignin peroxide and manganese peroxide, thereby increasing the rate of decolouriza-
tion (Singhal and Rathore 2001). These enzymes are produced by WRF during their
metabolism. Subsequently, lignin oxidation catalyses the degradation/transformation
of aromatic dyes either by precipitation or by opening the aromatic complex ring
structure, and therefore no energy source is required to the fungus (Husain 2010).
4.6.3 Effect of Carbon Source
Blánquez et al. (2008) showed that T. versicolor was able to continuously deco-
lourize a spent dyeing bath from a textile factory under non-sterilized conditions for
15 days in a 10-L air-pulsed bioreactor, attaining colour reduction levels between 40
and 60%. Nutrients were added at the start-up period (3 days) of the experiment,
and thereafter sterilized glucose was added. Therefore, although the author stated
that T. versicolor was able to treat successfully real industrial wastewater in con-
tinuous mode (HRT 48 h), the operation time was very short (15 days), and, in addi-
tion to this, bacterial contamination in the feeding tank was detected from day 10.
The ability of C. versicolor to decolourize the textile effluents collected from 5
different textile industries was tested. However, addition of starch (1% w/v) as a
carbon source was required to obtain significant decolouration levels (84% in
3 days) (Asgher et al. 2008). Phenol-degrading white rot fungus T. versicolor was
isolated from paper mill. 14C synthetic lignin mineralization assay showed that it
assimilated 24.3% of total label. During 5 days of incubation period, 71% of
p-hydroxybenzoic acid was utilized when glucose was used as a co-substrate and
56% degradation of protocatechoic acid using fructose (Udayasoorian and Prabu
2005).
4.6.4 Influence of pH and Temperature
Azo dye (RB5) wastewater is usually neutral to alkaline. However, though fungi
grow optimally under weakly acidic conditions, the azo dyes were degraded even in
a slightly alkaline pH range (pH 7–8). P. sordida PBU 0057 yielded 100% decolou-
ration in 72 hours over the pH range 6–8, while P. chrysosporium reached the maxi-
mum (96%) by 96 hours, though lesser (93%) at pH 8 (Forgacs et al. 2004).
Selvakumar et al. (2013) studied treated textile wastewater in a batch reactor with
Ganoderma lucidum. Under optimized conditions (pH 6.6; temperature 26.5 °C;
dye wastewater concentration 1:2; agitation speed 200 rpm), a maximum decoloura-
tion of 81.4% and a COD reduction of 90.3% were found. Therefore, dilution and
decreased pH of the original effluent were necessary for maximum decolouration
and COD reduction.
Hadibarata et al. (2013) assessed the effect of temperature on decolouration at

25, 30, and 35 °C. Complete RB5 decolouration by P. sordida PBU 0057 occurred
by 72 hours at 30 and 35 °C but was delayed until 96 hours at 25 °C. Overall, the
tropical isolates have the advantage of being active at higher temperatures – a practi-
cal attribute in tropical wastewater treatment facilities.
4.6.5 Concentration of Dye
Anastasi et al. (2010) reported the decolouration of wastewater from a dyeing-

textile factory by the white rot fungi Bjerkandera adusta packed in a fixed-bed
bioreactor. The fungus remains effective during 4 cycles of decolouration for a very
long period (70 days) under non-sterile conditions and with no nutrient addition.
Osorio Echavarría et al. (2011) reported the decolouration of wastewater from a
textile industry by the anamorph R1 of the white rot fungus Bjerkandera sp. Under
sterile conditions, the effluent was decolourized by 65% in 8 days, and its toxicity
was reduced by 58%, whereas under non-sterile conditions, the decolouration per-
centage was only 40% for the same time period. The authors stated that this lower
decolouration value was likely due to the presence of contaminant microorganisms
competing for the substrate.
Ma (2014) revealed that the Ganoderma sp. En3 had a strong potential and toler-
ance to decolourize and detoxify high concentrations of sulphonated azo dye
Reactive Orange 16 containing textile wastewater under in vitro conditions.
Maximum decolourization of 98% was achieved in synthetic dye bath effluent on
the third day under normal conditions by white rot fungus Phanerochaete chryso-
sporium. Experiment with different concentrations of effluent found that the increas-
ing in effluent concentration slows down the decolourization percentage and
optimized amounts of nutrients were found to be 0.5%, 0.1% and 0.5% of glucose,
manganese sulphate and ammonium salts, respectively. Adding inducers such as
lignin and starch augmented enzyme activity and the rate of decolourization
(Senthilkumar et al. 2014).
4.7 White Rot Fungi and Decolouration
Previous studies reported the ability of P. chrysosporium, Bjerkandera sp. and

T. versicolor to decolourize Remazol Orange, Reactive Blue, Remazol Brilliant
Blue and Tropaeolin O in agar plates. A basidiomycete fungus, P. chrysospo-
rium, was able to degrade the starch, cellulose, pectin, lignin and lignocellu-
loses complex compounds, which are characteristics of textile dye effluent
(Swamy and Ramsay 1999). Consequently, some strains including S. thermophi-
lum and T. trogii were reported to be able to decolourize and detoxify textile
effluents. Phlebia tremellosa and B. adusta showed a good efficiency to deco-
lourize textile effluent in N-limited conditions (Robinson et al. 2001). Tekere et al.
(2001a) reported the ability of T. versicolor, Trametes cingulata, Pycnoporus san-

guineus and Datronia concentrica to decolourize Poly R478 (Tekere et al. 2001b).
Conneely et al. (2002) found that, Remazol Turquoise Blue G133, Heligon Blue
S4, phthalocyanine dyes and Everzol Turquoise Blue are biosorbed by Phanerochaete
chrysosporium and also metabolized by its ligninolytic extracellular enzymes
resulting in the formation of free copper ions, dye decolourization and organic
metabolites with ultimate extensive phthalocyanine ring breakdown. It is assumed
that the ligninolytic extracellular enzyme laccase is involved in the initial produc-
tion of a metabolite M8 which involves break-up of the conjugated phthalocyanine
ring structure but which retains multi-negative charge. Another ligninolytic extra-
cellular enzyme, manganese peroxidase, is assumed to be involved in the release of
Cu2+ from the phthalocyanine structure to give a non-copper-containing phthalocya-
nine metabolite M1 with a slightly longer migration time than the parent dye and
absorption at 666 nm. The phthalocyanine ring structure is also broken up by meta-
bolic processes that involve oxidation and desulphonation to give phthalimide (M3)
and an unidentified electroactive metabolite M2.
WRF are most efficient in degrading synthetic azodyes. This property is due to
the production of extracellular LMEs, which, because of the lower substrate speci-
ficity, are able to degrade a wide range of xenobiotic compounds (Wesenberg et al.
2003). Mohorčič et al. (2006) found that Bjerkandera adusta was able to deco-
lourize the black-blue dye through red and violet to pale yellow via its extracellular
enzyme; MnP was also reported for its ability to decolourize Remazol Black B and
amaranth. Ben Younes et al. (2007) and Zouari-Mechichi et al. (2006) reported that
the crude enzyme as well as the purified laccase from Perenniporia tephropora was
able to decolourize dyes of the textile waste water, including Neolane Blue, Neolane
Pink and Remazol Brilliant Blue R (RBBR). The latter was also efficiently deco-
lourized by laccase from T. trogii. The ability of T. trogii laccase to decolourize tri-
arylmethane and azo dyes was approved in the absence of redox mediators, since
MG and BCG were completely degraded with crude laccase within 6 h of treatment.
This fungus appears to be the best choice, and it has the potential to degrade and can
be very useful in the treatment and disposal of textile and related effluents. P. chrys-
osporium can also be used to decolourize wastewater containing dyes with complex
structures directly (Kaushik and Malik 2009).
Manavalan et al. (2013) have first-time reported that Ganoderma lucidum lac-
case enzyme production using medium with 3% (v/v) ethanol improved the enzyme
production up to 14.1 folds. Moreira-Neto et al. (2013) studied on twelve
Basidiomycetes strains from the genus Pleurotus, Trametes, Lentinus, Peniophora,
Pycnoporus, Rigidoporus, Hygrocybe and Psilocybe on decolourization of the
reactive dyes Cibacron Brilliant Blue H-GR and Cibacron Red FN-2BL, both in
solid and liquid media. Among the evaluated fungi, seven showed great ability to
decolourize the synthetic textile effluent, both in vivo (74–77%) and in vitro (60–
74%), and laccase was the main ligninolytic enzyme involved on dyes decolouriza-
tion. Pleurotus ostreatus, Trametes villosa and Peniophora cinerea reduced near to
60% of the effluent colour after 1 h of treatment and the decolourization results
were still improved by establishing the nitrogen source and amount to be used during
the fungal strains cultivation in synthetic medium. For example their action on the
textile effluent, with yeast extract being a better nitrogen source than ammonium
tartarate. Rita de Cássia et al. (2013) study reported the LiP, Lac and MnP activities
of Curvularia lunata URM 6179 and Phanerochaete chrysosporium URM 6181 at
the end of textile effluent treatment in aerated and non-aerated bioreactors. Both
fungi showed higher activity of laccase but under different conditions: 2020 U/l for
the P. chrysosporium URM 6181 in aerated bioreactor and 2100 U/l for the
Curvularia lunata non-aerated bioreactor. Low LiP activity was observed for both
fungi strains. The activity of MnP was more evidenced in treatment with C. lunata
URM 6179. Activities of 7000 U/l for MnP and 8000 U/l for LiP in treatment
employing Lentinus strains, and 2000 U/l for Lac in treatment employing Coriolopsis
byrsina were found. The higher production of MnP and Lac was found in culture
medium containing wheat T. harzianum bran and glucose (Gomes et al. 2009). The
fungus Mucor racemosus CBMAI 847 produces 898.15 U/l of laccase in medium
containing 23% salinity and 4.5 mg/ml wheat bran while Cladosporium cladospo-
rioides CBMAI 857 produces 4.63 U/l.
El Monssef et al. (2016) study observed that the production of laccase enzyme
was higher at 35 °C and pH 5 after 6 days. The highest activity of laccase was
achieved at 35 °C and pH 5 during the reaction. FTIR analysis revealed that the
structure of extracted fungal pigments has aromatic ring and phenols group. Crude
laccase was capable to decolourize different pigment structures. The enzyme
showed great decolourization efficiency towards the extracted yellow pigment pro-
duced from Asp. terrus and Asp. ochareceous treated by 200 μl of partially purified
enzyme. Toxicity evaluation showed a final product detoxification (Ellouze and
Sayadi 2016). On the other hand, the fungal decolourization of RBBR has been
reported for other strains such as Ischnoderma resinosum, Dichomitus squalens, P.
ostreatus and Pleurotus calyptratus. Kunjadia et al. (2016) studied the role of ligni-
nolytic enzymes of Pleurotus spp. grown with azo dyes. The results indicated that,
WRF P. sapidus, P. ostreatus and P. florida were tested for ligninolytic enzyme
activity and their role in dye degradation. Percentage of decolourization clearly
showed higher removal of Coralene Golden Yellow (CGY) by P. ostreatus. Laccase
activity in cultures during dye decolourization was significantly higher compared to
MnP, suggesting the important role of laccase in dye degradation process.
Meanwhile, no LiP activity was found in any of the cultures. It is clear that enzymes
such as LiP, MnP and laccase play an important role in dye metabolism by WRF. P.
ostreatusis is the best fungal species out of all three studied organisms for lignino-
lytic activity and degradation of azo dyes.
4.8 hite Rot Fungi and Decolourization of Industrial Dye

W
Effluents
Reductions in the oxygen demand and carbon content of azo dye wastewaters, sub-
sequently treated with aerobic conditions, are well cited in previous studies (Horning
1977; Loyd 1992; McCurdy 1991). Due to the toxicity and xenobiotic nature,
degradation of azo dyes is indeed an uphill task. Now it had been well documented
that azo dyes can be degraded by microorganisms.
Due to their recalcitrant nature, azo dyes often pass through activated sludge
facilities with no or little reduction in colour (Cariell et al. 1995); however, some
researchers observed slight colour reductions in their findings (Zissi and
Lyberatos 2001). Effluents containing dyes are hardly decolourized by conven-
tional and biological wastewater treatments (Shaul et al. 1991; Willmott et al.
1998). In addition to their adverse impact and their visual effect, many synthetic
dyes are toxic with high chemical oxygen demand, carcinogenic and muta-
genic properties (Chung et al. 1978; Michaels and Lewis 1985). Also, the higher
volumetric discharge of industrial effluent in combination with severe legisla-
tion makes the searching of suitable treatment technologies an important prior-
ity (O'neill et al. 2000). Reduction of azo and other dyes with chemical and
physical methods requires highly expensive reagents and catalysts (Robinson
et al. 2001).
Usage of dyes and pigments are enormous in the paper, plastic, textile, cosmet-
ics, pharmaceutical and food industries (Levin et al. 2004). Based on toxicity and
carcinogenic nature of dyes and pigments, biodegradation of these synthetic dyes
may involve (Revankar and Lele 2006) WRF which are better dye-degraders than
prokaryotes. Extracellular non-specific LME system present in WRF plays a
major role in degrading a wide range of dyes (Christian et al. 2005). Most of the
former dye decolourization experiments were based mainly on Phanerochaete
chrysosporium and Trametes versicolor (Toh et al. 2003). However, other WRF
including Phellinus gilvus, Pleurotus sajor-caju, Pycnoporus sanguineus (Balan
and Monteiro 2001), Dichomitus squalens, Irpex flavus, Daedalea flavida,
Polyporus sanguineus (Chander 2007; Eichlerova et al. 2006; Gill et al. 2002),
Funalia trogii ATCC200800 (Özsoy et al. 2005), Ischnoderma resinosum
(Eichlerova et al. 2006) and Ganoderma sp. WR-1 (Revankar and Lele 2006) have
been experimented to have higher dye decolourization rates than P. chrysosporium
and T. versicolor.
At present, much attention has been concentrated on fungal decolourization
processes, especially on WRF due to their capacity of production of non-specific
enzymes, such as lignin peroxidase, manganese peroxidase and laccase, which act
as sorbents and detoxify many toxic aromatic compounds. Several research find-
ings indicate that WRF and their biodegradation/biotransformation capacity could
be an excellent candidate for dye removal (Gomaa et al. 2008; Rita de Cássia et al.
2013; Wesenberg et al. 2003). Among the different groups of fungi, Phanerochaete
chrysosporium is an effective dye-degrading microorganism. P. chrysosporium
has become known as a model system in textile, pulp and paper mill wastewater
bioremediation. P. chrysosporium is a basidiomycete fungus able to detoxify com-
plex compounds such as starch, cellulose, pectin, lignin and lignocelluloses in
textile dye wastewater (Senthilkumar et al. 2014). This fungus emerges to be the
best choice to degrade complex wastewater in the treatment process and disposal
of textile and related wastewater.
Much research on dye degradation has been reported by many scientists with indi-
vidual dyes or under simulated condition by WRF, but under dye industry, wastewa-
ter treatment was reported only in limited extent. Application of WRF at industrial
scale under non-sterilized condition is a big technical challenge. In addition, the
chemistry of intermediates formed during the degradation was also in its infant
stage. Bioassay studies of treated dye wastewater should also be established. Aquatic
fungi and their ability to produce several non-specific enzymes may serve as a new
resource to treat textile industry wastewater. Presence of degradative plasmid encod-
ing for such environmentally significant genotype of azo dye degradation opens up
the possibilities of genetic transfer of this character into other microorganisms,
which can act as a valuable tool in the remediation of azo dye-polluted habitat.
Increasing the contribution of small and medium enterprises of textile, chemical and
pharmaceutical in total exports of India is vital to India’s future economic growth.
Detailed scientific studies with natural dyes have established that in most cases their
properties are comparable to those of synthetic dyes. Therefore, if natural dyes have
to be commercialized, they need to conform to the same stringent standards of per-
formance that are applied to synthetic dyes. It thus follows that much more research
and developmental effort needs to go in this area.
Acknowledgement The authors are grateful to the Department of Environmental Sciences, Tamil
Nadu Agricultural University, Coimbatore, for providing laboratory facilities. The authors extend
their gratitude to Mrs. P. Divya and Mr. P. Sivasamy for providing textile wastes-related data.
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Chapter 5
Pleurotus ostreatus: A Biofactory
for Lignin-Degrading Enzymes of Diverse
Industrial Applications
Hesham El Enshasy, Farid Agouillal, Zarani Mat, Roslinda Abd Malek,

Siti Zulaiha Hanapi, Ong Mei Leng, Daniel Joe Dailin, and Dalia Sukmawati
5.1 Introduction
Mushrooms have been recognized as important food and medicine in many ancient
civilizations (El Enshasy et al. 2013). These are based on their high nutrient con-
tents of carbohydrates, proteins, vitamins, minerals, and many other growth-pro-
moting ingredients (Eleftherios et al. 2014; Maftoun et al. 2015). Nowadays, of
thousands types of mushroom studied, Pleurotus sp. is considered as one of the top
three economically important and widely grown mushrooms beside Agaricus and
Lentinula. Pleurotus ostreatus (widely known as oyster mushroom) gained more
interest in the recent years not only because of their high nutritional value but also
due to the high content of bioactive polysaccharides and other metabolites of high
medicinal values. These compounds exhibited immunomodulatory, antitumor,
antioxidant, anti-inflammatory, antihyperglycemic, antihypocholesterolemic,
H. El Enshasy (*)
Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM),
Johor Bahru, Malaysia
School of Chemical and Energy Engineering, Faculty of Engineering,
Universiti Teknologi Malaysia (UTM), Johor Bahru, Malaysia
City of Scientific Research and Technology Applications, New Burg Al Arab,
Alexandria, Egypt
e-mail: henshasy@ibd.utm.my
F. Agouillal
Research Unit on Analysis and Technological Development in Environment (URADTE),
Centre de Recherche Scientifique et Technique en Analyses Physico-Chimiques (CRAPC),
Tipaza, Algeria
Z. Mat · R. A. Malek · S. Z. Hanapi

102 H. El Enshasy et al.
antimicrobial, and antithrombotic properties (El Enshasy et al. 2012; El Enshasy

and Hatti-Kaul 2013; Mohamed and Farghaly 2014; Correa et al. 2016). The thera-
peutic effect of oyster mushroom extract and pure bioactive compounds has been
studied and proved in many in vivo and in vitro studies (Ryu et al. 2014; Elsayed
et al. 2014; Younis et al. 2015; Masri et al. 2017).
In addition, P. ostreatus gained more interest than other types of mushrooms
based on their ability to grow on large number of substrates and under different
environmental conditions (tropical and subtropical region), high capacity to pro-
duce a large number of hydrolases, and ease of cultivation both in solid-state fer-
mentation and in submerged cultivation system as well as higher growth rate
compared to other types of mushrooms (El Enshasy et al. 2010; Maftoun et al.
2013). However, to survive in nature, mushrooms should be able to degrade com-
plex lignocellulosic materials by different hydrolytic enzymes to break the complex
structure of wood and utilize the produced sugars as substrate for growth and metab-
olite production. Therefore, mushrooms are well known for their high capacity to
produce and excrete different types of enzymes of wide range of hydrolytic activi-
ties. Therefore, this chapter will provide the latest information about the enzyme
systems of this type of mushroom and their potential application in different
industries.
5.2 Understanding Lignin Structure
Lignins constitute with cellulose and hemicelluloses the three major components of
lignocellulosic biomass. They are the second most abundant terrestrial polymers
and carbon source after cellulose (Evers et al. 1999; Boerjan et al. 2003). In 1813,
A. P. de Candolle has firstly named the substance as “lignine” based on the Latin
word lignum which means “wood.” He described lignin as a fibrous, tasteless mate-
rial, insoluble in water and alcohol, but soluble in weak alkaline solutions and which
can be precipitated from solution using acid. The typical composition of the dry
weight of wood, considered as a whole, is about 50% cellulose, 25% lignin, 20–25%
hemicellulose, and 1–4% pectin (Camarero et al. 2014).
O. M. Leng
Harita Go Green Sdn. Bhd., Johor Bahru, Johor, Malaysia
D. J. Dailin
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi
Malaysia (UTM), Johor Bahru, Malaysia
D. Sukmawati
Faculty of Mathematics and Natural Sciences, Universitas Negeri Jakarta, Jakarta, Indonesia
5 Pleurotus ostreatus: A Biofactory for Lignin-Degrading Enzymes of Diverse… 103
Cellulose forms basically a skeleton that is bounded by hemicelluloses and lig-

nin (Sakakibari 1980). Lignin, a polyphenolic amorphous polymer, is the essential
natural glue that fills the spaces between cellulose and hemicellulose and acts like a
resin that holds the plants’ lignocellulose matrix together (Ritter 2008; Carmen
2009). Through this cross-linking with cellulose, lignin not only confers the
strength, rigidity, and flexibility as well as aids in water transport to the plant but
represents also the most significant barrier to wood decay by insects/microorgan-
isms attacks and prevents the access of low molecular weight diffusible agents
(Zakzeski et al. 2010).
In chemical point of view, lignin is a heterogeneous complex class of compounds
that changes according to biomass source and isolation technique (Johnson 2002).
However, many aspects in the chemistry of lignin still remain unclear due to its high
complex structure (Pouteau et al. 2003); However, the lignin classification as grass,
hardwood, and softwood is based on the ratios of three major phenylpropene mono-
mers which vary according to the plant species, plant tissue, individual cell types,
and cell wall layers (Faix 1991). In general, both native lignin (as present in biomass)
and technical lignin (isolated from biomass through various processes) have three-
dimensional amorphous polymer made up of methoxylated phenylpropane aromatic
units structures of p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Chakar
and Ragauskas 2004). These phenylpropene monomers are randomly cross-linked
polymers arising from an enzyme-mediated dehydrogenative polymerization of three
phenylpropane monomer precursors (cinnamyl alcohol: coniferyl, synapyl, and
p-coumaryl alcohols) (Kleinert and Barth 2008; Wong 2009). These three monoli-
gnols (phenylpropene units of lignins) shown in Fig. 5.1 are as follows:
Fig. 5.1 The chemical structure of the primary lignin building blocks and their corresponding
lignin polymer monomeric units (Zakzeski et al. 2010; Abdel-Hamid et al. 2013)
• Guaiacyl (G) units from the precursor trans-coniferyl-alcohol

• Syringyl (S) units from trans-sinapyl-alcohol
• p-Hydroxyphenyl (H) units from the precursor trans-p-coumaryl alcohol
Then, grass lignin is built up by the three monomeric units (G, S, and H), hard-
wood lignin contains roughly equal amounts of (G) and (S) units, and softwood
lignin is composed mainly of (G) units (Faix 1991).
5.2.1 Lignocellulose Processing and Industrial Applications
For more economic profitability, lignocellulosic biorefinery produces multiple

products, including fuels, and bulk or fine chemicals, from biomass, analogically
compared to a petroleum refinery, which produces fuels and chemicals from crude
oil (Zhang 2008; Rastegari et al. 2019). In order to achieve a high energy impact and
an economically viable biorefinery, the valorization of all components of lignocel-
lulosic biomass should be carried out (Abdel-Hamid et al. 2013). With various aro-
matic structures, many authors claim that lignin has a special industrial interest
offering the feasibility of replacing relevant aromatic polymeric and fine chemicals
(Duval and Lawoko 2014; Norgren and Edlund 2014; Laurichesse and Avérous
2014; Thakur and Thakur 2015; Thakur et al. 2014).
Lignins are usually considered as waste products of pulp and paper industry and
had limited industrial uses. However, in 1998, only 1% of the lignin produced was
used in valuable industrial processes. About 50 million tons of lignin were esti-
mated to be extracted annually from woody biomass for the pulp and paper industry
and applied as dispersants, adhesives, and surfactants (Cohen et al. 2002; Shah and
Nervd 2002; Karam and Nicell 1997). However, new technologies are currently in
use to valorization and converting lignin into value-added chemicals; then, a bio-
mass fractionation technology has been developed by PureVision Technology, Inc.,
to produce value-added low molecular weight lignin as a coproduct to the cellulose
stream and also as fuel substrate (Chheda et al. 2007).
In energy production process, the lignin fraction acts as a barrier against enzyme
or microbial penetration through lignocelluloses decreasing the fermentable sugar
yields and affecting negatively the overall biofuel development, making it an uneco-
nomical process (Margeot et al. 2009; Menon and Rao 2012). Essential mechanical,
thermomechanical, and thermochemical pretreatment strategies to overcome this
limitation have been reported by many authors (Margeot et al. 2009; Frigon and
Guiot 2010; Kumar et al. 2009) and could be summarized as follows:
• Mechanical particle size reduction including both of wet, dry, ball, or vibratory
ball milling and other forms of biomass grinding (Sarkar et al. 2012; Agbor et al.
2011).
• Thermochemical hydrolysis at temperatures between 140 °C and 180 °C, using
a dilute solution of sulfuric acid (0.5–2%) for 10–30 minutes as residence times
for rendering the carbohydrate fraction (Yang and Lu 2010).
• Thermomechanical steam explosion heating briefly the biomass to high tempera-

tures (~200 °C) under high pressure followed by a rapid pressure drop that makes
the biomass more penetrable for subsequent fermentation (Chandra et al. 2007).
• Microwaves heating at short time create localized hotspots and open up the lig-
nocelluloses biomass, facilitating enzyme access for saccharification (Canam
et al. 2013).
• Organosolv process modifying chemically and removing low molecular weight
lignin fractions from biomass using alcohols such as ethanol, methanol, or other
solvents at high-temperature extraction. In this process, some dilute acids like
hydrochloric and sulfuric acid are used as a catalyst (Agbor et al. 2011).
• As natural modification and degradation of the lignin component, biological pre-
treatments can reduce the severity requirements of obvious pretreatment strate-
gies. The exploitation of the capacity to access and to modify the lignocellulosic
biomass by microorganisms was studied by several authors such as Itoh et al.
(2003) who used a variety of lignin-degrading white-rot fungi pretreatments fol-
lowed by extracting lignin by an organosolv method from wood chips. This pro-
cess leads to saving of electricity up to 15% and increases the ethanol yield
obtained from the solid fraction.
The enzymatic degradation of lignocellulose by microbial consortia or lignin-
degrading fungi involves both oxidative and hydrolytic mechanisms based on OH
radical reactivities. Attack on the hydroxyl group in lignin subunits by abstracting
aliphatic hydrogens and adding to aromatic rings results in benzyl ketone produc-
tion and hydroxylated cyclohexadienyl radical generation occurring on subsequent
C-C bond cleavage and degradative chain reactions (Gierer 1990). This leads to
decreasing energy requirements, less producing of fermentation-inhibiting sub-
stances. Thus, biological pretreatment of lignocellulosic materials is very useful
when incorporated into any economical biorefinery strategies for biofuels and/or
metabolite production (Isroi et al. 2011; Chen et al. 2010). In pulp and paper indus-
tries, processed lignin is produced traditionally through three distinct processes:
kraft lignin, lignosulfonate lignin, and organosolv lignin (Doherty et al. 2011).
Other than renewable bioenergy resources and pulp and paper industry supplement,
significant progress is being made in research for the feasibility of a variety of appli-
cations of lignin or lignin-related product as a resource for chemicals and materials.
For this, two different strategies are followed for lignin conversion to value-added
components, either controlled depolymerization into small molecules (Li et al.
2015; Behling et al. 2016) or building block to synthesize new functional materials
(Kai et al. 2016; Upton and Kasko 2016). Possessing multiple functional groups
that can form inter- and intramolecular hydrogen bonding, lignin is considered as an
alternative source for the production of more value-added chemicals according to its
compatibility with host matrices (Karam and Nicell 1997; Doherty et al. 2011;
Wang et al. 2016). This compatibility which is improved by chemical modification
(oxyalkylation or hydroxyalkylation) minimizing lignin molecules’ auto-association
(Zhao et al. 2016; Feldman et al. 2001; Maldhure et al. 2012). Gallezot (2007)
indicated that three potential strategies for lignin valorization to produce fine chemi-
cals with a high degree of functionality can be adopted.
The first strategy acts on gasifying lignin allowed to synthesize gas or degraded
by pyrolysis to a mixture of small molecules. The second strategy turns, in the first
step, the functional groups present into the lignin monomers’ simple aromatic com-
pounds such as phenol, benzene, toluene, and xylene. In a second step, bulk and fine
chemicals are produced using catalytic technology developed for petroleum refiner-
ies. Finally, using highly selective catalysts in a one-pot approach, the third strategy
converses directly the biomass to valuable chemicals (Karam and Nicell 1997;
Doherty et al. 2011; Thakur et al. 2014; Chatterjee and Saito 2015; Liu et al. 2015).
Then, several value-added chemicals are produced including:
1. Lignin: as a filler or additive in polymers and biopolymers – usually at less than
20–30% of total weight
2. Lignin-derived functional materials such as carbon fibers, activated carbon,
adhesives, and foams
3. Lignosulfonates: as dispersants, water reducer in concrete, additive in coal-water
slurry, or viscosity reducer
In addition, in light of renewed interest in promoting value-added applications of
lignin, it is expected recently lignin nanoparticles will play a vital role in promoting
lignin valorization in polymer industry (Stark et al. 2015).
5.2.2 Lignin Degradation
Lignin is a complex polymer with very low degradation rate in nature. The biodeg-
radation which is carried out by wood-rotting fungi constitutes a key step for carbon
cycling in nature, as well as for the industrial use of plant biomass by increasing
accessibility to cellulose. It is an oxidative process that has been investigated for
decades as a model for biotechnological application in the pulp and paper indus-
tries, animal feeding, and bioethanol production. Ligninolytic oxidoreductases (lac-
cases and different types of peroxidases) secreted by wood-rotting fungi are the sole
enzymes able to oxidize the phenylpropane lignin units (Schwarze 2007; Yadav
et al. 2016, 2018). However, different enzymes have been reported for their role in
lignin degradation such as lignin peroxidase (LiP), manganese peroxidase (MnP),
aryl alcohol oxidase (AAO), and glyoxal oxidase (GLOX) as shown in Fig. 5.2.
The main enzymes associated with lignin degradation are laccases, lignin peroxi-
dases, and manganese peroxidases; while some white-rot fungi produce all the three
classes of enzymes, others produce only one or two (Hatakka 1994). Laccases are
multicopper enzymes, which catalyze the oxidation of phenolic compounds includ-
ing a range of dyes with concomitant reduction of oxygen (Eggert et al. 1996;
Chivukula and Renganathan 1995; Munoz et al. 1997). Recent interest in laccase is,
in part, a consequence of the findings that the substrate range of laccase can be
expanded to include non-phenolic dyes, eventually in the presence of suitable medi-
ators (Bourbonnais and Paice 1990).
Fig. 5.2 Lignin-degrading enzymes’ actions. LiP lignin peroxidase; MnP manganese peroxidase;
AAO aryl alcohol oxidase; GLOX glyoxal oxidase; VP versatile peroxidase; MED mediator.
(Modified from Abdel-Hamid et al. 2013)
5.3 Laccase (EC 1.1.2.2)
Mushrooms excrete different oxidative, hydrolytic, and non-hydrolytic enzymes

that act synergistically to hydrolyze the complex chemical structure which is com-
posed of cellulose, hemicelluloses, and lignin (Guerriero et al. 2015). Laccases and
class II peroxidases belong to the oxidative enzymes including lignin peroxidase,
manganese peroxidase, and hybrid lignin/manganese versatile peroxidase which
catalyze the cleavage of C-C and C-O-C bonds in a wide variety of organic com-
pounds, such as lignin and polyphenolic structures (Ertan et al. 2012; Siddiqui et al.
2014). Laccases (or phenoloxidase) were firstly described and identified in 1883 by
Yoshida, from the Japanese lacquer tree Rhus vernicifera. They are phenol-oxidizing
enzymes and listed as ecofriendly catalyzing variety of reactions involving one-
electron oxidation with water generation (Hao et al. 2007; Javed et al. 2017).
Laccases are widespread in nature and have been found in non-microbial sources
(plants and insects) and in microbial sources (Thurston 1994; Brijwani et al. 2010;
Mayer and Staples 2002; Santhanam et al. 2011; Yavuz et al. 2014). As shown in
Table 5.1, the genus Pleurotus showed strong laccase activity (Silva et al. 2012;
Alexandrino et al. 2007; Sethuraman et al. 1999; Ardon et al. 1996). P. ostreatus
produces many extracellular enzymes with strong ability for lignin degradation and
substrate oxidation. Therefore, this mushroom attracted attention because of its high
potential for biofactory laccase production for many biotechnology applications
(Karas et al. 2011; Faraco et al. 2009; Pezzella et al. 2013).
Table 5.1 Different enzymes produced by Pleurotus ostreatus
108
Enzyme Mushroom Substrate/media

(EC number) type composition Enzyme activity Cultivation conditions/parameters References
Laccase P. ostreatus Orange waste 75.0 U g−1 (laccase) Solid-state fermentation, cultivation at wide Alexandrino
(EC 1.10.3.2) 6.8 U g−1 (MnP) temperature range 20–35 °C for 20 days et al. (2007)
(laccase production), 30 days (MnP
production)
P. ostreatus Cotton stalk extract 0.95 U mL−1 Solid-state fermentation for 16 days at 28 °C Ardon et al.
Florida f6 (1998)
P. ostreatus Basic Broth Media 2.4 UmL−1 Submerged culture incubated at 28 °C Mansur et al.
strain V-184 agitated at 100 rpm for 14–16 days at pH 6.5 (2003)
P. ostreatus Wheat bran 803.3 U Submerged culture incubated at 28 °C and Krishna et al.
1804 pH 5.5 (2005a)
Production increased by addition of
(2,5-xylidene; 1.0 mM) as inducer after 96 h
P. ostreatus Glucose-based Free mycelia: 272.2 U Submerged culture in packed bed reactor Krishna et al.
1804 chemically defined Immobilized: 312.6 U incubating for 192 h at pH 5.6 (2005b)
(Immobilized medium Packed bed
on PUF) reactor:392.9 U
P. ostreatus Coffee husk 22.50 U mL−1 Submerged culture using CuSO4 (150 μM) as Silva et al. (2012)
inducer, incubation for 15 days at 28 °C
P. ostreatus Sugarcane bagasse 167 U g−1 Solid-state fermentation at 28 °C and pH 5.5 Karp et al. (2012)
P. ostreatus Three agro-industrial Maximal production of Solid-state fermentation, incubation at 20 °C Rodrigues dal
wastes (coffee husks, 16 μM min kg−1 in coffee for 20 days. Produce enzyme cocktail of Luz et al. (2012)
eucalyptus sawdust, and husks laccases, MnP, and cellulase
eucalyptus bark) with
20% rice bran
P. ostreatus Water polluted with 200 U L−1 Submerged cultures incubated for 24 days at Parenti et al.
wheat straw extracts 25 °C in the dark (2013)
Manganese P. ostreatus Semi-defined medium 451 U L−1 Submerged cultivation for 6 days at 26–28 °C Sarkar et al.
peroxidase (EC CBS 411.71 with glucose (1997)
H. El Enshasy et al.
1.11.1.13)
5
P. ostreatus Chemically defined 900 U L−1 Submerged cultivation for 25 days in dark at Kamitsuji et al.
ATCC 66376 glucose-based medium 28 °C and pH 4.5 (2005)
P. ostreatus Rice bran 7300 U L−1 Submerged cultivation for 28 days in dark at Tsukihara et al.
dikaryotic 28 °C (2006a)
strain #261
ATCC 66376
P. ostreatus Wheat straw 692 U mL−1 Solid-state fermentation for 7 days at 30 °C Ashger et al.
IBL-02 (2013)
Versatile P. eryngii Cotton stalks 178.9 U mL−1 Submerged cultivation at 25 °C Min et al. (2010)
peroxidase (accession
(EC 1.11.1.16) number
GIM5.280)
P. ostreatus Cotton stalks 0.16 U mL−1 Submerged cultivation in dark for 10 days at Salame et al.
(accession 28 °C (2012)
number
CECT20311)
Aryl alcohol P. eryngii Sterile wheat straw 0.04 U g−1 Cultivation using SSF method Camarero et al.
oxidase ATCC 90787 (1999)
(EC 1.1.3.7)
P. ostreatus Semi-synthetic medium 300 U L−1 Submerged cultivation for 24 days in dark at Feldman et al.
(accession supplemented with 30 °C (2015)
number different inducers:
CECT20311) tyrosine, VA, and benzyl
alcohol
P. ostreatus Saw dust-based medium 211 U L−1 Submerged cultivation in dark for 20 days Sannia et al.
(strain with addition of (1991)
Florida) inducers VA, olive oil,
Pleurotus ostreatus: A Biofactory for Lignin-Degrading Enzymes of Diverse…
and tween 80
(continued)
109
110

Pleurotus sp. Wheat straw 11.47 U mL−1 SSF is the best to secrete xylanase enzyme Getachew et al.
supplemented with after 4 days of incubation at 30 °C and pH 4 (2016)
xylan, peptone, and KCl
P. ostreatus 2.5% (corncob), 2.5% 24.98 U mL−1 Submerged cultivation at 26 °C Qinnghe et al.
SYJ042 (wheat bran), peptone (2004)
Xylanase P. ostreatus Pepper extract 155.8 Cultivation in submerged culture in dark for Morais et al.
(EC 3.2.1.8) (nmol/min × mL) 32 days at 22–26 °C (2005)
P. ostreatus Rice bran and saw dust 65 μM min−1 kg −1 of Fruiting bodies were produced after Rodrigues da Luz
(PLO 2 and substrate incubated for 20 days at 25 °C and 90% et al. (2012)
PLO 6)
P. ostreatus Spent mushroom 91.56 U g−1, SSF, optimal enzyme recovery was achieved Lim et al. (2013)
compost (SMC) when SMCs were extracted with 50 mM
sodium citrate (pH 4.5) buffer at 4 °C
Pleurotus 10% sisal leaf and sisal 3.73 U g−1 SSF cultivation for 20 days at 26–30 °C and Raymond et al.
HK-37 boles with ration humidity 78 ± 2% (2015)
(0:100) and cow dung
manure
P. ostreatus Oxo-biodegradable 0.51 U mg−1 SSF cultivation for 45 day at 25 °C Rodrigues da Luz
PLO6 plastic without physical et al. (2013)
treatment
P. ostreatus Saw dust 21.0 U g−1 SSF cultivation in dark for 50 days 20 °C. Sherief et al.
(2010)
P. ostreatus Cotton wastes (CW) as 48 U g−1, SSF, fruiting bodies formed after 49 days Elisashvili et al.
(Jacq.:Fr.) growth substrate (2003)
Kumm
P. ostreatus Modified Czapek 0.863 U ml−1 Submerged cultivation 30 °C for 7 days Karthikeyan,
medium (2015)
5
Cellulase P. ostreatus Rice straw 3.51 U ml Cultivation for 14 days at 37 °C and pH 5.5 Khalil et al.
(EC 3.2.1.4) (Jacquin ex (2011)
Fr.) Kummer
Endoglucanase P. ostreatus Avicel PH101 2.46 U ml−1 Submerged cultivation for 12 days at 27 °C Daba et al.
(EC 3.2.1.4) (Jacq.) and pH 5.5 (2011)
P. Kumm.
NRRL 366
Exoglucanase P. ostreatus Avicel PH101 1.80 U ml−1 Submerged cultivation for 12 days at 27 °C Daba et al.
(EC 3.2.1.4) (Jacq.) Rice straw and pH 5.5 (2011)
P. Kumm.
NRRL 366
P. ostreatus
Endoglucanase P. ostreatus Avicel PH101 4.02 U g−1 SSF cultivation for 20 days at 20 °C Sherief et al.
(EC 3.2.1.4) (2010)
Exoglucanase Rice straw 6.01 U g−1
(EC 3.2.1.4)
CMCase Saw dust 13.21 U g−1
Cellulase P. ostreatus What bran 1.25 U ml−1 Submerged cultivation system Radhika et al.
LCJ 183 (2013)
P. ostreatus Spent mushroom 1.67 U g−1 Submerged cultivation Lim et al. (2013)
compost
P. ostreatus Sisal leaf and Sisal bole 3.73 U g−1 SSF for 30 days at 26–30 °C and 87% Raymond et al.
HK-37 humidity (2015)
P. ostreatus Papain waste and rice 14.2 U g−1 SSF, for 7 days cultivation at 28 °C Rashad et al.
NRRL 366 straw (2009)
Pectinase Pleurotus 30% sisal leaf 8.28 U g−1 SSF for 30 days at 26–30 °C and 87% Raymond et al.
(EC 3.2.1.15) HK-37 humidity (2015)

P. ostreatus Sawdust 21.42 U g−1 SSF dark cultivation at 20 °C Sherief et al.
(2010)
(continued)
111
112

Pectin lyase P. ostreatus Lemon pulp waste 81.30 SSF for 4 days at 28–30 °C Rashad et al.
(EC 4.2.2.10) NRRL 366 (U mg−1 protein) (2011)
Polygalacturonase P. ostreatus Lemon pulp waste 158 U mg−1 SSF for 4 days at 28–30 °C Rashad et al.
(PG) NRRL 366 (2010)
(EC 3.2.1.15)
Exo- P. ostreatus Lemon peel 1750 U Submerged fermentation for 4 days Rashad et al.
polygalacturonase NRRL 366 (2009)
(exo-PGase)
(EC 3.2.1.67)
Cysteine protease P. ostreatus Commercial product 14.68 U g−1 Obtained from the fruiting bodies grown by Shin and Choi
SSF (1998)
P. ostreatus, Defined medium 8.10 U mg−1 Submerged cultivation for 6 days at 25 °C Liu et al. (2014)
No 4241 supplemented with
glucose and soymilk
ProA – serine P. ostreatus – 110 U Obtained from the fruiting bodies grown by Dohmae et al.
protease (Hiratake) 23,600 U SSF (1995)
(EC 3.4.22.9) Present (quantitative
ProB – analysis was not given in
metalloprotease this work)
(EC 3.4.99.32)
ProC –
metalloprotease
(EC 3.4.24.4)
Fibrinolytic P. ostreatus - Defined medium 8424 U Submerged cultivation for 5 days at 25 °C Shen et al. (2007)
protease supplemented with
(EC 3.4.21.5) glucose
5
Serine protease P. ostreatus Basal medium (potato 2.5 U mg−1 Submerged cultivation in dark for 3 days at Palmieri et al.
(EC 3.4.21.14) (Jacq.:Fr.) dextrose broth, yeast 27 °C (2001)
Kummer extract
(type:Florida)
Metalloprotease P. ostreatus Medium containing 120 U ml−1 Submerged cultivation for 7 days at 28 °C Genier et al.
Serine protease (PLO 06, (glucose 10 g /l; yeast (2015)
(EC 3.4.21) GenBank extract, 10 g/l;
accession K2HPO4, 5 g/l; and
number MgSO4, 0.10 g/l)
KC782771
P. ostreatus Potato peel waste 4 U L−1 SSF for 23 days at 25 °C Ergun and Urek
(Jacq.) (2017)
Pleurotus
Kumm.
(MCC16)
Amylase P. ostreatus Rice bran 220 μmol/min/mg Obtained from the fruiting bodies grown by Akinyele et al.
(EC 3.2.1.x) SSF (2010)
P. ostreatus Spent mushroom 2.97 U g−1 Obtained from the fruiting bodies grown by Lim et al. (2013)
compost (SMC) SSF
P. ostreatus Potato peel waste 6 U L−1 SSF for 23 days at 25 °C Ergun and Urek
(Jacq.) (2017)
Pleurotus
Kumm.
(MCC16)
Lipase P. ostreatus Using chemical defined 0,93 U mg−1 Produced in immobilized cell cultivation Wijayati et al.
(EC 3.1.1.3) medium system (2017)
P. ostreatus Medium supplemented 30 U L−1 Submerged fermentation (SF), 5 days of Guarino and
with olive mill cultivation Sannia (2013)

wastewater
113
5.3.1 Laccases Structure and Mechanism of Reaction
Laccase (EC 1.10.3.2), or benzenediol: oxygen oxidoreductases, belongs to a group

of phenol oxidizers enzymes called blue multicopper oxidases, which are consid-
ered generally as part of the monomeric glycoprotein group. This group of enzyme
is mainly exocellular enzyme that can oxidize phenols and aromatic compounds
including amines, esters, and ethers (Rathinasamy and Thayumanavan 2010;
Solomon et al. 1996). They act by reducing the molecular oxygen to water and cata-
lyzing the oxidation of ortho- and para-aminophenols, diphenols, aryl diamines,
polyphenols, polyamines, and lignin (Shleev et al. 2006a, b). Therefore, laccases
can involve in the degradation of a wide range of xenoaromatics substrates such as
textile dyes, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, pesti-
cides, and synthetic polymers (Mester and Tien 2000; Bezalel et al. 1997; Novotny
et al. 2000; Riva 2006; Yadav et al. 2019a, b). Regarding this high nonspecific oxi-
dation capacity, laccases derive interests and attraction from researchers to be used
as biocatalysts for many industrial and biotechnological applications, from food
processing to environmental technologies through textile and paper industry. Recent
research showed also the potential applications of laccases in nanobiotechnology
and biomedicine (Mayer and Staples 2002; Jurado et al. 2009; Zhuo et al. 2011;
Javed et al. 2017).
Based on genetic research, four different genes and their corresponding cDNAs
have been identified from P. ostreatus and named pox1 (laccase isoenzyme not yet
identified) (Giardina et al. 1995), pox2 or poxc (Giardina et al. 1996), poxa1b
(Giardina et al. 1999), and poxa3 (Palmieri et al. 2003). According to Mot and
Silaghi-Dumitrescu (2012), the structure of laccases is held up by monomeric units
consisting of three domains which are arranged in a sequence to form a barrel-type
structure. The same study reported that fungal laccases lack high-order oligomeric
assemblies making the crystal lattice and are found generally as monomers.
According to Hoegger et al. (2006), laccases have four catalytic copper atoms in
their structure organized in two individual sites that bind the reducing substrate and
O2. The substrate oxidation site contains a paramagnetic type 1 copper (T1 or T1Cu)
responsible for the characteristic blue color of the enzyme in the reduced resting
environment. Another copper (T2 or T2Cu) contributes in catalytic and redox activ-
ity site coordinated by three histidines. The two other coppers (T3 or T3Cu) are
responsible for the activation and O2 transport and substrate oxygenation and coor-
dinated by three histidines (Hoegger et al. 2006). Both T2Cu and the two T3Cu are
clustered at 12 Å from the T1Cu and form a tri-nuclear site as shown in Fig. 5.3,
where O2 is reduced to two molecules of water, receiving four consecutive electrons
from four independent mono-oxidation reactions at the T1Cu site through a strictly
conserved His-Cys-His electron transfer route (Mot and Silaghi-Dumitrescu 2012).
Beyond this molecular configuration, other enzymes can include also 2, 3, or 6
copper atoms (Gil et al. 2009; Messerschmidt and Huber 1990). The high potential
of copper T1 is of great interest to biotechnologists because of its geometry. In this
site, around the T1 copper atom, two histidines and one cysteine which are orga-
Fig. 5.3 Representation of a native fungus laccase from Trametes hirsute (Left), showing the cop-
per ions organization (Right). http://www.rcsb.org/pdb/explore/jmol.do?structureId=3FPXopt=3
nized three-dimensionally with two other residues are weakly coordinated from the
axial position. One of these residues is isoleucine, and the other can be phenylala-
nine, leucine, or methionine depending on the type of laccase characterized and
reduction potential (Rodgers et al. 2010; Rivera-Hoyos et al. 2013). Several authors
have reported that more than one laccase isozyme is secreted by fungi (Soden and
Dobson 2001; Hoshida et al. 2001; Palmieri et al. 2003; Rodrïguez et al. 2008). At
least, eight different laccase isoenzymes are produced by P. ostreatus and six of
which have been isolated and fully characterized (Giardina et al. 1999; Pezzella
et al. 2009; Palmieri et al. 1993, 1997, 2003). The most abundant protein present in
the P. ostreatus cultures is POXC (59-kDa with pI 2.7). The enzymes POXA1b and
POXA1w have a similar molecular weight around 61 kDa, while POXA2, POXB1,
and POXB2 isoenzymes are of higher molecular weight around 67 kDa. POXA3a
and POXA3b are heterodimers and composed of small (16 or 18 kDa) and large
(61 kDa) subunits (Palmieri et al. 1997; Giardina et al. 1999).
However, several studies reported that P. ostreatus produces mainly three laccase
isoenzymes called POXCs (Giardina et al. 1996): POXA1w, the white laccase iso-
enzyme with a singular metal content (Palmieri et al. 1997); POXA1b, the more
stable isoenzyme at alkaline pH (Giardina et al. 1999); and the heterodimeric lac-
case isoenzyme POXA3 (Palmieri et al. 2003; Giardina et al. 2007; Faraco et al.
2008) with two forms, POXA3a and POXA3b (Palmieri et al. 2003; Giardina et al.
2007), that exhibit unusual structural features, being heterodimeric enzymes
(Wahleithner et al. 1996; Yaver et al. 1996). These laccase isoenzymes are endowed
with quaternary structure, consisting of two subunits of different molecular weights
(Faraco et al. 2008). Palmieri et al. (1997) reported that the two laccase isoenzymes
POXA1 and POXA2 are monomeric glycoproteins containing 3% and 9% carbohy-

drate with pI values of 6.7 and 4.0, respectively. The sequencing of the N terminus
and three tryptic peptides of POXA1 reveals clear homology with laccases from
other microorganisms, while POXA2 showed a blocked N terminus. However,
POXA1 showed remarkable high stability at relatively wide range of temperature
and pH values, whereas POXA2 is less stable at high temperature (Palmieri et al.
1997). The molecular weights of the two laccase isoenzymes were determined with
three different methods. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
shown that the Mwt of POXA1 and POXA2 are of about 61 and 67 kDa; Gel filtra-
tion in native conditions shows a Mwt of about 54 and 59 kDa; Finally, matrix-
assisted laser desorption ionization mass spectrometry was applied for POXA1 and
give 61 kDa (Palmieri et al. 1997). Substrates with specific linkages and structural
similarity with lignin, an amorphous polyphenolic polymer, can induce laccase
activity. In addition, a wide range of aromatic compounds can be oxidized by lac-
cases with concomitant water generation by reduction of molecular oxygen
(Thurston 1994; Ullah et al. 2000).
In general, laccase is not able to oxidize substrates with lower redox potential
like non-phenolic compounds. However, Call and Mücke (1997) have reported that
the oxidation can be catalyzed in the presence of electron transfer mediators.
Oxidation of substrates with chemical structure smilar to lignin like amino-phenols,
orth- and para-diphenols, polyamines, aryl diamines, lignins and polyphenols is
also carried out using the same mechanism. Then, laccases can act on many sub-
stances such as agricultural inputs, pesticides and herbicides, and dyes used in many
industrial processes which must be degraded before being discarded (Morozova
et al. 2007).
According to Baldrian (2006), more than 100 fungal laccases are purified and
characterized and usually with optimal activity at acidic pH range with a tempera-
tures range from 50 to 70 °C. Table 5.1 summarizes some other characteristics of
laccase isoenzymes from Pleurotus ostreatus strains. As reported by Palmieri et al.
(1993, 1997), generally, for the P. ostreatus laccase POXC, the optimal pH is already
acidic for both ABTS and DMP degradation but around 6 for guaiacol and syrin-
galdazine with a range of optimum temperature from 50 to 60 °C (Palmieri et al.
1993, 1997). Studying the two laccase isoenzymes POXA1 and POXA2 from P.
ostreatus, Palmieri et al. (1997) reported that POXA2 acts with an acidic optimum
pH of 3 for ABTS as substrate but an optimal pH of around 6 with other substrates
like DMP, guaiacol, and syringaldazine, with optimum range of temperature
between 25 and 35 °C. Both POXA1and POXA2 isoenzymes oxidize ABTS and
syringaldazine, but POXA1 is unable to oxidize guaiacol (Palmieri et al. 1997).
5.3.2 Laccase Production and Bioprocessing
P. ostreatus grows naturally on tree stumps and can be easily cultured in the labora-
tory scale. However, most of mushroom cultivars usually use cereal straw-based
substrates (Arjona et al. 2009). Different parameters influence the laccase
production which is linked to a complex regulation of nutrients that affects directly

the molecular expression. These include the type and concentration of carbon and
nitrogen sources used, C/N ratio, and also the presence of some inducers and their
concentrations (Terrón et al. 2004). Beside the nutritional factors, the production of
laccase by P. ostreatus is influenced by other factors such as pH, the fermentation
technique applied, and cultivation conditions like agitation, aeration, cultivation
time, and many other factors (Terrón et al. 2004; Soden and Dobson (2001);
Periasamy and Palvannan 2010; Janusz et al. 2007).
Different studies were focused on the optimization of the fermentation medium
for laccase production by P. ostreatus to facilitate economic design of the full-scale
fermentation operation systems. Studies include the investigation of the effect of
carbon and nitrogen source (Stajic et al. 2006a; Liu et al. 2009), addition of some
metal ions (Soden and Dobson 2001; Collins and Dobson 1997; Galhaup and
Haltrich 2001; Galhaup et al. 2002; Baldrian and Gabriel 2002), and addition of
aromatic compounds as inducers (González et al. 2003; De Souza et al. 2004; Terrón
et al. 2004; Krishna et al. 2005a).
5.3.2.1 Media Composition and Cultivation Conditions
It has been reported that increasing the concentrations of glucose, wheat bran, urea,
yeast extract, KH2PO4 and inoculums can increase laccase production (Krishna
et al. 2005a; Fernández-Fernández et al. 2013). The works of Ardon et al. (1998)
and Krishna et al. (2005b) show that the maximal laccase production by P. ostreatus
was obtained within the pH range of 5.0–5.5 in submerged culture. Another study
carried out by Terrón et al. (2004) reported that the presence of nine different aro-
matic compounds, including p-coumaric acid, guaiacol, and ferulic acid, can
enhance laccase activity. Zhuo et al. (2017) studied the effect of aromatic com-
pounds on the transcript levels of laccase genes, and the results have confirmed the
previous results of Nyanhongo et al. (2002) who used ferulic acid as an effective
laccase inducer and show that ferulic acid and vanillic acid have the most pro-
nounced stimulatory effect on laccase gene transcription (Zhuo et al. 2017;
Nyanhongo et al. 2002). Krishna et al. (2005a) reported on the positive effect of
2,5-xylidene addition to culture media after 96 h on laccase production. However,
the inducers’ effects depend on the chemical structure, concentration, and time of its
introduction into the production medium (Pointing et al. 2000; Sethuraman et al.
1998). The production of laccases in P. ostreatus is regulated by the presence of
copper. Therefore, the two dimeric isoenzymes POXC and POXA1b have been
detected only in the presence of copper (Palmieri et al. 2000, 2003).
5.3.2.2 Cultivation Process and Purification
Solid-state fermentation is an interesting operational mode for the production of

laccases by P. ostreatus. For solid-state fermentation, Karp et al. (2012) have
reported a productivity of 151.6 Ug−1 after 5 days in sugarcane bagasse-based
medium. Several studies have showed high laccase productivity by P. ostreatus in

submerged culture conditions; a productivity of 3500 U L−1 was reported by Lenz
and Hölker (2004). Tlecuitl-Beristain et al. (2008) have reported 80 UmL−1 after
12 days in liquid culture without exogenous inducers (Lettera et al. 2011); also
12.2 UmL−1 has been reached after 18 days according to Tlecuitl-Beristain et al.
(2008). Optimizing the laccase production in submerged culture conditions, Krishna
et al. (2005a) and Rathinasamy and Thayumanavan (2010) have increased the lac-
case expression by 32.9%, from 538.8 to 803.3 U, and by 86.8%, from 485.0 to
906.3 U, using P. ostreatus 1804 and P. ostreatus IMI 395545, respectively.
Mansur et al. (2003) studied the secretion of laccase isozymes by P. ostreatus
V-184 with different substrate specificities. They showed that laccase activity
reached 2.4 U mL−1 using submerged culture for 14–16 days, at pH 6.5. Krishna
et al. (2005b) have also studied the feasibility of Pleurotus ostreatus 1804 immobi-
lization on polyurethane foam (PUF) cubes in the objective of laccase production in
three different cases of submerged culture system. After 192 hours of cultivation at
5.6 pH, the enzyme yields of free mycelia and immobilized and packed bed reactor
were 272.2 U, 312.6 U, and 392.9 U, respectively.
Rodrigues da Luz et al. (2012) reported also on the potential use of agrowastes
such as coffee husks, eucalyptus sawdust, and eucalyptus bark as substrate in com-
bination with rice bran for laccase production by P. ostreatus as substrates in com-
bination with 20% rice bran. Parenti et al. (2013) have used water polluted with
wheat straw extracts as substrate in submerged cultures incubated in the dark at
25 °C for 24 days with orbital shaking (150 rpm) and reported a laccase activity of
200 UL−1. To separate laccase isoenzymes, Palmieri et al. (1997) have fractioned P.
ostreatus culture broth after 70 h of growth using ammonium sulfate precipitation
followed by anionic exchange chromatography. Five different laccase fractions
were separated (POXA1, POXA2, POXB1, POXB2, and POXC). Elution with a
saline gradient at around 0.17, 0.18, and 0.32 M NaCl allowed to separate three
isoenzymes POXB1, POXB2, and POXC, respectively, whereas the major laccase
peak activity, corresponding to POXA1, and a fraction of POXA2 were recovered
using the equilibrating buffer (Palmieri et al. 1997).
5.3.3 Applications of Laccase
Since the nineteenth century, laccases are considered as the most effective green
catalyst by many researchers (Javed et al. 2017). Due to their nonspecific and high
oxidative capacity, laccases present a high potential in many industrial and biotech-
nological applications with a great market demand for commercial applications in
detergent, food, cosmetic, paper/pulp, and textile industries. In addition, they have
also many applications in soil bioremediation and wastewater treatment (Shah and
Nervd 2002). Recent studies showed also the potential use in pharmaceutical indus-
tries based on the enzyme ability for transformation of antibiotics and steroids
(Cohen et al. 2002; Wu and Nian 2014; Pezzella et al. 2013; Yadav et al. 2017a, b).
5.3.3.1 Bioremediation of Environmental Contaminants in Soil
Soil bioremediation is a process applied to recover pollutant from contaminated

soils using bacteria and fungi which degrade these organic compounds, transform-
ing them into compounds that are less or nontoxic. Bioremediation is considered a
safer, cheaper, and more efficient method compared to other physicochemical meth-
ods (Chibuike 2013). Acting on substrates, which are insoluble and very linked to
the soil particles and nonaccessible to bacteria, makes the application of laccases
more attractive. These exocellular enzymes are able to degrade some pollutant com-
pounds similar to lignin such as polyaromatic hydrocarbons, chlorophenols, and
nitrophenols (Chibuike 2013). Action of laccases on the xenobiotics releases inter-
mediate products with more bioavailability and less toxic, which can be more effi-
ciently removed by physicochemical and mechanical processes (Javed et al. 2017;
Viswanath et al. 2014; Zucca et al. 2015). Anthracene, benzopyrene, and organo-
phosphorus compounds, like the nerve agents VX or Russian VX, have been
removed by microbial and fungal laccases (Amitai et al. 1998; Zeng et al. 2016).
Other studies showed also that effluents from sewage treatment containing estro-
genic hormones have been treated using enzymatic systems containing laccases.
Fungal laccase has been used to oxidize the estrogens like estrone, 17b-estradiol,
and 17a-ethynylestradiol (Tanaka et al. 2009).
5.3.3.2 Wastewater Treatment
The appropriate wastewater treatment is important regarding that industrial sewage

can contain many carcinogenic, mutagenic, and teratogenic potential substances
with toxic effects to human, fish, microorganisms, and plant species (Aljeboree
et al. 2014). Using enzymatic processes in wastewater treatment is relatively a new
approach decreasing reagents’ consumption and allowing degradation of many per-
sistent and toxic substances such as dyes, solvents, inks, fertilizers, and pesticides
(Niebisch et al. 2014). One of the applications of laccases in combination with
peroxidases in wastewater treatment is related to the discoloration of wastewater
from textile industry that contains different types of dyes such as azo, triphenyl-
methane, and anthraquinone (Fig. 5.4). Laccases act on the degradation of the
chemical dyes before the final disposal. In this process, immobilized laccases in
alginate beads are commonly used (Niebisch et al. 2014; Peralta-Zamora et al.
1997). Another study by Zhuo et al. (2017) investigated the ability of laccase from
P. ostreatus HAUCC 162 to decolorize six synthetic dyes belonging to different
categories (methyl orange, crystal violet, malachite green, bromophenol blue,
Reactive Blue 4, and Remazol Brilliant Blue R (RBBR)). The results showed that
within 24 hours, and at an initial concentration of 100 mg/L, methyl orange, crystal
violet, malachite green, and bromophenol blue could be decolorized by 81.3%,
87.6%, 85.1%, and 98%, respectively. For Reactive Blue 4 and RBBR, the removal
of 64.6% and 89.1% was achieved, respectively, when using initial concentration up
to 800 mg/L (Rui Zhuo et al. 2017).
Fig. 5.4 Different synthetic dyes degraded by extracellular laccase from P. ostreatus
Decolorization of crystal violet by laccase from P. ostreatus MTCC142 has also

been studied by Kunjadia et al. (2012), and the results show that 92% of dye decol-
orization can be achieved at an initial concentration of 20 mg/L. Palmieria et al.
(2005) have reported the decolorization of the recalcitrant anthraquinonic dye
Remazol Brilliant Blue R (RBBR) by P. ostreatus on agar plate and also in liquid
media supplemented with veratryl alcohol, where RBBR was completely decolor-
ized in 3 days. Two isoenzyme laccases in mixture (POXC and POXA3) were found
to be responsible for this RBBR transformation under acidic conditions reducing its
toxicity by 95% (Palmieria et al. 2005). Another study by Hongman et al. (2004)
found that the anthraquinone dye SN4R (Remazol Brilliant Blue SN4R) decoloriza-
tion rate was increased by 90% in the presence of ABTS as a mediator of laccase.
The crude laccase alone in the concentration of 30 Uml−1 can effectively decolorize
the dye by 66%. Reactive Blue HFRL dye decolorization was also achieved by
using mushroom laccase after 3 days (Devi et al. 2012).
5.3.3.3 Food Industry
Laccases can catalyze homo- and hetero-polymerization reactions needed in fruit

juice processing, in sugar beet pectin gelation, and for wine/beer stabilization
(Osma et al. 2010). In addition, laccases are currently being utilized in baking to
cross-link biopolymers (Javed et al. 2017). It was also used to preserve and to
increase juice stability by reducing the oxidation of phenolic compounds, the for-
mation of polyphenol and protein is delayed, and there are many studies which
show that laccases can be used for food preservation/stabilization process (Osma
et al. 2010; Sammartino et al. 1998).
5.3.3.4 Paper and Textile Industries
In paper industry, the pretreatment of wood pulp with ligninolytic oxidoreductases

shows special attention to replace the conventional chlorine-based delignification
processes. Unlike peroxidase, the benefit of using laccase is that it requires O2 rather
than H2O2 (Javed et al. 2017). In addition, use of laccase for phenols grafting to flax
fibers in the production process of paper has been recently studied (Fillat et al. 2012;
Aracri et al. 2010; Virk et al. 2012). Laccases can be used in textile industry for
bleaching processes and they have now replaced the conventional peroxide bleach-
ing to enhancing the whiteness of cotton (Yavuz et al. 2014; Iracheta-Cardenas et al.
2016; Tzanov et al. 2003). Laccase-based products are also applied for denim
bleaching and against fabrics odor (Rodriguez-Couto 2012; Kunamneni et al. 2008).
Different textile dyes like phenoxazine and azo dyes have been produced using lac-
cases based on their ability to oxidize the aromatic compounds (Sousa et al. 2013).
5.3.3.5 Biofuels
Lignocellulosic materials are the most reliable feedstock for bioethanol and other
organic alcohol production in biofuel industries (Kour et al. 2019a). However, based
on the complexity of the material, it requires pretreatment of biomass to eliminate
lignin and thus expose the cellulose/hemicellulose to hydrolytic enzymes. An inter-
esting research showed that P. ostreatus enzymes have potential application in bio-
ethanol production process (Yu et al. 2009). The results showed that the combination
of ultrasonic pretreatment with enzymatic hydrolysis of rice hull showed a maximal
yield by using laccases followed by lignin peroxidase and Mn peroxidase (Yu et al.
2009).
5.3.3.6 Biomedical, Pharmaceutical, and Cosmetic Industries
Based on the ability of laccases to catalyze reactions by direct electron transfer,

developing biosensors based on laccases systems attracts scientists for many bio-
medicine application like in insulin, morphine, and codeine analysis (Rodriguez-
Delgado et al. 2015). In addition, this enzyme can play a potential role in
pharmaceutical industries in many processes such as antibiotic transformation,
amino acid derivatization, and synthesis of metabolically stable analogues
(Piscitelli et al. 2012). Complex medical products have been synthesized by
laccases, like immunosuppressors (e.g., cyclosporin A), antibiotics (e.g., penicillin

X dimer and cephalosporins), and anticancer drugs (e.g., vinblastine, mitomycin,
and actinomycin) (Kunamneni et al. 2008). By exploiting the oxidative potential of
laccases, many products have been developed in the field of cosmetic industry and
personal care products (Javed et al. 2017). In addition, laccases have been applied
for hair bleaching and dying. Therefore, some cosmetics and dermatological for-
mulations containing laccases were patented and used for skin lightening (Morel
and Christie 2011).
5.4 Manganese Peroxidase (EC 1.11.1.13)
One of the most widely used substrates for P. ostreatus cultivation is wheat
straw, especially in European country. As a white-rot fungus, it can be cultivated
of agro-industrial lignocellulosic wastes (Sanchez 2010). It also has been
described as selective and simultaneous lignocellulose degrader (Banfi et al.
2015). P. ostreatus start to grow at first by easily utilizable and soluble materials
from intra- and intercellular spaces from the plant substrate tissues. After nutri-
ent depletion, the fungal biomass starts to degrade the plant cellulose and lignin
polymers for further growth (Banfi et al. 2015. Lignin degradation is an oxida-
tive and nonspecific process usually carried out by white-rot fungi. Ligninolytic
enzymes, LiP, manganese peroxidase (MnP), and laccase, catalyze the one-elec-
tron oxidation of lignin units producing aromatic radicals that lead to non-enzy-
matic depolymerization. Ligninolytic enzymes, LiP, are the only enzyme able to
oxidize directly non-phenolic ones in the presence of certain compounds (Sarkar
et al. 1997). However, P. ostreatus produces two manganese peroxidase (MnP)
isoenzymes when grown in solid stationary condition on poplar sawdust
(Giardina et al. 2000).
5.4.1 Characterization of MnP
MnP was first described in P. chrysosporium (Kuwahara et al. 1984). Peroxidases

with Mn2-independent activity on phenolic and non-phenolic aromatic com-
pounds were first reported from Pleurotus species (Martínez et al. 1996; Camarero
et al. 1996; Sarkar et al. 1997; Palma et al. 2000). These enzymes have been
considered as MnP, despite differences in catalytic properties with P. chrysospo-
rium MnP, because they show the highest affinity for Mn and the existence of a
characteristic Mn2 interaction site has been shown in the molecular model of P.
eryngii Mn2-oxidizing peroxidase (Ruiz-Dueñas et al. 1999). Manganese peroxi-
dase (MnP) is the most common lignin-modifying peroxidase produced by
almost all wood-colonizing basidiomycetes causing white-rot and various soil-
colonizing litter-decomposing fungi (Hotrichter 2002; Hatakka 1994; Peláez
et al. 1995).
Fig. 5.5 3D Crystal

structure of manganese
peroxidase IV from
Pleurotus ostreatus.
(Source: http://www.rcsb.
org/pdb/explore.
do?structureId=4BM4)
MnP is developed by multiple forms of glycosylated heme protein with molecular

weight between 40 and 50 kDa and secreted by ligninolytic fungi (Fig. 5.5). From
that point, MnP preferentially oxidizes manganese (II) ions (Mn2+), commonly pres-
ent in wood and soils, into highly reactive Mn3+. The trivalent Mn3+ is then stabilized
by fungal chelators such as oxalic acid. Like other peroxidases, MnP is sensitive to
high concentrations of H2O2, but it can be rescued by Mn3+ ions but become quite
unstable in aqueous media. To overcome this drawback, they form complexes with
organic acids naturally secreted by the fungus, such as malonic or oxalic acid
(Hofrichter 2002; Wong 2009). There are three mnp genes, namely mnp1, mnp2, and
mnp3, that have been isolated, and their products were characterized from P. ostrea-
tus (Asada et al. 1995; Giardina et al. 2000; Irie et al. 2001). Besides, both MnP2 and
MnP3 have been purified (Kamitsuji et al. 2004; Sarkar et al. 1997). However, it is
worth to note that MnP2 from P. ostreatus (strain Florida) did not oxidize non-phe-
nolic compounds such as veratryl alcohol (Giardina et al. 2000). Therefore, the dif-
ference in enzymatic properties between these two MnP2 isozymes might be due to
the differences in posttranslational modification (Kamitsuji et al. 2005).
In fact, the homologous expression system developed by Tsukihara et al. (2006b)
reflects the posttranscriptional modifications, secretion, and stability in the p hysiological
condition during the enzyme production process in P. ostreatus. Irie et al. (2001) pointed
out the successful molecular breeding of a P. ostreatus strain with high MnP productivity,
using an expression system employing the promoter and terminator sequence of sdi1.
5.4.2 Mechanism of Action of MnP
Manganese peroxidase is a heme-containing glycoprotein which requires hydrogen

peroxide as an oxidant. Manganese peroxidase basically oxidases Mn2+ to Mn3+ in
the presence of H2O2 and organic acid chelators, such as lactic acid. Mn3+, in turn,
oxidizes a variety of phenolic substrates (Hotrichter 2002). MnP catalyze the oxida-
tion of several monoaromatic phenol and dyes but depends on both divalent manga-
nese and certain types of buffer. This enzyme has been shown to have the ability to
oxidize non-phenolic substrates in the presence of mediators. Due to the intense
activity in oxidizing a wide variety of aromatic compounds, it exhibited many
potential industrial applications. Mushrooms also produce aryl alcohol oxidase
(AAO), an enzyme participating in hydrogen peroxide production. MnP production
by different Pleurotus species, which were grown in both submerged and solid-state
culture, was shown (Stajic et al. 2006a, b). MnP are considered to be the key
enzymes in the lignin degradation system (Irie et al. 2001). The ability to synthesize
MnP is common among distinct taxonomical groups of basidiomycetes. In addition,
there are fungal MnP producers from rather exotic habitats such as decaying sea
grass, cooling tower wood, and brown coal. The molecular weight of MnP is usually
within the range between 38 and 62.5 kDa, but most purified enzymes have Mwt
around 45 kDa. MnP is often produced in multiple forms, and these isoforms differ
in their isoelectric points which are usually rather acidic (pH 3–4).
5.4.3 Applications of MnP
Manganese peroxidase (MnP) is one of the two extracellular peroxidases secreted

by the lignin-degrading fungus P. ostreatus. Manganese peroxidase has great poten-
tial for industrial waste remediation application such as degrading refractory indus-
trial pollutants (Asgher et al. 2013; Hotrichter 2002). As such, direct disposal of
olive mills waste to aquatic bodies’ results in environmental deterioration due to the
large amount of organic loading discharged (Fountoulakis et al. 2002). MnP was
very helpful in bioremediation of olive mills’ waste. In other applications, MnP is
also valuable for decolorization of synthetic dyes and significant potential applica-
tion for textile industry (Susla et al. 2008; Praveen et al. 2012; Saravanakumar et al.
2013). These complexes function as diffusible oxidants that, in turn, can oxidize
terminal phenolic substrates and also possibly non-phenolic substituents via a radi-
cal mediator (Giardina et al. 2000).
5.5 Versatile Peroxidase (EC 1.11.1.16)
The first study reported on the presence of versatile peroxidases (VP) in Pleurotus
eryngii was revealed by Martinez et al. (1996), and the complete purification of the
enzyme was carried out by Min et al. (2010). To date, little evidence has been found
associating versatile peroxidase in Pleurotus sp. except in few researches estab-
lished for P. ostreatus and P. pulmonarius (Moreira et al. 2007). Versatile peroxi-
dases are glycoprotein with hybrid properties, included in a group of H2O2-dependent
ligninolytic heme peroxidases (POXs) also known as lignin manganese peroxidases,
since it combines together with catalytic properties of lignin peroxidases (LiP, EC

1.11.1.14) and manganese peroxidases (MnP, EC 1.11.1.13) (Busse et al. 2013). It
is characterized by the similar properties of MnP in oxidizing phenolic and non-
phenolic aromatic compounds as well as its efficient oxidation of Mn2+ to Mn3+
(Tsukihara et al. 2006a). In another major studies, their capabilities in oxidizing
veratryl alcohol, the typical lignin peroxidase (LiP) substrate (Camarero et al. 1999)
and simple phenols, which are the substrates of Coprinopsis cinerea peroxidase
(CIP) has been reported (Ruiz-Dueñas et al. 2009). These could conceivably then
recognized that versatile peroxidase as a new family of ligninolytic peroxidases. In
consequence of this, while sharing almost identical heme environment together with
lignin peroxidase (LiP) and manganese peroxidase (MnP), VP differentiates in the
catalytic sites in their molecular structure. VPs form an attractive ligninolytic
enzyme group due to their dual oxidative ability to oxidize Mn(II) and also phenolic
and non-phenolic aromatic compounds (Dashtban et al. 2010). This dual substrate
specificity makes VP powerful to oxidize a variety of high and low redox potential
substrates (Ruiz-Dueñas et al. 2011).
5.5.1 Biochemical Characteristics of VP
The biochemical properties of only a few versatile peroxidases of Pleurotus sp. have
been analyzed and studied in details especially in P. eryngii, which is superior than
VP reported in P. chrysosporium (Camarero et al. 1999; Min et al. 2010). However,
the Pleurotus VP isoenzymes were first described as MnP isoenzymes due to their
similarity to Mn-oxidizing activity (Martínez et al. 1996). This has shown that VPs
have high affinity for manganese and dyes, and strongly oxidized 6-dimethoxyphenol
(DMP) and veratryl alcohol (VA) in a manganese-independent reaction (Moreira
et al. 2005). The catalytic cycle of VP consists of three steps as described clearly in
Bjerkandera adusta influenced by pH-independent and pH-dependent steps in the
conversion process (Ertan et al. 2012). The cleavage of H2O2 by heme Fe3+ to Fe4+
led to the formation and reduction of compound I and II intermediates. With the
excessive of H2O2 in the absence of reducing substrate at low pH (3.0-3.5), the con-
version of compound II to compound III will be carried out which lead to the inac-
tivation of the enzyme (Fig. 5.6). This supports the theory of involvement of LiP and
MnP in the catalytic cycle activities of VP (Wong 2009).
5.5.2 Structural Characteristics of VP
Comprehensive and complete genome sequences for P. ostreatus were done in JGI
(U.S. Department of Energy, Office of Science, Joint Genome Institute; http://
genome.jgi-psf.org). This facilitated the identification of nine genes encoding mem-
bers of short MnP- and VP-encoding gene family in the inventory of heme
H2O2 H2O O •+
N N N N
Fe(III) Fe(IV)
pH independent
N N N N
VP resting Mnp-1 / LiP-1 (compound I )

+
2H2O VA• Mn+2
VA
VAD Mn+3
VA
+ Mn+3
2VA• VA
•+
Mn+2
•+
O2 O
N N N N
Fe(III) Fe(IV)
N N N N
2H2O excess
2H2O2
VP-III (compound III) MnP-II / Lip-II (compound II)
Fig. 5.6 Catalytic cycle of VP involving the activities of LiP and MnP; VA veratryl alcohol; VAD
veratryl aldehyde (Ertan et al. 2012)
peroxidases of this mushroom (Ruiz-Dueñas et al. 2011). Gene-expression analysis

of P. ostreatus has revealed to have five Mn2+-dependent peroxidases encoded with
mnp3, 6, 7, 8, and 9, while for versatile peroxidases encoded with MNP1, 2, 4, and
5, VPs all have the related gene and protein structure (Salame et al. 2012). Among
these VPs, MNP3 and MNP2 were demonstrated to be unique in their ability to
oxidize high molecular weight compounds such as Poly R-478 and RNaseA
(Kamitsuji et al. 2005). According to Ruiz-Dueñas et al. (2011), two model types of
VPs (137760 and 137766) show genetic variations in only one or two amino acids
from the whole sequence of P. ostreatus MnP1 (GenBank AAA84396) (Asada et al.
1995) and MnP2 (GenBank CAB51617) (Giardina et al. 2000), respectively. A
recent review reported that P. ostreatus genome exhibited three versatile peroxi-
dases (VPs) and six manganese peroxidases (MnPs) in which two of these crystal
structures, VP1 and MnP4, designated dissimilarities at 1.0 to 1.1 Å (Fernández-
Fueyo et al. 2014). The significant differences between these two isoenzymes
involved not to their kinetic constants only, however including the activity T50 and
residual activity at both acidic and alkaline pH. The findings from the previous stud-
ies make several contributions to a growing body of literatures on VP in Pleurotus
sp. in which the presence of VP has strongly replaced the role of LiP in lignin deg-
radation for P. ostreatus. However, complete purification of VPs from P. ostreatus is
not fully reported yet. Versatile peroxidases from P. eryngii are gaining much atten-
tion so far as it is first revealed to have high redox potential compared to other
Pleurotus group. Generally, versatile peroxidase from P. eryngii was reported to
have 40 kDa molecular weight and isoelectric point of 4.1. ABTS (2-azino-bis-
(3-ethylbenzothiazoline-6-sulfonic acid) is the best to be used as a substrate, and the
maximal enzyme activity was achieved at 50 °C, pH 3.0 (Min et al. 2010).
5.5.3 Applications of VP
Pleurotus sp. has been described as being able to degrade lignin selectively. The
limited attack to cellulose makes them very interesting in different physicochemical
and biotechnological pretreatment applications related to the use of plant biomass,
including the integrated lignocellulose biorefineries for the future production of
chemicals, materials, and biofuels. The versatile catalytic of VP in biotechnological
and industrial application is due to its high redox potential and unique characteris-
tics in degrading the aromatic compounds without the use of redox mediators and
the presence of polyvalent catalytic sites (Ravichandran and Sridhar 2016). The
ability to direct degradation of a variety of recalcitrant compounds that other peroxi-
dases are not able to oxidize directly represents VPs as unique enzymes. Their
potential applications are not limited to Mn (II) oxidation only but also extend to
VA; phenolic, non-phenolic, high molecular weight compounds; as well as dyes in
Mn-independent reactions (Wong 2009).
In dye treatment, the capacity of P. ostreatus in the degree of decolorization is
depending on the type and concentration of dyes, the ligninolytic enzymes pro-
duced, and reaction conditions. An example of this is the study carried out by
Vishwakarma et al. (2012) in which the treatment of azo dye (direct blue 14) with
enzymes of P. ostreatus has been reported. They reported that immobilized enzymes
can reduce color up to 99% only in 18 hours. However, in contrast to Kahraman
et al. (2012), the color reduction of indigo carmine dye varied from 93% to 64% as
concentration was increased from 50 to 500 mg/L when using dead biomass. The
ability of P. ostreatus to degrade aromatic pollutants such as 2,4-dichlorophenol
(2,4-DCP) and benzo(a)pyrene ([B(a)P] was reported by Rodrıguez et al. (2004).
An increasing number of studies have found that versatile peroxidase from Pleurotus
represents an important preference with respect to LiP since no mediator such as
veratryl alcohol could be used.
5.6 Aryl Alcohol Oxidase (EC 1.1.3.7)
Aryl alcohol (AAO) is an extracellular flavoprotein (flavin adenine dinucleotide

(FAD)-dependent proteins) providing the H2O2 required by ligninolytic peroxidases
for fungal degradation of lignin (Xiao et al. 2017). Aryl alcohol oxidase (AAO)
which is thermodynamically favorable (Hamdane et al. 2015) has other names in
common use including veratryl alcohol oxidase and aromatic alcohol oxidase. Aryl
alcohol oxidase was first detected in the culture liquid of Polystictus versicolor (a
synonym of Trametes versicolor) (Farmer et al. 1960) before it is known as best

produced in three species of the genus Pleurotus: P. sajor-caju (Bourbonnais and
Paice 1988), P. eryngii (Guillen et al. 1992), and P. ostreatus (Sannia et al. 1991).
The isolation of the enzyme from basidiomycetes has been first carried out by
Janssen et al. (1965) which later designed this enzyme as “alcohol oxidase” included
as the prosthetic group of the enzymes (Janssen and Ruelius 1968).
Preliminary work in this field was focused mainly on alcohol oxidase in mycelial
extracts of a basidiomycete grown in submerged culture (Kerwin and Ruelius 1969).
In 2000, Varela et al. published a paper in which they described a gene of aryl alcohol
enzymes in the species of Pleurotus and Bjerkandera. Rodrıguez et al. (2004) define
evidence that supported the involvement of AAO in lignin degradation as they
observed the simultaneous production of AAO and VP in Pleurotus cultures. However,
previous studies have not studied AAO in much detail, and the molecular characteris-
tics are not yet clearly elucidated except in P. eryngii (Ferreira et al. 2009). P. eryngii
is the first Pleurotus species that has been cloned (Martínez et al. 1994), and a pro-
karyotic heterologous expression system has been developed in order to obtain fully
non-glycosylated AAO for further biochemical and structural studies (Ruiz-Dueñas
et al. 2006). However, further study within the species had discovered the best pro-
ducer of AAO is P. ostreatus compared to other species (Kumar and Rapheal 2011).
5.6.1 Biochemical Characteristics of AAO
Aryl alcohol oxidase cooperates with laccase and other peroxidase in the production
of hydroxyl radical, which is believed to be involved in the initial attack of lignocel-
lulosic materials. This is to prevent the repolymerization of laccase oxidation prod-
ucts to occur in order to sustain the lignin degradation process (Goswami et al.
2013). During degradation by Pleurotus species, H2O2 is generated from the expense
of benzylic or other p system-containing primary alcohols such as p-anisaldehyde
and p-anisyl alcohol into corresponding aldehydes (Ferreira et al. 2015). They point
out that in addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsatu-
rated primary alcohols; where flavin reduction by substrate oxidation and re-
oxidation of the reduce enzymes by oxygen before the release of the aldehyde
product. Aromatic radicals that are produced during the reaction then act to catalyze
subsequent degradation, which further generated potentially toxic molecules as
defensive tool for the cells from the environment (Li et al. 2015).
5.6.2 Structure of AAO
Depending on the substrate specificity, group of alcohol oxidase (alcohol: O2 oxido-

reductase; EC 1.1.3.x) may contain four different categories: AAO which was cat-
egorized as aromatic alcohol oxidase and others which are short-chain alcohol
oxidase (SCAO), long-chain alcohol oxidase (LCAO), and secondary alcohol oxi-
dase (SAO) (Goswami et al. 2013). However, SCAO and LCAO are reported as
intracellular in nature. The crystalline structure of AAO was revealed in P. eryngii
by Fernández et al. (2009) which confirmed to share similar fold topology with
other members of the glucose oxidase from Aspergillus niger (Frederick et al. 1990)
and choline oxidase from Arthrobacter globiformis (Quaye et al. 2008). The use of
single-wavelength anomalous diffraction of a selemethionine derivative obtained by
Escherichia coli expression and in vitro folding had identified that this monomeric
enzyme has two additional structural elements existing in the surroundings of its
active site that modulate the access of substrates, which is absent in the structure of
model GMC oxidoreductase glucose oxidase (Fernández et al. 2009). These two
domains were defined as FAD-binding domains, which interact non-covalently with
the proteins, and substrate-binding domain, with a type of funnel-shaped channel
that led the connection between the solvent and the flavin cofactor (Hernández-
Ortega et al. 2012). The unique properties of AAO associated with its active site,
which is able to bind over a wide range of aromatic ligands, include competitive
inhibitors such as chavicol (4-allylphenol) and p-anisic (4-methoxybenzoic) acid,
while 4-methoxybenzylamine was observed to be the best uncompetitive inhibitor
(Ferreira et al. 2005). Until recently, there has been little interest in AAO. Aryl alco-
hol oxidases from P. ostreatus monomeric glycoproteins were reported to have
67 kDa molecular weight, with optimum pH, and the temperature was found to be
around 6 and 40 °C, while Km value of AAO for oxidizing veratryl alcohol was
determined to be 0.6 mM, respectively (Kumar and Rapheal 2011).
5.6.3 Applications of AAO
The advantage characteristic of veratryl alcohol oxidase to oxidize various alcohols

irreversibly and selectively without the necessity of cofactors in the catalysis has
gained industrial interest for large-scale production (Goswami et al. 2013; Kerwin
and Ruelius 1969). It is widely known that cellulose and hemicellulose in lignocel-
lulose are the main carbon sources for P. ostreatus (Xiao et al. 2017). In a study by
Kumar and Rapheal (2011), AAO activity was observed to be induced by aromatic
amino acids and aryl alcohols up to a level of 289 U L−1. However, another compo-
nent of lignocelluloses, lignin, was reported to inhibit the growth P. ostreatus
(Barakat et al. 2012). The presence of lignin plays restricting roles in the efficiency
of enzymatic hydrolysis of cellulose and hemicellulose (Kumar et al. 2012). This is
supported by Feldman et al. (2015) who reported on the formation of toxic degrada-
tion products such as 5-hydroxymethylfurfural (HMF). However, by the involve-
ment of aryl alcohol oxidase and other yeast dehydrogenases via heterologous gene
expression, P. ostreatus was capable to metabolize and detoxify HMF while con-
verting it into 2,5-bis-hydroxymethylfuran (HMF alcohol) and 2,5-furandicarbox-
ylic acid (FDCA) (Feldman et al. 2015). The involvement of AAO in lignin
degradation requires either substrate from lignin-derived compounds; phenolic
aromatic aldehydes and acids that being reduced to alcohol substrates, and aromatic
fungal metabolites, respectively (Hernández-Ortega et al. 2012).
5.7 Heme-Thiolate Peroxidase (HTP)
Heme-thiolate (or haem-thiolate) proteins belong to the class of thiolate-containing

hemoproteins. Omura (2005) reported that heme-thiolate proteins contain six differ-
ent classes: cytochromes P450, chloroperoxidase (CPO), nitric oxide synthases,
cystathionine β-synthase, protein CoA, and eIF2α. The specialty for the heme-
thiolate proteins is based on their function as oxidoreductases in the biological sys-
tems and the most versatile biocatalyst. The thiolate group usually comes from
cysteine residue and is the axial ligand of heme iron, and the cysteine residue in
heme-thiolate peroxidases acts as peroximal axial ligand.
The only known heme-thiolate peroxidases (HTP) are CPO from ascomycete
Leptoxyphium fumago that has been extensively studied (Smith et al. 2015; Ruiz-
Dueñas et al. 2011). CPO was first discovered by Hager and team from marine
fungus Caldariomyces fumago (Shaw and Hager 1959; Morris and Hager 1966;
Hofrichter and Ullrich 2006). CPO is composed of 299 amino acids and uses hydro-
gen peroxide for catalytic process under acidic environment. Research in genome
sequences of ascomycete and basidiomycete has gathered many putative heme-
thiolate CPO sequences, where many were being included in PeroxiBase, a data-
base that has been created for heme and non-heme peroxidases (Ruiz-Dueñas et al.
2011; Koua et al. 2009). However, a second heme-thiolate peroxidase has been
reported. It is an aromatic peroxygenase (APO) and first reported in Agrocybe
aegerita (Anh et al. 2007; Ullrich et al. 2004). APO and CPO values are based on
their characteristics such as wide substrate specificity, ability to catalyze halogena-
tion reactions, shared catalases and cytochrome P450 monooxygenases, and ability
to oxygenate aromatic substrates.
Although they shared the same catalytic properties of peroxidases, these two
enzymes differ from typical heme peroxidases in terms of their amino acid sequence,
catalytic activity, and structure. Therefore, they were classified under new heme-
thiolate peroxidase superfamily. Three heme-thiolate peroxidase gene models were
also found in fungus P. ostreatus from genus Pleurotus. All genes were found at
sequences 2–4 with high amino acid identities that are similar to the unpublished
putative CPO of A. bisporus (GenBankCAC03461) (Ruiz-Dueñas et al. 2011).
5.7.1 Biochemical Characteristics of Structure of HTP
CPO of L. fumago have been identified with 21 amino acid N-terminal signal pep-
tide and 52 amino acid C-terminal propeptide (have chaperon-like function). Its
molecular structure has also been experimentally established and is available in
Protein Data Bank. Therefore, despite the low amino acid sequence (approximately
20%), its structure has been used as template to get the theoretical models for the
three putative P. ostreatus heme-thiolate peroxidases. Ruiz-Dueñas et al. (2011)
studied homology models of the three P. ostreatus heme-thiolate peroxidase and the
crystallographic model of CPO from L. fumago in detail. It has been shown that all
models have cysteine as axial heme ligand. The surrounding residues which stabi-
lize the cysteine-ligand loop by hydrogen bonding are conserved at the proximal
side. The presence of Ala and Cys residues contiguous to the axial Cys in 114464
could reinforce the hydrogen-bonds structure at this site. The models also show that
Glu, Ser, and His are responsible for cation coordination in CPO and suggest differ-
ent catalytic properties (activation rate and mechanism, substrate specificity, etc.)
from P. ostreatus heme-thiolate peroxidases (Ruiz-Dueñas et al. 2011).
5.7.2 Applications of HTP
Peroxidases and peroxygenases are beneficial biocatalyst and can play big role in
the chemical modification of wide range of organic substrates, including regioselec-
tive and stereoselective oxygenations, which are difficult to do using conventional
chemical reaction (Martínez et al. 2014). As a green technology, enzymes can act as
catalyst in chemical syntheses to perform reactions under mild conditions, environ-
mental friendly, and characterized by high specificity and selectivity compared to
traditional chemical methods. The advantages of this enzyme are involvement in the
production of fine chemical and the possibility to catalyze unspecific chlorination
reaction. In addition, it also suitable in bromination and iodation of electrophilic
organic substrates via hypohalous acid as actual halogenating agent (Hofrichter and
Ullrich 2006).
5.8 Other Enzymes Produced by Pleurotus ostreatus
Beside lignin-degrading enzymes, mushrooms have high capacity to produce a wide

range of biocatalysts which were not reported in one type of microorganism.
Previous studies reported on the production of different types of lipases, proteases,
amylases, and polyphenol oxidases (Deepalakshmi and Mirunalini 2014).
Nowadays, Pleurotus sp. has become a major cell factory in the enzyme cocktail
production based on the ease of cultivation and ability to grow on most of agro-
industrial wastes considered as sustainable and green substrate (Saini et al. 2014).
Pleurotus ostreatus can produce and excrete a large number of enzymes including
xylanases (Getachew et al. 2016; Masutti et al. 2015; Raymond et al. 2015;
Karthikeyan 2015; Rodrigues da Luz et al. 2013; Lim et al. 2013; Rodrigues da Luz
et al. 2012; Morais et al. 2005; Qinnghe et al. 2004; Elisashvili et al. 2003), cellu-
lases (Raymond et al. 2015; Lim et al. 2013; Radhika et al. 2013; Rodrigues da Luz
et al. 2013; Daba et al. 2011; Sherief et al. 2010), pectinases (Masutti et al. 2015;
Raymond et al. 2015; Morales Huerta et al. 2014; Sherief et al. 2010), proteases
(Ergun and Urek 2017; Genier et al. 2015; Lebedeva and Proskuryakov 2009;
Palmieri et al. 2001), amylases (Ergun and Urek 2017; Lim et al. 2013; Akinyele
et al. 2010; Rashad et al. 2009), and lipases (Wijayati et al. 2017; Guarino and
Sannia 2013).
This part describes other biocatalyst produced by P. ostreatus under different
cultivation strategies as well as the purifications process. The secretion of these
cocktail of enzymes is dependent on the medium composition, pH, and temperature
as well as the mycelial growth. For many researchers, solid-state fermentation (SSF)
is the most appropriate method for mushroom cultivation to scale up and the pro-
duction of extracellular enzymes such as laccase, manganese peroxidase, xylanase,
cellulose, and amylase (Koyani and Rajput 2015). This is supported by the utiliza-
tion of inexpensive lignocellulosic which can stimulate enzyme synthesis and sup-
porting fungal growth (Koyani and Rajput 2015).
5.8.1 Cellulases
Cellulases (EC 3.2.1.4) are known as hydrolase enzymes that cleave cellulose by
catalyzing a series of cellulolytic reactions. Cellulases can be classified under dif-
ferent types such as (i) endoglucanases, endo-1,4-β-glucanase, carboxymethyl cel-
lulose (CMCase), β-1,4-glucanase, endo-1,4-β-D-glucanase, β-1-4-endoglucan
hydrolase, β-1,4-glucanase, cellobiohydrolases (CBH), and exogluconases, exocel-
lulases, and β-glucosidases. Generally, cellulases were produced by most of the
fungi and have the potential to degrade cellulose; at the same time, the same fungus
can secrete other enzymes to hydrolyze lignin and hemicelluloses (Khalil et al.
2011). Different cellulases have been produced and isolated from P. ostreatus as
reported by many authors using SSF and submerged cultivation system and sum-
marized in Table 5.1. Several studies reported the potential use of green substrate,
which is in the form of agriculture wastes, for the production of cellulases by P.
ostreatus (Radhika et al. 2013; Rodrigues da Luz et al. 2012). However, growth
phase is critical factor governing the enzyme production in SSF. Singh et al. (2003)
reported that cellulase production was highest during fruiting phase of strain
Pleurotus sp. Furthermore, cellulases are widely used in textile industries, as well as
detergent industries. In addition, they have also many applications in paper/pulp
industries and pharmaceutical industries.
5.8.2 Xylanases
Xylanases are enzymes which are able to break down the linear polysaccharide
β-1,4-xylan into xylose monomer and were classified into two major families
based on their protein sequence alignment. The two protein sequence alignments
are Family 10 (F) and Family 11 (G). According to Alvarez-Cervantes et al.

(2016), the xylanases of GH10 family of P. ostreatus, BOFX60 are related to the
GH10 and GH11 families, bBased on the smilimarities in amino acids, 233–318
and 180–193, respectively. Xylanases have gained attention because of their high
potential applications in many industrial processes (Yadav et al. 2015). Xylan
plays very important role in insect nutrition and ruminant animal since xylan is
the major component of hemicelluloses of most of the plants biomass and the
polymer can be easily bioconverted into small digestible molecules. Applications
for xylanase can be found in the pulp, feed, food, and paper industries (El-Enshasy
et al. 2016; Kour et al. 2019b; Rana et al. 2019a, b). For instance, xylanases are
beneficial in improving the quality of bread. It enhances the digestibility of rumi-
nant feeds (El-Enshasy et al. 2016) and also applied widely in the pre-bleaching
of kraft pulp.
In general, the xylanase activities were higher when mushroom cultivated on
waste material is compared to medium containing glucose or other low molecu-
lar weight fermentable carbohydrates. Different researchers reported that xyla-
nase can be produced by P. ostreatus as summarized in Table 5.1. The study of
Hazra et al. (1997) showed that 90% of the xylanase enzyme from the fungus
was secreted out by mushroom T. clypeatus. Other research reported that P.
ostreatus SYJ042 was able to produce xylanase. This xylanase was character-
ized by optimum temperature (40 °C) and stability at pH range between 3.0 and
9.0 (Qinnnghe et al. 2004). Getachew et al. (2016) studied xylanase purification
by using ammonium sulfate in concentration between 30% and 80% (wt vol−1),
and the best yield was obtained at 40%. The produced enzyme exhibited maxi-
mal activity at 50 °C and pH 6.0. This enzyme showed high affinity toward sub-
strate used and can further be used for animal feed processing or other industrial
applications.
5.8.3 Pectinases
Pectinase (EC 3.2.1.5) or pectinolytic enzyme is a heterogeneous group of

enzymes which is used to hydrolyze pectin, pectic acid, and oligo-D-galacturo-
nate (Tapre and Jain 2014). In general, pectic substances can be found broadly
in the plant cell wall including fruits and vegetables. The pectic substances are
usually acidic and characterized as very high molecular weight (about
25–360 kDa) and negatively charged (Lakshminarasimha Reddy and Sreeramulu
2012). Pectinase is comprised of many groups and types. It is classified into
three major types which are pectin pectinesterases (PE), protopectinase, and
depolymerizing enzymes (Jayani et al. 2005; Garg et al. 2016). Usually, PE is
used for pectic acid formation, whereas protopectinase is used for soluble pectin
formation. Different research reported on the ability of P. ostreatus to produce
and excrete pectinases using different substrates and cultivation systems
(Table 5.1). Most of the studies were focused on increasing of the production
yield and secretion of pectinase.
The presence of extractive substances, derived from wheat bran and grape stalks
(Massutti et al. 2015), sawdust (Sherief et al. 2010), sisal leaf (Raymond et al. 2015)
as well as cell immobilization in polyurethane foam (PUF) (Morales et al. 2014), in
culture medium can increase the production of pectinase by P. ostreatus. Sherief
et al. (2010) reported on the cultivation of commercial strain of P. ostreatus on lig-
nocellulosic rice straw and sawdust in plastic bags. The highest pectinase obtained
was 21.42 U g−1 after 35 days in case of saw dust, and 13.80 U g−1 after 20 days
incubation in rice straw culture. However, pectinase production by P. ostreatus is
not widely studied like other enzymes in spite of its importance based on its wide
use in textile industry, food processing industries, and for wine and juice clarifica-
tion (Tapre and Jain 2014; Pasha et al. 2013; Jayani et al. 2005).
Pectinylase (PL) EC 4.2.2.10 can only be produced by a small number of micro-
organisms such as Aspergillus, Penicillium, and Fusarium (Rashad et al. 2011;
Buyukkileci and Fernandez-Lahore 2011). Pectinylase is a pectinase which is only
able to degrade highly esterified pectin into small molecules via β-elimination with-
out methanol production. It was reported that P. ostreatus has also the capacity to
produce PL. The produced enzyme had molecular weight of 23 KDa (Rashad et al.
2011). This pectin lyase showed high level of activity, with biochemical character-
istic with alkaline pH (7.5), high optimum temperature (60 °C), and good affinity
toward citrus pectin. Higher pectinase production was obtained by substrates from
food processing at different varieties which is orange peel, lemon peel, apple pom-
ace, and sugarcane bagasse using submerged fermentation by P. ostreatus NRRL-
366. The best substrate is lemon peel, giving 1750 U of exopolygalacturonase
(exo-PGase and 750 U of pectin lyase (PL) per 1 g wet substrate after cultivation for
only 4 days (Rashad et al. 2010).
5.8.4 Amylases
Amylases (EC 3.2.1.x) or amylolytic enzymes are widely used for starch hydrolysis
into glucose oligomers and glucose monomer. Amylases can be divided into four
types of enzymes which hydrolyze different linkages and form various products.
They are (i) α-amylase, β-amylase, glucoamylase, and pullulanase. Amylase can
secrete from plants, microorganism and animals and share at least 30% of the world
enzyme market (Deljou and Arezi 2016). However, enzymes from mushroom
sources have subjected applications in industrial part. Amylase has a great potential
application in industrial such as food, textile, paper, fermentation, detergent, and
pharmaceutical industries.
The production of β-amylase (Akinyele et al. 2010) and α-amylase (Ergun and
Urek 2017; Lim et al. 2013: Rashad et al. 2009) has been reported from the species
P. ostreatus (Table 5.1). α-Amylase is an enzyme with a potential to breakdown the
α-1,4-glucosidic linkage of starch to produce small molecules such as maltose and
malto-oligosaccharides (Deljou and Areze 2016). The isolation and characterization
of the β-amylase was reported from the edible mushroom, P. ostreatus by Akinyele
et al. (2010). The purification of amylase enzyme has been achieved by ion exchange
chromatography method. The final finding was proven that the purified α-amylase
by ion exchange chromatography on DEAE SephadexA 50 and gel filtration is ther-
mostable with high potential for industrial use. The recent study of Ergun and Urek
(2017) showed that the maximal amylase activities were obtained in SSF after
7 days of cultivation using dry potato peel waste after pretreatment with potassium
hydroxide.
5.8.5 Proteases
Proteases refer to a group of enzymes which catalyze hydrolytic reactions respon-

sible for the degradation of protein into peptides and amino acids. Proteases are
among widely used enzymes, which share about 65% of the global market based on
catalytic properties (Yin et al. 2014). It was reported that Pleurotus ostreatus can
secrete different types of proteases, which are metalloprotease (Shen et al. 2007;
Liu et al. 2014; Dohmae et al. 1995); serine protease (ProA, EC 3.4.22.9), amino
acid residues containing serine, aspartic acid, and histidine in the active center
(Lebedeva and proskuryakov 2009; Palmieri et al. 2001; Dohmae et al. 1995); cys-
teine protease or thiol protease, containing cysteine and histidine residues at active
center (Shin and Choi 1998); and aspartic protease (Yin et al. 2014). Researchers
isolated different types of proteases from fruiting bodies, mycelia, and fermentation
broth of P. ostreatus (Dohmae et al. 1995; Palmieri et al. 2001; Shen et al. 2007).
Interestingly, the aspartic protease showed modeling crystal structure containing
Po-Asp D110 and D295, with Asp110 and Asp295 in the active site. This study also
has clearly shown that the structure had specific N- and C-terminal sequence (Yin
et al. 2014). An aspartic protease from P. ostreatus (Pro-Asp) with Mwt of 35.3 KDa
and isoelectric point of 4.57 consisted of 1324 nucleotides with an open reading
frame (ORF) of 1212 bp encoding 403 amino acid residues (Yin et al. 2014). In
addition to that, the crude aspartic protease had milk clotting activity and can be
used in cheese making industry. MEROPS database defined that Aps was engaged
into 16 different families according to amino acid sequence homology. Cysteine
proteases or thiol proteases are enzymes that degrade protein containing a Cys-His-
Asn at the active site. These proteases have histidine residue at active site acting as
proton donor to enhance the nucleophilicity of the cysteine residue. Shin and Choi
(1998) successfully purified cysteine protease and did full characterization of the
enzyme. Cysteine protease from P. ostreatus is well known as leucine pNA (LPNA)
consisting of ASGLXXAIL amino acid. This enzyme showed thermal stability up to
37 °C and the pH optimum in the range of 5.5–6.5.
A serine protease (ProA, EC 3.4.22.9) and two metalloproteases, which are ProB
(EC 3.4.99.32) and ProC (EC 3.4.24.4), were isolated from the fruiting bodies of
P. ostreatus (Dohmae et al. 1995). Paminieri et al. (2001) reported that serine
protease, present in the submerged culture of P. ostreatus, is different from other
proteases purified from the fruiting bodies done by Dohmae et al. (1995).
Interestingly, these enzymes involved in POXA1b degradation and play a key role
in the regulation of laccase activity and thus involved in lignin degradation by P.
ostreatus. Furthermore, this finding was similar in the regulation of laccase enzyme
in cultures of Trametes versicolor and Phanerochaete chrysosporium (Dosoretz
et al. 1990). The purified serine protease (PoSI) was reported to have an isoelectric
point of 4.5 and Mwt of 75 kDa, which is larger than other fungal proteases (Palmieri
et al. 2001; Dohmae et al. 1995; Mellon and Cotty 1996).
Metalloprotease is defined as protease enzymes with catalytic mechanism
involve metal ions and mostly require zinc or cobalt for activity. PoFE (a fibrinolytic
protease) consisting of 19 amino acids with N-terminal sequence of
ALRKGGAAALNIYSVGFTS. Enzyme activity was increased by the addition of
Ca2+, Zn2+, and Mg2+ ions, which is similar to the metalloprotease, extracted from
fruiting body of P. ostreatus (GenBank Accession No. AAU94648.1) (Shen et al.
2007). In another study by Liu et al. (2014), they successfully purified fibrinolytic
protease enzyme which exhibited thermostability up to 40 °C and pH optimum of 7.4.
This result for optimum pH was similar to other fibrinolytic enzymes from Cordyceps
militaris, Armillaria mellea, and Tricholoma saponaceum and application for medi-
cal applications, for thrombolytic treatment (Liu et al. 2014). In general, proteases
from P. ostreatus have molecular weight between 18.2 and 75 kDA (Liu et al. 2014;
Yin et al. 2014; Lebedeva and Proskuryakov 2009; Shen et al. 2007; Palmieri et al.
2001; Shin and Choi 1998; Dohmae et al. 1995). However, different types of protease
produced by P. ostreatus are presented in Table 5.2.
Table 5.2 Characteristics and potential application of some proteases from Pleurotus ostreatus
Optimal
Purification conditions
Proteases of Yield Isoelectric Potential
Pleurotus sp. (%) Fold kDa point pH Temp application References
Metalloprotease 6.5 876 32 ND 6.5 35 °C Thrombolytic Shen et al.
therapy (2007)
Heart disease
Metalloprotease 7.54 147 18.2 8.52 7.4 40 °C Fibrinolytic Liu et al.
therapy (2014)
Serine protease 13 70 75 4.5 7–8 ND Acting as Palmieri
(PoSi) regulatory role et al. (2001)
for laccase
activity
Metalloproteases Maintenance Dohmae
ProA 8 262 30 9.0 6.5 cellular disorder et al. (1995)
ProB 8 34.8 19 8.35 5.6
ProC – – 42.5 – 5.6
Cysteine protease 4 238 ND ND 5.5– 37 °C Cell Shin and
6.5 germination, Choi (1998)
morphogenesis
Aspartic protease ND ND 35.3 4.57 ND ND Cheese making Yin et al.
industry (2014)
ND not determined or data not given in this study
5.8.6 Lipases
Lipases (EC 3.1.1.3) constitute a very important class of hydrolytic enzymes.

Lipases were classified into two types: (a) lipases those of enhanced enzyme activ-
ity in emulsion form and (b) lipases with the active site full of protein (Karigar and
Rao 2011; Sharma et al. 2011). Generally, lipases are varied in size and sequence of
amino acids in primary structure. Lipases are produced in all organisms, and natural
function of lipases is to hydrolyze the fats into glycerol and fatty acids (Sharma
et al. 2011). Therefore, they also play an important role in food, chemical, cosmetic,
detergent, and paper making industries. In addition, they were used as indicator to
determine hydrocarbon degradation for soil (Karigar and Rao 2011). Currently,
P. ostreatus genome was successfully isolated and characterized. Sixty seven puta-
tive lipase coding genes have been annotated in this species (Guarino and Sannia
2013). Moreover, the ability to produce lipase enzymes by P. ostreatus was inves-
tigated by many authors. The ability P. ostreatus to produce extracellular lipolytic
enzymes was studied by Guarino and Sannia (2013). They reported on the suc-
cessful production of lipase by using 5% olive mill waste as the main carbon
source to achieve a final titer of 30 U L−1 after only 5 days of cultivation. Mushroom
enzymes were extracted from the SSF cultures, and the extracted enzymes were
successfully immobilized using sodium alginate beads for continuous operation
(Wijayati et al. 2017).
As presented in this chapter, P. ostreatus have strong production and excretion

capacity for large number of enzymes from different groups. The process can be
easily shifted to the targeted enzyme through proper design of the cultivation
medium and optimization of the cultivation parameters. In addition, this mushroom
has long history as food without any previously reported negative impact on human
and animal health. Therefore, it received GRAS status from FDA which makes it
very attractive for any food, feed, cosmetic, and pharmaceutical industries. In addi-
tion, based on the high capacity for concomitant production of hydrolase enzyme
cocktail, P. ostreatus is considered as a very attractive biofactory in biorefinery for
the hydrolysis of complex lignocellulosic biomass structure to fermentable sugars
which is subsequently used for the economic production of different primary and
secondary metabolites. However, further research is required for better understand-
ing of the combined factors effect on the induction, production, and excretion of
mushroom enzymes during large-scale cultivation.
Acknowledgments The authors would like to express their sincere acknowledgment for the sup-
port of MOE and UTM-RMC (Malaysia) through HICOE grant no. R.J130000.7846.4J262.
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Chapter 6
Extracellular Fungal Peroxidases
and Laccases for Waste Treatment:
Recent Improvement
Shanmugapriya S., Manivannan G., Selvakumar Gopal,

and Sivakumar Natesan
6.1 Introduction
Fungi are widespread eukaryotic microorganism exploit subsidiary living conditions

through their unusual extracellular enzymes capable of utilizing variable sources as
substrates. Mostly, these extracellular enzymes degrade complex organic substances
including cellulose, hemicellulose, lignin, phenols, pesticides, hydrocarbons, etc.
into simple molecules for their carbon, energy, and nutrition (Burns et al. 2013).
Among the various organic substances, lignin, hemicelluloses, and phenolic com-
pounds are the major wastes as environmental pollutants. Due to the chemical com-
plexity, lignins take a long time to its natural degradation. In nature, majority of the
fungi in the phylum Basidiomycota have the enzymes such as laccases and peroxi-
dases to actively degrade the lignin containing polyphenol waste from the environ-
ment, which have potential biotechnological applications.
Three phenotypic groups of fungi specifically white-rot, brown-rot, and soft-rot
fungi are the predominant groups which degrade lignin compounds variably. Among
them, white-rot fungi execute complete lignin degradation with the ability to cleave
Cα- Cβ, β-aryl, and C1-Cα bonds, including aromatic C-C bonds degradation c, but
brown-rot fungi partially degrade lignin compounds by Fenton/Haber Weiss chem-
istry (Arantes et al. 2012). However, white-rot fungi produce a special group of
extracellular enzymes like laccases and peroxidases which entirely degrade lignin
Shanmugapriya S. · S. Natesan (*)

Department of Molecular Microbiology, School of Biotechnology,
Madurai Kamaraj University, Madurai, Tamil Nadu, India
e-mail: siva.biotech@mkuniversity.org
Manivannan G.
Department of Microbiology and Biotechnology, SVN College, Madurai, Tamil Nadu, India
S. Gopal
Department of Microbiology, Alagappa University, Karaikudi, Tamil Nadu, India

154 Shanmugapriya S. et al.
compounds. Also, fungal laccases and peroxidases have enormous potentials in

environmental detoxification and bioremediation of phenolic compounds. Using
these enzymes, the white-rot fungi can convert wood, paints, pesticides, and plastics
etc. into nutrients, and it has lots of industrial application. Laccases have been
regarded as a “Green Tool,” because they require molecular oxygen (O2) as the only
co-substrate for biocatalysis and not hydrogen peroxide (H2O2) (Surwase et al.
2016). In this chapter, the structure, functional properties, applications, and their
recent advancements are being discussed.
6.2 Laccases
Laccases (EC 1.10.3.2; 1,4-benzenediol: oxygen oxidoreductases) are extracellular

copper-containing monomeric glycoproteins, which come under multicopper oxi-
dase family (Solomon et al. 1996). It is otherwise called as polyphenol oxidase and
blue multicopper oxidases. It oxidizes several aromatic and non-aromatic com-
pounds especially phenols as well as diamines and hexacyanoferrate by using
molecular oxygen as an electron acceptor. It was first demonstrated in the sap of the
Japanese lacquer tree Toxicodendron vernicifluum (formerly Rhus vernicifera);
hence, it is named as laccase. Its molecular weight ranges from 50 kDa to 100 kDa
(Galhaup and Haltrich 2001).
6.3 Sources of Laccases
Laccases have been generally present as extracellular and intracellular enzymes in

several organisms ranging from microbes to higher plants. It was first discovered in
plants by Yoshida in 1883. It is widely distributed in all plants, but not yet studied
properly, because of the existence of several isoenzymes of laccase in lignified plant
tissues (Gavnholt et al. 2002) and difficulties of their detection and purification
from crude plant extracts (Ranocha et al. 1999), but it was well documented in
fungi. Fungi are unique important class of eukaryotic microorganisms and synthe-
size unusual enzymes capable of performing chemically tricky reactions (Viswanath
et al. 2008; Shraddha et al. 2011). Many fungal species are of great value and can
remove toxic recalcitrant compounds in an environment-friendly manner. Laccases
play an important role in bioremediation of toxic phenolic compounds (Singh et al.
2011) and degradation of recalcitrant xenobiotic compounds. The presence of lac-
case enzyme in fungi was first reported by Laborde in 1897 (Mayer and Harel
1979). Laccases are found in a wide range of fungi generally in white-rot fungi
(Brijwani et al. 2010; Mayer and Staples 2002).
Generally, paper and pulp industry effluents contain a large amount of chlori-
nated phenolic compounds that is formed from incomplete breakdown of lignin
during pulp bleaching process. Different groups of fungi can remove such lignin
6 Extracellular Fungal Peroxidases and Laccases for Waste Treatment: Recent… 155
and phenol from the effluent by producing extracellular enzymes like laccase,
manganese peroxidase, and lignin peroxidase. Several studies suggested that fila-
mentous fungi are the best choice than bacteria for the removal of soil pollutants
because fungi can reach the pollutant efficiently than bacteria (Rubilar et al. 2008;
Kour et al. 2019; Yadav et al. 2018). For example, laccase activity was detected in
the cultures of fungi belonging to Basidiomycetes, Ascomycetes, and Deuteromycetes
family (Table 6.1). The highest amount of laccase is produced by white-rot fungi
(Leonowicz et al. 1997). Laccase enzyme has been reported in many fungal species
such as Trichoderma reesei (Levasseur et al. 2010), Xylaria polymorpha (Nghi et al.
2012), Lentinus tigrinus (Pozdnyakova et al. 2006), Pleurotus ostreatus (Zhao et al.
2017), Cerrena unicolor (Kim et al. 2002) T. versicolor (Minussi et al. 2007;
Rogalski et al. 1991), Trametes pubescens (Shleev et al. 2007) Melanocarpus albo-
myces (Kiiskinen et al. 2002), Magnaporthe grisea (Iyer and Chattoo 2003),
Aspergillus flavus PUF5 (Priyanka and Uma 2017), Trametes hirsuta (Tapia-Tussell
et al. 2011), Trametes ljubarskyi (Goh et al. 2017), Aspergillus flavus (Kumar
et al. 2016), etc. Further, Abd El Monssef et al. (2016) reported that the genus
Alternaria, Aspergillus, Cladosporium, Penicillium, Rhizopus, and Trichoderma
also produce laccases.
Table 6.1 Examples of laccase producing fungi

Class and division Source References
Agaricomycetes Trametes versicolor Bourbonnais and Paice (1992)
Agaricomycetes Phanerochaete chrysosporium Srinivasan et al. (1995)
BKM-F-1767
Agaricomycetes Pleurotus pulmonarius Marques de Souza et al. (2002)
Sordariomycetes Chalara (syn. Thielaviopsis) Robles et al. (2002)
(Ascomycota) paradoxa CH 32
Agaricomycetes Trametes pubescens Galhaup and Haltrich (2001);
Rodriguez Couto et al. (2004);
Osma et al. (2007)
Agaricomycetes Phanerochaete chrysosporium Kapich et al. 2004
ME-446
Agaricomycetes Trametes hirsuta Rodríguez Couto et al. (2006)
Agaricomycetes Phanerochaete chrysosporium Gnanamania et al. (2006)
NCIM 1197
Agaricomycetes Pycnoporus sanguineus Vikineswary et al. (2006)
Agaricomycetes Ganoderma lucidum Murugesan et al. (2007)
Eurotiomycetes Aspergillus carbonarius Sanjay et al. (2007)
(Ascomycota)
Sordariomycetes Trichoderma harzianum WL1 Sadhasivam et al. (2008)
(Ascomycota)
Agaricomycetes Pleurotus ostreatus, Trametes Osma et al. (2011)
(Basidiomycota) pubescens, Cerrena unicolor, and
Trametes versicolor
Sordariomycetes Trichoderma spp. Kalra et al. (2013)
(Ascomycota)
Other than fungi and plants, laccase enzyme has been reported from bacteria
(Santhanam et al. 2011) (Yadav et al. 2016, 2019a, b), lichens (Laufer et al. 2009)
and sponges (Li et al. 2015a). Moreover, polyphenol oxidases with laccase-like
activity have been found in oysters (Luna-Acosta et al. 2010) and insect cuticles
(Lang et al. 2012). Functions of laccase enzymes are based on their source and the
stage of life of the organism producing them.
6.3.1 Structure of Laccases
Laccases are glycoproteins synthesized as monomer and containing four copper

atoms (Fig. 6.1). After synthesis, laccase was modified with mannose which is
accountable for about 10–50% of total weight of laccase. Carbohydrate may con-
tribute structural stability of laccases (Mayer and Staples 2002). Glycosylation of
laccases confers copper retention, susceptibility to proteolytic degradation, thermal
stability, and secretion. The copper atoms of laccase are divided into three types.
They are (i) Type 1 (T1) (ii) Type 2 (T2) and (iii) Type 3 (T3). Laccase contains one
T1 and T2 and two T3 copper atoms. The catalytic mechanism of the laccase starts
with the donation of an electron to the substrate by the T1 copper site, followed by
an internal electron transfer from the reduced T1 to the T2 and T3 copper sites.
During oxidation of substrate, molecular oxygen is reduced to water (Jones and
Solomon 2015). The reduction reaction takes place at trinuclear cluster which is
formed by the association of T2 and T3 copper atoms (Fig. 6.1). Type 1 copper
confers blue color to the enzyme because of maximum absorbance around at 600 nm
which is the result of the covalent copper–cysteine bond (Matera et al. 2008).
However, in fungal laccases, the axial ligand is leucine or phenylalanine, which pos-
sibly provides the mechanism for the regulation of enzyme activity (Claus 2004;
Kumar et al. 2003; Enguita et al. 2003; Garavaglia et al. 2004). Type 2 is a non-blue
copper and showed weak absorption in the visible spectrum (Niku-Paavola et al.
2004). Type 2 copper is coordinated by two histidine residues and is strategically
Fig. 6.1 Catalytic mechanism of laccase

positioned close to Type 3 copper. Type 3 copper is a binuclear center that showed
maximum absorbance at 330 nm in its oxidized form (Matera et al. 2008; Decker
and Terwilliger 2000). Laccase molecular weight was determined to be in the range
of 60–390 kDa (Kalme et al. 2009). The pH values vary between pH 4.32–6.51 and
pH 5.32–6.19 (Cázares-García et al. 2013; Moreno et al. 2017). The catalytic
domain of laccase is moderately conserved in diverse fungal species, and the rest of
the enzyme structure shows high diversity (Gochev and Krastanov 2007; Moreno
et al. 2017). However, laccases with variants in the active site are also reported in
Pleurotus ostreatus (Palmieri et al. 1997). In this fungus, enzymes lacking the maxi-
mum absorption around 600 nm are usually classified as “yellow” or “white” lac-
cases. Difference in the active center might confer these laccases have different
functional properties of interest. Similar white laccase has also been reported in
Deuteromycete fungus and Myrothecium verrucaria NF-05 (Zhao et al. 2012).
These white laccases contain only one Cu, one Fe and two Zn atoms (Palmieri et al.
1997; Zhao et al. 2012), but laccase enzyme of Phellinus ribis has one manganese
atom instead of T1 copper atom (Min et al. 2001). Many fungi have variable number
of laccase genes and they are typically inducible.
6.3.2 Mechanism of Laccase Activity
Laccases have wide range of substrate-specific activity on ortho- and para-diphenol

groups, as well as mono-, di-, and polyphenols, aminophenols, methoxyphenols,
ascorbate, and aromatic amines with the linked four-electron reduction of oxygen
to water (Bourbonnais and Paice 1990; Bourbonnais et al. 1995; Madhavi and Lele
2009). Oxidation of aromatic compounds occurs with the concurrent reduction of
one O2 molecule to H2O. After four cycles of single-electron oxidation of aromatic
compounds, it leads to formation of free radicals and reduction of one molecule of
oxygen into two molecules of H2O (Fig. 6.1). Initially, the free radical is unstable
and converted to a quinone in a second enzyme-catalyzed step. Alternatively, oxi-
dized phenol-containing polymers may be partially degraded by nonenzymatic
radical reactions. Partial degradation is due to the breaking of covalent bonds that
join the monomer (Strong and Claus 2011). In the presence of small molecules,
known as redox mediators, laccases improve their substrate specificity. Redox
mediators are low molecular weight and small-sized molecules that are used as
enhancer in the real electron transfer steps of enzymatic degradations process. It is
a stable and reusable molecule. It increases the capability of an enzyme to react
toward uncommon substrates (Majeau et al. 2010). The following mediators are
frequently used for laccase activity. (1) 2,2′-Azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS), (2) 1-hydroxy-benzotriazole, (HBT) (3) 1-nitroso-2-naph-
thol-3,6-disulfonic acid (NNDS), (4) syringaldehyde, (5) 4-Acetylamino-TEMPO
4-hydroxy-TEMPO, (6) violuric acid (VIO), and (7) p-coumaric acid (Majeau
et al. 2010).
6.3.3 Applications of Laccases
Laccases have several biological functions such as lignification of plant cell walls
(O’Malley et al. 1993), lignin biodegradation, detoxification of lignin (Baldrian
2006), virulence factors (Williamson et al. 1998), and copper and iron homeostasis
(Stoj and Kosman 2003). Further, laccases have potential applications in bioreme-
diation, paper pulp bleaching, finishing of textiles, biofuel cells, etc. Laccases
exhibit transformation reactions like oxidation of functional groups to the hetero-
molecular coupling for production of new antibiotics derivatives or the catalysis of
key steps in the synthesis of complex natural products (Xenakis et al. 2016).
However, fungal laccases are largely used for removal of phenols which present in
wastewater (Pang et al. 2016). The following are the significant areas of laccase
applications.
6.3.3.1 Environmental Applications
Extensive use of chemicals in agriculture and industrialization leads to release of

different persistent, hazardous, bioactive, and bioaccumulative chemicals to the
environment that causes pollution in land and water. These toxic chemicals create
adverse effects on both human and other flora and fauna of soil and aquatic environ-
ment. Naturally, phenol and its derivatives are ever-present pollutants that arrived as
wastewater from the effluents of industrial activities, such as pulp, petrochemicals,
coal refineries, pharmaceuticals, production of resins, paints, and textiles (Rastegari
et al. 2019; Yadav et al. 2017). They are highly toxic to aquatic organisms, including
fish and shellfishes. The toxic effects of phenol are based on its chemical complex-
ity and the range of free radical formation. It causes acute toxicity, with an effect of
damaging DNA or enzymes inducing mutagenicity, carcinogenicity, and hemato-
toxic and hepatotoxic effects toward humans and other living organisms
(Michałowicz and Duda 2007). Therefore, removal of phenol is essential to protect
the environment and individual. Most of the conventional oxidation method (chemi-
cal method) removes the chemicals but has several drawbacks such as (i) use of
hazardous chemicals for oxidation (ii) nonspecific, and (iii) undesirable side reac-
tions. In the present scenario, biological treatment methods (enzymatic oxidation)
are most suitable and widely used due to specific and biodegradable catalysts and
enzyme reactions are carried out in mild conditions (Rodríguez Couto and Toca
Herrera 2006).
Laccases are capable of oxidizing, polymerizing, or transforming different xeno-
biotics including phenolic pesticides into less toxic molecules. Hence, it is a more
apt enzyme in water (Majeau et al. 2010) and soil bioremediation. Laccase-based
bioremediation has been proposed to remove toxins from textile, paper and pulp,
food, distillery, pharmaceutical, printing, paint, and cosmetic industrial effluents.
For the remediation, laccase could be used as (1) free enzyme, (2) immobilized
enzyme, and (3) laccase containing cells to remove the pollutants from water
(Mugdha and Usha 2012).
6.3.3.1.1 Direct Use of Laccase Producing Fungi
The growing fungi are used for waste (harmful pollutants) treatment. In this method,
the fungal cells can adapt in the pollutant containing environment, utilize the harm-
ful pollutants as carbon and energy requirements by synthesizing the specialized
degradative enzyme, and digest or transform the pollutants to harmless. The intro-
duced organisms produced enzyme that co-metabolized the targeted contaminants
(Mugdha and Usha 2012). White-rot fungus Trametes versicolor is able to remove
humic acids from a real humic-rich industrial-treated wastewater of a food-
processing plant (Mostafa Zahmatkesh et al. 2017).
6.3.3.1.2 Cell-Free Laccase Enzyme
Enzymes extracted from organisms are used to treat toxic pollutants as a pure form
or crude extract. This method is advantageous, because of the following: (1) there is
no need of acclimatization of source organisms to the toxic environment, (2) addi-
tional nutrients are not essential, (3) growth supportive environment is not required,
(4) the growth rate of the source organism does not affect the amount of available
enzyme to treat the effluent, (5) usage of cell-free enzyme makes it easier to stan-
dardize optimum treatment conditions, and (6) it is easy to handle and monitor the
process (Karam and Nicell 1997). Crude enzyme extract is the least processed but
contains active form of the enzyme. It is used to treat large-scale effluent treatment.
Although usage of pure enzyme is highly expensive, crude enzyme preparation at
larger volume should be used for industrial effluent treatment. In general, enzyme
function is based on their conformation, under extreme conditions such as very high
or low pH and temperature, high ionic strength, high concentrations of reactants,
and presence of inhibitors; the structure of free enzyme may be modified and the
enzyme becomes nonfunctional (Karam and Nicell 1997). Besides, use of free
enzyme is hard to be taken from the residual reaction system for reuse (Wang et al.
2008). Therefore, immobilized preparation of enzymes and the whole-cell biomass
for repeated long-time usage have been developed.
6.3.3.1.3 Immobilized Laccase Enzyme
Immobilization of enzyme provides an increasing availability of enzyme to the sub-

strate with better turnover over a significant period of time. The practice of immo-
bilized enzymes in effluent treatment overcomes the cell-free enzyme because of
the following reasons: (1) high stability, (2) easy to handle, (3) reusability, and (4)
cost-effectiveness. Immobilization of laccase was done using different materials
and used for bioremediation process (Table 6.2). Several immobilization techniques
have been developed and adapted in enzyme immobilization for different applica-
tions. Theoretically, enzyme immobilizations are done by two basic methods:
they are physical (entrapment, encapsulation, and cross-linking) and chemical
Table 6.2 Immobilization of laccase enzyme and their applications

Organisms Method of
name Support immobilization Applications References
Trametes Porous glass beads Entrapment Dye decolorization Champagne
versicolor and Ramsay
(2010)
Trametes Microfibers Encapsulation Dye decolorization Dai et al.
versicolor (2010)
Aspergillus Green coconut fiber Adsorption Dye decolorization Cristovao et al.
(2011)
Trametes Carbon-based Adsorption Phenol removal Liu et al.
versicolor mesoporous (2012)
magnetic
composites
Trametes Gold electrode Covalent bonding Biosensor for Sarika et al.
versicolor Phenolic (2014)
compounds in
industrial effluents
Coriolopsis Calcium alginate Entrapment Remazol Brilliant Daassi et al.
gallica beads Blue R, Reactive (2014)
Black 5, and
Bismarck Brown R
T. versicolor Nanostructured Physical adsorption Biosensors and Chen et al.
bacterial cellulose and cross-linking establishment of (2015)
with glutaraldehyde bioreactors
Cyathus bulleri Polyvinyl alcohol Entrapment Decolorization of Chhabra et al.
azo dye acid red 27 (2015)
Cercospora sp. Alginate Entrapment Dye decolorization Vikram et al.
SPF-6 (2015)
Cyathus bulleri Polyvinyl alcohol Entrapment Acid red 27 Chhabra et al.
(2015)
Trametes ZnO/SiO2 Adsorption Remazol Brilliant Li et al.
versicolor nano-composite Blue B and Acid (2015b)
Blue 25
Trametes Poly(glycidyl Covalent bonding Bisphenol A Melo et al.
versicolor methacrylate-co- (2017)
ethylene glycol
dimethacrylate)
Trametes Chitosan Covalent bonding Anthracene Azzurra
versicolor macrobeads Apriceno et al.
(2017)
Trametes Magnetic Polymerization 4-chlorophenol Zhang et al.
versicolor nanoparticles (2017)
Trichoderma Sol–gel matrix Entrapment Dye decorization Zabin et al.
harzianum (2017)
strain HZN10
Cerrena sp Cross-linked Cross-linking Malachite green Yang et al.
enzyme aggregates (2017a)
Functionalized Covalent bonding Biosensor for Mazlan et al.
methacrylate– Tartrazine (2017)
acrylate
microspheres
(continued)
Fig. 6.2 Enzyme immobilization methods
(adsorption, covalent binding) interactions with enzyme-supportive matrix

(Matijosyte et al. 2010) (Fig. 6.2).
(i) Entrapment is defined as the preservation of enzymes in a porous solid matrix,
such as polyacrylamide, collagen, alginate, or gelatin (Dayaram and Dasgupta
2008; Lu et al. 2007; Niladevi and Prema 2008; Phetsom et al. 2009).
(ii) In encapsulation, enzymes are protected in a semi-permeable polymer materi-
als such as polyethyleneimine, sol–gel silica matrix, SiO2, and poly(GMA-co-
nBA) microspheres (Qiu and Huang 2010; Rochefort et al. 2008; Crestini et al.
2010; Mazlan and Hanifah 2017).
(iii) In the adsorption method, the enzyme immobilized onto a support is based on
ionic and/or other weak forces of attraction. Adsorption is based on the pH and
ionic strength of the medium and the hydrophobicity of the support (Xu et al.
2009; Fang et al. 2009; Forde et al. 2010). Adsorbents like Mobil Composition
of Matter (MCM), cyano-modified silica (CNS), and Santa Barbara Amorphous
(SBA-15) (Forde et al. 2010) and ion-exchange resins such as dextran, agarose,
and chitosan (Cordova et al. 2009; Çorman et al. 2010; Ibrahim et al. 2007) are
used for laccase immobilization.
(iv) Covalent attachment is widely used enzyme immobilization method in which
the chemical groups on the support surface are activated and react with nucleo-
philic groups on the protein (Arroyo 1998; Brady and Jordaan 2009). For
example, silica-based supports such as kaolinite or mesoporous silica nanopar-
ticles and GLU-activated silica nanoparticles (Champagne and Ramsay 2007;
Liu et al. 2008; Salis et al. 2009), epoxy-activated resins such as Eupergit and
Sepabeads (Berrio et al. 2007; Russo et al. 2008), Alumina and Granocel
(Crestini et al. 2010), and electrodes based on carbon, glass, gold, silver or
graphite (Balland et al. 2008; Rahman et al. 2008; Szamocki et al. 2009) have
been frequently used for laccase.
(v) In cross-linked method, enzyme immobilization is possible with the use of

bifunctional cross-linkers (Brady and Jordaan 2009). For example, dialde-
hydes, diiminoesthers, diisocyanates, and diamines activated by carbodiimide
(Arroyo 1998) have been used.
6.3.3.2 Textile Effluent Treatment
Colors and dyes are commonly used in textile, paper, food, cosmetics, and pharma-
ceutical industries. There are above 1,00,000 different human-made synthetic dyes
available on the market, and worldwide, its production is around 7,00,000 tons/year
(Hao et al. 2000). Wastewater from textile industries carries 10% of the dye stuffs
which has been a significant cause of environmental pollution. Most of the synthetic
dyes are lethal to living organisms due to their toxic and carcinogenic properties.
The removal of dyes from industrial wastewaters could be very important due to
their toxicity and carcinogenicity. The structural complexity of dyes makes effluent
treatment difficult by conventional physicochemical methods due to their high cost
and low effectiveness. Laccases are promising tools for the detoxification of dyes
(Table 6.3) because it has shown efficient decolorization of different industrial dyes
at low concentrations (Rodriguez et al. 1999; Reyes et al. 1999) without generation
of harmful aromatic amines (Chivukula and Renganathan 1995; Wong and Yu 1999).
Dye degradation ability of laccase depends on physiochemical parameters such
as cell aging, concentration of dye, immobilized cells, etc. (He et al. 2004; Kalyani
et al. 2008). Laccase from Polyporus rubidus showed efficient decolorization of
industrially important synthetic textile dyes in broad range of concentration without
the use of redox mediators (Bayoumi et al. 2014). Immobilized laccase of
Paraconiothyrium variabile has pH and thermal stability and exhibited efficient
decolorization of Acid Blue 25 and Acid Orange 7 (Mirzadeh et al. 2014). Complete
decolorization of malachite green was achieved with Cerrena sp. laccase CLEAs
(cross-linked enzyme aggregates) at 60 °C (Yang et al. 2017a). Trametes versicolor
CBR43 can decolorize different types of dyes such as acid disperse and reactive
textile dyes by producing laccase and Mn-dependent peroxidase (Yang et al. 2017b).
Laccase enzyme from Cerrena unicolor strain GSM-01has been purified and identi-
fied that laccase is a monomeric protein of 63.2 kDa, their optimal pH and tempera-
ture is 2.6 and 45 °C, respectively and effectively decolorize bromothymol blue,
evans blue, methyl orange, and malachite green (Wang et al. 2017).
6.3.3.3 Paper Industries
Large amount of phenolic compounds such as lignin and their derivatives contain-
ing effluent has been discharged from the paper industries. Commonly, chemical
bleaching method is used to remove lignin. In this process, chlorine is used, but
chlorine formed bond with lignin and produce toxic organochloro-complexes like
chlorolignins, chlorophenols, chloroguiacols, and chloroaliphatics. Large volume
Table 6.3 Decolorization of various dyes by laccase

Organism name Dye name References
Ganoderma lucidum Ramazol Black B and Ramzol Orange Murugesan et al.
16 (2009)
Trametes versicolor Orange G Casas et al. (2010)
Aspergillus ochraceus Methyl Orange Telke et al. (2010)
NCIM-1146
Paraconiothyrium variabile Bromophenol blue Vinoth Kumar
et al. (2011)
Trametes versicolor Reactive Black 5 Bibi and Bhatti
(2012)
Armillaria sp. F022 Reactive Black 5 Hadibarata et al.
(2012)
Trametes trogii Acid Orange 51 Dalel Daassi et al.
(2013)
Coprinopsis cinerea Methyl Orange Tian et al. (2014)
Aspergillus niger Basic fuchsin Rani et al. (2014)
Cerrena sp. circulans BWL1061 Malachite Green Yang et al. (2015)
Ganoderma sp. Direct Blue E Iyer et al. (2016)
Pleurotus ostreatus MTCC 142 Congo Red Das et al. (2016)
Paraconiothyrium variabile Acid Orange 67, Disperse Yellow 79, Forootanfar Hamid
Basic Yellow 28, Basic Red 18, Direct et al. (2016)
Yellow 107, and Direct Black 166
Talaromyces funiculosum (M2F) Reactive Magenta HB Ankita Chatterjee
et al. (2017)
Marasmiellus palmivorus Reactive blue 220 (RB)and Acid blue Cantele Cantele
80 (AB) et al. (2017)
Marasmius cladophyllus Remazol Brilliant Blue R, Orange G, Ngieng Ngui Sing
and Congo red et al. (2017)
of dark colored wastewater is produced at the end of bleaching process. Dark colored,
toxic wastewater of paper industry are highly hazardous and also create environ-
ment pollution. Physical and chemical methods such as ultrafiltration, ion exchange,
lime precipitation and aerated lagoons, and activated sludge methods are used to
treat wastewater, but they are ineffective and expensive. This triggers the use of
microbial laccase enzymes which fulfills the whole requirement and delignification,
separates wood into its constituent fibers and lessens the toxic wastewater forma-
tion. Laccase-mediated delignification was introduced in the 1900 and uses media-
tors to oxidize the phenolic compound, lignin.
The laccase enzyme itself can effectively break phenolic compound due to its
high redox potential. The incorporation of mediator along with laccase increases the
availability and dimension of the enzyme against non-aromatic ring-containing
compounds. Several mediators ABTS, HBT, N-hydroxyacetanilide (NHA), and vio-
luric acid have been used in delignification process. Effective mediators commonly
possess N-OH functional group, and it should be biodegradable, specific, and

economically feasible. Paper recycling reduces usage of source material and also
cost. Laccase facilitated bleaching of old newsprint pulp with improved brightness
by removing the lignin component (Hakala 2011; Xu et al. 2007, 2009). Combined
action of laccase and hemicellulolytic enzyme exhibited efficient deinking and
biobleaching of the pulp. The combination of xylanase and laccase is an effective
tool for lessening the amount of lignin and related molecules from the pulp (Valls
and Roncero 2009; Saxena and Chauhan 2016). Old newsprint is efficiently recy-
cled with high brightness and low effective residual ink concentration (ERIC) con-
tent through a combination of the physical methods like sonication and microwaving
and enzymatic method (laccase and xylanase) (Virk et al. 2013). Pure laccase
enzyme from T. versicolor decolorized the paper and pulp mills effluent to a clear
light-yellow solution (Karimi et al. 2010), and various structurally different indus-
trial dyes (Dhillon et al. 2012). The expression of delignifying enzymes only com-
menced complete glucose depletion (Girard et al. 2013). B. adusta and P.
chrysosporium have showed 100% delignification of industrial pulp and paper mill
wastewater in 8–10 days, independent from pH control, with a significant reduction
of total organic carbon (TOC) of the solution (Costa et al. 2017).
6.3.3.4 Bioremediation of PAHs
Polycyclic aromatic hydrocarbons (PAHs) are xenobiotic compounds and consist of

a benzene ring arranged linearly, angularly or in clusters (Zeng et al. 2011; Li et al.
2010; Yadav et al. 2018). Rapid industrialization and widespread use of pesticides
for better agricultural output liberates large amount of PAHs, the main pollutant of
soil, air, or aquatic environment. PAHs and their derivatives such as polychlorinated
biphenyls (PCBs); benzene, toluene, ethylbenzene, and xylene (BTEX); polycyclic
aromatic hydrocarbons (PAHs); trinitrotoluene (TNT); pentachlorophenol (PCP);
and 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) are highly toxic for
humans as well as carcinogenic to living beings. PAHs are less soluble in water and
are resistant to biodegradation (Ihssen et al. 2015). Laccase enzyme may convert
polycyclic aromatic hydrocarbons to their quinines and then carbon dioxide.
Laccase converts acenaphthylene to 1, 2- acenapthalenedione and 1,8-napthelic
acid when used along with mediator HBT (Madhavi and Lele 2009). Laccase-
mediated removal of PAHs is an economically feasible, ecofriendly, and efficient
bioremediation process. Polychlorinated biphenyls (PCB) are recalcitrant toxic sub-
stances, presently banned in most countries but used as pesticides and wood preser-
vatives. T. versicolor degraded PCP efficiently after the initial uptake by the mycelia
(Pallerla and Chambers 1998). Laccase-mediated degradation rate of PCBs is
inversely proportional to number of chlorine. The 4–6-chlorine substituted hydroxyl-
PCB is degraded by laccase in the presence of the mediator 2,2,6,6-tetramethylpipe
ridine-N-oxy radical (Keum and Li 2004). Heterologously expressed Trametes san-
guineus laccase in Trichoderma atroviride efficiently removed phenolic compounds
present in industrial wastewater, bisphenol A (an endocrine disruptor) from the
culture medium, benzo[a]pyrene, and phenanthrene (Balcázar-López et al. 2016).
Enzyme immobilized on mesoporous nanofibers that were prepared by Vinyl-

modified poly (acrylic acid)/SiO2 nanofibrous membranes exhibited a better triclo-
san removal (Xu et al. 2014). Laccase enzyme from Trametes versicolor and
Myceliophthora thermophile could degrade the hormones and endocrine disrupting
compounds (EDCs) (Dennis Becker et al. 2017). Phenolic compounds present in
industrial wastewater and bisphenol A (an endocrine disruptor) from the culture
medium was removed effectively by the heterologously expressed Trametes san-
guineus laccase in Trichoderma atroviride (Balcázar-López et al. 2016). Bjerkandera
adusta has the ability to degrade aromatic xenobiotics (Sodaneath et al. 2017) and
extractives (Kinnunen et al. 2017) have raised its biotechnological importance in
wastewater treatments for lignin removal.
The immobilization of Trametes versicolor laccase on carbon-based mesoporous
magnetic composites was done by an adsorbing laccase into bimodal carbon-based
mesoporous magnetic composites. Adsorption effects of the support were respon-
sible for the quick removal rate in the first hour, and up to 78% and 84% of phenol
and p-chlorophenol were removed in the end of the reaction, respectively, indicating
that the magnetic bimodal mesoporous carbon is a promising carrier for both immo-
bilization of laccase and further application in phenol removal (Liu et al. 2012).
6.3.3.5 Biosensor for Detection of Pollutants
Researchers concentrated to device a system for deduction of phenolic compounds

in the environment, food, and biomedical components by a user-friendly and cost-
effective approach. Biosensors are suitable for monitoring contaminated area con-
tinuously with high specificity and sensitivity. Among the various biosensors,
enzymatic biosensor has increased eventually, due to its substrate-specific catalytic
activities. There are more number of biosensors available to detect phenolic com-
pounds, in particular, horseradish peroxidase (Jaafar et al. 2006), and tyrosinase.
However, those enzyme biosensors have some disadvantages due to their lower
structural stability and sensitive to reaction products (Rodríguez-Delgadoa et al.
2015). On the other hand, laccase shows a strong claimant as a biosensor, having
selective advantages over other enzymes including stability, catalytic efficacy (elec-
tron transfer reaction), and oxidized phenol and related compounds in the presence
of O2 without any cofactors (Munteanu et al. 1998).
Laccase can react with wide range of phenolic compounds; therefore, it has been
used in biosensor technology to detect the presence of various phenolic compounds,
oxygen, aromatic amines, morphine, codeine, catecholamines, and plant flavonoids
even at low concentration (Leite et al. 2003; Jarosz-Wilkołazka et al. 2004; Ferry
and Leech 2005). The smaller and more efficient biosensors are developed through
controlled deposition and specific adsorption of laccase on different types of sur-
faces, at the micro and nanometer scale. There are two types of laccase biosensors:
the first type monitors spectrum variation (at an absorbance of 600 nm) of the
enzyme, and the second type monitors voltage changes from a modified oxygen
electrode (Madhavi and Lele 2009). Immobilized alkali-tolerant laccase on nitrocel-
lulose membrane can react with different substrates (syringaldazine, catechol,
catechin, and L- DOPA) at their low concentrations (Singh et al. 2010). Oktem et al.
(2012) immobilized laccase enzyme on Whatman filter paper No. 1 with coloring
agent MBTH (3-methyl-2-benzothiazolinone) that is used for the identification of
oxidation products of phenols by developing maroon-green colors.
6.4 Peroxidase
Peroxidases (EC 1.11.1.7) are glycoproteins with a hematin compound as cofactor.

This heme protein has iron (III) protoporphyrin IX as the prosthetic group. They
catalyze hydrogen peroxide (H2O2)-dependent oxidization of the different organic
and inorganic compounds. Its molecular weights range between 30 and 150 kDa
(Bansal and Kanwar 2013). It is widely distributed in all living organisms like
bacteria, fungi, algae, plants, and animals. Peroxidases have been applied to reduce
pollution in environment. They have the potential to oxidize phenols, cresols, and
chlorinated phenols and synthetic textile azo dyes in water. Phenolic compounds are
degraded by lignin peroxides (LiPs) in the presence of H2O2 (co-substrate) and vera-
tryl alcohol (mediator). In this degradation, H2O2 is reduced to H2O by accepting an
electron from LiP (which can oxidize itself). The oxidized LiP returns to its native
form (reduced) by gaining an electron from veratryl alcohol thereby veratryl
aldehyde is formed. Veratryl aldehyde gets reduced back to veratryl alcohol by
receiving an electron from the substrate (Karigar and Rao 2011).
6.4.1 Peroxidases Classification
Peroxidases are classified into two types based on the presence or absence of heme
group. They are (1) heme peroxidases and (2) non-heme peroxidases (Passardi et al.
2007a, b). Most of the known peroxidase are heme-containing peroxidases (>80%).
Small proportion of the non-heme peroxidases such as thiol peroxidase, alkylhydro-
peroxidase, and NADH peroxidase existed. Heme peroxidases have further been
classified into two superfamilies. They are (i) peroxidase-cyclooxygenase super-
family (PCOXS) and (ii) peroxidase-catalase superfamily (PCATS) (Passardi et al.
2007a, b; Zamocky and Obinger 2010) (Fig. 6.3).
6.4.1.1 Peroxidase-Cyclooxygenase Superfamily (PCOXS)
Animal peroxidases like myeloperoxidase (MPO), eosinophil peroxidase (EPO),

lactoperoxidase (LPO), and thyroid peroxidase (TPO) come under this peroxidase-
cyclooxygenase superfamily. They revealed major role in the innate immunity,
defense responses etc. (Dick et al. 2008; Soderhall 1999). In this superfamily per-
oxidase, the heme (prosthetic) group is covalently joined with the apoprotein
(Pandey et al. 2017).
Fig. 6.3 Classification of peroxidases
6.4.1.2 The Peroxidase–Catalase Superfamily (PCATS)
Non-animal (plant, fungal, and bacterial) heme peroxidases come under this super-
family. At first, based on the sources of peroxidase, this superfamily peroxidase was
called as the plant, fungal and bacterial heme peroxidase. But, the name of this
superfamily was altered as peroxidase–catalase superfamily after identification of
new cnidarians peroxidase. The non-animal peroxidases are further divided into
three classes. They are Class I, II, and III peroxidases (Pandey et al. 2017).
Class-I: They are intracellular peroxidases. It includes cytochrome c peroxidase
(CCP1), ascorbate peroxidases and catalase peroxidase.
Class-II: They are extracellular fungal peroxidases, like the lignin (LiP) and man-
ganese (MnP) peroxidase. Both are secreted by white-rot fungi and involved in
the degradation of lignin. Versatile peroxidases (VP; EC 1.11.1.16) displayed a
hybrid molecular structure between LiPs and MnPs (Pérez-Boada et al. 2005).
This group of peroxidases plays a major role in lignin biodegradation.
Class-III: They are extracellular plant peroxidases. This includes horseradish per-
oxidases (HRP), peanut peroxidase (PNP), soybean peroxidase (SBP), etc. They
play a major role in plant physiological processes such as cell wall metabolism,
lignification, suberization, auxins metabolism, wound healing, etc. Class II and
Class III peroxidases contain a N-terminal signal peptides, disulfide bridges, gly-
cans, and calcium in their structure (Pandey et al. 2017).
6.4.1.3 Fungal Peroxidase (Class II Peroxidase)
6.4.1.3.1 Peroxidase Structure
Fungal peroxidases have a high-spin protoporphyrin IX (heme b) prosthetic group.

It is located in-between the proximal (C-terminal) and distal (N-terminal) domains.
The Fe group of this peroxidase is pentacoordinated form which is associated with
the four pyrrole nitrogens in the imidazole group of the proximal histidine. The
active site containing Fe coordination of peroxidases is highly conserved. At the
active site, distal histidine assisted by an asparagine residue participate transfer of
electrons from H2O to the heme. Redox potentials of the enzyme are determined by
the length of Fe-imidazolic nitrogen (Fe–Ne2) bond. A higher basicity of the imid-
azole group gives a higher redox potential, except few (Choinowski et al. 1999). In
general, the change of basicity is dependent on the electron extraction from the
imidazolic nitrogen to the surrounding (proximal histidine) (Sinclair et al. 1992).
This may differ in peroxidases such as LiP and MnP. Active site residues such as
Ser177 and Asp201 weaken the basicity charges of imidazolic nitrogen bond.
The peroxidases enzymes have four disulfide bonds; it was identified in LiP,
ARP, and T. versicolor peroxidases (Kunishima et al. 1994; Limongi et al. 1995;
Poulos et al. 1993), but MnP have fifth SH linkage extracellular peroxidases con-
taining both N- and O-glycans; however, the glycosylation may be different in
various peroxidases, which determines its isozymes (Kjalke et al. 1992). In addi-
tion, extracellular peroxidases contain two highly conserved, Ca2+-binding sites
which have been located at the proximal and distal domains (Kunishima et al. 1994;
Poulos et al. 1993). Presence of Ca2+-binding sites gives structural stability of the
extracellular form of peroxidases (Banci 1997), and it gives more strength to the
active site. Being extracellular enzymes, fungal peroxidases are synthesized with an
N-terminal signal peptide. The LiP has eight Cys residues, all forming disulfide
bridges. The enzyme molecule consists of eight major and eight minor α-helices
and a limited β structure in the proximal domain.
6.4.1.3.2 Mechanisms of Peroxidase Activity
Peroxidase catalyzes the oxidation of several of organic and inorganic compounds

by using hydrogen peroxide which acts as the electron acceptor. The native form of
enzyme (E) is oxidized to an active intermediate enzymatic form termed compound
I (EI) with concurrent reduction of hydrogen peroxide (H2O2) to water molecule.
Compound I oxidizes a phenol molecule to phenol-free radical and becomes com-
pound II (EII). Compound II oxidizes another one phenol molecule to phenol free
radical and returns to its original state (E) (Fig. 6.4). The formed free radical polym-
erizes and forms insoluble polyaromatic products which are precipitated by solid–
liquid operations (Nicell 1994).
Fig. 6.4 Steps involved in peroxidase catalytic activity
6.4.1.4 Lignin Peroxidase
Lignin peroxidase (EC 1.11.1.14) comes under oxidoreductases family (Higuchi

2004; Martínez et al. 2005; Hammel and Cullen 2008). It was first observed in
the basidiomycete fungi Phanerochaete chrysosporium by Burdsall in 1983 (Glenn
et al. 1983; Tien and Kirk 1988). LiP is an extracellular H2O2-dependent heme pro-
tein (Gold and Alic 1993; Haglund 1999; Piontek et al. 2001; Erden et al. 2009).
LiP enzyme contains 343–345 amino acids preceded by a 27-or 28-residue leader
sequence (Gold and Alic 1993). LiP has less substrate specificity, reacting with dif-
ferent phenolic compounds. LiP is capable of oxidizing a variety of reducing sub-
strates including polymeric substrates. It can oxidize methoxylated aromatic rings
without a free phenolic group and produce cation radicals that undergo ring open-
ing, demethylation, and phenol dimerization (Haglund 1999). LiP needs H2O2 to
initiate the reaction, but not mediators to decompose high redox potential com-
pounds. It is used for various industrial application and bioremediation process
because of their wide substrate specificity and high redox potentials (Erden et al.
2009). Phenolic compounds are degraded by lignin peroxidase (LiP) in the presence
of H2O2 (co-substrate) and veratryl alcohol (mediator). In this degradation, H2O2 is
reduced to H2O by accepting an electron from the LiP (which can oxidize itself).
The oxidized LiP returns to its native form (reduced) by gaining an electron from
veratryl alcohol; thus veratryl aldehyde is formed. Veratryl aldehyde gets reduced
back to veratryl alcohol by accepting an electron from the substrate (Fig. 6.5).
White-rot fungi secreted lignin and manganese peroxidases degrade lignin. Lignin-
degrading peroxidases are identified in a number of basidiomycetous fungi:
Phanerochaete chrysosporium, Trametes versicolor, Pleurotus spp., Phlebia radi-
ata, Coprinus spp., Bjerkandera adusta, Ceriporiopsis subvermispora, Dichomitus
Fig. 6.5 Catalytic cycle of a LiP-mediator oxidation system. VA-OH veratryl alcohol, VA-CHO
veratryl aldehyde
squalens and Arthromyces ramosus, Cylindrobasidium evolvens, and Daedaleopsis

septentrionalis (Kimura et al. 1990; Pelaez et al. 1995; Varela et al. 2000; Kinnunen
et al. 2016).
6.4.1.5 Manganese Peroxidases
Manganese peroxidases (EC 1.11.1.13) also belong to oxidoreductase family (Higuchi

2004; Martínez et al. 2005; Hammel and Cullen 2008). It is a lignin-degrading enzyme
and was discovered in the fungus Phanerochaete chrysosporium following the dis-
covery of LiP (Glenn and Gold 1985). MnP is present in all white-rot fungi than lignin
peroxidase (Hammel and Cullen 2008). MnPs are present mostly in white-rot fungi,
such as Phanerochaete chrysosporium, Ganoderma sp., Pleurotus sp., Trametes sp.,
and Irpex lacteus (Manavalan et al. 2015; Janusz et al. 2013), Phyllosticta, Aspergillus,
Fusarium, and Penicillium (Pant and Adholeya 2007), Hyphodontia sp., Pleurotus
pulmonarius and Trametes ochracea (Kinnunen et al. 2016).
The MnP enzyme is made up of 330–370 amino acids and has a leader peptide
that consists of 21–29 amino acids (Li et al. 1999). It is a glycosylated heme protein;
molecular weight is ranging from 38 to 62.5 kDa, and averaging at 45 kDa
(Hofrichter 2002). Compared to LiP, MnP redox potential is low and oxidizes the
substances with the use H2O2 which act as oxidant. Manganese acts as a mediator in
the MnP catalytic cycle. Manganese peroxidase (MnP) activity involves the oxida-
tion of Mn2+ ions to Mn3+. The Mn3+ is highly reactive and chelated with organic
molecules such as oxalate and malates which are produced by the fungus (Kishi
et al. 1994; Galkin et al. 1998; Mäkelä et al. 2002). Chelated Mn3+ oxidizes phenolic
structures to phenoxyl radicals (Hofrichter 2002).
6.4.1.6 Versatile Peroxidase
Versatile peroxidase (VP) (EC 1.11.1.16) is a heme-containing ligninolytic peroxi-

dase and, as the name suggests, has the catalytic activities of both MnP and LiP and
is able to oxidize Mn2+ similar of MnP and high redox potential non-phenolic
compounds like LiP. It was first identified in the white-rot fungus Pleurotus eryngii
(Martinez et al. 1996). It was first purified from the fungi Bjerkandera (Moreira
et al. 2007) and can transform lignin even in the absence of an external mediator. It
is found in Physisporinus vitreus (Kong et al. 2017), Phlebia radiata, P. pulmo-
narius, and Galerina marginata (Kinnunen et al. 2016). VPs can oxidize wide range
of substrates with low and high redox potentials. Generally, VPs have hybrid molec-
ular structures of LiP and MnP and provide multiple binding sites for the substrates
(Camarero et al. 1999). VPs are superior than other peroxidases, because VPs effi-
ciently oxidize phenolic compounds without the use of veratryl alcohol or Mn(II)
that are needed for LiPs and MnPs activity, respectively (Ruiz-Duenas et al. 2009).
Because of the catalytic versatility, VPs have been involved in the different biotech-
nological applications. VP can oxidize not only Mn (II), but also veratryl alcohol,
phenolic, non-phenolic and high molecular weight compounds, including dyes in
Mn-independent reactions (Asgher et al. 2008; Wong 2009). Like MnP, commercial
applications of VPs are limited, because of their unavailability in large quantities
which can be overcome by the use of DNA recombinant technology (Ruiz-Duenas
et al. 2009).
6.4.2 Applications of Fungal Peroxidases
Ligninolytic extracellular enzymes especially lignin peroxidase and manganese

peroxidase have shown capability toward the degradation of various xenobiotics
including dyes, chlorophenols, polycyclic aromatic hydrocarbons (PAHs), organo-
phosphorus compounds, and phenols (Wesenberg et al. 2003), improve the digest-
ibility of wood or straw for animal feed (Valmaseda et al. 1991), and reduce costs
for the pulp and paper industry (Martinez et al. 1994) (Table 6.4). The other applica-
tions of LiP are delignification of feedstock for ethanol production, textile effluent
treatment and dye decolorization, coal depolymerization, treatment of hyperpig-
mentation, and skin-lightening through melanin oxidation. The lignin peroxidase–
graphite electrode biosensor systems have been established for recognition of
recalcitrant aromatic compounds because of their effective bioelectrocatalysis
(Ferapontova et al. 2006). The applications of peroxidases on various industries are
given below.
6.4.2.1 Textile Industry
Dye is a synthetic colored substance and is used by various industries to color paper,
cotton, polyester, nylon, silk, leather, plastics, hair, etc. to which the dye binds and
becomes an integral part. When unbound synthetic dyes are released into water,
they cause pollution and cause skin allergy, cancer, and chromosomal aberrations
for human beings and also affect plants that reduce photosynthetic activity by
reflecting sunlight and affect germination rate of plants. Fungi can rapidly become
Table 6.4 Applications of LiP and MnP enzymes

Enzyme
Organism type Compound removal References
Phanerochaete LiP Anisyl alcohol (Monomethoxylated Valli et al.
chrysosporium Aromatic Compounds) (1990)
Trametes versicolor MnP Pulp Bleaching (oxidation of phenolic Paice et al.
lignin substructures) (1993)
Phanerochaete MnP & Bentazon (3-isopropyl-1H-2,1,3 Castillo (1997)
chrysosporium LiP benzothiadiazin-4(3H)-one 2,3-dioxide)
and MCPA (4-chloro-2-
methylphenoxyacetic acid)
Phanerochaete LiP Procion Brilliant Blue HGR, Ranocid Fast Verma and
chrysosporium Blue, Acid Red 119, and Navidol Fast Madamwar
Black MSRL (2002)
Fungal strain L-25 MnP Azo, diazo, and anthraquinone dyes Kariminia
et al. (2007)
P. chrysosporium LiP Catechol derivative Cohen et al.
Burds BKM-F-1767 (2009)
Phanerochaete MnP Orange II Sharma et al.
P. chrysosporium LiP & Azo dyes Ghasemi et al.
RP78 MnP (2010)
Anthracophyllum MnP Phenanthrene, anthracene, fluoranthene, Acevedo et al.
discolor pyrene and benzo(a)pyrene (2011)
P. floridensis LiP Coracryl brilliant blue Chander and
Kaur (2015)
Phanerochaete LiP Paper and pulp industry effluent treatment Singh et al.
chrysosporium (Color and lignin removal) (2016)
Phanerochaete MnP Congo Red Bosco et al.
accustomed to varying nutritional sources because they can produce a significant

number of intra- and extracellular enzymes that are needed to degrade several com-
plex organic pollutants such as dye stuffs, polyaromatic compounds, organic waste,
and steroids (Gadd 2001; Humnabadkar et al. 2008). The fungal system can be uti-
lized in the treatment of colored and metallic textile effluents (Ezeronye and
Okerentugba 1999) because they can produce nonspecific enzymes such as lignin
peroxidase (LiP), manganese peroxidase (MnP) and laccase (Christian et al. 2005)
that can mineralize dyes. Direct participation of fungal ligninolytic enzymes is nec-
essary for the mineralization of dyes (Park et al. 2007). Versatile peroxidases (VPs)
have shown effective direct oxidation of high redox potential dyes Reactive Black
5. Reactive Black 5 is oxidized by LiP only in the presence of veratryl alcohol,
redox mediators (Heinfling et al. 1998). It can oxidize phenols, including hydroqui-
nones (Gomez-Toribio et al. 2001). Fungi produce enzymes extracellularly that
confer decolorization ability of dyes. Lignin peroxidase of P. prosopidis degrades
scarlet RR dye (Fernandes et al. 2008). LiP degrades dye by the following steps
initial asymmetric cleavage, demethylation and denitrification and form

N-ethyl-1 l3-chlorinin-2-amine which is further degraded by laccase. Pleurotus
ostreatus can decolorize Remazol Brilliant Blue by producing peroxidase extracel-
lularly (Shin et al. 1997).
Ganoderma lucidum, a white-rot basidiomycete, could be capable of the decol-
orization of four dyes (Drimaren Blue CLBR, Drimaren Yellow X-8GN, Drimaren
Red K-4B and Disperse Navy Blue HGL) and degradation of phenol with the aid of
manganese peroxidase. Manganese peroxidase (MnP) from Ganoderma lucidum
was expressed in Pichia pastoris and recombinant MnP can also degrade four textile
dyes and phenol (Xu et al. 2017). Similarly, Pleurotus species have been reported
for the production of lignin peroxidases, manganese peroxidases, and laccases
enzymes, which play a vital role in the biodegradation and bioremediation (Pandey
et al. 2012) of textile effluents. White-rot fungi, Pleurotus flabellatus, P. ostreatus,
and P. citrinopileatus, are used effectively and efficiently for dye decolorization and
bioremediation of recalcitrant substances (Singh and Srivastava 2016).
MnP of Pleurotus pulmonarius could be able to decolorize the anthraquinonic
dye Remazol Brilliant Blue R and the azo dye Congo Red. The enzyme is strictly
dependent on Mn2+ for oxidizing phenolic and non-phenolic compounds. MnP of
Pleurotus pulmonarius can be used for textile dye effluent treatment (da Silva et al.
2017). Agrawal et al. (2018) reported that Ganoderma lucidum will be an effective
phenanthrene and pyrene degrader by producing ligninolytic enzymes (laccase,
lignin peroxidase, and manganese peroxidase).
6.4.2.2 Paper and Pulp Industry
Humic substances (HS) are formed from microbial breakdown of dead plant matter,
mainly from lignin. HS tend to be polydisperse polymers of aromatic and aliphatic
units that have been synthesized from the polymerization of intermediate lignin
degradation products (Abdel-Hamid et al. 2013), and the polymer is physically and
chemically structurally complex (Niladevi 2009). HS are existing in soil, marine,
and groundwater environments and wastewater from industrial and municipal water
treatment (Abdel-Hamid et al. 2013). In the pulp and paper industry, HS are pro-
duced from the chemical treatment of wood and removed using membrane filters
during wastewater treatment, but they form biopolymer and produce blockage of
filter leads to decrease of filtration flux rates (Sutzkover-Gutman et al. 2010). The
enzymes are applied to remove the HS in ecofriendly method with low cost
(Cavicchioli et al. 2011). Peroxidases catalyze H2O2-dependent oxidation of aro-
matic polymers, including HS, by generating radicals which can break aromatic
rings, ether and carbon–carbon bonds, and by causing demethoxylation (Wong
2009; Abdel-Hamid et al. 2013). Versatile peroxidase oxidizes complex polymeric
humic substances (HS) derived from lignin (humic and fulvic acids) and industrial
wastes (Siddiqui et al. 2014).
6.4.2.3 Bioremediation of Toxic Agrochemicals
The herbicide atrazine was converted to the less toxic compounds desethyl atrazine
and hydroxyatrazine (N-dealkylated and hydroxylated metabolites, respectively),
by the fungus Phanerochaete chrysosporium. Atrazine removal corresponded to the
production of LiP and MnP from the fungus (Mougin et al. 1994). LiP and MnP of
white-rot fungus P. chrysosporium can degrade the herbicide and isoproturon in
in vitro and in vivo conditions (Del Pilar et al. 2001). MnPs from P. chrysosporium
have the ability to break bentazon in the presence of mediators like Mn(II) and
Tween 80. The herbicide glyphosate was oxidized by MnP that is produced by
Nematoloma frowardii (Pizzul et al. 2009). This information evidently indicates the
prospective application of lignin-degrading enzymes in the treatment of herbicides
contaminated soil and water. Polycyclic aromatic hydrocarbons (PAHs) such as
anthracene and pyrene are highly hydrophobic, but they are oxidized by MnP and
LiP of wood rotting fungus Nematoloma frowardii. In the presence of low molecu-
lar mediator substances, the substrate range and the oxidation rate of LiP, MnP is
increased (Günther et al. 1998). When endocrine-disrupting chemicals and trace
organic contaminants like pharmaceuticals and personal care products are released
into water, it leads to bioaccumulation, acute, and chronic toxicity to aquatic living
organisms and also causes severe effect on human health. Podoscypha elegans
degrades lignin and organic pollutant by producing nonspecific extracellular ligni-
nolytic enzymes such as laccase, lignin peroxidase (LiP) and manganese peroxidase
(MnP). It can be used for the removal of pollutants from the environment (Nikki
Agrawal et al. 2017). MnP from Pleurotus ostreatus could detoxify aflatoxin B1
(AFB1) depending on the enzyme concentration and incubation period (Yehia
Ramy Sayed 2014). Non-lignolytic filamentous fungus Penicillium sp. CHY-2 can
degrade different aliphatic and aromatic hydrocarbons. Penicillium sp. CHY-2 effi-
ciently degrades decane than octane, dodecane, ethylbenzene, butylbenzene, naph-
thalene, acenaphthene, and benzo[a]pyrene by producing MnP enzyme. The relative
molecular mass of MnP enzyme from Penicillium sp. CHY-2 is estimated to be
36 kDa, and the native form of MnP is a monomer (Govarthanan et al. 2017).
Fungal laccases and peroxidases are a promising biocatalyst, used as a better alter-
native for conventional chemical processes in the treatment of lignin degradation,
wastewater treatment, decolorization, and detoxification of textile dyes and bio-
sensor preparation to detect the environmental pollutant. Their substrate range is
fairly wide and immobilization technology increases enzyme stability and to
achieve its reuse.
Acknowledgments The authors acknowledge to DST-PURSE Phase-II for providing computer

facilities to prepare this review. There are no conflicts of interest.
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Chapter 7
Fungal Enzymes for Bioremediation
of Contaminated Soil
Prem Chandra and Enespa
7.1 Introduction
Fungi are varying in size and shape and are known as eukaryotes. Individual cells of
fungi vary in its size such as yeasts. The chains of elongated cells stretch for miles
(Alberts et al. 2013). The saprophytic fungi grow on dead organic matter, whereas
others are parasites. These are absorptive heterotrophs that secrete digestive enzymes
and break down the organic matters into substrates, and then absorb easily available
molecules of organic matters (Sankaran et al. 2010). The fungal hyphae have large
surface areas and small volumes which improve the absorptive capability of the
fungi (Wright and Upadhyaya 1998). The bioremediation (mycoremediation) has
been progressively renowned by the potential of fungi (Igiehon and Babalola 2017).
Other explanation also describes why the fungi are perfect organisms for the myco-
remediation (bioremediation), and why they may be superior to other microorgan-
isms such as bacteria, algae, and plants under various situations (Harms et al. 2011).
The mycelial network of fungal organizations has capability to penetrate into soils
and contact spaces of soil pores (Boswell et al. 2007). The fungal mycelium can
performance as a component, and mature surrounding the hurdles, recycled dead
hyphae and reallocate properties and development to resource-rich zones of the soil.
In comparison to various other eukaryotes, the cells of fungal hyphae display the
indeterminate growth, so the division of cells takes place continuously in the hyphae
of a mycelium, providing the mineral components accessible in the substrate (Miller
and Fitzsimons 2011). The fungal mycelium in drier soils or soils holding the
P. Chandra (*)
Department of Environmental Microbiology, School for Environmental Sciences, Babasaheb
Bhimrao Ambedkar (A Central) University, Lucknow, Uttar Pradesh, India
Enespa
Department of Plant Pathology, School of Agriculture, MPDC, University of Lucknow,

190 P. Chandra and Enespa
nutrient patches locally may be benefitted. This is because their mode of growth
permits them to associate with soil openings for their growth (Tsing 2012). The
fungi have more potential to tolerate high concentrations of toxins compared to
bacteria (Gonzalez-Chavez et al. 2004).
Furthermore, several secondary metabolites are produced by the fungi for the
bioremediation of polluted soils, and the enzymes secreted are particularly signifi-
cant, and several of them lack substrate specificity and they are released as exoen-
zymes into the substrate (Cohen and Hadar 2001). Generally, the fungi depend on
the degradation of macromolecules extracellularly, and a large quantity of enzymes
is produced and secreted into the substrate (Girish and Kemparaju 2007). In the last
years, emergent pollutants are of great interest (Bosco and Mollea 2019). Among
them, endocrine-disrupting chemicals (EDCs) and pharmaceutical personal care
products (PPCPs) generated from human being chemicals are significant due to
their biological effects on nontarget organisms; particularly, the endogenous hor-
monal effects, antagonized or simulated to EDCs, are toxic at very low concentra-
tions to the organisms (Duarte et al. 2018). Bisphenol A, Estrone, 17 β-estradiol, 17
α-ethinylestradiol, and triclosan are detected in soil and studied mostly. Due to the
irrigation of contaminated wastewater, EDCs and PPCPs enter mainly into the soil
ecosystem (Dodgen et al. 2014; Ying and Kookana 2005; Chen et al. 2010). The
ligninolytic fungi have the capability to convert EDCs, permitting a decrease in
their ecotoxicity or the endocrine-disrupting activity; furthermore, the capability of
degradation by the heterogeneous class of PPCPs is also reported to occur broadly
by unspecific enzymatic systems (Cajthaml 2015; Rodarte-Morales et al. 2011;
Yadav et al. 2018).
7.2 Fungal Diversity
In any geophysical area, the variation in living organisms taken together abundantly
is known as biodiversity (Sogin et al. 2006; Rana et al. 2019b). However, most of the
fungi are not observable by the naked eye and must be considered under a micro-
scope. The cultivable fungi can be recognized due to the sporulation (Tibbett and
Carter 2008; Yadav et al. 2017). The fungi are superior to bacteria, viruses, and other
smaller forms of life such as viroids (Parlevliet 2002). Approximately, 72,000 named
species of fungi are recognized, and some new species are being added at the rate of
about 700 to 1500 each year (Tortella et al. 2005; Yadav et al. 2019a, b). About
63,500 species of fungi have been labeled, in which 13,500 are found to be associ-
ated with algae as lichens. Sometimes, these organisms are termed the lower fungi
such as Zygomycetes and Chytridiomycetes or higher fungi such as Ascomycetes
and Basidiomycets (Blackwell 2011). They are classified as enzymatic machinery.
The name white rot is given for rot fungi that decay the lignin and brown rot for those
that decay only the cellulose. This difference is reflected in the overall appearance of
the rotten wood (Rytioja et al. 2014). The brown rot fungi are known as soft rot fungi,
which decompose only the cellulose, but they attack damper wood, and decay
7 Fungal Enzymes for Bioremediation of Contaminated Soil 191
ordinarily occurs only near the wood surface (Enespa and Chandra 2017). Currently,
the fungal potential for bioremediation (mycoremediation) has been gradually
renowned. The bioremediation of hydrocarbons is based on biodegradation, and so it
is involved in a partial or a complete breakdown of the relevant pollutants (Alvarez
et al. 2017; Rastegari et al. 2019). Fundamentally, this mechanism is different from
bioaccumulation which designates the capability of some fungi to accumulate or
hyperaccumulate various metals inside the cell from the soil (Alvarez et al. 2017).
7.3 Fungal Enzymes
Generally, based on catalytic reaction activity, these enzymes are divided into six
classes: oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), lyases (EC
4), isomerases (EC 5), and ligases (EC 6) as expressed in (Table 7.1) (Alexander
et al. 2017; Kour et al. 2019b; Rana et al. 2019a; Suman et al. 2016; Yadav 2019).
The fungal extracellular enzymes mostly belong to two classes of enzymes. They
are oxidoreductases and hydrolases. The transfers of hydrogen or oxygen atoms or
Table 7.1 Classification of enzymes and types of reactions

Classification of
enzymes and their
number Reaction profile References
Oxidoreductases The transfer of electrons from one molecule to another Pearce et al.
(EC1) involved in oxidation reactions. We usually see the removal (2003)
of hydrogen from the substrate in biological systems.
Dehydrogenase is the typical enzyme in this class such as,
alcohol dehydrogenase. R–CH2OH + A R–CHO + H2A
(where, A is an acceptor molecule). If A is oxygen, the
relevant enzymes are called oxidases or laccases; if A is
hydrogen peroxide, than they are known as peroxidases
Transferases (EC2) The enzymes catalyze the transfer of groups of atoms from Clodoveo
one molecule to another molecule. The transfer of an amino et al. (2014)
group from an amino acid to an alpha-oxoacid is promoted
by aminotransferases or transaminases
Hydrolases (EC3) The cleavage of peptide bonds in proteins, glycosidic bonds Goodman
in carbohydrates, and ester bonds in lipids by hydrolases. (2010)
Generally, the hydrolases breakdown the larger molecules to
smaller fragments
Lyases (EC4) The formation of double bonds through the removal of Munoz-
groups by the catalyzation of lyases. The splitting of the Munoz et al.
glycosidic linkages by beta-elimination in pectate lyases (2017)
Isomerases (EC5) Isomerases catalyze the transfer of groups from one position Wu et al.
to another in the same molecule. Catalyze isomerization (2011)
Ligases (EC6) Ligase enzymes participate in biosynthetic reactions where Nelson et al.
new groups of bond are formed by the joining of molecules (2008)
together with covalent bonds. These reactions require the
energy input in the form of cofactors such as ATP
electrons from one substrate to another are oxidation and reduction reactions,
known as oxidoreduction reactions and are catalyzed by oxidoreductases catalase
(Lucas et al. 2008). The cleavages of chemical bonds by the addition of water are
hydrolysis reactions and are catalyzed by hydrolases catalase. In the growing phase
of fungi, various extracellular of enzymes are produced, and they degrade various
polymers (Leonowicz, et al. 2001). In the lignin degradation, the oxidoreductases
enzymes are involved, since lignin cannot be utilized as a source of energy or car-
bon by the fungi. The cellulose and hemicellulose enzymes have the capability for
growth after degradation of complex lignocellulose structure in the plant cell wall
(Kirk and Farrell 1987). The hydrolases catalyze lignin degradation and further
break down to cellulose and hemicellulose (Langston et al. 2011).
7.3.1 Oxidoreductases (EC 1)
Lignin is the most complex natural polymer formed from the random polymeriza-
tion of phenyl propanoid units. Lignin-modifying enzymes (LMEs) are known as
extracellular nonspecific oxidative enzymes, which have the capability of degrada-
tion to lignin by radical reactions (Joy et al. 2015). Four copper ions per molecule
are found in laccases known as phenol oxidases. The oxygen is reduced to water due
to catalysis by laccases and supplemented by the oxidation of various phenols
(Galhaup and Haltrich 2001). A low redox potential has been seen in the laccases,
but other organic compounds are also oxidized in the presence of redox mediators,
such as 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and
1-hydroxybenzotriazole (HBT) (Camarero et al. 2007). The peroxidase enzymes are
MnPs, LiPs, and VPs, which have heme protein (protoporphyrin IX containing Fe2+-
ion as a central atom) as their prosthetic group (Table 7.2). The Mn2+ ions are oxi-
dized to Mn3+ ions which are catalyzed by MnPs, and stabilized by chelation with
the organic acids. The one-electron oxidation of both phenolic and non-phenolic
aromatic compounds is catalyzed by LiPs (Ye et al. 2010). VPs have both catalytic
activities of MnP and LiP, and are known as hybrid peroxidases. The non-ligninolytic
heme-containing peroxidases are also produced by the Coprinopsis cinerea, which
are dye-decolorizing peroxidases (DyP) and peroxidase (CiP). The smaller dye
molecules and phenols are oxidized by the CiP, whereas the DyP also have the abil-
ity of oxidizing complex dye molecules (Liers et al. 2013). Moreover, the lignin
degradation is also shown by intracellular cytochrome P450 monooxygenases. The
lipophilic compounds are also catalyzed by the monooxygenation of P450 enzymes,
and they may degraded to lignin and other organic compounds together with peroxi-
dases (Wittich 1998). The catalytic similarities have been shown in extracellular
aromatic peroxygenases (APO) and intracellular P450s. In Agrocybe aegerita and
Coprinellus radians, the APOs activities have been described (Pecyna et al. 2009).
LMEs used in several applications are potential industrial enzymes used in process-
ing industries of paper and pulp, for example, in the functionalization of lignocel-
lulosic materials, biobleaching, biopulping, pitch removal, and alteration of wood
Table 7.2 Extracellular oxidative enzymes of fungi and their environmental applications
Enzyme
activity Reaction mechanism Occurrence in fungi References
Laccase O2-dependent one-electron Basidiomycota and Ray et al. (2014)
oxidation of various phenols. Ascomycota, in most
Extended substrate range in the WRF and LDF
presence of redox mediators.
Mostly acidic and rarely in
neutral pH range
Manganese H2O2-dependent one-electron Basidiomycota, Zhang et al.
peroxidase oxidation of Mn2+ to Mn3+, common in WRF and (2014)
(MnP) chelated Mn3+ oxidizes phenolic LDF
compounds, acidic pH range
Dye- H2O2-dependent one-electron Basidiomycota and Merckx (2013),
decolorizing oxidation of organic compounds, Ascomycota Salvachúa et al.
peroxidase decolorizes Reactive Blue 5, (2013)
(DyP) additional hydrolyzing activity,
acidic pH range, also very low
pH
Coprinopsis H2O2-dependent one-electron Basidiomycota, only in Floudas et al.
cinerea oxidation of various phenols, C. cinerea (2012), Millati
peroxidase acidic and alkaline pH range et al. (2011)
Versatile Reaction mechanism of both Basidiomycota, only in Datta et al. 2017,
peroxidase MnP and LiP, acidic pH range Pleurotus sp., Kinnunen et al.
(VP) Bjerkandera sp. and (2017), Pollegioni
Trametes versicolor et al. (2015)
fibers (Ferhan 2016), improving the contaminated soil from effluents and its biore-
mediation by enhancing the enzymatic hydrolysis of lignocellulosic substrates
(Pérez et al. 2002). The laccases and MnPs are the strategic fungal enzymes for the
bioremediation (Reddy 1995). All white rot fungi produce laccase and MnP; they
are more widespread among LiP produced by WRF (Christian et al. 2005).
Particularly, the activity of LiP has been identified only in a few experiments from
the soil. Moreover, Pleurotus sp., Bjerkandera sp., and Trametes versicolor pro-
duced only versatile peroxidase (VP) (Hofrichter 2002).
7.3.2 Hydrolases (EC 3)
For the degradation of cellulose and hemicellulose from wood or plant litters, the
fungi utilize various extracellular hydrolytic enzymes. The chains of single cellulose
are connected together with the bonds of hydrogen to form microfibrils (Lynd et al.
2002). Three different types of cellulases are required for the degradation of cellulose
namely endoglucanase (EC 3.2.1.4), exoglucanase (cellobiohydrolase, EC 3.2.1.91),
and β-glucosidase (EC 3.2.1.21), and they act synergistically (Schwarz 2001).
Endoglucanases hydrolyze β-1, 4-glucosidic linkages in noncrystalline sections of
the cellulose chain internally, which results in a decrease in the chain length and an
increase in the number of free ends. Cellulose is degraded by exoglucanases starting

from the free ends and splitting off cellobiose, a disaccharide formed of two glucose
units (Eriksson et al. 1990). Lastly, the cellobiose hydrolyzes to glucose by the
β-glucosidase. In recent times, cellulose-breaking accessory enzyme has been dis-
covered (Várnai et al. 2010). This enzyme catalyzes the oxidative cleavage of crystal-
line cellulose and increases its accessibility to classical hydrolytic enzymes
(cellulases) catalyzed by the lytic polysaccharide monooxygenase (also known as
GH61 enzyme) (Quinlan, et al. 2011). Branched heteropolysaccharides composed of
various hexoses (D-glucose, D-mannose, D-galactose), pentoses (D-xylose,
L-arabinose, D-arabinose), deoxyhexoses (Lrhamnose, L-fucose), and uronic acids
(4-O-methyl-D-glucuronic acid, D-galacturonic acid, D-glucuronic acid) are hemi-
celluloses. The hemicelluloses composition in softwood, hardwood, and annual
plants varies with different plants (Oliveira et al. 2007). Several enzymes are required
for the breakdown of hemicelluloses due to its various compositions (Pérez et al.
2002). The xylan (in hardwood) and glucomannan (in softwood) are the two most
common hemicelluloses. The chains of 1, 4-linked β-D-xylopyranose units are situ-
ated in xylan and have homopolymeric backbone (Zhou et al. 2017). Xylan contains
on average one glucuronic acid besides xylose and the side group is attached to every
tenth xylopyranose unit. β-D--mannopyranose and β-D-glucopyranose units are
found in the backbone and β-D--galactopyranose units as side groups in glucoman-
nan, which is a branched heteropolysaccharide (Alamgir 2018). In the soft wood
(glucomannan), the ratio of galactose: glucose: mannose is 1:2:7 (Lechat et al. 2000).
7.4 Environmental Contamination
7.4.1 Common Source of Pollutants in Soil
In soils, the persistent toxic compounds, chemicals, salts, radioactive materials, or

disease-causing agents and heavy metals are present due to the industrial move-
ments and pollution (Förstner and Wittmann 2012) and have adversarial effects on
the growth of plants and animal health. Soil can become polluted by the various
routes, such as seepage from a landfill, industrial waste discharge into the soil, con-
taminated water that percolated into the soil, rupturing of underground storage
tanks, overuse of pesticides, herbicides, or fertilizers. (Quint 1998).
7.4.2 Contaminated Land and Its Issues
The removal of pollutants from the soil and water by the fungal microorganisms is
known as bioremediation. There are two methods, namely, in situ and ex situ, to remove
pollutants from various sites. If the contaminated material is treated onsite, the method
is known as in situ method, and if the treatment of contaminated material is done
elsewhere, the method is known as ex situ method (Mueller et al. 1989). The bioreme-
diation methods are known to degrade toxic organic materials, for example, from oil
spills, pesticides, and industrial waste and heavy metals at the molecular level and
convert them into less toxic compounds (Boopathy 2000). The bioremediation is the
full mineralization of contaminants, that is, their transformation to CO2, H2O, N2, HCl,
by the various fungal strains (Rhodes 2014). The heavy metals and radioactive cations,
of course, cannot be decayed, but can be reduced into forms of low solubility, for
example, by a change in the oxidation state, such as U (IV) (in UO2), so that it may be
less harmful in the soil or might be physically unconcerned by mycoremediation,
which involves the harvesting of plant or fungus (Gavrilescu et al. 2009; Glasser 2001).
7.4.3 Heavy Metal Contamination in Soil
The elements with metallic properties and have atomic number > 20 are known as
heavy metals. Certainly, the metal conversion is the normal mechanism in the soil.
However, the high concentrations of heavy metals can be toxic for the living cells
such as plants, animal, and microbes (Lasat 1999). The most common heavy metal
pollutants in the environment are As, Sr, Cs, U, Cd, Cr, Cu, Hg, Pb, and Zn. Some
of these metals are micronutrients and are necessary for the growth and develop-
ment of plants, for example, Zn, Cu, Mn, Ni, and Co, while others have unknown
biological function, such as Cd, Pb, and Hg (Alloway 2013). Heavy metals enter
into the environment or soil by both natural and anthropogenic sources (Duruibe
et al. 2007). The weathering minerals, erosion of rock, and volcanic activity are the
most significant natural sources, and the mining, smelting, electroplating, use of
pesticides and fertilizers, as well as biosolids in agriculture, sludge dumping, indus-
trial discharge, atmospheric deposition are the anthropogenic sources (Ali et al.
2013; Barsainya et al. 2016). The anthropogenic and manmade sources of several
heavy metals in the environment or soil are presented in Table 7.3.
7.5 Bioremediation of Heavy Metals by Fungi
Various species of filamentous fungi such as Trichoderma sp., Penicillium sp.,

Aspergillus sp., and Mucor sp., have the capability to tolerate and remediate heavy
metals from the soil by the various activities (Deng et al. 2011). Due to the presence
of negative charge on the various functional groups, the fungal cell walls have excel-
lent metal-binding properties such as carboxylic, amine or sulfhydryl, and phos-
phate in several components of the cell wall (Wang and Chen 2009). The most
prominent soil microorganism is arbuscular mycorrhizal fungus (AMF). It is estab-
lished by the physical link directly between the soil and plant roots which increases
the surface area of the root surface and smoothing nutrient absorption by the plants
(Chapin 1980). In the alleviating of metal toxicity to the host plant, the AM fungi
Table 7.3 Anthropogenic and natural sources of heavy metals in the soil
Heavy Effects on
metals Sources microorganisms Effects on human being References
Zinc (Zn) Mining, oil Inhibits growth, Kidney and liver failure, Karwade
refinery, death, decrease in lethargy, macular et al. (2018)
plumbing, Brass biomass degeneration, metal fume
manufacturing fever, prostate cancer,
seizures, vomiting Ataxia,
depression, gastrointestinal
irritation, hematuria,
icterus, impotence
Lead (Pb) Battery Inhibit enzyme Learning deficits, reduced Shawai
manufacture, activitiesand fertility, renal system et al. (2017)
herbicides and transcription, and damage, risk factor for
insecticides, aerial denatures nucleic Alzheimer’s disease,
emission from the acid shortened attention span,
combustion of anorexia, chronic
lead petrol nephropathy, damage to
neurons, high blood
pressure, hyperactivity,
insomnia
Chromium Fly ash, paints Growth inhibition, Irritation of the skin, Dhal et al.
(Cr) and pigments, inhibition of itching of respiratory tract, (2013)
tannery industry, oxygen uptake, liver diseases, lung cancer, Chandra
steel industries elongation of lag nausea, renal failure, and Singh
phase reproductive toxicity, (2014)
vomiting
bronchopneumonia,
chronic bronchitis,
diarrhea, emphysema,
headache
Mercury Release from Disrupt cell Dizziness, dysphasia, Ali et al.
(Hg) Au–Ag mining membrane, inhibits gastrointestinal irritation, (2013)
and coal enzyme function, gingivitis, kidney problem,
combustion, decrease population loss of memory, pulmonary
medical waste, size, denature edema, reduced immunity,
Power plants protein sclerosis ataxia, attention
emissions deficit, blindness, deafness,
decrease rate of fertility,
dementia
Nickel Nonferrous metal, Inhibit enzyme Chest pain, dermatitis, Bhatnagar
(Ni) paints, porcelain activities, oxidative dizziness, dry cough and and
enameling, stress, disrupt cell shortness of breath, Minocha
electroplating, membrane headache, kidney diseases, (2010)
lung and nasal cancer,
nausea, cardiovascular
diseases
(continued)

Heavy Effects on
metals Sources microorganisms Effects on human being References
Arsenic Atmospheric Enzyme Cardiovascular and Matschullat
(As) deposition, deactivations respiratory disorder, (2000)
mining, conjunctivitis, dermatitis,
pesticides, rock skin cancer and brain
sedimentation, damage
smelting,
Pesticides and
wood
preservatives
Cadmium Electroplating of Transcription, Itai-Itai, kidney diseases, Fleischer
(Cd) cadmium inhibits carbon and lung and prostate cancer, et al. (1974)
containing nitrogen lymphocytosis, microcytic
plastics, mineralization, hypochromic anemia,
phosphate damage nucleic testicular atrophy,
fertilizer, paints acid, denature vomiting, bone disease,
and pigments, protein, inhibit cell coughing, emphysema,
plastic stabilizers division headache, hypertension
Cooper Pesticides and Inhibits enzyme Liver and kidney damage, Branham
(Cu) fertilizers activities, Disrupt metabolic disorders, et al. (1995)
cellular function nausea, vomiting
Abdominal pain, anemia,
diarrhea, headache
are also involved. The arbuscular mycorrhizae showed specific role in the host plant
on exposure to heavy metal and depend on various environmental factors, such as
the ecotypes and the plant species, the fungal species and the ecotype, the availabil-
ity of metal, edaphic conditions of soil, such as the fertility of soil, and the growth
conditions of plants like intensity of light or density of root (Khan 2005; Hall 2002).
Likewise to PGPR, numerous mechanisms have been assumed for the direction of
toxic metal and the allocation in plant roots in the presence of AMF, including the
binding of heavy metals to the cell wall and their deposition in the vacuoles of AMF
(Chandra and Enespa 2016), the sequestration of metals with the help of sidero-
phores in the soil or into the root apoplasm, the binding of metals to metallothio-
neins or phytochelatins inside the fungal and plant cells, and the transporters of
metals at the tonoplast of both plants and fungi catalyze the transport of metals from
the cytoplasm (Neagoe et al. 2013; Jan and Parray 2016).
7.5.1 Heavy Metals and Enzyme Regulation
For the utilization of complex nutrients, the saprotrophic basidiomycetes utilized a

variety of extracellular enzymes such as ligninolytic enzyme (Kaur 2016). The con-
trolling factors for the production enzyme among white rot fungi have been widely
researched (Sanchez and Cardona 2008). The nutrients availability, inhibitory
compounds, temperature, pH, and interrelationships with other fungi are the main
factors which influence the production of enzymes (Mata et al. 2010). The lignino-
lytic and cellulolytic extracellular enzymes are controlled at the transcription level
by the heavy metals and during the course of their action (Lee et al. 2012). The
energy flux in the ecosystem influences the effect of heavy metals on the enzyme
activities. A positive regulation of laccase and isoenzymes on the copper application
has been reported in P. ostreatus study (Baldrian 2003). With increasing Cd concen-
tration, the Mn-peroxidase activity decreased, and the activities highly increased of
endo-1, 4-l-glucanase, 1, 4-l-glucosidase and laccase in the presence of metal
(Kapahi and Sachdeva 2017). The P. sajor-caju laccase isozyme genes (phenol oxi-
dase A1b (POXA1b), POXA2, and POXC) that are regulated at the transcriptional
level in response to copper and manganese have been reported. The energy flux in
the ecosystem influences the effect of heavy metals on the enzymes activities
(Goudopoulou et al. 2010). A positive regulation of laccase and isoenzymes on the
copper application has been reported in P. ostreatus study (Palmieri et al. 2003).
With increasing Cd concentration, the Mn-peroxidase activity decreased, and the
activities highly increased of endo-1, 4-l-glucanase, 1, 4-l-glucosidase and laccase
in the presence of metal (Kapahi and Sachdeva 2017). The P. sajor-caju laccase
isozyme genes (phenol oxidase A1b (POXA1b), POXA2, and POXC) that are regu-
lated at the transcriptional level in response to copper and manganese have been
reported (Goudopoulou et al. 2010). It has also been reported that the activity of
laccase decreases immediately and reduces the stability of the enzyme after the
addition of Hg. Stimulatingly, Cu and Hg increased the activity of MnP slightly
(Zeng et al. 2012). At low concentrations of Cd, Cu, and Hg, the activity of MnP
decreased when it is incubated in the presence of all three metals, due to the syner-
getic effects of the heavy metals (Aragay et al. 2011; Dwivedi and Enespa 2014).
And Mn has also been found to affect MnP gene transcription, and the activity of
enzyme in a positive way in some Pleurotus spp. has been reported (Cohen et al.
2002).
The progeny of Pleurotus eryngii was incubated in the Zn-, Cu-, Co-, Cd-, and
Ni-enriched substrate and assessed for the effect on morphology and physiology.
During the incubation stage in Ni and Cu, the concentration of laccase activity
decreased, and completely inhibited during the fruiting stage (Baldrian 2003). The
effects of inhibition were more noticeable when multi-metal solution exposure
takes place. For the consideration of a fungal species as a biosorbent, the desorption
of the adsorbed metal ions and their successive reuse and the productivity of the
biomass in biosorption need to be taken into account (Volesky and Holan 1995).
The desorption in the acidic solution has been described to be more effective than
that in the alkaline desorption solution. Under acidic conditions, the protons partici-
pate for the sites releasing metal ions in the medium (Mohan and Pittman 2007). A
97% desorption of the adsorbed Hg from the immobilized and heat-treated P. sajor-
caju resulted when eluted with HCl. P. ostreatus, using HCl for a contact period of
1 h only showed that 99% Pb could be desorbed (Akar et al. 2008). P. florida could
be regenerated and reused for the biosorption of Pb for six times using biomass. The
59% generation rate of Cu has been reported for P. mutilus (Kapahi and Sachdeva
2017). However, they can be upgraded by involving chemical desorption technique

with the recovery of copper; the capacity of regenerated biomass for 10 g/l content
has a maximum adsorption capacity that is smaller, but still significant 59.75 mg/g
(Malyarenko et al. 2005).
7.5.2 Mechanisms of Mycoremediation
The fungi are very resourceful and have a wide range of adaptability and quick
responsiveness to stress condition, environmental adversities, and exciting climatic
conditions. The reduction of complex hydrocarbons and the chains of hazardous
substances into simpler, nontoxic, biodegradable form to tidying the environment
can be achieved with the help of fungal strain (Dubos 1987). Various fungi also have
excellent capability to binding with metallic ions, which comprise the efflux of
metal ions outside the cell and buildup, and construct the metal ion complex inside
the cell; later, they reduce the toxic metal ions to a nontoxic state (Hajipour et al.
2012). Various mechanisms have progressed by which they can immobilize, mobi-
lize, or renovate the metals and make them inactive or tolerate the uptake of heavy
metal ions (Fig. 7.1) (Prado et al. 2015). The mycoremediation mechanism adopted
by fungi includes
Exclusion: Due to the formation of a permeable barrier, the metal ions are kept away
from the target sites.
Extrusion: By the active transport, the metal is pushed out of cells.
Fig. 7.1 Bioremediation mechanism of heavy metals by fungi (modified and adopted from
Chandra and Enespa 2019)
Fixation: By the formation of a complex with metal-binding proteins or other cell

components like enzymatic detoxification, intra- and extracellular sequestration,
dissolution of metal by acid production, chelation, and precipitation through the
production of organic bases, extracellular metal precipitation fixes the metals.
Biotransformation: The toxic metals are reduced to less toxic forms like methyla-
tion, demethylation, volatilization, oxidation, and reduction. Generally, the
immobilization, mobilization, biosorption, and biotransformation are considered
main approaches used for mycoremediation of hazarders in the agro ecosystem
in order to avail good air and water quality for future generations (Sardrood et al.
2013).
7.6 iodegradation and Bioremediation of Pesticides

B
by Fungi
7.6.1 Organic Contaminants in the Soil
The hazardous organic compounds are released by the industry and other sources to
soil in the form of wastewater and the solid waste (Wuana and Okieimen 2011). This
hazardous substance may be heavy metal, pesticides, and herbicides and they can accu-
mulate in other organisms through the food chain and have the capability of long-range
transport and are classified as Persistent Organic Pollutants (POPs) by the Stockholm
Convention, 2001 (World Health Organization 2010). After its 5th meeting, there are
22 organic compounds that are currently classified as POPs; some are directly pro-
duced by the chemicals and some are generated as a form of by-products of reactions
(Schwarzenbach and Gschwend 2016). The by-products formed in the industrial pro-
cesses are polychlorinated biphenyls (PCB), polychlorinated dibenzo-p-dioxins
(PCDD), polychlorinated dibenzofurans (PCDF), hexachlorobenzene (HCB), carcino-
genic polyaromatic hydrocarbons (PAHs), and certain brominated flame-retardants
(Roots 2004). The POPs include some pesticides also, such as dichlorodiphenyltrichlo-
roethane (DDT), and some organometallic compounds, for example, tributyltin (TBT).
In transformers and capacitors, the PCBs were widely used as coolants and insulating
fluids (Darbre 2015). PCBs are common contaminants in many former industrial soils
due to their widespread use. PCBs are problematic to be degraded by bacteria because
due to the formation of toxic chlorobenzoic acids (CBAs), the PCBs are not easily
degraded by bacteria which have a tendency to accumulate in soils polluted with PCBs
(Mikszewski 2004). However, the ligninolytic fungi have capability to further reduce
CBAs while instantaneously transforming PCBs (Peu 2014). So, the “emerging con-
taminants” increased in the environments (Petrie et al. 2015).
Previously, these chemicals were not detected in the natural water, but discovered
currently such as plasticizers (also known as Bisphenol A (BPA), flame-retardants,
pesticides, pharmaceuticals products such as antibiotics and other personal care
products (Loraine and Pettigrove 2006). They naturally disrupt the endocrine system
or cause other long-term effects to human being or other living organisms in very low
concentrations (Colborn et al. 1993). For instance, Bisphenol A has estrogenic activ-
ity even at pico- to nano-molar concentrations (Wetherill et al. 2007). BPA is known
not for the estrogenic activity since 1993, but it is still used to create polycarbonate
plastics and epoxy resins, and reactions can be found in many other consumer prod-
ucts such as water bottles and coatings inside food and beverage cans (Vandenberg
et al. 2007). During washing with hot water or in alkaline or acidic conditions, BPA
leaches out from these materials (Howdeshell et al. 2003). Furthermore, BPA is not
completely degraded in the treatment of wastewater, and thus BPA can spread into
the aquatic environment through the wastewater effluent. BPA and other emergent
contaminants could be degraded in municipal wastewaters with the help of LMEs
(Tijani et al. 2013). In the degradation of BPA with the help of MnP, it was observed
that the treatment after 90 min. with MnP resulted in 100% reduction of estrogenic
activity and 98% degradation of BPA (Kabiersch et al. 2011).
In the arctic environment where the natural reduction is extremely slow, the long-
range transport is the most important source for pollutants. The sources of long-range
transport are previously used chemicals and already-existing environmental pollution
due to their long life spans after the restriction and the production of many POPs
(Wania 2003). So, it is necessary to treat the soils affected with POPs with special
treatment techniques to remediate the contaminants. The fungal enzymes can provide
new approaches to treat polluted soils or wastewaters (Gomes et al. 2013). There is a
potential to use fungal mycelia to treat POP soils, including PCB, PAH-, and PCDD/
F-contaminated soils, and fungal enzymes to clean up effluents, to degrade emerging
contaminants in municipal wastewaters, synthetic dyes in textile industry wastewa-
ters, and lignin in pulp mill wastewaters (Anasonye et al. 2014; Anasonye et al. 2015).
7.6.2 Bioremediation of Pesticides by Fungal Enzymes
Based on their morphological, physiological, and genetic features, the fungi are
considered unique organisms. They are able to colonize in natural environments
(soil, air, water) and are known as omnipresent, and they maintain the ecosystem’s
equilibrium (Barea et al. 2005). Fungi develop their unique bioremediation proper-
ties due to adaptation to their environments. Based on laboratory studies, it is known
that all the natural organic compounds can be degraded by various fungal species
due to the production of enzymes such as amylases, lipases, and proteases and that
allow them to use substrates as fats, proteins, and starches (Shah et al. 2008). The
pectin, cellulose, and hemicelluloses can be used by some other species as a carbon
source. The natural complex polymers are degraded by some main fungal strains
and may be resistant to microbial attack, such as keratin, chitin, and lignin (Walker
and White 2011). Generally, on the basis of reactions, the enzymes are divided into
six classes such as catalyze: oxidoreductases (EC 1), transferases (EC 2), hydro-
lases (EC 3), lyases (EC 4), isomerases (EC 5), and ligases (EC 6) (Andreini et al.
2008). Mostly, the extracellular fungal enzymes are categorized into two enzyme
classes: oxidoreductases and hydrolases. The oxidation or reduction reactions, that
is, transfer of hydrogen or oxygen atoms or electrons from one substrate to another
are carried out by oxidoreductases (Madhavi and Lele 2009). The cleavage of chem-
ical bonds by the addition of water is hydrolysis reaction that is done by the hydro-
lases. In their growth environment, extracellular enzymes are produced by the fungi
to degrade various polymers (Lucas et al. 2008). In lignin degradation, oxidoreduc-
tases enzymes are involved. The lignin cannot be used as a source of energy or
carbon by the fungi. The complex lignocellulose structure of the plant cell wall has
capability to use cellulose and hemicellulose for the growth. The breakdown of cel-
lulose and hemicellulose is catalyzed with hydrolases (Pérez et al. 2002).
7.6.3 Biodegradation of PAHs
The PAHs are also formed due to the incomplete combustion of carbon-containing
fuels, including fossil fuels, wood, or other biomass, and are also present in crude
oil and coal (Huber et al. 2006). The PAH contamination can be either long-winded
contamination in urban surface soils, which receive a continuous input of pyrogenic
PAHs from the emissions to air, or the contamination from a point source like gas-
works soil or oil-polluted soil. In sawmill soil, PAH contaminations originate from
coal-tar creosote, which completely consists of PAHs, and is commonly used to
preserve wood and in the making of waterproof crossties and power line poles
(Winquist et al. 2014). The United States Environmental Protection Agency (US
EPA) (Article IV) has listed 16 PAHs of special concerns. The α-benzopyrene is
considered as the most carcinogenic PAH-compound associated with five benzene
rings (Ramesh et al. 2004). The PAHs are resistant to microbial degradation due to
their complex structure with two or more fused benzene rings and low water solubil-
ity (Haritash, and Kaushik 2009). The diffuse contamination of PAH in surface soils
has been observed and PAHs like phenanthrene, fluoranthene, and pyrene can be
degraded by soil microorganism, while the benzo (a) anthracene and benzo (a)
pyrene are examples of more recalcitrant high molecular weight (HMW) PAHs that
are biodegraded very slowly in the soil (Tian et al. 2008). The ligninolytic fungi
have a capability to degrade and mineralize the PAHs and the mechanism is thought
to be similar to that of lignin degradation.
The oxidation of PAHs is catalyzed by peroxidases or laccases and results in the
formation of PAH-quinones that can be further oxidized (Haritash, and Kaushik
2009). LMEs might have an important role in the initial attack on HMWPAHs in
soil. Since the LMEs are extracellular, they have capability to diffuse effectively to
the highly immobile HMW PAHs. The resulting metabolites are more water-
soluble, and thus, more bio-accessible (Turja et al. 2013). The formed compounds
can be substrates for many bacteria, but they may also be further degraded by fun-
gal intracellular enzymes, such as cytochrome P-450 monooxygenase (Saratale
et al. 2011). When the degradation of several PAHs by Irpex lacteus was deter-
mined, the structures of some of the metabolites suggested the involvement of both
LMEs and cytochrome P-450 monooxygenase. In addition to mineralization, a
significant fraction of PAHs are incorporated into humic substances during the
bioremediation (Covino et al. 2016).
7.6.4 Biodegradation of PCDD/Fs
Persistent Organic Pollutants (POPs) belong to the most toxic compounds such as
polychlorinated dibenzo furans (PCDF) and dibenzo-p-dioxins (PCDD). Two aro-
matic rings with one to eight chlorine atoms are found in PCDD/Fs (Seo et al. 2009).
Chlorine groups have toxic congeners in all of the 2,3,7,8 positions (7 PCDDs and
10 PCDFs), and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (2, 3, 7, 8-TCDD) is the most
toxic congener (Safe 1993). By multiplying their concentration with a Toxic
Equivalency Factor (TEF) (Article III), the toxicity of other congeners is compared
to that of 2, 3, 7, 8-TCDD. By adding up all the 2, 3, 7, 8-TCDD equivalents of all
the individual congeners, the total toxic concentration (expressed as World Health
Organization – Toxic Equivalent, WHO-TEQ) can be calculated (Giesy and Kannan
1998). The compounds PCDD/Fs are chemically stable in structure and poor bio-
availability, so the degradation rate of these compounds is very slow in nature.
PCDD/Fs are tightly adsorbed on soil particles, and absorbed into organic matter in
soils and sediment due to its highly hydrophobic nature (Anyasi and Atagana 2011).
In industrial processes, the PCDD/Fs are almost exclusively produced as by-
products, for example, the municipal waste incineration, chlorine bleaching of
paper and pulp, manufacturing of pesticides, herbicides, and fungicides (Mezcua
et al. 2012). In Finland, the main source of PCDD/F pollution was the production
and use of a chlorophenol-containing wood preservative (Ky-5) during 1940–1984
(Salo et al. 2008). The composition of Ky-5 in chlorophenols is 2, 3, 4,
6-tetrachlorophenol (55%), 2, 4, 6-trichlorophenol (36%), and pentachlorophenol
(7%) (Anasonye et al. 2014). Furthermore, in Ky-5, the PCDD/Fs were found as
impurities. Over the past several decades, most of the chlorophenols in these con-
taminated sawmill soils have volatilized, leached, and biodegraded (Josefsson et al.
2016). In the top soil with hepta and octachloro dibenzo furans (1, 2, 3, 4, 6, 7,
8-HpCDF and OCDF) as the main congeners, still, the PCDD/Fs continue to be the
main source for the contaminations (Seike et al. 2007). Additionally, severe PCDD/F
contamination is also found in Kymijoki River sediments due to sawmill soils.
The Plant situated of Ky-5 along the river and a fire in the plant in 1960 caused a
large spill in the environment (Karademir et al. 2013). It is observed that several
white rot fungi (WRF) to degrade all congeners of PCDD/Fs have been reported,
even the ones with maximum amount of chlorine atoms (Anasonye et al. 2015).
Additionally, to extracellular LMEs, some WRF have also intracellular P450, and
the LMEs attack co-metabolic lyon-chlorinated dioxins under aerobic conditions.
P450 enzymes of fungi, together with peroxidases, are also involved in the degrada-
tion of lignin. Probably, P450 enzymes and extracellular enzymes are involved with
later stages of degradation of chlorinated dioxins by WRF (Anasonye et al. 2014).
Several fungi used for the biodegradation of pesticides are given in Table 7.4.
Table 7.4 Bioremediation of pesticides using fungus

Name of chemical
compounds Name of the fungi References
PAH White rot fungi: Pleurotus ostreatus Trametes Mohapatra et al. (2018)
Diphenyl ether versicolor Kumar (2017)
Anthracene Armillaria sp. Saiu et al. (2018)
Naphthalene Pleurotus eryngii
Bleached kraft pulp Rhizopus oryzae or Pleurotus sajor-caju Purohit et al. (2018)
mill effluent
Fungicide: Metalaxyl Gongronella sp. and R. stolonifer Pandey et al. (2018)
and Folpet
Petroleum products A. niger, Rhizopus sp., Candida sp., Babič et al. (2017)
crude oil Penicillium sp., Mucor sp.
Gasoline Exophiala xenobiotica
Polychlorinated Doratomycesnanus sp., D. purpureofuscus, Mouhamadou et al.
biphenyles, POPs, D. verrucisporus, Myceliophthora (2013), Takada et al.
Polychlorinated thermophila, Phoma eupyrena, and (1996), Papanikolaou
dibenzofurans Thermoascus crustaceus, Phanerochaete et al. (2004)
Phenylurea herbicide sordida, Mortierella isabellina
diuron
Dye decolorization of Aspergillus niger, A. foetidus, T. viride, Jebapriya and
textiles A. sojae, Geotrichum candidum, Penicillium Gnanadoss (2013)
sp., Pycnoporus cinnabarinus, Trichoderma
sp.
Endosulfan Penicillium sp. Romero-Aguilar et al.
(2014)
Pesticide P. ostreatus Palmieri et al. (2000)
Chlorinated Aspergillus terreus Abraham et al. (2013)
hydrocarbons;
Heptaclor
Chloropyriphos
Leather tanning Aspergillus flavus, Aspergillus sp. and Bhadbhade et al.
effluent A. niger, Aspergillus jegita (2002)
Heptachlor O Trichoderma viridae, Roberti et al. (2006)
Toxaphene O
7.6.5 Biodegradation of Naphthalene
The enzymes are categorized into six general groups according to the Commission on
Enzymes of the International Union of Biochemistry. The enzymes catalyzing oxidation–
reduction reactions are known as oxidoreductases, and those catalyzing a chemical group
from one molecule to another are known as transferases; the hydrolytic enzymes are
known as hydrolases; those catalyzing the addition of functional groups to double bonds
or vice versa are known as lyases; those catalyzing the intramolecular rearrangements are
known as isomerases; and those enzymes catalyzing the condensation of two molecules
coupled with the cleavage of a pyrophosphate bond of ATP or similar triphosphate are
known as ligases (Karigar and Rao 2011; Faber and Faber 1992). Hydroxylation of aro-
matic rings is catalyzed by oxygenases and is initiated by various enzymes. One oxygen
atom is inserted into their substrates from the monooxygenase types, in another process,
H OH COOH
OH OH
OXYGENASES
H
ENZYME
NAPHTHALENE CIS-NAPHTHALENE SALICYLIC ACID

DIHYDRODIOL
OH
ACETYL-CO ENZYME A OH
O COOH
CH3 – CO – SCoA
COOH
ACETIC ACID 3 - OXO - ADIPIC ACID CATECHOL

CH3 –COOH
CO2 + H2O
NAPHTHALENE DEGRADATION BY FUNGAL ENZYMES
Fig. 7.2 The biodegradation process of naphthalene
two oxygen atoms are inserted into their substrates by dioxygenase-type approach
(Ullrich and Hofrichter 2007). Consequently, in a series of reactions, they are converted
into 2-ketoadipate or another compound and the ring is cleaved. And these compounds
can be consumed by the fungi or other microorganisms (Fan and Krishnamurthy 1995).
The biodegradation process of naphthalene is displayed in Fig. 7.2.
7.7 Soil Bioremediation and Its Benefits
For the bioremediation, the use of microbes is tormented with several rate-limiting fac-
tors. For the detoxifications of hazardous substance, various limitations apply to using
microbes. To the production of microbial culture, an expensive and long-approaching
process may be used (Hamelinck et al. 2005). Moreover, severe surroundings such as
chemical shock, pH and temperature, hazardous toxins, predators, and high dose of the
pollutants or their products may permanently damage or metabolically deactivate
microbial cells (Burridge et al. 2010), and the difficulty in continuing active cells dur-
ing the conveyance to the contaminated sites also limits the use of whole-cell purifica-
tion machineries (Raafat et al. 2008). The other factors that could control the use of
microbes include restricted mobility of the cells within the soil, the alternate carbon
sources, and the weakness of the inoculated microorganisms in competition with the
native population (Nannipieri et al. 2003). The biotransformation is involved in a series
of catalyzed enzymes reactions. If the enzyme reactions (preferably immobilized) are
used instead of the microbes, most of these hostile factors can either be eliminated or
mitigated (Kumar et al. 2015; Kour et al. 2019a). The bio-oxidation catalyst causes a
host of reactions by the enzymes. Specificity is shown by the enzymes and is character-
ized as they show an ideal temperature and pH for their actions (Denard et al. 2013).
The other catalysts, similarly, accelerate the reaction rate of chemical by lowering the
activation energy for a specific reaction.
Currently, in the world, contamination and its remediation have received considerable
attention due to the fact that numerous heavy metals cannot be degraded in the whole
environment and these metals lie in the soil. Various approaches have been followed
successfully to generate various plants and sites that have the capability to propagate in
polluted soils of metals and accumulate or tolerate the stress of metals. Naturally, the
microbial approaches for the remediation of heavy metal tolerance are an eco-friendly
and economical approach. Subsequently, the heavy metal uptake by plants and their
tolerance depend on various environmental factors, and interactions between plant and
fungi can play an important role in successful survival and growth of plants in contami-
nated soils. The plant growth-promoting microbes also assist plant growth by changing
the bioavailability of heavy metal due to various enzymatic activities of fungi. These
beneficial effects demonstrated by fungus, together with the interrelationship between
heavy metal tolerance and plant growth-promoting capability, indicate that their exploi-
tation in remediating metal-contaminated soils might have significant potential in the
near future. The synergistic approach of plant and fungi and their metal mobilization
mechanism, transformation, and detoxification have been observed. Further, observing
and handling the fungal and heavy metal remediation involve categorization of the fate
and behavior of the compounds of interest in the environment. So, these highlights rep-
resent the importance of a consistent link among the research and development for the
valuation and management of unindustrialized metal contaminants and the tools, appa-
ratus, and knowhow that contribute toward the contentment of these experiments.
Acknowledgments The authors are grateful to the Department of Environmental Microbiology,

Babasaheb Bhimrao Ambedkar University, Lucknow, and Department of Plant Pathology, School
for Agriculture, MPDC, University of Lucknow, Lucknow, for providing valuable support to write
this chapter. There are no conflicts of interest.
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Chapter 8
Bioremediation of Polycyclic Aromatic
Hydrocarbons (PAHs) Contaminated Soil
Through Fungal Communities
Ulises Conejo-Saucedo, Darío R. Olicón-Hernández, Tatiana Robledo-Mahón,

Haley P. Stein, Concepción Calvo, and Elisabet Aranda
8.1 Introduction
Polycyclic aromatic compounds (PAC), including the well-known subgroup of

polycyclic aromatic hydrocarbons (PAHs) and the heterocyclic aromatic com-
pounds, include several thousand individual compounds (Achten and Andersson
2015). In particular, polycyclic aromatic hydrocarbons (PAHs) are benzene rings
fused pollutants widely distributed in nature. They are considered small-PAHs when
they have from two to four rings (such as naphthalene, anthracene, phenanthrene,
and pyrene) and large-PAHs with more than four rings (benzopyrene, chrysene,
benzoanthracene, etc.) (Table 8.1). PAHs are listed by EPA (Environmental
Protection Agency) as priority pollutants and considered to be mutagenic, terato-
genic, and carcinogenic compounds (Luch 2009). The physicochemical properties
of PAHs depend on the number of rings, as well as the presence of substituent
(alkylated). Thus, vapor pressures and water solubility decrease when the number of
rings increases; however, the n-octanol-water partition coefficient log KOW increases
(Achten and Andersson 2015).
PAHs are distributed in all environments worldwide. PAHs present in carbon-
containing organic materials, such as coal tar and charcoal, are of petrogenic origin,
while those released during combustion of fossil fuels or natural combustions (for-
est fires, volcanic eruption) are of pyrogenic origin. In addition, PAHs can also be
synthesized during the degradation of organic matter or biologically by bacteria and
plant life, although this fact has not yet been clarified (Parlanti 1990).
The largest contributor of PAHs in the environment is from incomplete com-
bustion (both natural and anthropogenic) varying geographically around the Earth
U. Conejo-Saucedo · D. R. Olicón-Hernández · T. Robledo-Mahón · H. P. Stein ·

C. Calvo · E. Aranda (*)
Department of Microbiology, Institute of Water Research, University of Granada,
Granada, Spain
e-mail: earanda@ugr.es

218 U. Conejo-Saucedo et al.
Table 8.1 Polycyclic aromatic hydrocarbons including some heterocyclic and their details health
risk, and environmental data
Chemical Ranking Molecular Environmental
Compound structure positionb formula/(Mw) Health riska dataa
Small-PAHs
Naphthalene 81 C10H8/128.174
Anthracene 9 C14H10/178,23 Toxic; Very toxic to

(PAHs) noncarcinogenic aquatic
organisms, it
may cause
long-term
adverse effects
in the aquatic
environment;
hazardous to the
environment
Phenanthrene 252 C14H10/178,23 Toxic; specific Very toxic to
toxicity to some aquatic
organs; organisms, it
noncarcinogenic; may cause
non-mutagenic long-term
adverse effects
in the aquatic
environment;
hazardous to the
environment
Acenaphthene 171 C12H10/154.2
Fluorene 9 C13H10/166.223
(PAHs)
Benzo(a) 38 C18H12/228,2879
anthracene
Pyrene 260 C16H10/202,25 Toxic; Very toxic to

non-genotoxic aquatic
organisms, it
may cause
long-term
adverse effects
in the aquatic
environment;
bioaccumulation
may occur in
crustacea, fish,
milk, molluscs,
and algae
Fluoranthene 138 C16H10/202.256
(continued)
8 Bioremediation of Polycyclic Aromatic Hydrocarbons (PAHs) Contaminated Soil… 219

Chemical Ranking Molecular Environmental
Compound structure positionb formula/(Mw) Health riska dataa
Large-PAHs
Benzo[a] 230 C20H12/252,31 Toxic; Very toxic to
Pyrene carcinogenic; aquatic
may cause organisms, it
reproductive may cause
difficulties; long-term
teratogenic adverse effects
in the aquatic
environment;
hazardous to the
environment
Benzo(b) 10 C20H12/256.34
fluoranthene
Benzo[g,h,i] 9 C22H12/276.338
perylene (PAHs)
Indenol 176 C22H12/276.338

(1,2,3-cd)
pyrene
a
Data obtained from the US Agency for Toxic Substances and Disease Registry (http://www.atsdr.
cdc.gov/substances/index.asp). 2017 CERCLA Priority List of Hazardous Substances
b
Ranking position in the CERCLA Priority List of Hazardous Substances
(Zhang and Tao 2009). Once in the atmosphere, small PAHs and gaseous PAHs
can be volatilized and transported over long distances, influenced by certain mete-
orological conditions. Small PAHs can then be degraded by different reactions
such as photodecompositions and/or chemical oxidation, defining the PAH half-
life in the atmosphere, until they are absorbed into particles, where further degra-
dation could be completely inhibited. At this point, the small PAHs are deposited
onto terrestrial, lacustrine, and marine surfaces (Fig. 8.1). Heavy PAHs can be
transported in settling atmospheric particles which contribute to the depositions in
soils and sediments (Wilcke 2000). Thus, PAHs can be dispersed locally and inter-
continentally via atmospheric and aquatic transport (Becker et al. 2006). In soils,
PAHs result mostly from atmospheric deposition and tend to be absorbent to soils
particles. This absorption is strongly influenced by the soil composition, particle
size, and organic carbon content. Retention of PAHs in organic matter and soil
particles contributes to the aging of soils with PAHs, and as a result, the ability for
PAH-degrading microorganisms decreases and they are accumulated (Loehr and
Webster 1996).
Fig. 8.1 Main sources of PAH in the environment: (1) forest fires, (2) volcanic eruptions, (3)
combustion of fossil fuels, and (4) accidental spills during storage, transport, and disposal of
fossil fuels
8.2 Fungi as Key Drivers in PAHs-Polluted Soils
Microorganisms are found in all ecosystems and are essential biological compo-
nents to their functioning. Microorganisms are involved in the nutrient cycling
which contributes to biogeochemical processes in the biosphere. Microbial metabo-
lism which drives degradation and transformation processes depends on the interac-
tions and the relationships between microbes and substrate availability. The
combination of microorganisms which comprises the microbial community includes
bacteria, archaea, fungi, and microeukaryotes (Fuhrman 2009; Yadav et al. 2019a,
b). The first person to observe and discuss the microbial community was Clements
(1916), who considered the community as a supraorganism with levels of
organization, in which the stratified interactions involved allowed different proper-

ties to the system. Gleason (1926) defined the terms as an individualist concept: a
microbial community exists as a space where an assortment of species cohabit in a
balance, due to the level of tolerance to physical and chemical substances that they
produce themselves. In this sense, more recently, Konopka (2009) defined microbial
community as “multi-species assemblages, in which organisms live together in a
contiguous environment and interact with each other.” Thus, the key factor is that
the communities involve a group of microorganisms which share a habitat and
engage in different interactions and roles in relation to the biological and the psy-
chochemical conditions. Particularly, these interactions contribute to the challenge
in defining the limits of an ecosystem. It is likewise a challenge to define specific
actions of the microbial interface, due to feedback systems between the various
microorganisms and their effect on the environment. Microbial ecology aims to
study these interactions of each community in a specific environment. However, to
outline the specific bioreactions in the ecosystem is not an easy task. More often,
these interplays are studied at the scale of the micron, an exception being the hyphae
of fungi, which can be considered on a larger scale.
The dynamics, structure, and succession of microbial communities are useful in
understanding the complexity of ecosystems, this being the main goal of observa-
tional ecology (Garland 1997). Additionally, just as diversity is related to the stabil-
ity of an ecosystem, a higher ecosystem diversity involves a wide variety of
microorganisms with varying functions and requirements. This broad metabolic
profile will favor the community more adapted under unfavorable chemical and
biological conditions. As such, the cohabitation of communities with different eco-
logical functions act as a buffer when faced with a change in the environmental
conditions, thereby regulating the connections between different microbes and
maintaining the balance of the ecosystem (Konopka et al. 2015). In this context,
polluted environments make for fascinating ecosystems of study, due to the higher
potential of the inhabitants to develop biodegradation and biotransformation pro-
cesses. In observation of the metabolic interactions between microbial cohabitants
and the abiotic environmental factors, researchers may gain significant insights into
the ecological functions of each diverse microbial community.
Fungal communities are ubiquitous, colonizing terrestrial and aquatic habitats,
and even extreme ecosystems such as polar zones, deserts or hypersaline environ-
ments (Margesin and Schinner 2001; Rana et al. 2019; Sharma et al. 2019; Yadav
et al. 2017a, 2018). Fungi are well-known to play a major role in nutrient cycling
and biogeochemical cycles, in both aquatic and terrestrial environments (Kubicek
and Druzhinina 2007; Suman et al. 2016). In addition, fungi are frequently found in
hostile environments, polluted ecosystems, or environments which have otherwise
been altered by anthropogenic activity, characterized by the presence of toxic or
recalcitrant compounds, such as heavy metals or PAHs. Fungi have several mecha-
nisms of adaptation to these conditions, including the thick cell wall, the production
of spores, the production of intracellular and extracellular unspecific enzymes, and/
or a developed enzymatic system to counteract the effect of reactive oxygen species
(ROS) (Harms et al. 2011). In addition, in polluted environments, the fungal com-
munity is involved in complex interactions with other microorganisms. In fact, the

close interaction between fungi, plants, and bacteria, called the rhizospheric effect,
has been well documented in scientific literature (Kotoky et al. 2018). The rhizo-
spheric effect is mediated by mutual interactions of plant and microbes, mainly
during phytoremediation processes, as well as having a role in highways of fungi
mycelial tissue for the movement of bacteria through the soil (Kohlmeier et al.
2005). Despite the representation of fungi in these environments, fungal communi-
ties, and their specific roles in these ecosystems remain poorly studied.
8.2.1 Culture-Dependent Techniques
Diverse techniques have been used to analyze fungal communities in PAH-

contaminated soil over the last decades. Traditionally, culture-techniques based on
the direct isolation of fungal strains, and conventional microbial techniques (plate
counts, community level physiological profiling or Biolog), have been used as the
necessary optimal media to growth and conserve the strains.
However, it is known that cultured fungi represent only a fraction of the entire
fungal community, and favors fast growing species that produce large quantities of
spores (O’Brien et al. 2005). Despite these disadvantages, there is no doubt about
the great interest in using culture-techniques as a source of fungal cells with ability
to remove pollutants for bioremediation purpose and for general studies focused on
deeper knowledge of these specific microorganisms.
8.2.2 Culture-Independent Techniques
In the last decades, a number of approaches have been developed based on genetic
diversity. These include omics technologies, which are useful in understanding the
cellular and molecular behavior of the communities, especially during a bioremedia-
tion event. Thus, if metagenomics supply the genetic profile of the fungi involved in
a polluted soil, metatranscriptomics reveal information related to gene expression
profiles in active populations of the mycobiome involved, for example in signaling,
or in the degradation and metabolism of PAHs. Metaproteomics then show the pro-
tein expression profile as a response to a specific compound and function, integrat-
ing intermediates and final products in pathways through metabolomics (Kachienga
et al. 2018; Kotoky et al. 2018). In addition, sequencing technologies have allowed
for the elucidation and annotation of several genomes at increasing rates. The “1000
Fungal Genome project” represents a strong push to obtain a number of new genomic
sequences to include in a large-scale catalogue of fungal species (https://genome.jgi.
doe.gov/programs/fungi/1000fungalgenomes.jsf) (Grigoriev et al. 2014).
The application of these technologies has become an indispensable tool in bacte-
rial soil ecology, but its use in studies of fungal communities has so far been limited.
Fig. 8.2 Principal barcode-primer pair combinations used in fungi
Fungal databases display limitations related to the number of sequenced and

well-annotated fungal genomes and have largely failed to apply the sequence data
to phylogenetics, as a way of potentially reorganizing fungi according to the new
taxonomic classifications (O’Leary et al. 2016). Nevertheless, the collaboration of
the different databases marks an advance in the creation of a complete and update
database (Huzefa et al. 2017).
On the other hand, the absence of a universally accepted DNA barcode is a
restriction in ecological and biological studies, which has generated the necessity to
use different biomarkers. Since the 1990s, taxonomic studies of fungi have been
based on ribosomal genes of 18S or small subunit (SSU), the 5.8S subunit, and the
28S or large subunit (LSU) genes, being the LSU and SSU more efficient at high
taxonomic levels (Cuadros-Orellana et al. 2013). Currently, the intergenic region
(ITS) has been considered the universal DNA barcode markers for fungi as suitable
barcode. Different fungal studies have shown the efficiency of the ITS region as the
barcode marker with the highest probability of correct identification, for a broad
analysis of sampled fungi (Dentinger et al. 2011; Kelly et al. 2011; Schoch et al.
2012). Likewise, most diversity studies on environmental samples were using the
ITS region to identify fungi (Bellemain et al. 2010; Wang et al. 2011; Rosling et al.
2011; Ihrmark et al. 2012) (Fig. 8.2).
However, it is recommendable to use secondary region if there is a low variabil-
ity of interspecific region, in order to obtain a high genetic precision. It must be
considered that the use of a biomarker may lead to biased amplification of different
taxonomic groups (Schoch et al. 2012; Blaalid et al. 2013).
8.3 Fungal Mechanisms in PAH Transformation
The isolation of fungi-dwelling polluted environments provides important informa-

tion about metabolic capability, for further application in PAH-polluted soils. Fungi
are strict heterotrophic organisms, which involve the transformation of external car-
bon sources to obtain energy for basal metabolism as well as biomass production.
Keeping in mind this degradative capacity and ecological niche, fungi have been
supplied with an array of intrinsic bioprocesses to colonize natural matrices, survive
in extreme conditions, degrade a variety of substrates, and adapt within adverse

habitat (Anastasi et al. 2013; Selbmann et al. 2013; Zafra et al. 2015). As a biotech-
nological tool, the morphological components, type of growth, and enzymatic sys-
tems, among others, are important elements for fungi to be considered as excellent
models to implement new technologies to, for example, develop and improve biore-
mediation processes.
In the context of the elimination of PAHs, it was demonstrated that fungi can
degrade complex aromatic carbon derivatives such as anthracene (Hadibarata
et al. 2013; Marco-Urrea et al. 2015), pyrene (Bhattacharya et al. 2014; Mineki
et al. 2015), and phenanthrene (Lee et al. 2014; Fu et al. 2018) as well as simple
ring compounds and linear hydrocarbons such as benzene and methyl tert-butyl
ether (Thomas 2013). In order to adapt the xenobiotic carbon source into their
aerobic metabolism pathway and/or eliminate it by nonenzymatic methods, fungi
can interact with contaminants by two main mechanisms: intra- and extracellular
processes. In terms of the capability to secrete extracellular enzymes or not, as
well as possessing elements for biosorption and/or bioaccumulation, fungi differ
in the action mechanisms. However, it seems clear that both systems are func-
tional and works together for the elimination of contaminants (Mougin et al. 2013;
Deshmukh et al. 2016).
8.3.1 Extracellular Mechanism
Extracellular mechanism for elimination of PAHs in fungi involves the use of low-
specific enzymes as main component (Morelli et al. 2013). White-rot fungi (WRF)
are the most important representatives of this kind of fungal elimination systems
due to their ability of producing a set of extracellular lignin-modifying enzymes
such as laccase, manganese peroxidase (MnP), and lignin peroxidase (Winquist
et al. 2014; Yadav and Yadav 2015) and unspecific peroxygenases, versatile peroxi-
dases, and decolorizing-type peroxidases (DyP-type) (Kües 2015). These kinds of
enzymes can transform and/or mineralize the phenolic polymer components in
wood that have complex and heterogeneous matrices, making them ideal tools for
the degradation of a variety of aromatic compounds (Anastasi et al. 2013). From
this point of view, WRF have shown promise for application in the bioremediation
of PAH contaminated soil and water by employing these enzymes and focusing on
improving the removal rates, while continuing to evaluate kinetic parameters, reus-
ability and efficiency (Xuanzhen et al. 2014; Lee et al. 2014; Bautista et al. 2015;
Godoy et al. 2016). However, studies in fungal diversity by culture independent
techniques show that in polluted environments, some of the main fungal populations
are fungi lacking of these enzymes (D'Annibale et al. 2006). Another element of
extracellular systems in the elimination of PAHs is the use of cell wall and mem-
brane components as biosorbent systems (Harms et al. 2011). This physicobio-
chemical surface phenomenon considers the electrostatic interaction of biomass
with contaminants involved to gauge the removal of the pollutant and could be
complemented by bioremediation (Ding et al. 2013; Tony et al. 2014). The use and
production of fungal biosurfactants to improve extracellular uptake of xenobiotic
compounds by biomass is one branch of bioaugmentation, and may play a role as an
element that can improve the overall degradation of the xenobiotics in question
(Lladó et al. 2013a). In this context, an emulsifying effect was observed in the bio-
degradation of ring-PAHs by Pleurotus ostreatus D1, due to the production of natu-
ral biosurfactants. The author suggested that the presence of this element increases
the solubility and bioavailability of the pollutants and/or increase the production of
extracellular enzymes for the elimination of the PAHs (Nikiforova et al. 2009).
8.3.2 Intracellular Enzymatic Pathways
Intracellular enzymatic pathways for the biotransformation of PAHs show that

xenobiotic entry into the cell occurs by several mechanisms and results in the for-
mation of hydroxy, dihydroxy, dihydrodiol, and quinone derivatives. This process
(phase I) is mediated by the cytochrome P450 (CYP) system and epoxide hydro-
lases (EHs). The next step, the conjugation process occurs when sulfate, methyl,
glucose, xylose or glucuronic acid groups are linked to oxidized metabolites (Phase
II); this process is mediated by transferase enzymes. During phase III, these metab-
olites are secreted or stored in organelles. The final step for the elimination of oxy-,
methoxy-, or sulfate PAHs has not been fully discerned (Aranda 2016) (Fig. 8.3).
CYP enzymes are elements present in the degradation of aromatic and aliphatic
compounds in WRF and Ascomycetes such as Aspergillus sp. and Penicillium
sp. (Aranda et al. 2017; Camacho-Morales et al. 2018; Huarte-Bonnet et al. 2018).
Fig. 8.3 Proposed phenanthrene degradation pathway in fungi (Marco-Urrea et al. 2015)
In fact, CYP fungal enzymes involved in hydrocarbon assimilation are dependent

on phylogeny (Huarte-Bonnet et al. 2018). These enzymes participate in the initial
hydroxylation of aromatic and alkane derivatives in order to oxidize the xenobiotic
and produce energy and are the main component in intracellular PAHs degradation
(Huarte-Bonnet et al. 2018). In this context, it was described that CYP52 and
CYP53 clans play a significant role in fungi detoxification, however, being a com-
plex superfamily of enzymes the participation of these proteins is not fully clear
(Godoy et al. 2016; Olicón-Hernández et al. 2017; Huarte-Bonnet et al. 2018).
Bioaccumulation of PAHs in cytoplasm or vacuoles, also complemented the intra-
cellular systems of elimination (Verdin et al. 2005; Gu et al. 2016).
8.4 Case Study of Fungal Communities on PAH Degradation
In the last two decades, the interest in the use of biotechnologies, destined to recover
contaminated environments from hazardous aromatic pollutants such as PAHs, has
increased. The analyses of metagenomics in hydrocarbon-polluted areas have pro-
vided information about the richness and diversity of the autochthonous community
in this type of environment, the genes involved in the degradation, the protein
expression, and the final product generated. Several investigations have focused
their attention on the study of the fungal diversity in PAH contaminated soils
(Cerniglia and Sutherland 2010; Mineki et al. 2015; Sawulski et al. 2015; Siles and
Margesin 2018; Yadav et al. 2017b).
Fungi, as a part of a community focused on PAH degradation, have an important
role as the main degraders or/and complement of communities of bacteria and com-
plex eukaryotes for elimination of hydrocarbons aromatic derivatives (Zafra et al.
2014; Vasudevan et al. 2018). Borowik, et al. (2017) analyzed the effect of diesel oil
contamination on the diversity of fungal communities in soils. After 270 days, the
degradation of the PAHs with four rings was 64%; in PAHs with five rings was 28%;
and in PAHs with six rings was 16%. However, the colony development index for
fungi increased significantly, while the diversity index decreased, indicating a direc-
tional selection. In recent studies, Biache et al. (2017) analyzed the impact of PAH
contamination on the abundance of microbial communities, and the communities’
degradation capacity for the PAHs. The results showed differences in the degrada-
tion rates according to the distribution, the availability of PAHs, and the three soils
that were evaluated; reaching 98%, 76%, and 34% of degradation, respectively
(Biache et al. 2017).
In nature, the biodegradation of PAHs occurs among different microbial com-
munities and is not restricted to single microbial species. Thus, cometabolism of
PAHs will result in multitudes of high-polarity metabolites more easily degradable
by lower species in the taxonomic class. Consequently, in a mixed microbial popu-
lation, initial cometabolic transformation may pave the way for subsequent attack
by another organism, resulting in a synergism of fungi-bacteria/bacteria-fungi for
the elimination of PAHs (Vasudevan et al. 2018). This step is essential for the
c omplete mineralization of xenobiotics, although in a community is not clear if

bacteria or fungi works as final biodegradator. The consortiums isolated from PAHs-
contaminated environments demonstrated high tolerance of concentration of PAHs;
however, in the case of fungi, it was observed the same tolerance, even without the
fungi being previously exposed to environments contaminated with aromatic com-
pounds (Simarro et al. 2013). This is an advantage for the use of fungi in bioreme-
diation proposes.
Bioremediation using fungal-bacterial cocultures generally favored bacteria in
increased log phase and lag periods, utilization of PAHs as carbon source, and toler-
ance to heavy metals, among others. Thus, in the absence of fungal communities,
initial oxidation of PAHs are seldom possible by bacteria, and on the other hand, in
the absence of bacterial communities, there is a huge accumulation of dead-end
fungal metabolites which may be toxic (Vasudevan et al. 2018). Regardless, Zhou
et al. (2017), found a significant correlation of PAH contents with fungal commu-
nity structure, but not with fungal diversity; interestingly, the community structure
of bacteria might be more sensitive to soil PAH contamination than those of fungi
and archaea (Zhou et al. 2017).
Still, fungi have several characteristics that favor their use in consortium
(Bacterial-Fungal) for bioremediation processes. Mainly, fungi can spread through
soils, allowing bacteria to extended through the soil and access to contaminants
(Nazir et al. 2010; Banitz et al. 2011); mycelium can act as dispersion networks for
bacterial transport and actively mobilizing PAHs, called “fungal high-ways”
(Kohlmeier et al. 2005; Schamfuß et al. 2013). In addition, in recent studies, it has
been shown that bacteria adapt to these environments due to the release of nutrients
and signaling molecules by fungi (Nazir et al. 2010). On the other hand, it has been
found that these mycelia can increase the mobility of a wide range of PAH due to
their translocation during cytoplasmic transmission (“fungal pipelines”) (Furuno
et al. 2012); in this way, the access of bacteria and plants to soil pollutants and their
degradation is favored (Banitz et al. 2013).
8.5 ioremediation Performed by Consortia

B
in Contaminated Soils
Two of the most used fungal bioremediation technologies for PAH removal in situ
are bioaugmentation and biostimulation. While the process of bioaugmentation
implies the introduction of a group of microbes that have the ability to survive in a
contaminated environment and carry out the bioremediation process, during bios-
timulation, the autochthonous microorganisms receive nutrients for an adequate
growth and metabolic rate in order to increase the efficiency of degradation (Vidali
2001).
Contaminated soils are an excellent source of adapted microorganisms (Björklöf
et al. 2009; Bourceret et al. 2016). For this reason, several studies have been carried
out on native fungal communities in contaminated soils and their subsequent

isolation and selection based on their ability to grow, efficiency to degrade different
PAHs or use them as a source of carbon and energy (Li et al. 2008; Alrumman et al.
2015; Marco-Urrea et al. 2015; Marchand et al. 2017) for bioaugmentation pur-
poses. In this context, some recent investigations showed the isolation of nine non-
ligninolytic fungal strains native from soil contaminated with Maya crude oil. The
results showed strains of the genera Fusarium, Neurospora, Aspergillus,
Scedosporium, Penicillium, Neosartorya, and Talaromyces. However, Aspergillus
terreus, Talaromyces spectabilis, and Fusarium sp. present the best tolerance to
2000 mg kg−1 of a mixture of phenanthrene and pyrene soil in a solid-state micro-
cosm system for 2 weeks. The elimination of PAHs was close to 21% (Reyes-César
et al. 2014). Similar examples were found in native fungi represented mainly by
Ascomycota phylum, Mucoromycotina subphylum and Basiodiomycota phylum
isolated from a historically pyrogenic PAH-polluted soil in Spain. Out of the 23
isolated species, 12 species were able to oxidize anthracene at different rates (Godoy
et al. 2016).
In a study done by Anastasi et al. (2009), the fungal consortium (T. versicolor,
Bjerkandera adusta, Bjerkandera fumosa, and Lopharia spadicea) were performed
in a contaminated soil composting system. These researchers concluded that these
consortia eliminated approximately 56 out of every 100 mg kg−1 dry weight of
pyrene in 28 days (Anastasi et al. 2009); and reduced a high concentration of naph-
thalene (500 mg kg−1) in three weeks.
Nevertheless, several studies have shown that a microbial consortium (Fungal,
Fungal-Bacterial) are more effective (Jacques et al. 2008; Lladó et al. 2013b; Balaji
et al. 2014; Maddela et al. 2015). In the studies carried out by Zafra et al. (2014), the
consortium was composed of 12 fungal and bacterial PAH-tolerant isolates; the
results shows a removal of 48.18% pyrene, 56.55% benzo[α]pyrene and 87.76%
phenanthrene after 14 days. In addition, a study from Sharma et al. (2016), reported
a new microbial consortium composed of Serratia marcescens L-11, Streptomyces
rochei PAH-13 and Phanerochaete chrysosporium VV-18. Under controlled condi-
tions, the consortium degraded nearly 70% of PAHs in 7 days; also, the degradation
rate of PAHs significantly increased between 56% and 98% under natural conditions
(in situ) in 7 days, and almost complete degradation was observed on the 30th day.
Recently, Zafra et al. (2017) have constructed a new, tolerant PAH-degrading
microbial consortia, composed of six fungal and seven native bacterial strains with
the ability to degrade up to 92% of phenanthrene, 64% of pyrene, and 65% of
benzo(α)pyrene out of 1000 mg kg−1 after 2 weeks of incubation and used these
compounds as a sole carbon source.
The evidence shows that the microbial community (bacterial-fungal) plays a key
role in the degradation of PAHs in soils. However, in conjunction with plant life
(Plant-Fungal-Bacterial), microbes offer a novel type of consortium that makes the
degradation more efficient (Fan et al. 2008; Tejeda et al. 2012; Aranda et al. 2013).
For example, Storey et al. (2014) found that in the presence of tomato plants, the
degradation of fluoranthene was greater, degrading close to 80.2% and 68.1% in
concentrations of 500 and 5000 mg kg−1 fluoranthene over 30 days, respectively.
Interestingly, fluoranthene in combination with the absence or presence of plants

had a significant impact on bacterial and fungal community structures.
Biostimulation technologies have been studied using compost/organic substrates
in contaminated soils, that helps to improve the degradation, the texture of the soil,
favors the transfer of oxygen and provides energy to microbial populations (Tyagi
et al. 2011; Li et al. 2012; Lladó et al. 2013a; Bastida et al. 2016). Recent studies
examine the addition of both fungal inoculum and composted vegetable waste to a
PAH-contaminated sawmill soil in treatments at the laboratory and field scales.
Several fungi, such as Agrocybe dura, Agrocybe praecox, Gymnopilus luteofolius,
Irpex lacteus, Mycena galericulata, Phanerochaete velutina, Physisporinus rivu-
losus, Stropharia aeruginosa, Stropharia rugosoannulata, and Trametes ochracea,
were found to be potential players in the degradation of PAHs; 96% of the four-ring
PAHs and 39% of the five- and six-ring PAHs were degraded in 3 months (Winquist
et al. 2014).
Andreolli et al. (2015) compared bioagmentation and biostimulation in a burned
woodland soil contaminated with high molecular weight hydrocarbons. The biore-
mediation protocol included a mycelial suspension of a Trichoderma sp. strain and
a microbial growth promoter formulation. The results showed that the biostimula-
tion approach was the best treatment; about 70% of the initial concentration of high
molecular weight hydrocarbons was degraded after 60 days (Andreolli et al. 2015),
indicating the important role of native microorganisms in the bioremediation pro-
cess. However, in this study, the shift in the microbial community has not been
assessed. Zafra et al. (2016) show that bioaugmentation with a fungal-bacterial con-
sortium and biostimulation of native microbiota using easy-to-assimilate carbon
sources and texturizer produced appreciable changes in the microbial diversity of
polluted soils. The combination resulted in a shift in the native microbial communi-
ties to favor degrading specific contaminants such as PAHs.
Other studies of biostimulation with NPK fertilizer, characterized soil microbial
communities (bacterial, archaeal and fungal communities); the results showed high
removal rates of total petroleum hydrocarbon obtained with agricultural inorganic
NPK fertilizer in treatments at 10 °C and 20 °C during 15 weeks and resulted in
changes in bacterial and fungal community structures (Siles and Margesin 2018).
The abundance and diversity of bacterial and fungal communities did not play a
decisive role on the effectiveness of soil bioremediation; these results were more
influenced by changes in temperature and fertilization, indicating the effect of a cor-
rect biostimulation of the native microorganisms.
Zafra et al. (2016) have reported a new consortium composed of four fungal spe-
cies (Aspergillus nomius H7, Aspergillus flavus H6, Trichoderma asperellum H15,
Rhizomucor vari-abilis H9) and five bacterial species (Bacillus cereus B4, Klebsiella
pneumoniae B1, Klebsiella sp. B10, Pseudomonas aeruginosa B6, Stenotrophomonas
maltophilia B14). Results showed that phenanthrene (84.29%), pyrene (59.66%),
and benzo[α]pyrene (58.98%) degradation was significantly higher in soils inocu-
lated with this consortium than control. These studies concluded that the consortia
used produced appreciable changes in the microbial diversity, changing native
microbial communities in favor of the degradation of specific populations.
With the even growing emphasis on green technologies, the novel field of
bioremediation has introduced opportunities for discovery in biological sciences.
The inclusion of and focus on fungi in studies of microbial bioremediation can serve
to add to understanding not just xenobiotic metabolism, but also aid in the under-
standing of microbial interactions in both laboratory and natural settings.
Importantly, scientists may better be able to observe how fungal communities func-
tion and impact their environment on a larger scale. Isolating specific fungi can have
its place in discovering the single players in a systemic whole, but ultimately, the
consensus shows that fungi, bacteria, and plants work best all together in contami-
nant purification.
Nonetheless, individually and within these systems of microbial communities,
through applying surveys using omics techniques, scientists may be able to uncover
levels of enzymatic processes behind PAH degradation. Additionally, studying
fungi in polluted settings can carry insights that explicitly speak to the ability of
species to adapt to stress in their habitats, as fungi have shown to be more resilient
and adaptive in polluted settings. Researchers using omics techniques could ulti-
mately pinpoint specific molecular adaptations that operate to augment chances of
survival in PAH contaminated environments. Perhaps genetic engineers can even
come to apply these mutations to organisms in degradation consortiums, to design
whole, synergistic microbial communities for the complete removal of anthropo-
genic PAHs in contaminated habitats. In this way, fungi serve an important role in
the biological and chemical rehabilitation of terrestrial and aquatic environments
for a restoration of native ecological equilibrium in contaminated sites.
Acknowledgments The authors gratefully thank the Ministry of Economy and Competitiveness
(MINECO) and European Regional Development Fund (ERDF) funds for the Ramón y Cajal con-
tract of EA (RYC-2013-12481). UC and DRO thank CONACyT Mexico for the postdoctoral fel-
lowship (230592/209148/473970; 231581/454815, respectively). HPS acknowledges the Fulbright
Program (PS00247479) for the Open Study/Research Grant. We would like to acknowledge the
Environmental Microbiology Research Group [RNM-270] of the University of Granada (Spain).
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Chapter 9
Role of Fungal Enzymes
for Bioremediation of Hazardous
Chemicals
Nitika Singh, Abhishek Kumar, and Bechan Sharma
9.1 Introduction
Hazardous chemicals may be defined in terms of any waste material that would
be a present or future threat to organism or to the environment. It can also be
defined as unwanted materials that exhibit hazardous characteristics (Wu et al.
2008; Gnanasalomi et al. 2013). Agricultural practices, industrial processes, and
the use of variety of chemicals in different extents of our routine life result in the
deliberate or accidental release of potent chemicals (i.e., lethal to humans) into the
environment. These hazardous chemicals can be transported via atmosphere and
water as well and, in many cases, find their way into sediments and soils after their
release in environment. Environmental hazardous chemicals of particular interest
are mainly pollutants including petroleum hydrocarbons, halogenated solvents from
industrial sources, endocrine-disrupting agents and drugs, explosives, chemicals
from agricultural activities, heavy metals, metalloids, and radionuclides (Harms
et al. 2011; Kumar and Sharma 2018; Kumar et al. 2018). A number of activities
including anthropogenic activities (e.g., agricultural practices) and industrial pro-
cesses (e.g., mining and metal processing, petrochemical and industrial complexes,
industry effluents, chemical weapons production, pulp and paper industries, dye
industries, and industrial manufacturing) are responsible for the pollution in the
environment. These chemicals may affect the health of humans, plant, and animals
and also affect the environment for several causes (Rao et al. 2010). Among all envi-
ronmental hazardous chemicals, some substances are potentially toxic and present
in the environment and affect microbial organisms, plants, animals, humans, soil,
sediments, water, and air. These toxic substances are very often present not only as
mixtures of different organic compounds but also of organic and inorganic ones.
Because the number of industries increases day by day, a large amount of waste
N. Singh · A. Kumar · B. Sharma (*)

Department of Biochemistry, Faculty of Science, University of Allahabad, Allahabad, India

238 N. Singh et al.
materials has been let out into the environment. These wastes are very toxic and
persist in environment for a long time. They can enter into the food chain from
soil and water even after their use. These chemicals have also the capability to bio-
accumulate within the cells of organism. Thus, biomagnifications occur over the
period because of the increasing levels of toxic compounds within the body. These
hazardous pollutants are flammable, explosive in nature, corrosive, toxic, and bioac-
cumulative. Sometimes, eutrophication takes place due to the presence of inorganic
pollutants, and acute and chronic diseases occur due to exposure to these pollutants
(Gnanasalomi et al. 2013).
Bioremediation is a process mediated by microorganisms that transform or
degrade contaminants into nonhazardous or less hazardous substances. The efficient
capability of bioremediation of pollutants in many organisms like bacteria, fungi,
algae, and plants has been reported (Vidali 2001; Leung 2004; Karigar and Rao
2011). The bioremediation process primarily depends on action of microorganisms
and which convert hazardous chemical to innocuous products by enzymatically
attacking on the hazardous chemical. An effective bioremediation can only exist
where environmental conditions favor microbial growth and activity. Often, the
applications of bioremediation involve the manipulation in the environmental con-
ditions which allow microbial growth and rapid degradation of chemical. Generally,
degradation of pollutants is a very slow process. Only some certain fungal species
have been reported as potent pollutant degraders. Some of these strains are working
as some effective bioremediation agents only under laboratory conditions. The envi-
ronmental factors like pH, temperature, oxygen, soil structure, moisture and appro-
priate level of nutrients, poor bioavailability of contaminants, and presence of other
toxic compounds limit the fungal growth. However, the microorganisms can grow
in environmental conditions, but most of them prefer optimal ideal condition that is
difficult to maintain outside the laboratory (Dua et al. 2002; Dana and Bauder 2011).
Most bioremediation systems operate under aerobic conditions, but anaerobic envi-
ronments may also permit microbial degradation of recalcitrant molecules. Fungi
rely on the participation of different intracellular and extracellular enzymes, respec-
tively, for the remediation of recalcitrant and lignin and organopollutants (Hammel
1997; Karigar and Rao 2011) (Table 9.1).
9.2 ccurrence and Morphological Features of Fungi Used

O
in Bioremediation
Fungus belongs to a large group of eukaryotic organisms like microorganisms

including yeasts, mushrooms, and molds. Fungi are heterotrophic, eukaryotic, and
absorptive organisms that typically develop mycelium (a branched, tubular body),
and reproduction takes place by sporulation. Less than 100,000 of the 1.5 million
estimated fungal species have been described in Stajich et al. (2009). Up to 75% of
fungal species account for the soil microbial biomass, i.e., total soil microbial
9 Role of Fungal Enzymes for Bioremediation of Hazardous Chemicals 239
Table 9.1 Fungal enzymes and their role in bioremediation

Enzyme Fungal taxa Mechanism Applications References
Laccase Ascomycota and O2-dependent Food industry, paper Baldrian
Basidiomycota one-electron and pulp industry, (2006),
oxidation of textile industry, Majeau et al.
organic nanotechnology, (2010)
compounds synthetic chemistry,
bioremediation,
cosmetics, and so
forth.
Lignin Basidiomycota H2O2-dependent Food industry, paper Hofrichter
peroxidases one-electron and pulp industry, (2002),
oxidation of textile industry, Ruiz-Dueñas
aromatic pharmaceutical et al. (2009),
compounds industry, Hammel
bioremediation, and (1995)
so forth.
Manganese Basidiomycota H2O2-dependent Food industry, paper Hofrichter
peroxidase one-electron and pulp industry, (2002),
oxidation of Mn2+ textile industry, Ruiz-Dueñas
to Mn3+, which pharmaceutical et al. (2009),
subsequently industry, Hammel
oxidizes organic bioremediation, and (1995),
compounds so forth Hofrichter
(2010)
Tyrosinases Ascomycota, O2-dependent Cosmetic Halaouli
Basidiomycota, hydroxylation of applications, et al. (2006),
and monophenols to production of dyes, Ullrich and
Mucoromycotina o-diphenols biosensors, Hofrichter
(cresolase activity) biosynthesis and (2007)
Oxidation of o medical
diphenols to applications, and
catechols food applications
(catecholase
activity)
Dye-decolorizing Basidiomycota H2O2-dependent Dye decolorization, Ullrich and
peroxidases one-electron Oxidation of Hofrichter
oxidation of anthraquinone dyes (2007)
organic with high redox
compounds potentials
Additional
hydrolyzing
activity
Cytochrome Ascomycota, Incorporation of a Pharmaceutical Kasai et al.
P450 Basidiomycota, single atom from industry and (2010),
monooxygenases Mucoromycotina, O2 into a substrate bioindustrial Yadav et al.
and molecule, with applications (2006)
Chytridiomycota concomitant
reduction of the
another atom to
H2O
(continued)
240 N. Singh et al.

Enzyme Fungal taxa Mechanism Applications References
Nitroreductases Ascomycota, NAD(P) Reduction of TNT Rieble et al.
Basidiomycota, H-dependent to hydroxylamino (1994),
and reduction of dinitrotoluene and Esteve-
Mucoromycotina nitroaromatics to amino- Nunez et al.
hydroxylamino dinitrotoluenes (2001),
and amino(nitro) Formation of Bhushan
compounds and of mononitroso et al. (2002),
nitro functional derivatives and ring Crocker et al.
groups of cleavage products (2006),
N-containing from cyclic Scheibner
heterocycles nitramine et al. (1997)
explosives,
biomass, and dry weight is 50–1000 μg per g and 2–45 t per ha, respectively. The
hyphae length for arable, pasture and forest top soils can be up to 102 m per g,
103 m per g, and 104 m per g, respectively (Ritz and Young 2004).
Some classes of fungi tolerate optimal environmental conditions such as tem-
peratures (−5 to +60 °C) and pH (1–9) and grow at a water activity of only 0.65, or
with 0.2% oxygen. Morphologically, fungal species vary in shape and size widely
from unicellular (microscopic organisms) to multicellular forms (easily seen with
the naked eye). Individual cells range from 1 μ to 30 μ. Molds and yeasts are exam-
ple of microscopic fungi. The kingdom fungi is estimated to contain about 80,000–
100,000 described species. Most of them are pollutant degraders and belong to the
phyla Ascomycota and Basidiomycota, followed by the subphylum mucoromyco-
tina (Harms et al. 2011).
9.2.1 Ascomycota Fungi
Among all the fungal groups, meiosporic ascomycetes and mitosporic ascomycetes
account for about 64% of all described fungal species and also the largest fungal
group in terrestrial and aquatic environments (Shearer et al. 2007). Numerous mem-
bers of the subphylum Pezizomycotina (the most diverse group of the filamentous
ascomycetes) attack environmental organic hazardous. For example, species of the
genera Cladophialophora and Exophiala belong to order Chaetothyriales and
assimilate the toluene (Prenafeta-Boldú et al. 2006). Aspergillus and Penicillium
spp. belong to order Eurotiales and degrade pollutants such as aliphatic hydrocar-
bons, chlorophenols, polycyclic aromatic hydrocarbons (PAHs), pesticides, syn-
thetic dyes, and 2,4,6-trinitrotoluene (TNT) (Hofrichter et al. 1994; Pinedo-Rilla
et al. 2009; Prince 2010).
This diverse group includes microorganisms that can exist in both reproductive
states, i.e., anamorph and teleomorph states, making their classification extremely
difficult (Hibbett and Taylor 2013). This phylum contains parasitic, symbiotic, or
saprotrophic lifestyles of microorganisms and morphologies such as unicellular in
yeast and multicellular in filamentous, and dimorphic fungi can exist as mold/
hyphal/filamentous form or as yeast. Studies on the fungal diversity in some anthro-
pogenically polluted samples, including activated sludge or wastewater-treatment
plants indicate ascomycota as the dominant phylum (Weber et al. 2009; Evans and
Seviour 2012; Maza-Márquez et al. 2016). These groups of fungi have been studied
for the involvement of the intracellular enzymes such as Cytochromes P450 (CYP),
manganese peroxidase, or laccase that have degradative capability of aromatic sub-
stances such as PAHs, chlorinated hydrocarbons, and diverse xenobiotic compounds
(Marco-Urrea et al. 2015; Aranda 2016; Bovio et al. 2017). However, ascomycetes
are able to degrade a large number of compounds.
9.2.2 Basidiomycota Fungi
The phylum Basidiomycota constitutes roughly 34% of all described fungal species.
Basidiomycetes are mostly inhabiting in terrestrial environments and are rarely in
aquatic environments (Lynch and Thorn 2006). The group of macro- and microor-
ganisms having characteristic features such as formation of basidia, a bottle-shaped
cell structure containing haploid and sexual basidiospores, is the basidiomycota
division of fungi. The most fungi of basidiomycota group pass between a dikaryotic
state and diploid growth to asexual reproduction by conidia with the subsequent
formation of basidiospores during their life cycle (Alexopoulos et al. 1996). Due to
ecosystem balance and their environmental relations, these fungi have established
and enhanced different systems such as cycling of carbon and nitrogen sources. The
main mechanisms involved in the degradation of hazardous chemicals including
PAHs and aromatic compounds by using basidiomycota fungi are groups of extra-
and intracellular oxidoreductases like laccases, peroxidases, and CYPs. These
enzymes break down and modify the bonds of different compounds, mainly by
extracellular pathways (Schmidt-Dannert 2016). Several kinds of LME and fungal
mediators (Phanerochaete chrysosporium, Phlebia ochraceofulva, Pycnoporus
sanguineus, Pleurotus ostreatus, and T. versicolor) have been studied in relation to
the biodegradation of PhACs in the white-rot fungi (Yadav et al. 2006; Cajthaml
et al. 2009; Díaz-Cruz et al. 2015) (Fig. 9.1).
9.2.3 Mucoromycotina Fungi
Mucoromycotina incertae sedis, formerly known as Zygomycota, represents a het-

erogeneous group. The characteristic feature of these fungi is the formation of zygo-
spores and aplanospores in the sexual phase and asexual phase, respectively (Benny
et al. 2014). Among all the lifestyles present in this group including mutualists,
242 N. Singh et al.
Fig. 9.1 The biochemical pathway of polycyclic aromatic hydrocarbon biodegradation by fungi
(Harms et al. 2011)
saprophytes, and pathogens, members of Mucorales group (core group of the tradi-
tional Zygomycota) are facultative parasites/saprobes in nature. Cunninghamella
elegans, member of Mucorales, have the ability to reproduce regioselective and
stereoselective transformations in mammal enzymatic systems due to this nature; it
has been extensively used as model fungi in different studies on the metabolism of
xenobiotics. The subphylum Mucoromycotina part of the basal fungal lineage
accounts for less than 1% of all described. In this subphylum, the genera
Cunninghamella, Mucor, and Rhizopus (members of the order Mucorales) include
degraders of PAHs, pesticides, textile dyes, and TNT (Scheibner et al. 1997; Pinedo-
Rilla et al. 2009; Prince 2010). The high ability of this fungus group to transform a
broad range of compounds has been studied in detail. The mechanism involved in
the biotransformation of PAHs by C. elegans includes phase I and phase II reac-
tions. The phase I reaction includes oxidation, reduction, and hydrolysis that gener-
ate hydroxylated metabolites such as 2-hydroxycarbamazepine, hydroxyflumequine,
hidroxywarfarin, etc. and sulfoxidated products such as chlorpromazine sulfoxide.
These reactions are highly regio- and stereoselective. Sometimes, these compounds
undergo phase II reactions to form conjugated metabolites such as fluoresomidearyl
glucoside. Other representatives of Mucoromycotina such as Umbelopsis ramanni-
ana and Mucor rammanianus have been investigated in microbial transformations
of carbamazepine (Kang et al. 2008) and fluoroquinolones, such as sarafloxacin and
enrofloxacin, respectively (Parshikov et al. 2000, 2001).
9.3 Fungal Enzymes Involved in Remediation
As a type of bioremediation technology, microbial remediation, including bioaug-

mentation or biostimulation, biodegrades petroleum hydrocarbons contained in
petroleum-contaminated soil by using the growth activities of microorganisms
(Wang et al. 2012; Kuppusamy et al. 2016a; Rana et al. 2019; Yadav et al. 2017a,
2018). In remediation studies of the petroleum-contaminated soil, investigators
have found several microorganisms which can be used in biodegradation of petro-
leum hydrocarbons (Kuppusamy et al. 2016b; Liu et al. 2017). White-rot fungi have
been reported to degrade petroleum hydrocarbons by secreting some enzymes like
lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Canet et al.
2001; McErlean et al. 2006). Fungi play a major role as decomposers and symbionts
in all ecosystems (soil and aquatic habitats) owing to their robust morphology and
diverse metabolic capacity, and because of that, they are best suited for the purpose
of bioremediation. In bioremediation, when fungi are used as degrader to decon-
taminate contaminated areas, it is termed as mycoremediation (Shearer et al. 2007).
There has been increasing interest in the special capacity of fungi to degrade pollut-
ants by employing a number of extracellular and intracellular enzyme systems such
as peroxidases and cytochrome P450, respectively, for the biodegradation and
detoxification (Jebapriya and Gnanadoss 2013; Morel et al. 2013; Durairaj et al.
2015). In contrast to bacteria, white-rot fungi require a higher nutrition level for
growth but have a higher-grade oxidase system presenting better degradation ability
for complex petroleum hydrocarbons (D'Annibale et al. 2006; Mohan et al. 2006;
Liu et al. 2017). Figure 9.2 represents the mechanism of bioremediation of hazard-
ous chemicals used by fungi.
Fig. 9.2 Mechanism of bioremediation of hazardous chemical used by fungi (Deshmukh et al.
2016)
244 N. Singh et al.
9.3.1 Laccase
Laccases constitute a family of multicopper oxidases belonging to group of blue

oxidases which use copper as cofactor and molecular oxygen as cosubstrate. It is
produced by certain plants, fungi, insects, and bacteria. Laccases catalyze mainly
the oxidation of a wide range of reduced phenolic and aromatic substrates with
concomitant reduction of molecular oxygen to water (Mate and Alcalde 2017;
Yadav et al. 2016, 2017b). There are multiple isoenzymes of laccases that are known
to occur which are encoded by a separate gene (Giardina et al. 1995). The nonspeci-
ficity of laccases makes them ideal for a variety of substrate catalysts for a variety
of industrial applications of which these enzymes have been extensively explored
for their effective potential for bioremediation (Baldrian 2006; Hildén et al. 2009).
However, the mechanism of action of laccases under extreme conditions is less
explored. The crystal structure of only a few laccases from ascomycetes
(Melanocarpus albomyces (MaL) and Thielavia arenaria (TaLcc1) is fully known
(Arora and Rampal 2002). In spite of their incredible potential of bioremediation,
the utility of laccases is limited by their low shelf life. Among the biological agents,
laccases represent an interesting group of ubiquitous, oxidoreductase enzymes that
show promise of offering great potential for biotechnological and bioremediation
applications (Gianfreda et al. 1999).
9.3.2 Catalase
As reported in other biological systems, generation and accumulation of reactive

oxygen species (ROS) mainly result in damage to macromolecules (cellular), which
is deleterious for cellular integrity. Primary defense mechanism to ROS generation
in fungi consists of monofunctional catalases and bifunctional peroxidase/ catalase
enzymes. It has been suggested that catalase activity could be used as monitoring
tool for monitoring bioremediation efficiency since their study revealed that cata-
lase activity decreased with increasing oil concentration during bioremediation of
oil contaminated soil (Deshmukh et al. 2016).
Thus, considering the significance of catalases in providing heavy metal toler-
ance capacity to fungi, fungi producing this enzyme can be promising candidates
for bioremediation of metal contaminated sites (Marco et al. 2013).
9.3.3 Tyrosinases
Tyrosinases are produced by some fungal species including Ascomycota,

Basidiomycota, and Mucoromycotina. It is a natural enzyme and is often purified to
only a low degree and it is involved in a number of functions which primarily
catalyze the o-hydroxylation of monophenols into their corresponding o-diphenols

and further the oxidation of o-diphenols into o-quinones using molecular oxygen,
which finally polymerizes to form brown or black pigments (Zaidi et al. 2014).
According to Ba and Kumar (2017), tyrosinases has been investigated as “green
products” in the removal of several chemical contaminants in waters as well as soils.
Different fungal groups have been studied for the isolation of tyrosinase and
obtained from Agaricus bisporus, Neurospora crassa, Amanita muscaria, Lentinula
edodes, Aspergillus oryzae, portobello mushrooms, Pycnoporus sanguineus, and
Lentinula boryana (Zaidi et al. 2014).
9.3.4 Peroxidase
Peroxidase catalyzes the oxidation of a wide variety of chemical compounds.

This enzyme occurs in plants, animals, and microorganisms. The biological func-
tions and specificity of peroxidases vary with sources of the enzyme. Some fun-
gal groups such as Basidiomycota and Ascomycota secreted extracellular
enzymes that play important role in bioremediation (Silva et al. 2016). On the
basis of source and activity of peroxidases, it is classified into lignin peroxidase
(LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Among per-
oxidases, LiP, MnP, and VP have been studied the most because of their high
potential to degrade toxic substances in nature. LiP are heme proteins mainly
secreted during secondary metabolism by the white-rot fungus. LiP plays a cen-
tral role in the biodegradation of the plant cell wall constituent lignin. Degradation
of lignin and other phenolic compounds is mediated by LiP in the presence of
cosubstrate H2O2 and mediator like veratryl alcohol. LiPs have higher redox
potential to oxidize aromatic compounds, and the exact redox mechanism is still
poorly understood. MnP is an extracellular heme enzyme that oxidizes Mn2+ to
the oxidant Mn3+ in a multistep reaction from the lignin-degrading basidiomyce-
tes fungus. It has been reported that white-rot and basidiomycetes fungi degraded
most of toxic compounds. On the other hand, VP enzymes are broad substrate-
specific enzymes having capability of oxidizing both phenolic and nonphenolic
compounds and are highly valued for biotechnological processes such as biore-
mediation (Hiner et al. 2002; Koua et al. 2009).
There are some other peroxidases (Coprinopsis cinereal peroxidase, dye-
decolorizing peroxidases, and Caldariomyces fumago heme-thiolate chloroper-
oxidase) secreted extracellularly by Basidiomycota and Ascomycota. Among
these, Coprinopsis cinereal peroxidase and dye-decolorizing peroxidases cata-
lyze H2O2- dependent one-electron oxidation of organic compounds.
Caldariomyces fumago heme-thiolate chloroperoxidase catalyze H2O2-dependent
peroxygenation (two- electron oxidation), leading to epoxidation of (cyclo)
alkenes, hydroxylation of benzylic carbon, and sulphoxidation of S-containing
organic compounds (Liers et al. 2011).
246 N. Singh et al.
9.3.5 Fungal Unspecific Peroxygenases
A secreted heme-thiolate peroxidase with promiscuity for oxygen transfer reactions

was discovered in the basidiomycetous fungus, Agrocybe aegerita. The enzyme
turned out to be a functional monoperoxygenase that transferred an oxygen atom
from hydrogen peroxide to diverse organic substrates like aromatics, heterocycles,
linear and cyclic alkanes/alkenes, fatty acids, etc. (Karich et al. 2017). Later, similar
enzymes were investigated from other genera of mushroom such as Coprinellus and
Marasmius (Hofrichter et al. 2014). This new enzyme type was classified as unspe-
cific peroxygenase (UPO, EC 1.11.2.1) and placed in a separate peroxidase sub-
class. Furthermore, UPOs and related heme-thiolate peroxidases such as well-studied
chloroperoxidase (CPO) represent a separate superfamily of heme proteins on the
phylogenetic level. The reactions catalyzed by UPOs include hydroxylation, epoxi-
dation, O- and N-dealkylation, aromatization, sulfoxidation, N-oxygenation,
dechlorination, and halide oxidation (Hofrichter et al. 2015; Peter et al. 2013).
9.3.6 Monooxygenases
Monooxygenases are classified into two subclasses based on the presence cofactor:
flavin-dependent monooxygenases and P450 monooxygenases. Oxygenases are grouped
into two categories, the monooxygenases and dioxygenases, on the basis of number of
oxygen atoms used for oxygenation. The enzymes of this group are generally cell
bound. They play a key role in the metabolism of organic compounds by increasing
their reactivity or water solubility or bringing about cleavage of the aromatic ring.
Oxygenases have a broad substrate range and are active against a wide range of com-
pounds, including the chlorinated aliphatic. The desulfurization, dehalogenation, deni-
trification, ammonification, hydroxylation, biotransformation, and biodegradation of
various aromatic and aliphatic compounds are catalyzed by monooxygenases. These
properties have been explored in recent years for important application in biodegrada-
tion and biotransformation of aromatic compounds (Arora et al. 2010).
9.3.7 Phenol 2-Monooxygenases
Phenol 2-monooxygenases are studied under flavin-dependent monooxygenases

that contain flavin as prosthetic group and require NADP or NADPH as coenzyme.
Monooxygenases act as biocatalysts in bioremediation process and synthetic chem-
istry due to their high region selectivity and stereoselectivity on a wide range of
substrates. Majority of monooxygenases studied previously are having a cofactor,
but there are certain monooxygenases which function independently of a cofactor.
These enzymes require only molecular oxygen for their activities and utilize the
substrate as reducing agent (Cirino and Arnold 2002; Arora et al. 2010).
9.3.8 Cytochrome P450 Monooxygenases
Fungi possess complex oxidative and hydrolytic enzymatic systems for detoxifying
toxic compounds in the environment. Besides these systems, certain fungi possess
intracellular networks which constitute the xenome, consisting of cytochrome
(CYP) P450 monooxygenases for dealing with diverse range of pollutants. The fun-
gal cytochrome P450 system can serve as versatile catalyst for region- and stereo-
specific oxidation of nonactivated hydrocarbons and can be ideal substitutes for
chemical catalysts (Urlacher and Girhard 2012). Ichinose (2013) has highlighted
the significance of cytochrome P450 systems in metabolism of series of endogenous
and exogenous compounds. CYP63A2 P450 monooxygenase from white-rot fun-
gus P. chrysosporium oxidized crude oil aliphatic hydrocarbon n-alkanes, endocrine-
disrupting long-chain alkylphenols (APs), and mutagenic/carcinogenic fused-ring
high molecular weight PAHs (HMW-PAHs) (Syed et al. 2013). Preinduction of the
P450 monooxygenase before application in degradation studies could result in
enhanced PAH removal. Enhanced removal of pollutants also achieved by molecu-
lar tools aimed at rapid and over production of cytochrome P450 monooxygenase
(Bhattacharya et al. 2013; Guengerich and Munro 2013; Theron et al. 2014)
9.3.9 Nitroreductases
Among the enzymes identified as involved in the degradation of TNT are the nitro-
reductases. This group of enzymes reduces a wide range of nitroaromatic com-
pounds such as nitrofurazones, nitroarenes, nitrophenols, and nitrobenzenes
including explosives such as TNT, RDX, and GTN.
TNT (2,4,6-trinitrotoluene) degradation by fungi is initiated by mycelia bound
nitroreductases which reduce TNT to hydroxyl amino dinitro toluene and aminodi-
nitrotoluenes. Further degradation of these products and mineralization is achieved
through the activity of oxidative enzymes especially lignin degrading enzymes (lig-
nin and manganese peroxidases). Nitroreductases have generated a significant
amount of interest recently for application in bioremediation of nitroaromatic com-
pounds (Spain 1995).
9.3.10 Quinone Reductases
It has been reported the involvement of quinone reductases in the degradation of

several chlorinated aromatics by P. chrysosporium. Occurrence of quinone reduc-
tases in white-rot and brown-rot basidiomycetes has been reported. It catalyzes
NAD(P)H-dependent reduction of quinones and is a cell-bound enzyme
(Akileswaran et al. 1999).
248 N. Singh et al.
9.3.11 Reductive Dehalogenases
Reductive dehalogenation proceeds via the replacement of a halide ion by a hydro-

gen atom and requires the transfer of two electrons. The role of electron donor can
be fulfilled by a reduced organic substrate or H2 (Habash et al. 2004).
9.3.12 Miscellaneous Transferases
Various transferases act on hydroxyl groups of pollutants and their metabolites to

catalyze the formation of conjugates. which are typically not subject to further fungal
degradation. These are also cell-bound enzymes. Detoxification through the excretion
of water-soluble conjugates is well documented for the fungal metabolism of PAHs
and other organic pollutants (Hundt et al. 2000; Cerniglia and Sutherland 2010).
9.4 Fungal Degradation of Hazardous Chemicals
Certain hazardous chemical compounds, including polycyclic aromatic hydrocar-

bons (PAHs), penta-chlorophenols (PCPs), polychlorinated biphenyls (PCBs), poly-
chlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs),
1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT), benzene, toluene, ethylben-
zene, and xylene (BTEX), trinitrotoluene (TNT), and heavy metals, are persistent in
the environment and have carcinogenic and/or mutagenic effects. Environment is
being loaded with a large quantum of such types of contaminants and recalcitrant
compounds. Many conventional physicochemical methods of treatment/removal of
these compounds, though effective, are not feasible for application on large scale
(Akcil et al. 2015; Rastegari et al. 2019; Yadav et al. 2019a, b). Other toxic materials
include polychlorinated biphenyls, dioxins, phenols, chlorophenols, pesticides, efflu-
ents from pulp and paper mills, dyestuffs, and heavy metals that have been success-
fully degraded by using white-rot fungi (Bennett 2007). Bioremediation does not
involve only the degradation of pollutants, but also it is sufficient to remove the pol-
lutant from the environment without degrading it (Broda 1992).
However, bioremediation by fungal species has been reported to recognize as
environment-friendly and economical. In the bioremediation, the efficient conver-
sion of toxic and recalcitrant compounds into nontoxic or less toxic products takes
place by applying natural biological processes, especially in the case of contami-
nated land and water. The technique involved in this process is use of suitable
microbes in the polluted area which perform different physical and chemical reac-
tions (as a part of their metabolism) resulting in degradation and removal of pollut-
ants (Gillespie and Philip 2013; Mishra and Malik 2014). Bioremediation of
hazardous chemicals can involve either natural attenuation, biostimulation, and bio-
augmentation processes or a combination thereof. These processes have been appro-
priately demonstrated during the bioremediation of atrazine (Sagarkar et al. 2013),

petroleum hydrocarbons, and TNT in soil microcosms (No˜lvak et al. 2013; Lien
et al. 2015). Bioremediation can be considered as a “green technology” due to its
dependency on biological organisms and processes, and it does not require any
treatment such as chemical addition or heating (Juwarkar et al. 2010).
Bioremediation technology for microbial remediation involves bioaugmentation
and/or biostimulation that biodegrades petroleum hydrocarbons contained in
petroleum-contaminated soil. Several biodegrades of petroleum hydrocarbons have
been found in remediation of petroleum-contaminated soil. Of these, there are some
bacterial species (isolated from petroleum-contaminated soil) that grow with petro-
leum hydrocarbons by using their carbon source, and white-rot fungi also degrade
petroleum hydrocarbons by secreting enzymes such as LiP, MnP, and laccase. In
contrast to bacteria, white-rot fungi have a higher grade of oxidase system and deg-
radation ability for complex petroleum hydrocarbons. White-rot fungi also need a
higher nutrition level (Liao et al. 2012).
A number of researchers reported the widespread use of pesticides in public
health and agricultural programs has caused severe environmental pollution and
health hazards. The use of fungal species in degradation of pesticides has been
reported in the literature (Singh et al. 2017). A large number of fungal species have
been identified for their ability to degrade phenylurea herbicides (Khadrani et al.
1999). Sometimes, the fungal species capable of degrading pesticide have been
shown to utilize the compound as a source of carbon and nitrogen (Kulshrestha and
Kumari 2011). The filamentous fungus belonging to the group of nonligninolytic
fungus, i.e., Cunninghamella elegans, a typical soil fungus, has been studied exten-
sively for its ability to transform PAHs that migrate to the sediment in aquatic eco-
systems due to their hydrophobic nature and low water solubility (Cerniglia 1997;
Moody et al. 2004). The same fungi can rapidly oxidize PAH compounds such as
benz-anthracene, 6-nitrochrysene, 6-nitrobenzo-pyrene, 3-nitrofluoranthene,
2-nitrofluorene, dibenzofuran, 1-nitropyrene, fluoranthene, naphthalene, acenaph-
thene, fluorene, phenanthrene, and nitrochrysene into an array of metabolites. Some
other species, such as Cyclothyrium sp., Penicillium simplicissimum, and Psilocybe
sp., transformed pyrene, anthracene, phenanthrene, and benzo[a]pyrene (Silva et al.
2003; Silva and Esposito 2004; Hammel et al. 1991). Degradation of the benzene-
toluene-ethylbenzene-xylene (BTEX) group of organopollutants by the white-rot
fungus P. chrysosporium was studied (Oh et al. 1998; Yadav and Reddy 1993).
9.5 Recent Advances
With recent advances in biomolecular engineering, the bioremediation of persistent

organic pollutants using genetically modified microorganisms has become a rapidly
growing area of research for environmental protection. Biomolecular engineering
holds opportunities for the rapid advancement of bioremediation technology and
offers the prospect of degrading some of the most recalcitrant. Most of the examples
250 N. Singh et al.
focused on the redesign of various features of the enzymes involved in the bioreme-
diation of persistent organic pollutants, including the enzyme expression level,
enzymatic activity, and substrate specificity. Now, with the recent advances in bio-
molecular engineering, the prospect of short circuiting the process of natural evolu-
tion to degrade environmental xenobiotic pollutants has been created.
Environmental hazard is growing largely due to the indiscriminate and frequently

deliberate release of hazardous, harmful substances into the atmosphere. Chemicals
that are used in domestic, industrial, and agricultural processes may lead to environ-
mental complications when they deteriorate the quality of soil as well as groundwa-
ter. Among biological agents, enzymes have a biochemical potential that can be
effectively transform and detoxify polluting substances. Simply, bioremediation is
the use of biological organisms for cleaning up hazardous chemical by decreasing
the toxicity levels of chemical compounds and restoring in its natural conditions.
Fungal enzymes are capable of metabolizing, immobilizing, or absorbing toxic
compounds from their environment. However, a major advantage of these systems is
that they are less harmful to the environment with minimum or no by-products. Thus,
evaluation of earlier research work may contribute to understanding that microbes
are capable of completely removing pollutants from the environment and also leads
to synthesis of useful compounds as by-products. Bioremediation is a cost-effective
and eco-friendly biotechnological treatment that is capable of reducing and even
eliminating pollution powered by microbial enzymes.
Acknowledgments NS and AK are grateful to the University Grant Commission, New Delhi, for
providing financial assistance in the form of a Research Fellowship. The authors acknowledge
UGC-SAP and DST-FIST for the support to the Department of Biochemistry, University of
Allahabad, Allahabad, India. The authors declare no conflict of interest.
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Chapter 10
Biotechnological Applications
of β-Glucosidases in Biomass Degradation
Sushma Mishra, Deepika Goyal, Amit Kumar, and Prem Kumar Dantu
10.1 Introduction
Cellulose, the chief chemical constituent of primary cell walls, forms the most
abundant group of carbohydrates produced by plants. For example, the cellulose
content forms ~90% in cotton fibres, 40–50% in wood and approximately 57% in
dried hemp. In contrast to starch, cellulose is an unbranched linear polymer of
D-glucose units linked by β(1→4) glycosidic linkages between C#1 of one glucose
and C#4 of the next glucose (Shahzadi et al. 2014). In fact, it is these beta linkages
present between the monomer units that enable the formation of long, rigid chains
of cellulose microfibrils that bear numerous intra- and intermolecular hydrogen
bonds. The chains are oriented in parallel and form highly ordered, crystalline
domains that are responsible for high tensile strength of plant cell walls (Beguin and
Aubert 1994; Shahzadi et al. 2014). In nature, cellulose is degraded mainly by fungi
and bacteria. The degradation of cellulose to glucose molecules is catalysed by the
synergistic activity of three individual enzymes (Fig. 10.1): endoglucanase
(1,4-β-D-glucan hydrolase, EC 3.2.1.4), exoglucanase (1,4-β-D-glucan glucohy-
drolase EC 3.2.1.74) and β-glucosidase (β-D-glucoside glucohydrolase EC3.2.1.21)
(Dashtban et al. 2010; Tiwari et al. 2013; Seo et al. 2013; Lambertz et al. 2014).
Exoglucanases, also known as cellobiohydrolase, hydrolyse cellulose polymers
from the ends releasing mainly cellobiose, a disaccharide consisting of two
β-glucose molecules. Endoglucanases hydrolyse glucosidic bonds at random posi-
tions in cellulose chains to generate oligosaccharide chains of different length, also
S. Mishra (*) · D. Goyal · P. K. Dantu

Department of Botany, Dayalbagh Educational Institute, Deemed University,
Dayalbagh, Agra, India
A. Kumar
Host Plant Section, Central Muga Eri Research & Training Institute,
Central Silk Board, Lahdoigarh, Jorhat, Assam, India

258 S. Mishra et al.
CELLULOSE CHAIN
CELLOBIOSE
GLUCOSE
Exoglucanase
Endoglucanase
β 1,4-glucosidic bond
β-glucosidase
Glucose monomer
Fig. 10.1 Cellulose degradation by the synergistic action of endoglucanase, exoglucanase and
β-glucosidase
producing new sites to be attacked by exoglucanases. Finally, β-glucosidase breaks

down cellobiose and short oligosaccharides into glucose units (Kumar et al. 2008;
Sukumaran et al. 2005). In other words, in the enzymatic hydrolysis of cellulose,
endoglucanases and exoglucanases are responsible for degrading cellulose to
cellobiose, after which β-glucosidases hydrolyse cellobiose to free glucose mole-
cules. In this process, the step catalysed by β-glucosidases is generally the rate-
limiting step and hence is responsible for the regulation of the entire cellulose
degradation process. This inhibition is mainly caused due to the inhibitory effects
by cellobiose on both endoglucanase and exoglucanase activities (Bok et al. 1998;
Kruus et al. 1995).
The most widely accepted system of classification of β-glucosidases is based on
their nucleotide sequence identity (NCI) and hydrophobic cluster analysis (HCA).
In this system, enzymes with overall amino acid sequence similarity and well-
conserved sequence motifs are placed in a family (Henrissat 1991; Cairns and Esen
2010). According to the data collected in June 2018, 153 glycoside hydrolase (GH)
families are listed in Carbohydrate Active enZYme website (http://www.cazy.org).
This is the most widely accepted classification, and β-glucosidases are placed in
glycoside hydrolase (GH). Most of the β-glucosidases are reported in GH1, GH3,
GH5, GH9, GH30 and GH116 families. HCA system of classification is believed
to reflect structure, evolutionary relationship and catalytic mechanisms of this
10 Biotechnological Applications of β-Glucosidases in Biomass Degradation 259
enzyme (Cairns and Esen 2010). β-Glucosidases belonging to GH family 1 are

mainly reported from archaebacteria, plants and animals, whereas β-glucosidases
belonging to GH family 3 are from bacteria, fungi and yeast.
β-Glucosidases could also be classified on the basis of substrate specificity into
three classes: aryl-β-glucosidases that hydrolyse only aryl-β-glucoside linkage,
cellobiases that hydrolyse only disaccharides and broad substrate specificity
β-glucosidases that hydrolyse wide range of substrates with different bonds such as
β(1→4), β(1→3), β(1→6), α(1→4), α(1→3) and α(1→6) (Singh et al. 2016).
Cellulose recycling forms an important part of the carbon cycle in biosphere, as
it is the major carbohydrate synthesised by plants. As mentioned above, this degra-
dation process is brought about by the synergistic activity of a series of enzymes,
where the terminal steps catalysed by β-glucosidases form the rate-limiting step.
Consequently, the entire cellulolytic process is limited by the activity of this enzyme.
Hence, an increased understanding of the factors affecting β-glucosidase activity
would promote efficient conversion of the otherwise abundant cellulose into the
much needed biofuels and other economically important products. With this objec-
tive in mind, the authors have tried to present an overview of β-glucosidase enzyme
and its biotechnological applications, followed by major rate-limiting components
and possible solutions for large-scale conversion of lignocelluloses into ethanol.
10.2 Sources of β-Glucosidases
β-Glucosidases are a class of hydrolytic enzymes produced by various organisms,

ranging from microorganisms to higher plants and animals. Wood-degrading organ-
isms like termites and wood-decomposing fungi have been generally targeted by the
researchers for isolating cellulolytic enzymes (Sanderson 2011). Among plants,
β-glucosidases have been well characterised from Arabidopsis thaliana, rice, cherry,
wheat, sorghum and maize (Tiwari and Verma 2017; Sue et al. 2006; Dharmawardhana
et al. 1995; Seshadri et al. 2009; Kittur et al. 2007). Some of the in-planta functions
of β-glucosidases include chemical defence, plant–microbe interactions, cell wall
remodelling, alkaloid metabolism and phytohormone regulation (Seshadri et al.
2009; Singh et al. 2016).
However, for industrial production of β-glucosidases, fungi are the best source
due to several advantages like high yield, fast growth, cost-effectiveness, etc. (Kour
et al. 2019b; Yadav et al. 2015, 2016a, b, 2017b, 2019a, b). Many fungi such as
Trichoderma reesei (Chen et al. 1992), the filamentous fungus Acremonium persici-
num (Pitson et al. 1997), Aspergillus oryzae (Riou et al. 1998), Thermoascus auran-
tiacus (Parry et al. 2001), Chaetomium thermophilum (Venturi et al. 2002),
Penicillium purpurogenum (Karnchanatat et al. 2007), Daldinia eschscholzii
(Kaur et al. 2007), Melanocarpus sp. MTCC 3922 (Chen et al. 2012), Neocallimastix
patriciarum W5 (Daroit et al. 2008), Monascus purpureus and brown-rot basidio-
mycete Fomitopsis palustris (Yoon et al. 2008) produce β-glucosidase (Tables 10.1
and 10.2). In addition, this enzyme has recently been produced from Penicillium
Table 10.1 Large-scale production methods of β-glucosidases from fungi

Fungal species Fermentation method References
Tolypocladium cylindrosporum Submerged fermentation (SMF) Bai et al. (2013)
Syzx4
Penicillium simplicissimum Submerged fermentation Elyas et al. (2010)
H-11
Aspergillus strain SA 58 Solid-state fermentation (SSF) Ng et al. (2010)
Penicillium citrinum YS40-5 Solid-state fermentation Bhatti et al. (2013)
Fusarium proliferatum Submerged fermentation Gao et al. (2012)
Fusarium solani Solid-state fermentation Raza et al. (2011)
Aspergillus niger + A. oryzae Solid-state fermentation Vaithanomsat et al. (2011)
Fomitopsis palustris Submerged fermentation Yoon et al. (2008)
Aspergillus niger SOI017 Submerged fermentation Qian et al. (2012)
Flammulina velutipes Submerged fermentation Mallerman et al. (2015)
Monascus sanguineus Solid-state fermentation Dikshit and Tallapragada
(2015)
Phoma sp. KCTC11825BP Submerged fermentation Choi et al. (2011)
Thermomucorindicae- Solid-state fermentation Ling et al. (2011)
seudaticae N31
Aspergillus niger HDF05 Solid-state fermentation Cassia et al. (2015)
Gongronella butleri Solid-state fermentation Ling et al. (2011)
Penicillium miczynskii Submerged fermentation Beitel and Knob (2013)
Fusarium oxysporum Submerged fermentation Santos et al. (2016)
Aureobasidium pullulans Submerged fermentation Saha et al. (1994)
Candida peltata Submerged fermentation Olajuyigbe et al. (2016)
Kluyveromyces marxianus Submerged fermentation Rajoka et al. (2004)
Aureobasidium sp. Solid-state fermentation + Saha and Bothast (1996)
submerged fermentation
Saccharomyces cerevisiae Submerged fermentation Iembo et al. (2002)
purpurogenum KJS506, Phoma sp. KCTC11825BP (Choi et al. 2011), Aspergillus

fumigatus Z5 (Liu et al. 2012), Penicillium italicum (Park et al. 2012), Fusarium
proliferatum NBRC109045 (Gao et al. 2012), Aspergillus saccharolyticus (Sorenson
et al. 2014), Aspergillus niger A20 (Abdel-Naby et al. 1999), Fusarium solani
(Bhatti et al. 2013), Flammulina velutipes (Mallerman et al. 2015), Monascus san-
guineus, Sporothrix schenckii (Hernández et al. 2016), Gongronella butleri (Santos
et al. 2016) and Fusarium oxysporum (Olajuyigbe et al. 2016). The fungal species,
Aspergillus niger, is the major source of commercial β-glucosidase under the name
of Novazym188 (Sorenson et al. 2013).
β-Glucosidase has been identified, purified and characterised from several
bacterial species as well, such as Clostridium thermocellum (Ait et al. 1982),
Pyrococcus furiosus (Kengen et al. 1993), Bacillus circulans subsp. Alkalophilus
(Paavilainen et al. 1993), Flavobacterium johnsoniae (Okamoto et al. 2000), actino-
mycete Thermobifida fusca (Spiridonov and Wilson 2001), Lactobacillus brevis
(Michlmayr et al. 2010), Caldicellulosiruptor saccharolyticus (Hong et al. 2009)
Table 10.2 Optimum physical parameters for industrial production of β-glucosidases from
microbial sources
Preferred Preferred
carbon nitrogen Preferred Preferred
Microorganisms source source temp. pH References
Aspergillus niger Wheat bran Ammonium 30 °C 5.5 Raza et al. (2011),
sulphate Vaithanomsat et al.
(2011)
Penicillium Sucrose Sodium nitrate 28 °C 4.0 Jeya et al. (2010), Jeya
purpurogenum and Lee (2013)
Candida peltate Glucose and – 50 °C 5.0 Saha and Bothast
xylose (1996), Rajoka et al.
(2004)
Fusarium Corn stover Urea 25 °C 5.0 Gao et al. (2012)
proliferatum and wheat
bran
Chaetomium Cellulose Peptone, yeast 45 °C 5.5 Venturi et al. (2002)
thermophilum extract
Aspergillus Glucose Ammonium 30 °C 3.0 Yadav et al. (2016a, b)
protuberus sulphate
Penicillium Maltose Ammonium 70 °C 5.0 Ng et al. (2010)
citrinum sulphate
and Terrabacter ginsenosidimutans (An et al. 2010). Thermoanaerobacterium

thermosaccharolyticum is known to produce a glucose-tolerant β-glucosidase
(Pei et al. 2012).
10.3 Biotechnological Applications of β-Glucosidases
The use of cellulases in biotechnology began in the early 1980s in animal feed and
food industry (Chesson 1987; Thomke et al. 1980). Subsequently, these enzymes
were used in textile, laundry as well as in pulp and paper industries (Godfrey et al.
1996; Wong and Saddler 1992). Today, β-glucosidases are also used for production
of biofuels, detoxification of cassava cyanogenic glucosides and in the treatment of
Gaucher’s disease (Cairns and Esen 2010; Prasad et al. 2012). The catalytic activity
of β-glucosidases include hydrolysis of β(1–4), β(1–3), β(1–6) and β(1–2) gluco-
sidic linkages in aryl-, amino-, or alkyl-β-D-glucosides, cyanogenic glucosides, and
disaccharides, polysaccharides and glucose-substituted molecules. Apart from
hydrolysis of sugars, some mutant β-glucosidases have been reported to catalyse
synthetic reactions of sugars by reverse hydrolysis and transglycosylation
(Mackenzie et al. 1998). Due to the diverse types of reactions and substrates of
β-glucosidases, they have several industrial applications, some of which have been
described below.
10.3.1 Conversion of Lignocellulose into Biofuels
Lignocellulose refers to the woody parts of plants, mainly agricultural residues like
wheat stems, corn stalks and leaves, and forestry wastes like wood shavings from
logging. The lignocellulose, which is generally considered to be ‘plant waste’ and
discarded, could be converted into energy sources. These so-called second-
generation biofuels have many advantages in comparison to first-generation biofu-
els that were derived mainly from food crops (Sanderson 2011; Guo et al. 2015;
Prasad et al. 2014; Rastegari et al. 2019; Prasad et al. 2019; Yadav et al. 2017a,
2018). Apart from being a renewable and sustainable way of energy production, the
bioconversion of lignocellulose into ethanol has an additional advantage of solving
the problem of waste disposal of agricultural residues and other biomass
(Khan et al. 2018, 2019). Lignocelluloses, chemically made up of cellulose,
hemicellulose and lignin, which when converted into fermentable sugars, could be
used to produce liquid fuels like ethanol or oil and gaseous fuels like biogas and
electricity (Menon and Rao 2012; Prasad et al. 2009, 2013; Kour et al. 2019a; Rana
et al. 2019a, b). Briefly, the lignocellulose is first subjected to heat and acid or
ammonia to separate lignin, thereby exposing cellulose and hemicelluloses.
Thereafter, the combined action of exoglucanase, endoglucanase and β-glucosidases
converts this polymer into glucose sugar, which could then be fermented to give rise
to ethanol or the longer chain alcohol butanol. The pentose and hexose obtained
after cellulose degradation could be fermented to ethanol by the action of yeast
and certain enzymes. The preferred physical conditions required by some of the
commonly used microbial sources of β-glucosidases for lignocelluloses degradation
are mentioned in Table 10.2.
The bioconversion of lignocellulose involves two steps: hydrolysis of cellulose
in lignocellulosic biomass to produce reducing (fermentable) sugars, and fermenta-
tion of the sugars to ethanol (Sun and Cheng 2002). An outline of steps involved in
biomass degradation is presented in Fig. 10.2. Briefly, plant parts are first cut into
small size by either milling or chipping, followed by a pre-treatment step using
either physical or chemical agents. The pre-treatment step mainly disrupts the close
inter-component association between main constituents (cellulose, hemicellulose,
lignin) of the plant cell wall (Jönsson and Martín 2016). Some of the most com-
monly used pre-treatment methods include acid hydrolysis with mineral acids/
organic acids, steam heating followed by sudden decompression, hydrothermal pro-
cessing and oxidative methods (Jonsson and Martin 2016). These methods basically
aim to remove hemicelluloses and/or lignin from the lignocellulosic matrix, thereby
facilitating the subsequent enzymatic degradation of cellulose to D-glucose.
Together, endoglucanases, exoglucanases and β-glucosidases make a potent sys-
tem for cellulose degradation. These three enzymes could be present either as mul-
tienzymatic complex called cellulosome or exist as individual enzymes (Bae et al.
2013). Being the terminal enzyme in cellulose degradation pathway, β-glucosidases
play a critical role in this process (Bhatia et al. 2002). If β-glucosidases are not pres-
ent in sufficient amounts, not enough glucose will be produced, and cellobiose will
Plant debris (lignocellulosic biomass)

Cellulose Hemicellulose Lignin
PRE-TREATMENT OF LIGNOCELLULOSIC BIOMASS
Physical methods Chemical methods Biological methods

Ball milling Sodium hydroxide Pleurotus florida
Compression milling Perchloric acid Pleurotus cornucopiae
Cryomilling Acetic acid Streptomyces viridosporus
Steam treatment Ammonia freeze explosion Cyathus sp.
Ultrasound Organic solvents
Microwave
ENZYMATIC HYDROLYSIS OF PRE-TREATED CELLULOSE
FERMENTATION OF SUGARS
BIOETHANOL PRODUCTION
Fig. 10.2 Various steps involved in degradation of plant biomass. The pre-treatment of lignocel-
lulosic biomass could be done either by physical, chemical or biological methods. Subsequently,
the plant biomass is subjected to cellulase degradation to convert cellulose to sugars, which could
be fermented to yield ethanol
accumulate. Since cellobiose is an inhibitor of endo- and exoglucanases, this would

negatively affect glucose formation, making it the rate-limiting step of the pathway
(Dekker 1986). Therefore, the activity of β-glucosidase could be regulated to
increase the efficiency of conversion of cellulose to glucose (Dashtban et al. 2010;
Lambertz et al. 2014). This aspect has been dealt with in detail in the next section
on ‘Challenges in Lignocellulose Bioconversion’.
10.3.2 Production of High-Value Bioproducts
Apart from ethanol, which forms the primary product of lignocellulose degradation,
the by-products could be used to generate a number of organic chemicals. For
example, the fermentation of hexoses and pentoses obtained after cellulose degrada-
tion could be used to produce lactic acid by using the bacteria, Bacillus coagulans
(Patel et al. 2006). Likewise, the lignocelluloses conversion of palm waste produces
xylose, which could be further processed to form xylitol, a sweetening agent

(Rahman et al. 2007). Another by-product, furfural, obtained from hydrolysis of
hemicelluloses, is reported to be used in plastic, varnishes and herbicide preparation
(Montane et al. 2002).
10.3.3 Hydrolysis of Isoflavone Glycosides
Phenolic compounds like flavonoids, flavonones, flavones and isoflavones form a

class of secondary metabolites in plants that have antioxidant, anticancerous, antial-
lergic, anti-inflammatory and antihypertensive properties (Kabera et al. 2014;
Karimi et al. 2012; Servili et al. 2013). Majority of these metabolites are present in
the form of glycosides, which increase their water solubility and stability, but limit
their absorption. The release of non-carbohydrate part requires the action of specific
enzymes such as arabinosidases and β-glucosidases. For example, daizdin, genistin
and glycitin are some of the glucosidic isoflavones in soybean and soy (a soybean-
based food), which are generally found in an inactive state. Aglycone forms of these
isoflavones, produced by β-glucosidases, exhibit phytoestrogenic properties and
hence are useful in the treatment of various diseases like prostate cancer, breast
cancer, cardiovascular disease and menopause treatment (Izumi et al. 2000; Hati
et al. 2015). The different microbial sources of β-glucosidases for the hydrolysis of
isoflavone or flavonoid compounds are represented in Table 10.3.
10.3.4 Flavour and Nutrition Enhancement
In plants, flavour compounds generally occur in the form of glycoconjugates, in

order to suppress the flavour and make them non-volatile. The β-glucosidase
enzyme releases the glycoconjugate form of flavour compounds, thereby impart-
ing the unique flavour to plants. For example, β-glucosidase isolated from
Sporidiobolus pararoseus and Aureobasidium pullulans have been found to hydro-
lyse terpenyl glycosides and improve aroma of wines (Baffi et al. 2013). Likewise,
it has also been reported to improve the organoleptic properties and reduce the
bitterness of citrus fruit, which is caused by the glucosidic compound, naringin
(Roitner et al. 1984). β-Glucosidase isolated from Bacillus subitilis is used for
improving sugarcane juice quality (Singh et al. 2016) by immobilising it on algi-
nate beads for industrial production. β-Glucosidases could also be used in the
release of some nutritionally important components, such as vitamins and antioxi-
dants from their glycoside-conjugated form. For example, vitamin B6 of rice could
be released from pyridoxine glucoside form by the application of β-glucosidase
(Opassiri et al. 2004).
Table 10.3 β-Glucosidases with the ability to hydrolyse flavonoid compounds
Source of β-glucosidase Flavonoid glycoside Product Biological activity References
L. acidophilus LA-5 Delphinidin-3-glucoside, Gallic, Syringe Antioxidant Ávila et al. (2009)
malvidin-3-glucoside homogentisic acid
Paecilomyces thermophila Daidzin, genistin, glycitin Genistein, Daidzein, Anticancer, osteoporosis, Yang et al. (2009)
J18 Glycitein antihypercholesterolaemia
Thermoanaerobacter Daidzin, genistin Genistein, Daidzein Anticancer, antipostmenopausal Song et al. (2011)
ethanolicus JW200 syndrome
Pseudomonas ZD-8 Genitin and daidzin Genistein, Daidzein Anticancer, osteoporosis, etc. Yang et al. (2004)
Bacillus subtilis 18 Genistin and daidzin Genistein, Daidzein Anticancer, osteoporosis, etc. Kuo et al. (2006),
Kuo and Lee (2007)
Gongronella sp. Daidzin and genistin Daidzein, Genistein, Anticancer, osteoporosis Fang et al. (2014)
Saccharomyces cerevisiae Ginseng Ginsenoside Rd, F2 Anti-inflammatory, anti-cancer, Choi et al. (2014)
HJ014 Compound K (CK) anti-aging, antioxidant activities
Paecilomyces bainier sp. Ginsenoside Rb1 Compound K Tonic, adaptogenic, Yan et al. (2008)
229 immunomodulatory, anti-aging
effects
Mucilaginibacters Protopanaxatriol-type ginsenoside (S)-Rh1 (S)-Rg2 Antineoplastic, antistress, Cui et al. (2013)
mixture (PPTGM) antioxidant activities
Paenibacillus sp. KB0549 2,6-O-di(β-D-glucopyranosyl)-β-D- Sesaminol Antioxidants Nair et al. (2013)
glucopyranosylsesaminol (STG)
Pyrococcus furiosus Hesperidin, neohesperidin, naringin, Hesperetin, Hesperetin, Antiallergic, antioxidant, anti- Shin et al. (2013)
poncirin, diosmin, neoponcirin, rutin Haringenin, Naringenin, inflammatory, antihypertensive
Quercetin, Rutinose
10 Biotechnological Applications of β-Glucosidases in Biomass Degradation
Bifidobacterium bifidum Daidzin, genistin Daidzein, Genistein Anticancer, osteoporosis, You et al. (2015)
antihypercholesterolaemia
Bacteroides Daidzin, genistin, glycitin Daidzein, Genistein, Anticancer, osteoporosis, Byun et al. (2015)
thetaiotaomicron Glycitein antihypercholesterolaemia
VPI-5482
Aspergillus terreus Daidzin, genistin, glycitin Daidzein, Genistein, Anticancer, osteoporosis, Yan et al. (2016)
265
Glycitein antihypercholesterolaemia
10.3.5 Detoxification of Cyanogenic Glycosides in Cassava
Cassava is a carbohydrate-rich food and a source of food for ~500 million people in
the world. Cassava fruit contains cyanogenic glycosides like linamarin and lotaus-
tralin, which have been reported to cause Konzo syndrome, a human central nervous
system disorder (Vasconcelos et al. 1990). Although certain glucosidases are natu-
rally present in cassava roots, their insufficient expression leaves a significant part
of the cyanogenic glycosides in the processed food. Therefore, an additional treat-
ment of cassava with β-glucosidases has the potential of detoxifying and liberating
these cyanogenic glycosides, thereby improving its nutritional quality (Gueguen
et al. 1997; Maduagwu 1983).
10.3.6 Deinking of Waste Paper
Waste paper causes environmental pollution; its recycling can solve the two-
dimensional problem of forest wood consumption and waste management. Removal
of ink from paper is the most challenging obstacle, which could be overcome by
using enzymes. The enzymatic method for waste paper recycling has been reported
to be efficient in solving this problem. The enzyme preparations for waste paper
recycling contain cellulase, β-glucosidase and hemicellulase (Prasad et al. 1992;
Pathak et al. 2011; Lee et al. 2013).
10.3.7 Applications Based on Synthetic Activity
Apart from the hydrolytic activity, β-glucosidases also exhibit synthetic activity
(through reverse hydrolysis and transglycosylation), leading to the production of
oligosaccharides and alkyl and aryl β-glucosides (Ahmed et al. 2017). Alkyl glu-
cosides like hexyl, heptyl and octyl glucosides are biodegradable and, therefore,
find wide applications as emulsifier and antimicrobial agents (Bankova et al.
2006). Synthetic glucosides are also used in the preparation of therapeutic drugs
like heparin and acarbose. They act as probiotic agents and increase the number of
useful microorganisms in human gut. Some of the in-planta functions of these
oligosaccharides include fertilisation, embryogenesis and cell proliferation (Singh
et al. 2016; Krisch et al. 2010). Galacto-oligosaccharides and isobutyl-galacto-
sides are synthesised from lactose in water-organic media by the action of
β-glucosidase produced by Aspergillus oryzae (Bankova et al. 2006). Arbutin-β-
glycoside was found to be synthesised via the transglycosylation reaction of
β-glucosidase produced by Thermotoga neapolitana to manufacture a skin whiten-
ing agent, and these products were checked for their melanogenesis inhibitory
activities (Jun et al. 2008).
10.4 Challenges in Lignocellulose Bioconversion
10.4.1 Complex Structure of Plant Biomass
The primary challenge in lignocellulose conversion to fermentable sugars is its

complex structure: constituting approximately 33–40% of cellulose, 20–25% of
hemicellulose and 15–20% of lignin (Hess et al. 2011). Although cellulose is a
simple homopolysaccharide composed of D-glucose residues, the linear cellulose
microfibrils are associated with several hydrogen bonds that make the macromole-
cule highly crystalline and difficult to hydrolyse (Jørgensen et al. 2007). In addition,
cellulose exists in complex with hemicelluloses, the heterogenous polysaccharides
composed of variety of sugars, making it difficult to be converted into a single
product, ethanol. In addition, the cellulose and hemicellulose are complexed with
lignins, which form extensive cross-linking, making it resistant against microbial
degradation.
10.4.2 I nhibition of Enzyme Activity Due to Pre-treatment

Methods
The pre-treatment of lignocellulosic biomass is often required to promote the sub-

sequent step of enzymatic conversion of cellulose to sugars (Jørgensen et al. 2007).
This step is basically aimed at removal of hemicelluloses and/or lignin from the
lignocellulosic matrix A drawback of the pre-treatment step is formation of by-
products like carboxylic acids, gluconic acid and glucaric acid, phenolic com-
pounds, furfurals, benzoquinones, etc. that inhibit downstream processes by
interfering with microbial activity (Jönsson and Martín 2016). For example, acid
hydrolysis of lignocellulose results in corrosion of pre-treatment equipment and
release of heavy metal ions like copper, nickel, chromium and iron, which can be
inhibitory to fermenting microorganisms (Watson et al. 1984; Garrote et al. 2008).
10.4.3 End-Product Inhibition
End-product inhibition is a method of negative feedback regulation, where the final

product in a series of reactions inhibits an enzyme from an earlier step in the
sequence. The product binds to an allosteric site of the enzyme and temporarily
inactivates the enzyme via non-competitive inhibition. This mode of regulation is
also seen in enzyme-mediated lignocellulose degradation. The enzymes, cello-
biohydrolase and β-glucosidase, are subjected to end-product inhibition by cellobi-
ose and glucose, respectively (Qing et al. 2010; Teugjas and Väljamäe 2013; Kumar
and Wyman 2014). This limits the turnover number of these enzymes, leading to
decline in product formation. This problem gets adverse when lignocellulosic

degradation is performed at high solid concentrations in order to reduce the con-
sumption of water and running cost of the process (Kristensen et al. 2009).
10.4.4 Other Challenges
In addition to the above limitations, the activity of cellulases could also be inhibited
by non-productive binding to lignins and residual hemicelluloses (Rahikainen et al.
2013; Pareek et al. 2013). Other factors limiting plant biomass degradation include
relatively low activity of currently available hydrolytic enzymes, high cost of
enzyme production and thermal inactivation of enzyme (Sun and Cheng 2002). The
optimum fermentation conditions vary with species and other controlling parame-
ters like source of carbon and nitrogen used in media. The carbon source may con-
tain some contaminants in the form of secondary metabolites and chemicals that can
interfere with the rate of β-glucosidase enzyme production. Therefore, a thorough
screening of these secondary metabolites/inhibitors and their subsequent degrada-
tion or inactivation is crucial for the optimum enzyme production. Research should
also be focused on the possibility of temperature stress on the yield and activity of
β-glucosidase. Thermal inactivation of β-glucosidase is a major roadblock towards
achieving high enzyme efficiency. The thermal stability of β-glucosidases could be
enhanced by recombinant DNA technology and genetic modification of microbial
strains. Precise genome editing using site-specific nucleases like CRISPR/Cas9 is a
suitable option to achieve this goal. The other major hurdles in the commercial
β-glucosidase production are product inhibition, low product yields and high cost of
enzyme production. The search for better alternatives to the currently available
enzyme preparations should be highly promoted. Isolation of novel fungal species
having higher β-glucosidase activity would contribute towards revolutionising the
field of lignocellulose-mediated production of biofuels.
10.5 Approaches for Enhancing β-Glucosidase Activity
Researchers are trying to engineer cellulases with high specific activity, high thermal
stability, high adsorption capacities, high catalytic efficiency and lower end-product
inhibition. Some of the major limitations in cellulase- or β-glucosidase-mediated
biomass degradation have been addressed by using approaches like increasing the
production of β-glucosidase through strain improvement by mutagenesis, co-cultiva-
tion of microbes in fermentation to increase the quantity of desirable components of
cellulase complex, improving the performance of existing lignocellulose-degrading
enzymes by genetic engineering, and finally, metagenomics approach that involves
identification of novel β-glucosidases by DNA analysis of environmental samples.
These approaches have been described below.
10.5.1 Co-culturing
As mentioned above, cellulose degradation requires synergistic action of three

enzymes: exoglucanase, endoglucanase and β-glucosidase; however, no native micro-
bial strain produces optimum amounts of all three enzymes under the same condition.
For instance, while T. reesei produces exoglucanase and endoglucanase in abundant
amounts, it produces β-glucosidase in very low amounts (Peterson and Nevalainen
2012). Likewise, Aspergillus niger produces large quantity of β-glucosidase, but lim-
ited amount of exoglucanase and endoglucanase (Stockton et al. 1991; Kumar et al.
2008). Therefore, co-cultivation of T. reesei and A. niger using paper mill sludge as a
cellulosic substrate has proven to be a solution for efficient hydrolysis of cellulosic
residues (Maheshwari et al. 1994). Other successful cases include co-culturing
Aspergillus ellipticus with A. fumigatus (Gupte and Madamwar 1997) and T. reesei
with A. phoenicis using bagasse and corncobs as cellulose substrate in solid-state
fermentation (Duenas et al. 1995). Different strains of Trichoderma fungus are used
for production of beta glucosidase and are represented in Table 10.4.
Table 10.4 Studies done on β-glucosidase isolated from different strains of Trichoderma
Trichoderma strain β-Glucosidase Isolation strategies References
T. citrinoviride Extracellular Protein purification, biochemical Chandra et al.
β-glucosidase and proteomic characterisation (2013)
T. reesei TrBgl2 Mutational studies involving active Lee et al. (2012)
site residues of the enzyme
T. reesei QM9414 bgl1 Overexpression of bgl1 from Dashtban and
Periconia sp. in T. reesei QM9414 Qin (2012)
under T. reesei tef1 promoter
Recombinant T. bgl1 Construction of T. reesei strain Nakazawa et al.
reesei strain, expressing Aspergillus aculeatus (2012)
X3AB1 bg1 under control of xyn3 promoter
T. reesei bgl I Molecular cloning and expression in Chen et al.
Pichia pastoris (2011)
T. reesei CL847 BGL1 Protein purification and kinetic Chauve et al.
characterisation (2010)
T. reesei β-Glucosidase Molecular cloning and expression in Murray et al.
(cel3a) T. reesei (2004)
T. ressei β-Glucosidase Molecular cloning, expression in E. Saloheimo et al.
BGLII (Cel1A) coli and characterisation (2002)
T. harzianum C-4 Protein purification and biochemical Yun et al. (2001)
characterisation
T. reesei BGL2 Molecular cloning and expression in Takashima et al.
Aspergillus oryzae (1999)
T. harzianum 1,3-β-Glucosidase Protein purification and Lorito et al.
strain P1 characterisation (1994)
T. reesei QM9414 Aryl-β-D- Protein purification and Chirico and
glucosidase characterisation Brown (1987)
T. viride β-Gluc I Protein purification and biochemical Beldman et al.
characterisation (1985)
10.5.2 Genetic Manipulation
Genetic engineering approach involves introduction of specific desirable genes from

one species to another species using recombinant DNA technology (Sticklen 2008).
This strategy could be used to generate novel β-glucosidases with desirable proper-
ties like high efficiency, thermotolerance and high specificity for plant biomass deg-
radation (Blumer-Schuette et al. 2014). T. reesei, the most commonly used source of
cellulases, being mesophilic loses its enzyme activity at higher temperatures.
Transformation of thermotolerant β-glucosidase genes into T. ressei from thermo-
philic fungus like T. emersonii was found to confer higher specific activity and tem-
perature tolerance of up to 71.5 °C (Dashtban and Qin 2012; Druzhinina and Kubicek
2017). Similar results were obtained in Paenibacillus polymyxa where single amino
acid substitution contributed increased thermal resistance (Garvey et al. 2013). Apart
from targeting β-glucosidase, chimeric proteins have been constructed by the fusion
of endoglucanase from Acidothermus cellulolyticus and exoglucanase from T. reesei,
which resulted in improved saccharification (Chandel et al. 2012).
Another attractive option of increasing cellulase production is to express cellu-
lase from heterologous systems (Garvey et al. 2013). This involves codon optimisa-
tion, use of strong and inducible promoters and elimination of inhibitory sequences
to enable efficient protein expression from heterologous systems. Cellulases were
originally produced from anaerobic bacteria isolated from animal digestive systems
(Chandel et al. 2012). In addition, recombinant systems like E. coli and Bacillus
subtilis are being increasingly used for protein production because of the increased
enzyme yields from these systems. Apart from bacterial expression hosts, yeasts
like Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces marxianus
have been employed due to its superior post-translational modification of secreted
proteins (Tanaka et al. 2012).
10.5.3 Mutagenesis
The inherently low β-glucosidase activity of T. reesei has been improved by muta-
genesis through the use of chemical mutagens and UV radiations. The T. reesei
RUT-C30 mutant was reported to produce 4–5 times higher β-glucosidase than wild
T. reesei (Montenecourt and Eveleigh 1979). In another study, T. atroviride were
modified through mutagenesis by the use of N-methyl-N′-nitro-N-nitrosoguanidine
and UV light, and these mutants were found to have high cellulolytic activity than
wild types (Kovacs et al. 2008). Apart from random mutagenesis through the use of
physical and chemical mutagens, site-directed mutagenesis has also been used to
enhance cellulase activity. In one of the study, Mahadevan et al. (2008) altered the
amino acids around the active site of endoglucanase of Thermotoga maritima creat-
ing a mutant which displayed 10% higher activity than the wild-type enzyme.
Similarly, mutation of the conserved residue F476 to Y476 from Cel9A of
Thermobifida fusca displayed 40% improved cellulase activity. This was achieved
through the integration of computer modelling with site-directed mutagenesis
(Escovar-Kousen et al. 2004).
10.5.4 Metagenomics Approach
A relatively recent approach involves analysis of DNA collected from environmen-

tal samples, enabling identification and quantification of microbial species that
inhabit the natural environment. Metagenomics of microbial communities from cow
rumen (Hess et al. 2011), termite hindgut (Warnecke et al. 2007) and mangroves
(Simões et al. 2015) have provided detailed insights into the diversity of
lignocellulose-degrading enzymes through the identification of uncultivable bacte-
ria. Bergmann et al. (2014) have isolated two novel β-glucosidases from soil of
Amazon forest. In addition, new genes could be discovered that encode novel ligno-
cellulolytic enzymes.
10.5.5 ther Strategies for Enhancing Lignocellulose

O
Degradation
One of the simple ways to prevent end-product inhibition of lignocellulose degra-

dation is continuous elimination of end-products through sophisticated reactor
designs (Andric et al. 2010). Another method to relieve end-product inhibition is
simultaneous saccharification and fermentation, a process in which fermenting
microorganism is added along with hydrolytic enzymes (Teugjas and Väljamäe
2013). This prevents accumulation of cellobiose and glucose in the reaction mixture
that may interfere or inhibit cellulase activity. This method, however, has a major
drawback that different conditions are required for optimal hydrolysis and fermen-
tation. While the optimum temperature for yeast fermentation is approximately
35 °C, the optimum temperature of ~50 °C is optimal for cellulase activity. This
issue could be addressed by the use of thermostable enzymes involved in fermenta-
tion of sugars, produced after cellulose hydrolysis, into ethanol.
10.6 Conclusion and Future Perspectives
The first-generation biofuels, obtained primarily from food crops such as grains,
sugar beet and oil seeds, have raised a number of concerns in terms of food security,
climate change mitigation, economic growth and sustainability. Most of these con-
cerns could be addressed through the use of second-generation biofuels that involve
the use of non-food biomass like cereal straw, bagasse, forest residues and other
lignocellulosic materials. This would also serve as an attractive alternative for dis-
posal of non-edible portions of plants. However, compared with the production of
ethanol from food crops, the use of lignocellulosic biomass is more complicated
because the polysaccharides are more stable and the pentose sugars are not readily
fermentable by Saccharomyces cerevisiae. Several biotechnology-based approaches
are being used to overcome such problems, including the development of microbial
strains, use of alternative yeast species that naturally ferment pentose sugars and the
engineering of enzymes that are able to break down cellulose and hemicellulose into
simple sugars. Many fungal species are reported to produce various isoforms of
β-glucosidases. Thus, it is of utmost importance to screen the best yielding isoform
for a particular species. In addition, the thermal stability of β-glucosidases could be
enhanced by recombinant DNA technology and genetic modification of microbial
strains. Precise genome editing using site-specific nucleases like CRISPR/Cas9 is a
suitable option to achieve this goal. The other major hurdles in the commercial
β-glucosidase production are product inhibition, low product yields and high cost of
enzyme production. The search for better alternatives to the currently available
enzyme preparations should be highly promoted. Isolation of novel fungal species
having higher β-glucosidase activity would contribute towards revolutionising the
field of lignocellulose-mediated production of biofuels. To conclude, the lignocel-
lulosic biomass holds a large potential to meet the energy needs of the world
without compromising food security.
Acknowledgements The authors would like to thank Director, DEI, for his continuous support
and encouragement. SM is grateful to Dayalbagh Educational Institute, Deemed University, Agra,
for sanctioning the Research Project, DEI/Minor Project/2017-18 (iv), as a start-up grant.
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Chapter 11
Role of Fungi in Climate Change
Abatement Through Carbon Sequestration
Sandeep K. Malyan, Amit Kumar, Shahar Baram, Jagdeesh Kumar,

Swati Singh, Smita S. Kumar, and Ajar Nath Yadav
11.1 Introduction
The United Nations Environment Programme and the World Metrological

Organization established the Intergovernmental Panel on Climate Change (IPCC) in
1988. The aim of setting up IPCC was to evaluate technical, scientific, and socio-
economic information related to climate change, the potential impact of climate
change, and its possible mitigation measures. IPCC published a report in the year
2014 which made it clear that climate change is not a myth. To deal with climate
change is a challenge for the worldwide scientific, political, and economic commu-
nity. Greenhouse gases (GHGs), viz., carbon dioxide (76%), methane (16%), nitrous
S. K. Malyan (*) · S. Baram

Institute of Soil, Water and Environmental Science, The Volcani Research Center,
Agricultural Research Organization (ARO), Rishon LeZion, Israel
e-mail: sanm@volcani.agri.gov.il; shaharb@volcani.agri.gov.il
A. Kumar
Host Plant Section, Central Muga Eri Research and Training Institute, Central Silk Board,
Lahdoigarh, Jorhat, Assam, India
J. Kumar
Department of Hydrology, Indian Institute of Technology Roorkee,
Roorkee, Uttarakhand, India
S. Singh
Department of Environmental Science, Chaudhary Charan Singh University,
Meerut, Uttar Pradesh, India
S. S. Kumar
Center for Rural Development and Technology, Indian Institute of Technology Delhi,
New Delhi, India
A. N. Yadav
Department of Biotechnology, Akal College of Agriculture, Eternal University,
Baru Sahib, Sirmour, Himachal Pradesh, India

284 S. K. Malyan et al.
420
Atmospheric CO2 concentration (ppm)
400
380
360
340
320
300
1984
1988
2014
2018
1960
1962
1964
1966
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1970
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1976
1978
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1994
1996
1998
2000
2002
2004
2006
2008
2010
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2016
Fig. 11.1 Trends of CO2 concentration in the atmosphere at Mauna Loa. (Source: NOAA 2019)
oxide (6%), and chlorofluorocarbons (2%), are likely to affect agricultural produc-
tivity and food security adversely through climate change (Ranjan and Yadav 2019;
Malyan 2018; Fagodiya et al. 2017a; Kumar et al. 2016, 2017b; Pathak et al., 2016;
Gupta et al. 2015, 2016; Kumar and Malyan 2016; IPCC 2014; Bhatia et al. 2013a,
b). Malyan et al. (2016a, b) quoted that the rise in atmospheric greenhouses gases
may result in rising of global mean temperature up to 1.5 °C by the end of the
twenty-first century. Among all greenhouse gases, CO2 only accounts for 76%, so it
is considered most important. At the beginning of industrial revolution, atmospheric
CO2 concentration was 280 ppm (Rastogi et al. 2002), and it has now increased to
410 ppm in March 2019 (Fig. 11.1). The annual mean growth rate (differences in
CO2 concentration between the last month of the year [December] and the first
month [January] of that year) was monitored by Global Monitoring Division of
National Oceanic and Atmospheric Administration (NOAA) at Mauna Loa site. The
rate of growth was 0.54 ppm/yr. in 1960 and it increased to 2.10 ppm/yr. in 1983
(Fig. 11.2). The growth rate of CO2 in the atmosphere was 2.98 ppm/yr. in the year
2016, and it might rise over 3 ppm/ yr. in few upcoming years (NOOA 2019). The
CO2 growth rate is increasing continuously due to many anthropogenic actions,
including fossil fuel combustion, deforestation, forest fire, automobile, etc.
Soil, plant, and ocean are the major natural sink for atmospheric CO2. Recently,
the scientific community enhanced the work on mitigating climate change and
global warming by reducing atmospheric CO2 concentration through carbon seques-
tration (Bhattacharyya et al. 2018; Mukherjee et al. 2018). Soil acts as both source
(soil respiration) and sink (carbon sequestration) for CO2, and soil respiration con-
tributes to 20% of the total CO2 emission to the atmosphere (Kumar et al. 2017a, b;
Gupta et al. 2016; Rastogi et al. 2002). The global soils hold 3.3 times (2500 Gt C)
as compared to carbon present in the atmosphere (Lal 2014). Out of 2500 Gt, soil
11 Role of Fungi in Climate Change Abatement Through Carbon Sequestration 285
3.5 2.98
3 2.1
2.5
ppm per year
1.5
0.5
0
1959
1961
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2011
2013
2015
2017
Fig. 11.2 Growth rate of carbon dioxide at Mauna Loa, Hawaii. (Source: NOAA 2019)
organic matter (SOC) and soil inorganic carbon retain 1550 Gt and 950 Gt, respec-
tively (Lal 2014). The world soil pool of C is 4.5 times the size of C present in
biomass (Lal 2014). In the soil carbon sequestration, biological soil crust plays a
significant role. Soil fungi especially arbuscular mycorrhizal fungi enhance the soil
carbon sequestration in different types of ecosystems. The objective of this study is
to assess the carbon sequestration potential of fungi in soils and the factors affecting
thereof.
11.2 Fungi and Carbon Sequestration
Carbon is the major building block in all living organisms. It exists in many forms
such as soil organic matter, plant biomass, and CO2 in the dissolved form in water
and gas in the atmosphere. Carbon in soil is more than the total carbon in the atmo-
sphere and plants biomass. Numerous small fungi in the soil, consist of hyphae that
use carbon as a building block (Fig. 11.3). When this hypha dies, it is easily decom-
posed and its carbon is stored as soil organic matter for a long time (Treseder and
Holden 2013). The process of long-term storage of carbon in the soil, terrestrial
biomass, or the ocean so that the buildup of CO2 in the atmosphere will be slowed
down or reduced is known as carbon sequestration. In other words, carbon seques-
tration is defined as “the capture and secure storage of carbon that would otherwise
be emitted to or remains in the atmosphere” (FAO 2000). In carbon sequestration,
fungi play a significant role in the northern hemisphere of the Earth which help to
combat global warming. In soil, fungi do symbioses association (mycorrhizal fungi)
with plant roots and help the plants to utilize the nutrients from the soil. As a result,
mycorrhizal fungi stimulate the plant’s growth which results in the faster removal of
atmospheric CO2 through its conversion into plant biomass. There is a different
pathway of converting the atmospheric CO2 to plant biomass (Fellbaum et al. 2012).
Fig. 11.3 Showing mycorrhizal fungi hyphae in soil
Fig. 11.4 Simplified

method of carbon Carbon
sequestration in soil di-oxide
organic matter by fungi
Biological
soil crust Plants
Fungi
Carbon sequestration in
organic matter of soil
The photosynthates of the host plant are transferred to the intraradical hyphae
followed by extraradical hyphae before it releases to the soil (Fig. 11.4). Carbon
sequestration in soil depends on the volume and hyphal biomass of fungi (Solaiman
2014; Zhu and Miller 2003).
Fungus is a nonchlorophyllous organism and is heterotrophic (requires an organic
source of carbon) in nature, i.e., it obtains its food from either dead organic matter
(saprophytic) (Chaubey et al. 2019) or from the autotrophic/heterotrophic associates
(parasitic and symbiotic). Agaricus, Aspergillus, Morchella, Mucor, Penicillium,

Rhizopus, Saprolegnia, etc. are the example of the saprophytic mode of nutrition.
The parasitic fungi are Peronospora, Puccinia, Fusarium, Pythium, Melampsora,
etc. The associate may be animal, plants, and microbes. If an associate is a plant, the
autotrophic association can be stated as true symbiosis. The fungi can use a variety
(e.g., yeast used the acetate) of the organic matter as a source of energy but the
majority of them prefer carbohydrates. Glucose is a preferred carbohydrate for
almost all the fungi as compared to fructose. Starch and cellulose are also used by
some of the fungi which are capable of synthesizing hydrolytic enzymes. Organic
acids are the least preferred sources of energy by most of the fungi. Basidiomycetes
are able to utilize lignin as an energy source. Saprolegniaceae and Blastocladiales
can grow only with organic nitrogen, i.e., amino acids. The organic matter is first
dissolved by the extracellular enzymes, and then it is directly absorbed by diffusion
either through the hyphal walls of the hyphae that penetrate the substratum or by the
rhizoidal hyphae.
Lichens (a single composite thallus plant) are the best-explained association of
fungi with the alga where the fungus provides the minerals, water from the substra-
tum, and space for alga which in return supplies food. Mycorrhiza is the association
of fungi with the roots of higher plants and can be ectomycorrhiza, endomycorrhiza,
and ectoendomycorrhiza based on the positional association.
The input of the plant’s organic matter into the soil carbon is both aboveground and
belowground litter in the form of leaves, stem, flower, and roots. The plant also con-
tributes some organic compounded inform of exudates to the soil. This contribution is
known as rhizo-deposition. The rhizo-deposition is also an important carbon contribu-
tion of higher plants which is based on plant root architecture, environment, physiol-
ogy, biochemistry, and chemical composition of the deposited matter. The litter’s
input is varying in quantity and depends on the climax community of the ecosystem.
The belowground input mainly contributes to the organic matter and stabilizes their
medium to long duration based on the physicochemical properties of soil and micro-
biota (Dignac et al. 2017; Clemmensen et al. 2015; Kuzyakov and Domanski 2000;
Mendez-Millan et al. 2010).
Carbon sequestration entirely depends on the photosynthetic rate of the auto-
trophs and the respiratory losses of the autotrophs and symbionts (mycorrhizal
fungi). These fungi utilize 5 to 20% of the net primary productivity of the symbiotic
system (Luo and Zhou 2010). The change in the carbon assimilating or degradation
rate can entirely change the carbon sequestration potential of the association until
the change in both the processes is in the same direction and potential. The degrada-
tion of rhizo-deposited carbon is done by microorganisms. These microbes are pres-
ent or associated with the specific plants and soils. The quantity of the rhizo-associate
is also contributed majorly. The quantified contribution in the respiratory losses of
these rhizo-associates especially mycorrhizal fungi is not reported in different asso-
ciations separately. Thus, the exact carbon sequential potential of mycorrhizal fungi
still remains to be estimated. However, few studies have confirmed in controlled and
field experiments that carbon sequestration depends on plant photosynthesis and
respiration through mycorrhizal fungi (Luo and Zhou 2010; Bahn et al. 2008;
Friedlingstein, et al. 2006; Ussiri et al. 2006). Global forest soils efflux into the
atmosphere through belowground respiration has been estimated to be approximately
24 Pg C y−1 in which mycorrhizal fungi contribute about 16% (Zhang et al. 2007;
Hu et al. 2005). Thus, increasing the sequestration potential of the mycorrhizal
fungi can reduce the contribution drastically.
11.3 Factors Affecting Fungal Growth in Soil
Fungi have high plasticity and they easily adapt to adverse conditions in the soil. A
diverse range of fungi is found in almost every type of ecosystem owing to the fact
that fungi can adapt to a wide range of temperature and pH (Frac et al. 2018; Frac
et al. 2015). Fungi activity in soil is affected by abiotic (physical disturbance, soil
temperature, soil pH, soil texture, moisture, and salinity) and biotic (plant and other
organisms interaction) factors, and it may influence the carbon sequestration activity
of fungi (Kour et al. 2019; Rana et al. 2019; Yadav et al. 2017, 2018, 2019; Farc et al.
2018; Rouphael et al. 2015; Posada et al. 2008; Treseder and Allen 2000) (Table 11.1).
Some factors affecting fungi activity in the soil are discussed below in brief.
11.3.1 Temperature
The Northern hemisphere consists of 67.3% of total Earth’s landmass; Moritz et al.
(2002) reported that global warming has led to a rise in temperature by ~1.5 °C.
The IPCC (2007) predicted that by the end of 2100, the temperature may elevate to
Table 11.1 Factor affecting the fungi in soils

Factor Remarks References
Temperature Soil warming results in rapid fungal respiration which Hawkes et al.
results in carbon losses to the atmosphere (2008)
No effect on fungal carbon sequestration under soil Rosenstock et al.
warming (2018)
P-Fertilizer On addition of N fertilizer fungal biomass increase which Aliasgharzad
helps in carbon sequestration et al. (2018)
To an optimum level (20 mg kg−1) shows a positive Aliasgharzad
response while at a higher dose (40 mg kg−1) shows a et al. (2018)
negative impact on fungal root growth
Application of phosphorous fertilizer has a negative effect Smith et al.
on fungal diversity in soil (2011)
Pesticides Have a direct and indirect negative impact on fungi Willis et al.
diversity and carbon sequestration in soil (2013)
Physical Biocrust removal drastically affects inoculum potential of Jasper et al.
disturbances AM (Arbuscular Mycorrhiza) fungal mycelial due to web (1989)
fragmentation
additional 4–7 °C which will affect all the ecosystem services. There are contradictory
reports on the effect of soil temperature on the fungal diversity and activity
(Rosenstock et al. 2018; Solley et al. 2017; Allison and Tresseder 2008; Fujimura
et al. 2008; Zogg et al. 1997; Yadav et al. 2018). On one hand, some studies have
reported significant changes in fungal activity; to the contrary, some others could
not assess any change related to temperature. This suggests that the effect of tem-
perature is species-specific. In Tundra, Fujimura et al. (2008) observed no change in
fungal diversity even after soil warming. Warming of soil samples taken from the
environment resulted in a significant change in fungal community of soil (Zogg
et al. 1997). Rosenstock et al. (2018) observed that the soil warming from a range
of 0–5.5 °C above the control has no or limited effect on the growth of ectomycor-
rhizal and no effect on community composition and fungal carbon sequestration in
Picea sitchensis forest soils (Rosenstock et al. 2018).
11.3.2 Physical Disturbance
Disturbance in several ecosystems is a universal process and it affects all levels of

biological organisms prevailing in that ecosystem. The causes of disturbance may
be natural or anthropogenic. Anthropogenic physical disturbance in top soil (20–
30 cm) results in fragmentation of fungal mycelia (Jasper et al. 1989) which results
in a lower rate of soil carbon sequestration. Korb et al. (2003) reported that in the
soil, fungi diversity recovered rapidly after a forest fire. On the other hand, in an
agricultural field, tillage practices disturb the fungal diversity and they recover at a
slow rate. However, in the Gangetic Plains of India, the fungal diversity was
observed to recover at a rapid rate. The high rate of fungal recovery in Indian plains
was due to a large diversity of spores (Oehl et al. 2005).
11.3.3 Soil pH
Soil pH is the primary factor affecting fungal activity in the soil and provides an
environment that is essential for carbon sequestration in soil. Minor increases in pH
are associated with higher root colonization by fungi in acidic soils with less phos-
phorus availability (Ge et al. 2017; Oehl et al. 2005).
11.3.4 Fertilizer
Phosphorus(P)-based fertilizers have environmental concerns related to eutrophica-

tion of fresh waterbodies. Generally, soil has an abundant amount of both inorganic
and organic P in soil, but its bioavailability is very low for the crop and plants
(Sarabia et al. 2017). Mycorrhiza increased the P availability for host plant, and in
return, fungi get the photosynthetic product and result in more development of
hyphae which increases the soil carbon sequestration. Aliasgharzad et al. (2018)
reported that application of 20 mg-P kg−1 soil showed positive fungal root growth
while increasing the P dose to 40 mg kg−1negatively affected the growth rate of
fungi (Table 11.1).
11.3.5 Pesticide
Fungi in agricultural soils are exposed to different type of pesticides. Application of

pesticide directly or indirectly affects the host plant and thus indirectly suppresses
the rate of carbon sequestration by fungi in soils (Table 11.1). Zocco et al. (2011)
reported that fungicides like fenpropimorph inhibit the growth and development of
fungi in soil. Nevertheless, pesticides like dimethoate, fenamiphos, and aldicarb
were not found to inhibit the fungal diversity (Karpouzas et al. 2014; Schweiger and
Jakobsen 1998; Nemec 1985). Spokes et al. (1981) studied the impact of eight chem-
icals on the development of fungi. It was observed that aldicarb had negligible impact
on fungi development, while fungicide chloroneb acted as development stimulator
for fungal population in the soil. Then again, triadimefon and benomyl fungicides
application had an inhibitory impact on fungal development (Spokes et al. 1981).
11.3.6 Moisture
Soil moisture is an important constituent for the growth of the soil fungi. The soil
fungi and degradation of the rhizo-deposition material is highly influenced through
the moisture percentage. The decrease in the moisture percentage can directly limit
the availability of organic matter to the fungi, so that the rate of degradation can
decrease and ultimately the growth of soil fungi can also get limited. Therefore,
moisture also directly controls the soil temperature and soil heat flux. Moreover, the
higher the soil temperatures, the lower is the soil moisture, especially in the topsoil
horizon, and ultimately the lower will be the soil fungi due to the scarcity of the
food material.
11.4 Research Gap and Future Recommendation
At certain places and in some of the ecosystems, fungi can play an important role in
the sequestration of carbon dioxide present in the atmosphere by playing principal
roles in fixation of organic matter in the soil and degradation. However, the mechanism
of sequestration is not yet fully understood. There are certain avenues that need to
be explored in order to have a deeper understanding of this phenomenon. Some of
the gaps that have been observed from literature to direct the future research have
been summarized as follows:
(i) The quantified contribution of soil and mycorrhizal fungi in different ecosys-
tems and various associations has not yet been evaluated and reported. This
quantification can serve a major role to define the direction of the soil and
mycorrhizal fungi as a carbon sequestrate.
(ii) The minimizing of the respiratory losses of soil and mycorrhizal fungi can also
enhance the carbon sequestration potential. Therefore, future research should
be conducted in this direction.
(iii) The role and potential of different fungal communities along with different
types of soil and mycorrhizal fungi can also serve to enhance the carbon
sequestration in different ecosystems.
(iv) The factors influencing growth of fungi such as soil moisture, soil temperature,
CO2 enrichment, quality of rhizo-deposition, and precipitation changes control
the heterotrophic respiration. Thus, the impact of these factors with respect to
the soil and mycorrhizal fungi can also lend a hand to understand their behavior
in changing the environment.
(v) Relevant studies in different interactions, i.e., moisture vs mycorrhizal diversity,
soil temperature vs mycorrhizal composition and diversity, and soil tempera-
ture vs moisture, are very limited in respect of the soil fungal respiration and
also poorly understood.
(vi) Contribution of mycorrhizal association on carbon storage in contaminated/
mineral/nutrient-rich soil is not available.
Therefore, future research in this direction can help in improved understanding
of the mechanism of fungal carbon sequestration.
11.5 Conclusions
Based on this study, the following conclusions can be drawn. The global soil carbon
is more than the carbon present in the atmosphere, but the rising temperature due to
global warming has the potential to affect the fungal diversity and fungal activity. The
rate of soil carbon sequestration by fungi depend upon direct (quality and quantity of
hyphae) and indirect (soil pH, texture, moisture, soil management, physical distur-
bance, pesticide application, etc.) factors of the ecosystem. There are three important
strategies to combat climate change: development of less or zero carbon fuel, limiting
of fossil energy use, and carbon dioxide sequestration from atmosphere or emission
point source through abiotic (engineered) or biotic methods (photosynthesis).
Soil carbon sequestration by fungi in soil is the environmentally sound method to
reduce the carbon dioxide levels in the atmosphere.
Acknowledgments The financial support to the first author, Sandeep Kumar Malyan, provided
by Ministry of Agriculture and Rural Development, Israel, under ARO-Postdoctoral
Fellowship-India and China, is highly acknowledged. The authors are also very thankful to the
Central MugaEri Research and Training Institute, Central Silk Board, Lahdoigarh, Jorhat-785700,
India, for having provided the necessary support.
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Chapter 12
Microbial Enzymes and Their Application
in Pulp and Paper Industry
Abdulhadi Yakubu, Upasana Saikia, and Ashish Vyas
12.1 Introduction
Industrial utilization of wastepapers in the production of new one is increasing

globally. Currently, pulp and paper industry is one of the largest consumer of wood.
Based on their demands due to global economic growth, more trees will be har-
vested and waste will be consumed and disposed in the environment (Pathak et al.
2011). Chemical agents such as sodium hydroxide, hydrogen peroxide, sodium
carbonate, diethylenetriaminepentaacetic acid, sodium silicate, and surfactants are
used in large quantities by paper industries as conventional methods of deinking
wastepaper which lead to expensive wastewater treatment to meet environmental
regulations (Pathak et al. 2011; Saxena and Chauhan 2016). Enzymes such as
lipase, xylanase, pectinase, cellulase, hemicellulase, amylase, and esterase are
used as substitute to chemical conventional methods of deinking wastepapers
(Yadav et al. 2015, 2016). These enzymes are reported to be environmentally
friendly as compared to conventional method. It was realized several decades ago
that microbial enzymes might be useful in processing of papers since it is com-
posed of natural polymers such as cellulose, hemicellulose, and lignin. Microbial
enzymes have been commercially used in pulp and paper industry only in the pre-
vious decade, while microorganisms are presently used in other industrial process-
ing steps, though long been used in the treatment of wastewater. This is due to the
fact that wood and pulps which act as substrates are difficult to degrade. In addi-
tion, most research now focuses on lignin biodegradation since it is lignin that is
A. Yakubu
Department of Science Laboratory Technology, College of Science and Technology,
Jigawa State Polytechnic, Dutse, Nigeria
U. Saikia · A. Vyas (*)
Department of Microbiology, School of Bioscience and Bioengineering, Lovely Professional
e-mail: ashish.vyas@lpu.co.in

298 A. Yakubu et al.
removed from wood in chemical pulping and pulp in bleaching. Lignin likely
evolved in part as a deterrent to microbial degradation, and it continues to be an
impediment to biotechnological processing of wood and pulps. During the last
decade, the number of possible applications of enzymes in paper and pulp indus-
tries in which many are of commercial quantity has grown rapidly. These include
xylanase for enzymatic bleaching, lipase for pitch removal, as well as cellulase and
hemicellulase for freshness enhancement (Kirk et al. 1996).
Globally, our lives are somewhere governed by little inscriptions laid on paper in
one way or the other. The major domains of public sector are reliant on paper and
paper products directly or indirectly. The history of paper dates back almost
2000 years, while the manufacturing process happened to be reported in China
when inventors imprint their writings on crafted cloth sheets. The knowledge of
papermaking then became popularized through westward and eventually reached
India around 605 AD. A wide variety of raw materials used in the papermaking
process include cellulose originated from agriculture waste, forests, and wastepa-
per, noncellulosic coal, talcum powder, etc. However, raw materials like wood logs
and its waste, bamboo, wastepaper, bagasse, and agricultural residues like bran of
wheat, rice straw, grasses, and seaweed are majorly used in the process. The basic
product coming out during paper processing is the cellulosic pulp which is used in
papermaking and as animal feedstock. Pulp being a lignocellulosic fibrous material
separates the cellulosic fibers from wood logs, fibers, wastepaper, and rags. Two
steps are involved in pulp production: wood pulping and pulp bleaching (Fig. 12.1).
Both processes require high-energy input and reagents which leads to the produc-
tion of significant amount of gaseous, liquid, and solid wastes that need to be treated.
Biotechnological approaches provide substantial solution of the aforementioned
problem. Lignin degradation by white-rot fungi has been intensively studied for
biotechnical applications such as biopulping, biobleaching, and treatment of pulp
mill effluents. Also, lignolytic enzymes and hemicellulases provide a prospective
way to decrease the usage of hazardous bleaching reagents and to make an improve-
ment in the quality of bleached pulp (Tavares et al. 2014). The application of
Fig. 12.1 The stages of enzymatic deinking process

12 Microbial Enzymes and Their Application in Pulp and Paper Industry 299
icrobial enzymes in paper and pulp industries has increased rapidly which lead to
m
the decrease of adverse effect on ecosystem due to the increased awareness of sus-
tainability issues (Yadav et al. 2019a, b). Utilization of these microbial enzymes
also leads to the decrease in energy consumption, processing time, and amount of
chemicals in deinking wastepaper. They are equally used to aid deinking and bleach
in paper and pulp industry as well as waste treatment by increasing biological oxy-
gen demand (BOD) and chemical oxygen demand (COD).
It is difficult to deink laser-printed office papers by employing the conventional
method of deinking. Due to higher demand of laser printers and copy machines
every year, there has been an increase in the amount of nonimpact printed papers
entering the recycled papers. It is challenging for the inventors to remove ink from
these papers. The reason is primarily due to the strong adherence of the toner par-
ticles to the paper surface. The photocopier printers are indulged in using thermo-
setting toners made up of synthetic polymers as ink. Due to applying high amount
of heat during inking, the ink particles become physically bonded to the paper mak-
ing it difficult as well as expensive to remove by conventional method.
A biological process using enzymes had been evaluated which implied positive
results in deinking process of different types of wastepaper. One of the major
advantages of using enzymes is production of minimum treatment effluent, and it
is also less harmful to the environment. The most important step involved in recy-
cling process is removal of ink from the paper (Table 12.1). Several enzymes like
cellulase, hemicellulase, α-amylase, lipase, xylanase, and other lignolytic
enzymes are involved in the biological process of deinking. The enzymatic treat-
ment is favorable because enzymes are eco-friendly in nature and during its pro-
cessing ink detachment occurs without any discharge of harmful chemicals, thus
rendering our environment green. Most of the cited studies reported deinking of
mixed office waste consisting of photocopier paper using commercially available
enzymes (Roushdy 2015). India generates approximately 36.5 million tons of
municipal solid waste annually, of which 14.6 million tons consist of paper
wastes. The Indian Agro and Recycled Paper Mills Association (IARPMA) esti-
mated that India is among the countries with low recycling of waste of wastepa-
pers (26%) as compared to countries like China (38%), Thailand (45%), and
Germany (80%). Based on the shortage of raw materials, resources, and high
demands being imposed on green plants, Indian paper industries are facing many
challenges on daily basis. One of the attractive solution to these problems in India
is the recycling of municipal office waste (MOW) papers but still very difficult to
remove nonimpact ink. Deinking process which involves the removal of printed
ink from used paper involves dislodgement of ink particles from fiber surface and
the separation of dispersed ink from the fiber suspensions by washing and flota-
tion (Tavares et al. 2014).
In chemical deinking process, industries used high quantity of chemicals
such as chlorine, chlorine-based derivatives, sodium hydroxide, sodium car-
bonate, sodium silicate, hydrogen peroxide, hypochlorite, and chelating
agents, which lead to hazardous effluent disposal problems (Vega et al. 2012;
Pala et al. 2004). For these reasons, biological deinking by using microbial
Table 12.1 Microbial enzyme and their applications in pulp and paper industry
Microorganism Enzymes Application References
Commercial enzymes Cellulase Ink removal, freshness, and Pathak et al.
reduction of drainage time (2011)
Trichoderma Cellulase and Improved drainage, high deinking Pathak et al.
harzianum xylanase efficiency, brightness, and reduction (2014)
of drainage time
Commercial enzymes Cellulase Not good if specks surface of the Tsatsis et al.
deink paper are used (2017)
Commercial enzymes Cellulase Detach significant amount of ink Zhang et al.
from ONP/OMG (2008)
Aspergillus niger Cellulase and Enhanced optimum deinking Lee et al.
hemicellulase efficiency (2007)
Streptomyces sp. Xylanase Biobleaching effect Li et al.
L22001 (2010)
Bacillus altitudinis Xylanase Potential for biodeinking and Adhyaru
biobleaching et al. (2017)
Bacillus sp. CKBxID Xylanase Deinking agent of recycled Maity et al.
wastepaper (2012)
Alkalothermotolerant Xylanase and Commercially viable with better Singh et al.
pectinase paper quality (2012)
Aspergillus niger Xylanase Deink old newspaper with improved Desai and
brightness, removal of surface ink Iyer (2016)
particles from ONP pulp
Aspergillus nidulans Xylanase Reduction of ink and increased Taneja et al.
brightness of recycled paper (2002)
Commercial enzymes Laccase and Deink old newspaper Xu et al.
hemicellulase (2011)
Commercial enzymes Cutinase and Increase brightness and ink removal Wang et al.
amylase (2018)
Commercial enzymes Amylase and Improve brightness Gil et al.
cellulase (2013)
enzymes which act directly either on the fiber or on the ink film becomes more
attractive. For example, hydrolysis of cellulose and hemicellulose brings
detachment at fiber/ink interbonding regions and finally releases ink particles
into the suspension when treated with cellulase/hemicellulase enzymes (Lee
et al. 2007). Microbial enzymes can also detach small fibrils from the surface
of the ink particles; hence, they can change the usual hydrophobicity of the
particles, which brings about their separation in the flotation/washing step.
These enzymatic technologies have been described as especially advanta-
geous in deinking high-quality wastepapers, whose reuse is usually limited as
the nonimpact inks (toners) polymerize onto the paper surface using thermo-
plastic binders during the high-temperature printing process. In the chemical
deinking process, the toner particles usually remain large, flat, and rigid and
separate very poorly from papers during the fiber/ink separation stages (Jiang
and Ma 2000).
12.2 Enzyme Producing Microbes
Twelve bacterial culture has been isolated from alkaline sediments from Lonar Lake
in Maharastra India. Based on 16S rRNA sequencing, biochemical characterization,
and phylogenetic analysis, all the isolates were identified. For the first time in Lonar
lake, bacteria such as haloalkaliphilic Marinobacter excellens, Alkalimonas dela-
merensis, Roseinatronobacter monicus, and Rhodobaca bogoriensis were identified
(Borgave et al. 2012). In another research conducted by Kladwang et al. (2003),
about 490 alkaline-tolerant fungi from a natural environment using petri dishes con-
taining potato dextrose agar medium buffered at pH 11.0 were identified. Many of
the alkaline tolerant fungi has been isolated from fifty one samples from different
habitats of Thailand. This research indicated that a good source of alkaline enzyme
production can be found from alkaline-tolerant fungi isolated from tree holes in
alkaline and acidic environments. A bacterium Bacillus pumilus SV-205 produces
xylanase in an optimized fermentation condition. A medium containing a mixture of
yeast extract and peptone yields high xylanase as compared to other combinations.
High pH stability over a range of 6–11 in 24 h is the major characteristic feature of
this enzyme which can also maintain 65% activity after 2 h incubation at 60 °C
(Nagar et al. 2012).
In Barabanki district of Uttar Pradesh, India, George et al. (2001) isolate a novel
alkalothermophilic actinomycete with optimum growth at pH 9 and 50 °C from self-
heating compost. Thermomonospora sp. was able to produce 23 IU/ml carboxy-
methyl cellulase (CMCase) enzyme purified under fractional ammonium sulfate
precipitation followed by cellulose affinity chromatography and Sephacryl S-200
gel filtration. The enzyme retained 100% activity at 50 °C for 72 h and had half-
lives of 7 and 3 h at 60 and 70 °C, respectively. The enzyme activity was tested as
an additive to laundry detergents based on its stability in the presence of commercial
detergents viz. Ariel, Henko, and Surf Excel. During an investigation exploring pos-
sible sources of novel thermophilic species in natural products, a novel thermophilic
and alkaliphilic actinomycete capable of producing alkaline cellulase from soil of a
tropical rain forest in Yunnan province, China, was isolated and identified (Wu et al.
2018). The whole-cell hydrolysates were found to contain glucose and ribose. The
organism was identified as Genus Streptomyces based on 16S rRNA gene sequence
analysis. Organism formed a distinct phyletic line together with closely related type
strain called Streptomyces burgazadensis ZIR7 T. Strain named Streptomyces ther-
moalkaliphilus represent a novel species in the genus Streptomyces based on its
phenotypic, chemotaxonomic, and phylogenetic characteristics.
Bilanenko et al. (2005) reported an isolate representing the group Ascomycete
from saline soda soils of Central Asia and Africa. The bacterium described as
Heleococcum alkalinum sp. novel was isolated on alkaline agar with carboxymethyl
cellulose (CMC) and was a dominant species in samples of soda soils with pH >10
and relatively high salinity. It shows an alkali-tolerant adaption by growing within the
pH range of 6.7–10.8. This cellulolytic activity of an alkaliphilic obligate anaerobic
bacterium, Z-7026, which was isolated from the microbial community of soda-lake
sediments belonging to the cluster III of Clostridia with low G + C content was
investigated by Zvereva et al. (2006). The bacterium has the ability of growing in
media with cellulose or cellobiose as the sole energy source. The maximum growth
rate on cellobiose was found at a pH range of 8.5–9.0, while that of cellulase synthe-
sis, assayed by using a novel fluorimetric approach, was observed at pH 8–8.5. In the
laboratory, bacterium Penicillium citrinum (MTCC 6489) was previously isolated
from soil producing an alkali-tolerant and thermostable cellulase (Dutta et al. 2008).
The study reports the presence of alkali-stable cellulase from alkali-tolerant fungus
Penicillium citrinum for the first time, which may have potential effectiveness as
additives to laundry detergents.
According to Vyas and Lachke (2003), two extracellular alkali-stable 1,4-β-d-
glucan-4 glucanohydrolase (EC3.2.1.4) fractions, namely, EndoA and EndoB, were
separated from the culture filtrate of an alkalotolerant Fusarium strain. These
enzymes are found to be suitable for deinking mixed office wastepapers leading to
the increase in brightness with decrease in ink counts of the recycled paper. Probable
mechanism of enzymatic deinking process was schematically presented based on
the distinct properties of endoglucanases. Picart et al. (2007) reported one fungal
strain from subtropical soils on the medium supplemented with rice straw exhibit-
ing high cellulase activity. Using isoelectric focusing, zymography, and sodium
dodecyl sulfate polyacrylamide gel electrophoresis, these new strains were identi-
fied as Penicillium sp. CR-316 and Penicillium sp. CR-313 which indicated that the
strains secreted multiple enzymes that hydrolyze cellulose. Crude cellulose pro-
duced by Penicillium sp. CR-316 has potentials in industrial applications since it
showed activity and stability at high temperature and produced a thermostable cel-
lulase. Kalpana and Rajeswari (2015) has isolated Streptomyces from agricultural
waste capable of producing enzymes for degrading xylan. Streptomyces sp are vital
source of enzymes involved in lignocellulosic degradation. Isolate was reported to
grow on different types of feedstuffs such as oat spelt xylan, sugarcane molasses,
tomato pomace, rice bran, wheat bran, and sawdust under submerged fermentation
conditions.
The xylanase activity in each production medium was confirmed by measuring
the amount of reducing sugars liberated from the medium by the DNS method using
crude extract which was found to have an application in deinking of newsprint.
Nadagouda et al. (2016) isolate cellulase enzyme from Trichoderma viride GSG12
under solid-state fermentation technique using cheap and readily available agricul-
tural waste material called rice bran. This indicates the possibility of using rice bran
to produce cellulase using Trichoderma viride under solid-state fermentation. The
finding shows that cellulase production can be influence by optimal pH, initial
moisture level of the medium, incubation temperature, inoculum size, and incuba-
tion time. The optimum pH, initial moisture level, incubation temperature, and
inoculums size were 5.5, 70%, 32 and 2 × 108 spores/flask, 120 h, respectively.
Increased enzyme production was favored by supplement of lactose and corn-steep
solid to the rice bran.
From various mangrove sites in the Philippines, conventional as well as analyti-
cal profile index (API) were used to characterize and phenotypically identify five
promising cellulase producing bacterial strains. The finding provide data regarding
species of Bacillus producing cellulase enzyme and additional knowledge regarding
the bacterial diversity of mangrove forests in the Philippines (Tabao and Monsalud
2014). Makky and Abdel-Ghany (2009) described old newspaper (ONP) waste as a
carbon source for growing Bacillus subtilis where avicelase and carboxymethylcel-
lulase (CMCase) enzymes are estimated in the culture filtrate. Bacillus subtilis
CMCase has more activity at optimal temperature and pH than Avicelase. Deinking
with these enzymes brings about an increase in brightness of the sheet effective
removal of ink particles and also prevents redeposition onto the fiber surfaces. These
findings indicate that enzymatic deinking can perform better than the conventional
chemical method. Bacillus halodurans was purified to homogeneity to produce an
extracellular haloalkaline cellulase by bioconversion of lignocellulosic waste by
(Annamalai et al. 2013).
The enzyme has retained up to 80% activity at 80 °C, 12% and 35% temperature,
pH and NaCl with optimum of 60 °C, 9.0 and 30% respectively. When detergents
and organic solvents such as n-hexane, acetone and acetonitrile are present, the
enzyme was found to be stable. This indicates that a purified cellulase produce from
Bacillus halodurans utilizing ligncellulosic biomass could be of great potentials in
industrial process. Maitan-Alfenas et al. (2016) used Aspergillus nidulans to isolate
and characterize xylanase in Pichia pastoris. At 60 °C and 7.5 as well as 50 °C and
6.0 temperature and pH, respectively, this enzyme has its optimum activity which is
completely inhibited by SDS and HgCl2. Another important bacterium capable of
producing an extracellular and thermostable xylanase enzyme is Bacillus pumilus
ASH when grown on solid-state fermentation (SSF). When wheat bran is moistened
with deionized water at a substrate to moisture ratio 1:2.5 (w/v), higher xylanase
activity is obtained with optimum production temperature of 37 °C. The enzyme
activity was slightly lower in solid-state fermentation (SSF) than in submerged fer-
mentation technique, but the ability of the organism to produce such a high level of
xylanase at room temperature, with deionized water and with no addition of any
mineral salts in SSF, could lead to substantial reduction in the overall cost of enzyme
production.
12.3 Cellulase and Its Applications
Microbial cellulases have been focused as the important biocatalysts being multi-
plex in nature and bearing extensive applications. Cellulase and hemicellulase
enzymes are both synthesized by fungi and bacteria as seen in Table 12.2. These
microorganisms can be aerobic, anaerobic, mesophilic and thermophilic. Among
them, the most commonly studied fungal genera are Thermomonospora,
Trichoderma, and Aspergillus. Fungal and bacterial cellulases are structurally simi-
lar (Fig. 12.2). Fungal cellulase having two domains: a catalytic domain and a cel-
lulose binding module. Commercially, cellulase enzymes have been accessible for
30 years or more, and these enzymes have been used for study purpose as well as
Table 12.2 Microbial enzymes and their sources

Temp
Sample Location Organism Enzyme pH (°C) References
Soil Pulp and paper Bacillus subtilis Cellulase 4.0 60 Pala et al.
industries, (2004)
India
Soil Macuya rain Aspergillus sp. Cellulase 4.8– 28 Vega et al.
forest, LM-HP32 and 9.4 (2012)
Penicillium sp.
Pucallpa, Peru
LM-HP33 and 37
Soil Iguazú Penicillium sp. Cellulase 4.5 65 Picart et al.
rainfalls, CR-313 and (2007)
Argentina CR-316
Wastepaper USM Campus, Aspergillus niger Cellulase, 6.0 50 Lee et al.
Penang, hemicellulose (2013)
Malaysia
Agricultural Cairo, Egypt Bacillus Cellulase, 3–7.6 60– Abo-State
waste thuringiensis xylanase 80 et al. (2013)
MAM-29 and
MAM-38
Waste Medellin, NA Cellulase, 7.0 40 Gil et al.
photocopy Colombia amylase (2013)
paper
Wild Wayanad, Escherichia coli Cellulase, NA 37– Kumar
herbivore, Kerala, India SD5 xylanase 39 et al. (2018)
rain deer
Soil, Jeju Island, Bacillus subtilis CMCase, 5.0 50 Kim et al.
compost, South Korea C5–16 and S52–2 avicelase, (2012)
animal waste xylanase
slurry
Wastepaper NA NA Cutinase, 9–11 50 Wang et al.
amylase (2018)
Water Lonar Lake, Many Lipase, 10.5 23 Kanekar
Buldhana, haloalkalihpilic amylase, et al. (2008)
Maharashtra, bacteria caseinase,
India cellulose
Soil Vellore, Tamil Streptomyces sp. Xylanase 7.5 37 Kalpana
Nadu, India and
Rajeswari
(2015)
Old Chandigarh, Bacillus Xylanase 8–9.5 65 Virk et al.
newsprint, Punjab, India halodurans (2013)
magazine, FNP135
inkjet, Xerox
Soil Ambala Cantt, Bacillus pumilus Xylanase 6–11 60 Nagar et al.
Haryana, India (2012)
Soil Tianshan Streptomyces Xylanase 5–8 70 Li et al.
Xinjiang, rameus L2001 (2010)
China
(continued)

Temp
Sample Location Organism Enzyme pH (°C) References
Industrial Shreyans Paper Aspergillus Xylanase 8–8.5 55 Taneja et al.
effluents Industry, nidulans KK-99 (2002)
Ahmedgarh,
Punjab, India
Compost pit BREC Sadra, Bacillus Xylanase 8.0 45– Adhyaru
Gujarat, India altitudinis DHN8 55 et al. (2017)
Wastepaper Chandigarh, Bacillus Xylanase and 8–9.5 65 Virk et al.
Punjab, India halodurans laccase (2013)
Soil Effluents of Bacillus pumilus Xylanase, 8.5 55 Singh et al.
paper AJK10414 pectinase (2012)
industries,
India
Fig. 12.2 A simplified schematic representation of the enzymatic action of cellulase, involving
exoglucanase, endoglucanase, and β-glucosidase
industrial researchers. The different applications of cellulase enzyme in this indus-

try have reached a considerable increase in the last decade. The conventional method
of the woody raw material using refining and grinding happens to pulp fines with
high content, bulks, and stiff. However, the enzymatic pulping process using cellu-
lase leads to decrease in the utilization in the energy during refinement and improv-
ing the strength content of handsheet.
In Peru, a soil from an undisturbed forest was investigated for fungi capable of pro-
ducing alkaline cellulase. At different PH value, plate clearing assay and carboxymethyl
cellulase as substrate, 11 of 50 morphological colonies were selected. These 11 fungal

strains produced cellulase of high alkaline PH values in a liquid culture media. The best
producers of cellulase in highest productivities are the Penicillium sp. LM-HP33,
Penicillium sp.LM-HP37 as well as Aspergillius sp. LM-HP32. These fugal strains are
found to be suitable production of alkaline cellulase (Vega et al. 2012). Pathak et al.
(2011), conducted a study on deinking photocopier papers using chemicals and com-
mercial cellulase enzyme where they optimized all the parameters of deinking experi-
ment for hydro pulping. Ink removal efficiency and freshness were improved by 24.6%
and 12.6%, respectively, along with reduction of drainage time of 11.5% as compared
to chemical deinking.
As compared to fungi, bacteria have a high rate of cellulase enzyme production
rate due to its advantage of high bacterial growth rate. The most important param-
eters for successful production of cellulase enzyme are the screening of the
organism, optimization of fermentation conditions and selection of substrates.

Using carboxymethyl cellulase as substrate, (Ariffin et al. 2006) produced enzyme
cellulase from local isolate of Bacillus pumilus EB3.This enzyme screened from
this bacterium was purified using ion exchange chromatography using anion
exchanger for cellulase characterization. Rawat and Tewari (2012) isolated and
identified microorganism which hydrolyzed carboxymethyl cellulose (CMC) as
Bacillus subtilis strain LFS3. Gel filtration chromatography, ion exchange, and
sodium sulfate precipitation are the methods used to isolate and screen enzyme
carboxymethylcellulase with overall recovery of 15%. Optimum temperature and
pH for active profile of this enzyme was 60 °C and 4.0 respectively. A fungi called
Coprinopsis cinerea was found to have the ability of producing a crude cellulase
and xylanase enzymes with potentials of deinking photocopier wastepaper deink
photocopier wastepapers as reported by Pathak et al. (2014). In their view to achieve
maximun and possible efficiency without affecting paper and its strength propertis,
enzyme dose, point of enzyme addition, pulp consistency, and reaction time were
investigated which also confirmed the potential of crude enzyme of C. cinerea for
deinking of photocopier wastepapers.
Effects on the use of cellulase for deinking of office wastepaper were investi-
gated by Tsatsis et al. (2017). Better results were achieved by the use of enzyme in
deinking experiment as compared to those in which enzymes are inactive. It was
discovered that enzyme application has disadvantage if specks surface of the deink
paper sheets was uses as compared conventional deinking. Based on their finding,
more research is needed in formulations of enzyme with better performance under
alkaline conditions as well as the types of paper printed in different photocopier and
laser printers.
Abo-State et al. (2013) isolated Bacillus strains from agricultural waste and iden-
tified as Bacillus thuringenesis which have the ability to produce cellulase and xyla-
nase based on their pH and temperature. Stability at different temperatures
(60–80 °C) at separate duration was also investigated. Zhang et al. (2008) evaluate
three commercial cellulase enzymes for their application on deinking artificially
aged old newspaper (ONP) mixed with fresh old magazine (OMG) in a ratio of 7:3.
At the start of repulping, these enzymes were added followed by incubation for 3 h.
Despite the fact that cellulase enzymes were able to remove a significant amount of
ink from ONP/PMG, they have low efficiency than using conventional methods of
either sulfite or alkaline deinking chemistry. None of the three cellulase enzymes
tested were able to separately deink aged ONP/OMG, and a poor deinkability was
also observed by using either sulfite or alkaline chemistry. However, the research
indicates a significant increase of deinking when a combination of enzyme and sul-
fite is applied which provide a potential strategy of achieving effective deinking of
old newspapers at neutral pH.
Enzymatic deinking has added advantages over conventional deinking viz.
Reduced alkali usage, improving fiber brightness, and greater strength property.
Moreover, enzymatic deinking process also prevents alkaline yellowing and reduces
environmental pollution. However, excessive use of enzymes can be degradable as
it might lead to significant hydrolysis. With the aim of increasing the rate of produc-
tion, cellulase has been pursued by several mills in improving the drainage. These
enzymes are also used in the production of easily biodegradable stationary objects
including paper towels and sanitary paper (Kuhad et al. 2011). Laccase mediator
system was used in a study conducted to identify the similarity on the application of
cellulase/hemicellulase for deinking printed fibers from newspapers and magazines.
In this regard, commercially available endoglucanase and endoxylanase activities
and a commercial laccase were evaluated in the presence of synthetic or natural
mediators. They concluded that factors to be considered for the application of enzy-
matic deinking processes in addition to enzymes include ink types, printing m ethods,
and fiber/ink separation process (Ibarra et al. 2012). Lee et al. (2007) also developed
a laboratory procedure for enzymatic deinking of wastepapers using cellulase and
hemicellulase enzymes produced from Aspergillus niger. Using an optimized floa-
tation system of 6.0 and 45 °C pH and temperature, respectively, an optimum deink-
ing efficiency with these enzymes was enhanced to 95%. The deinked papers are
found to have similar properties with commercial papers indicating the effective-
ness of the developed enzymatic process.
12.4 Xylanase and Its Applications
Xylanase comprises the hydrolytic enzymes which are capable of breaking the β-1,4
backbone of the multiplex plant cell wall polysaccharide. Xylan is the second larg-
est polysaccharide after cellulose (Yadav et al. 2017, 2018). A diverse array of
microorganisms like bacteria, actinomycetes, yeast and fungi are involved in the
hydrolysis of hemicellulose as indicated in Table 12.2. Wood is processed for
debarking, chipping and screening. Then a chip undergoes steaming process so that
the microorganisms become lesser in number. After this, the chips are allowed to
cool down and inoculated with biopulping fungus. The biopulping process is cost
effective and technologically feasible. The main advantage is the decrease in con-
sumption of energy as well as the increase in mill consumption. The processes also
lead to enhancement in the properties such as strength of the paper, and reduced
environmental impacts (Khonzue et al. 2011). From the past studies, it has been
concluded that pre bleaching method with xylanase enzyme is cheaper and eco-
friendly. It also decreases the significant amount of chemicals which are indulged in
order to get brightness in chemically bleaching process. In conventional method of
paper making process, the manufacturers use hazardous chemicals which impart
negative impact to the environment.
A high amount of xylanase enzyme was produced from Bacillus pumilus SV-205
using optimized fermentation conditions. The bacterium secretes maximum amount
of cellulase free xylanase in combination with yeast and peptone which also
enhanced highest xylanase production that differ from other combinations. The
enzyme maintained a thermal stability of 65% activity after incubation at 60 °C for
2 h (Nagar et al. 2012). Li et al. (2010) isolate xylanase with biobleaching potentials
on wheat straw pulp from Streptomyces L2001 with stable optimum temperature of
70 °C and pH of 5.3. High production of xylanase from another bacterium called
Bacillus altitudinis DHN8 followed by its purification and application was pre-
sented by Adhyaru et al. (2017). Using response surface technology, enzyme yield
was improved by optimizing submerged fermentation conditions which includes
incubation time, temperature, agitation speed, sorghum straw, inoculum size, and
gelanin. This leads to twofold increase in activity based on the abovementioned
optimized conditions as compared to activity in one factor at a time optimization.
The research indicates a potential use of Bacillus altitudinis for biodeinking and
biobleaching.
For pollution free environment, the recently employed technique is the recycling
of civic paper waste by enzyme based technology. In these regards, a newly isolated
bacterium for recycling of laser jet paper waste was isolated for its potential ability
to purify xylanase enzyme by Maity et al. (2012).This potent xylanase producing
bacterium from microbial consortia of termite gut was identified as Bacillus sp
CKBx1D based on 16S rRNA sequence. Response surface methodology was the
technique used to optimize all operational parameters, while purified enzyme mix-
ture was used for laser printed paper waste deinking potentials. This deinking poten-
tial was detected from the enzyme at a pH of 6.8 with 72 h continuous shaking at
constant temperature of 35 °C. Hence, the bacterial isolate and its xylanase enzy-
matic system could efficiently be used in recycling paper waste as deinking agent.
Using cheap agricultural residue, pectinase and xylanase enzymes were isolated for
the first time from alkalo thermotolerant bacterial strain with potentials of deinking
capabilities. The enzymes may also play important role in making enzymatic deink-
ing an eco-friendly having 50% decrease of chemicals, commercially viable with
better paper quality (Singh et al. 2012). According to (Gessesse and Mamo 1999),
overall cost of xylanase enzyme production from Bacillus sp. AR-009 can be greatly
reduce using solid-state fermentation technique. This bacterium has the ability to
produce dry bacterial bran xylanase activity when grown in solid-state fermentation
and produced a high bacterial bran xylanase activity with wheat bran as substrate.
The ability of the organism to produce high xylanase activity at alkaline pH and
lower wheat bran to moisture ratio could have a potential advantage in minimizing
the risk of contamination. In addition, the cost of downstream processing during
product upgrade and that of waste treatment steps can be greatly reduce since the
enzyme can be produce with a least quantity of liquid.
Dutta et al. (2007) studied the culture filter of Penicillium citrinum grown on
wheat bran bed in solid-state fermentation to purify an extracellular xylanase
enzyme. Moderately thermostable xylanase showed optimum activity at 50 °C at
pH 8.5. Purification and characterization of this novel endoglucanase free alkaliphi-
lic xylanase from the alkali tolerant fungus P. citrinum were discovered for the first
time which may have potential implications in paper and pulp industries. Desai and
Iyer (2016) isolate cellulase free xylanase enzyme from fungi for deinking of Old
News Paper (ONP) pulp. Aspergillus niger strain DX-23 from the total 16 fungal
producing enzyme isolates had a maximum xylanase of 48.9 ± 0.02 Uml−1. The
enzyme deinked ONP pulp efficiently with improved brightness after optimization
and subsequent H2O2 treatment as compared to untreated pulp. According to (Taneja
et al. 2002), an alkaline thermostable xylanase was produced by alkalophilic fungi
called Aspergillus nidulans KK-99 in an alkaline medium consisting of wheat bran,
KNO3, pH 10.0 and temperature of 37 °C. this partially purified enzyme was stable
at a pH range of 4.0–9.5 and temperature of 55 °C and reach optimum activity at a
pH 8.0 and same temperature of 55 °C where decrease of ink and brightness of
recycled paper was achieved by this enzyme treatment. An investigation in to xyl-
ano pectinolytic enzymes for deinking of school wastepaper was conducted by
Shatalov and Pereira (2008). This biodeinking in combination with conventional
deinking approach leads to an increase in BOD and COD values of effluent as
compare to use of only conventional deinking method. The usage of xylanase
enzymes in deinking process has some limitations depending on different parame-
ters. These comprise different factors like narrow pH ranges, thermal instability, no
proper end product, and cost of production of enzyme.
Biological treatment using xylanase enzyme has proved to be helpful in both
reducing the costs and also improved the quality of the fiber. Xylans are highly
available to hydrolytic enzymes as they are not having a complex structure.
Therefore, its specific activity becomes two to three times higher as compared to
crystalline cellulose form (Shatalov and Pereira 2008). In order to obtain whiter and
brighter pulpy matter to process better quality paper, it becomes necessary to sepa-
rate the residual lignin with the use of bleaching method (Azeri et al. 2010). The
most important advantages of biobleaching include: (a) Reduction in the use of the
bleaching chemicals, (b) Enhanced quality of the paper and pulp, (c) Improving
whiteness and brightness of the pulp, and (d) Decrease effluent toxicity and pollu-
tion load.
12.5 Laccase and Its Applications
Laccase is the oldest and widely studied enzymatic system discovered for the first
time in 1883 by Yoshida from the exudates of Japanese lacquer tree called Rhus
vernicifera. In 1985, Bertrand discovered it as a metal containing oxidase, while in
1896, it was demonstrated by both Bertrand and Laborde to be present in fungi. The
redox potential is found to be higher in fungal laccases rather than bacterial or plant
laccases up to 800 mV. Therefore, these are found to be beneficial in lignin degrada-
tion or in the eradication of phenolic toxins arising during lignin degradation.
Laccase is found to be present in higher plants and fungi such as basidiomycetes,
white root fungi and ascomycetes. White rot basidiomycetes are responsible for
efficient degradation of lignin and the enzymes involved in lignin degradation
includes lignin peroxidases, manganese dependent peroxidases and laccases. Also,
fungal laccases have been involved in cellular processes in many ways including
sporulation, plant pathogenesis and other specificity. Woody chips when pretreated
with lignolytic fungus leads to increase in the strength of the pulp while decrement
in the requirement of energy for the mechanical pulping process.
The most widely studied application of laccase in this industry is bleaching of
kraft pulp. It was studied that when SL4, a lignolytic fungal strain, was applied, it
reduced 25% usage of chlorine during bleaching of kraft pulp and produced 1.8 unit
brightness (Kaur and Nigam 2014). C. albidus responsible for producing laccase
helps in the reduction of lignin content found in the eucalyptus wood and happen to
be useful in the process of biopulping. Fungal laccases also efficiently remove toxic
effluents coming from the pulp mills which contain a significant number of phenolic
compounds and chlorolignins. Laccase mediated biobleaching process is a friendly
way to improve pulp and paper production.
The ink removal capability of laccase and xylanase enzymes from an alkalo-
philic bacteria on recycled old newspaper (ONP) in combination with physical
deinking method of sonication and microwaving were investigated. Parameters such
as PH, enzyme dose as well as treatment time of these enzymes were optimized
statistically using Response Surface Methodology in which any mediator supple-
mentation for deinking was not required. Optimization of these deinking parameters
were conducted statistically using response surface methodology in which for the
first time laccase did not need any mediator supplementation (Virk et al. 2013). An
enzyme showing alkaliphilic laccase activity was purified from the culture superna-
tant of Myrothecium verrucaria 24G-4 by (Sulistyaningdyah et al. 2004). The
enzyme was very stable in alkaline conditions with an optimum pH of 9.0 and was
able to remove synthetic dyes under the same conditions which confirm the function
of this enzyme in alkaline environment.
12.6 Amylase and Their Applications
Amylases hydrolyze starch molecules and give diverse array of products including
dextrin and glucose units (Fig. 17.2). Microbial amylase enzymes are available in
market in large number and they are replacing chemicals involving hydrolysis of
starch. The first commercially produced microbial enzyme was amylase which was
of fungal origin in 1894 and used as therapeutic aid to cure digestive disorder.
Nearly 25% of the global enzyme comprises amylase. Enzymatic pretreatment of
wastepaper pulp was conducted for deinking process at neutral conditions using
amylase and cellulase enzymes and ethoxylated fatty acids as surfactant. At a tem-
perature of 40 °C, this enzymatic deinking process was conducted by flotation con-
sistency of 0.8% within 6 min (Gil et al. 2013). In a future work, effectiveness of
combine enzymes in removal of waste photocopy paper and other wastepapers mix-
tures is expected to be discussed.
Amylases are distributed in plants, animals and microbes. However, amylases
produced by microbes are replacing the chemical processing methodology indulged
in paper industries due to cost effectiveness and technical advantages. Amylases
classified into two types on the basis of catalysis, endoamylase and exoamylase.
Endoamylases carry out hydrolysis in a random manner while exoamylases hydro-
lyze from the nonreducing end. Endoamylases results in oligosaccharides with
branching of different chain length and exoamylases forms short end products.
Main fungi involve in the production of industrially important enzymes are
Aspergillus niger, A. oryzae, and A. flavus. Also these fungi are capable of produc-
ing large quantities of amylases which can be used commercially. Aspergillus niger
having significant amount of hydrolytic capacities in the production of α-amylase
(Sahni and Goel 2015)
Sakthivel et al. (2010) isolated and screened some bacteria that inhabit decaying
vegetables for the production of amylase enzyme. Enterococcus pseudoavium is the
only specie identified to have a relatively higher amylase activity of the bacterial
species tested. Four days after growth, the organism deinked pulp completely when
grown with paper pulp. When cultured with paper pulp, the bacterial culture immo-
bilized in sodium alginate beads can decolorize this paper within 4 days. This shows
the ability of extracellular amylase produced from Enterococcus pseudoavium to
effectively deink and decolorize paper pulp within 4 days of incubation. The role of
α-amylases in the paper industry as shown in Table 17.4 is to modify starch of
coated mixed paper which comprises production of lower viscous, starch with high
molecular weight, deinking, drainage and cleaning of paper. The coating tends to
make the paper smoother and stronger, to improve the quality of writing. Since the
viscosity of natural starch remains high enough for paper sizing and this can be
changed by degradation of the polymer with the use of α-amylases in the batch or
continuous culture. Starch being good sizing agent helps in giving fine finish of the
paper, improvement of the paper quality, and reusability, besides behaving as a per-
fect paper coating (Singh et al. 2012).
12.7 Lipase and Its Applications
Lipases are the enzymes which hydrolyze long chain triglyceride and constitute as
one of the most focal biocatalysts for biotechnological applications. It was first
found to be present in pancreas by J. Eberle in 1834 and during 1856 by C.I Bernard.
Under aqueous conditions, they are able to release fatty acids and glycerol by acting
on carboxyl ester bonds present in triacylglycerol (Gupta 2004). These are serine
hydolases and are able to work independently without the use of any cofactor.
Microbial lipases play an important part in the field of biotechnology due to its
versatility and ease of mass production. It has got wide enzymatic properties. Both
bacterial lipase and fungal lipase are widely used in different industries. Lipase
producers include bacteria, fungi, yeast, and actinomycetes. Examples of fungi pro-
ducing lipase are Acinetobacter radioresistens, Aeromonas hydrophila, Aspergillus
oryzae (Andualema and Gessesse 2012).
Wood is the cheap source of paper pulp and pitch describes the hydrophobic
contents of wood (triglycerides and waxes). These are appearing to create serious
problem in the paper processing. The problems might be sticky deposits and holes
or spots in the finish product. However, lipases remove the pitch from the pulpy
matter during the process of paper making process. Around 90% of triglycerides
present in the pitch get hydrolyzed into glycerol, monoglycerides, and fatty acids by
lipase enzymes which are having less stickiness and more hydrophilic character
(Jaeger and Reetz 1998). In Japan, a paper industry called Nippon Paper industries
developed a method to control pitch that utilizes the Candida rugosa fungal lipases
to hydrolyze up to 90% of the woody triglycerides present. Lipases have many
advantages in general as its utility increase the rate of pulping, enhancing whiteness
and intensity power, decrement in the usage of chemical, equipment with prolong
life, reduction in the level of pollution in the wastewater, saving energy utility, and
time and reduction in the composite cost. Pseudomonas sp. (KWI-56) has an added
advantage as addition of its lipase to the deinking process leads to increase in the
whiteness of the paper and reduce usage of residual inking. A thermophilic isolate
of Bacillus coagulans BTS-3 can be used to screen alkaline lipase. The bacterium
can be enhanced substantially when parameters like nitrogen source, carbon source,
and initial pH of culture medium were consecutively optimized. Enzyme activity of
culture medium was obtained in 48 h at 55 °C and pH 8.5 with refined mustard oil
as carbon source and a combination of peptone and yeast extract as nitrogen sources.
Maximum activity of the enzyme was achieved at 55 °C temperature and 8.5 pH and
was also stable between the pH range of 8.0 and 10.5 at temperatures up to
70 °C. This purified lipase enzyme indicates a variable hydrolytic activity toward
various 4-nitrophenyl esters (Kumar et al. 2005).
12.8 Other Microbial Enzymes and Their Applications
Cutinase and other microbial enzymes have also play a vital role in recycling of
wastepaper (Table 17.2). For the first time, Wang et al. (2018) reported the effect of
cutinase enzyme on the deinking of mixed office wastepaper (MOW). Combination
of cutinase, amylase, and some complicated surfactants can be used to replace the
conventional chemical deinking methods at neutral deinking process. When these
enzymes are treated in combination of surfactants mixed with pulp and office waste-
paper at a temperature of 50 °C for 30 min, the brightness, ink removal rate, tensile
index, and tear index of the deink paper will increase significantly. Hemicellulase
was combined with laccase mediator system (LMS) by Xu et al. (2011) to deink old
newsprint (ONP). The result indicates the effective residual ink concentration was
lower as compared to pulp deink with hemicellulase or LMS individually. According
to this study, an environmentally friendly and effective deinking method can be
achieved when cutinase and amylase enzymes combine with cardanol polyoxyeth-
ylene ether and other surfactants.
Industrial utilization of wastepapers in the production of new one is increasing glob-

ally. Currently, pulp and paper industry is one of the largest consumers of wood.
Based on their demands due to global economic growth, more trees will be har-
vested, and waste will be consumed and disposed in the environment. With increas-
ing demand of paper globally and high cost of conventional methods for deinking
and recycling of wastepapers, attentions are now focused on the use of enzymes for
eco-friendly deinking of wastepapers especially extremozymes such as cellulase,
hemicellulase, xylanase, amylase, and laccase that are able to deink wastepapers at
optimum environmental conditions. Combination of enzymatic and chemical meth-
ods can greatly increase the brightness of deink wastepaper, ink removal, and qual-
ity of recycled wastepapers. However, biological deinking method of wastepaper
with the help of varied microbial enzymes comprises an eco-friendly way that can
produce high-quality paper without relying upon the traditional usage of harmful
chemicals. Thus, more efforts should be efficiently directed to still tap the wide
underlying potential of these fungal lignolytic enzymes.
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Chapter 13
Arbuscular Mycorrhizal Fungi-Mediated
Mycoremediation of Saline Soil: Current
Knowledge and Future Prospects
Dileep Kumar, Priyanka Priyanka, Pramendra Yadav, Anurag Yadav,

and Kusum Yadav
13.1 Introduction
The explosive rise of global population led to the increased exploitation of natural
resources in response to the high demands for food, energy, and other things. Soil is
the ultimate source of nutrients for the plant kingdom. The weathering of rocks
present in the earth’s crust forms soil. The rocks are composed of minerals and the
process of weathering occurs continuously. The soil has different concentrations of
these minerals. If any specific type of minerals has a greater concentration, then it
changes the quality of soils. For example, the saline soil holds a greater salt concen-
tration; the acidic soil has low pH and sodic or alkaline soil possess high pH. The
soil salinization is a serious problem for arable lands, which decreases the produc-
tivity of agricultural products. According to some reports, the soil salinization is
gradually increasing in many parts of the world, mostly in arid and semiarid regions
(Giri et al. 2003; Al-Karaki 2006). The amount of evaporation causes increased salt
concentration or precipitation amount leading to a decrease in salt concentration in
a given land area (Mahajan and Tuteja 2005). Salinization of land is a global prob-
lem, which is rapidly increasing. The United Nations Environment Program (UNEP)
estimated that approximately 20% of agricultural land and 50% of cropland in the
world is facing salt stress (Flowers and Yeo 1995). The extent of salt-affected soils
is highest in the Asia Pacific region including Australia. The countries like Argentina,
Australia, China, Egypt, India, Iran, Iraq, Pakistan, Thailand, former Soviet Union,
and the United States of America (USA) are predominantly affected by soil saliniza-
tion. Salt-affected soils are occupied about 7% of the earth’s land surface
D. Kumar · P. Priyanka · P. Yadav · K. Yadav (*)

Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India
A. Yadav
Department of Microbiology, College of Basic Science and Humanities, Sardarkrushinagar
Dantiwada Agricultural University, Banaskantha, Gujarat, India

320 D. Kumar et al.
(Ruiz-Lozano and Azcon 2000) and about 5% of the total cultivated land around the
world, i.e., 1.5 billion hectares (Sheng et al. 2008).
In India, about 30 million hectares of coastal land is barren and uncultivable due
to salinity. Soil salinization in arid and semi-arid regions is one of the major prob-
lems, which impose a challenge for sustainable agriculture in irrigated production
systems. Plant growth is directly affected by high levels of sodium chloride and
other salts in the soil (Yadav et al. 2019a; Yadav and Saxena 2018; Yadav et al.
2015). The area of saline soil is rapidly increasing because of climate change, saline
irrigation, and high evaporation, which induce salt accumulation in the soil, thus
causing a significant decrease in irrigation practices. According to Kohler et al.
(2009), saline soils and saline irrigation constitute a serious production problem for
vegetable crops, as saline conditions are known to suppress plant growth particu-
larly in arid and semiarid areas. The high salt concentration is deposited in the soil
to generate a low water potential zone in the soil, making it difficult for the plant to
absorb water and nutrients.
The basic physiology of salt and drought stress overlaps, therefore salt stress
essentially results in a water-deficit condition in the plant that takes the form of a
physiological drought (Mahajan and Tuteja 2005; Yadav 2017; Yadav and Yadav
2018). Wang et al. (2003) anticipated that the increased salinization of arable land
would result in 50% land loss by the middle of the twenty-first century. The signifi-
cance of soil salinity in agricultural yield is crucial (Tester and Davenport 2003)
which negatively affect the establishment, growth, and development of plants, lead-
ing to huge losses in productivity (Mathur et al. 2007). Irrigated land is only 15% of
the total cultivated land but has at least twice the productivity of rainfed and accounts
for one-third of the world’s food supply (Munns 2005). The three main direct effects
of salt on plant growth are (a) reduced osmotic potential of soil, causing a decrease
in the amount of water availability to plant, leading to physiological drought; (b)
toxicity of excessive Na+ and Cl− ions in the cell, causing disruption of enzyme
structures and other macromolecules, damage to cell organelles and plasma mem-
brane, as well as disruption of photosynthesis, respiration, and protein synthesis
(Feng et al. 2002); and (c) nutrient imbalance in the plant caused by nutrient uptake
leading to ion deficiencies (Adiku et al. 2001). The plants must maintain lower
internal osmotic potentials to prevent water movement from roots into the soil (Feng
et al. 2002).
The general response of plants to high soil salinity is reduced growth (Ghoulam
and Foursy 2002). At low or moderate salt concentration, plants adjust osmotically
and maintain the potential for water influx (Gunes et al. 1996). High salt concentra-
tions disturb membrane integrity and interfere with internal solute balance and
nutrient uptake, causing nutritional deficiency symptoms similar to drought (Grattan
and Grieve 1999). Grattan and Grieve (1999) reported that high NaCl uptake com-
petes with the uptake of other nutrient ions, such as K, Ca, N, and P, resulting in
nutritional disorders and eventual reduction in yield and quality. In most cases,
salinity decreases the concentration of P in plant tissues (Kaya et al. 2001). Osmotic
stress, ion imbalances, and direct toxic effects of Na+ and Cl− ions on the metabolic
processes are most important and widely studied physiological impairments caused
13 Arbuscular Mycorrhizal Fungi-Mediated Mycoremediation of Saline Soil: Current… 321
by salt stress (Munns 2005). Cusido et al. (1987) revealed that salinity inhibits the
plant growth by affecting water absorption, protein biosynthesis, and by N as well
as CO2 assimilation.
Certain halophytes are also known to remediate soil salinity. Gallagher (1985)
and Glenn and O’Leary (1985) searched for new salt-tolerant crop plants that could
deal with saline soils and minimize the crop loss. Development of salt-tolerant crop
varieties through breeding programs is underway. To challenge the detrimental
effects of salinity, a salt-tolerant perennial rye-grass (Lolium perenne) was obtained
by transforming rice vacuolar membrane Na+/H+ antiporter gene via Agrobacterium-
mediated transformation. The salt tolerance of perennial ryegrass was improved by
overexpression of the Na+/H+ antiporter gene (Wu et al. 2005). Most of such suc-
cessful methods are costly and beyond the economy of several developing nations
(Cantrell and Linderman 2001). Hence, an alternative attempt is needed to chal-
lenge the deleterious effects of saline soils by inoculating salt-tolerant AMF in the
agricultural crops.
External and internal microorganisms in their natural environment colonize
plants. Under stress environment, plant performance could be improved by the
deliberate introduction of some microorganisms in rhizosphere. The AMF associa-
tion with plants is one such association that could mitigate several types of plant
stress. The AMF is associated with the roots of over 80% terrestrial plant species
including halophytes, hydrophytes, and xerophytes (Mathur et al. 1999; Yadav et al.
2019b, c; Hejiden et al. 1998). The AMF form a symbiotic relationship with the
plants in which the plant supplies carbohydrate to the fungi while the mycorrhizal
fungi extend the surface area of roots, thus increasing their ability to absorb nutri-
ents and water from the soil. These fungi constitute an integral component of the
natural ecosystem and are known to exist in saline environments (Giri et al. 2003).
The AMF holds the potential to reduce the impact of salinity-induced stress on
plants. The AMF associations often result in greater yield of crop plants even under
saline conditions. The AM fungi widely exist in salt-affected soils (Juniper and
Abbott 1993). To some extent, AM fungi are considered as bio-ameliorators of
saline soils (Azcon-Aguilar and Barea 1997; Rao 1998). The AMF is increasingly
being considered for mycoremediation of soil salinity. According to Kohler et al.
(2009), the use of plant symbiotic microorganisms, especially AM fungi, is useful
in developing strategies to facilitate plant growth in saline soils.
The AMF maintains physiological and biochemical processes of the host plant.
In salt-stressed soil, PO43− ions usually precipitate along with Ca2+, Mg2+, and Zn2+
and remain less available to plants (Azcon-Aguilar et al. 1979). However, AMF
symbiosis in plants enhance the uptake of less mobile phosphorus by extending
their external hyphal network beyond nutrient depletion zones. Apart from salt
stress mitigation, AMF also improves plant growth and hormonal status, increases
nutrient acquisition, maintains osmotic balance and reduces ion toxicity (Juniper
and Abbott 1993). It also stabilizes soil for plant growth by producing glomalin, a
substance that binds soil aggregates (Wright and Upadhyaya 1998). The purpose of
this chapter is to outline the current state of knowledge on soil salinity mycoreme-
diation. This chapter highlights interaction mechanisms between AMF and
322 D. Kumar et al.
MF-colonized plants in which AM fungi ameliorate the deleterious effects of

A
salinity. It includes a brief discussion on how this knowledge is currently being used
for mycoremediation of soil salinity with future prospects.
13.2 orphological Features of AMF and Development

M
of Fungal Symbiosis
The AMF are soil fungi that form a mutualistic symbiosis with the plant roots. It is
reported that AMF occurs in most of the plant species and taxonomically the sym-
biotic associations of AMF with the plants are widespread in nature (Schwab et al.
1991). The AMF occurrence is too diverse from geography and plant taxonomy
point of view. However, it has become possible to define two morphological types,
the Arum-type and the Paris-type. These types were named after the plants in which
they were first described for Arum maculatum and Paris quadrifolia (Cavagnaro
et al. 2001). Smith and Smith (1997) found that the morphology of the AMF sym-
biosis with the angiosperms depends on plant taxa (plant species). It was concluded
that 30 families belong to Arum-type, 41 to Paris-type and 21 families form either
an intermediate morphology or had members with both Arum and Paris-types. The
plant largely controls AMF morphology through the presence or absence of exten-
sive air-spaces in roots (Brundrett and Kendrick 1990).
The general morphology of AMF can be differentiated into six different types:
1. The intracellular hyphae forming coils often found in the outer layers of cortical
parenchyma
2. The intercellular hyphae
3. The intracellular hyphae with numerous ramifications, i.e., the arbuscules
4. The inter- or intracellular hypertrophied hyphae, i.e., the vesicles
5. The extracellular ramified hyphae, i.e., branched-absorbing structures (BAS)
6. The resistance propagules, i.e., the spores
The AMF spores are capable of independent germination. However, AMF devel-
opment is hindered for their inability to complete the life cycle in the absence of
host and the fungus start producing vigorous mycelia. This is because the AM fungi
coevolved along with host plants and require each other for mutual development
(Remy et al. 1994; Redecker et al. 2000). The AMF has different survival strategies
like regulation of infection structure (Giovannetti et al. 1994), the ability of multiple
germinations (Koske 1981), induction of energy-saving mechanism of spores ger-
minate in the absence of the host (Logi et al. 1998), etc. In addition, the ability to
form wide hyphal networks by pre-symbiotic and symbiotic mycelia represents a
fundamental mechanism for increasing the chances of AM symbionts to contact
host roots (Giovannetti et al. 1999).
The hyphae of AM fungal soil propagules and asexual spores or mycorrhizal
roots begin the establishment of AMF symbiosis by colonizing on compatible plant
roots. Even dead roots from annual plants might be a good source of AMF growth
because roots protect the fungus from environmental hazards until new hyphae
grow out of the roots and colonize plants (Requena et al. 1996). The hyphae of fun-
gus penetrate the plant cortex through an aspersorium to form distinct morphologi-
cally special structures like inter- and intracellular hyphae, coils, and arbuscules.
Out of these structures “arbuscules” are specialized hyphae similar to haustoria of
plant pathogenic fungi. They are presumed to be the main site of nutrient exchange
between the plant and the fungus (He and Nara 2007). After host colonization, the
fungal mycelium grows out of the root, exploring the soil in search of mineral nutri-
ents to colonize other susceptible roots (Breuninger and Requena 2004). The fungal
life cycle completes after the formation of asexual chlamydospores on the external
mycelium. The life cycle and different morphological parts of AMF are shown in
Fig. 13.1.
Fig. 13.1 Showing the life cycle of AM fungi and different morphological parts of AMF. (From
Porcel et al. 2012)
324 D. Kumar et al.
13.3 Ecophysiology Mechanisms of AMF
The interactions between plants and AMF affect the plant ecophysiology and have
the following consequences on nutrition, growth, competition, stress tolerance, fit-
ness, and soil structuring:
1. Enhanced uptake of low mobile ions
2. Improved quality of soil structure
3. Enhanced plant community diversity
4. Improved rooting and plant establishment
5. Improved soil nutrient cycling
6. Enhanced plant tolerance to biotic and abiotic stress
13.3.1 Development of Extraradical AMF Networks
A network of AM fungal hyphae is present in almost every terrestrial ecosystem

but our knowledge lacks the proper understanding of morphology and develop-
ment of AM fungal network. The reason for this knowledge gap lies in method-
ological difficulties to extract and observe AM mycelia from the soil. The common
mycelia extraction method includes blending, wet sieving, and decanting of soil
samples (Jakobsen et al. 1992). The soils rich in organic or porous particles may
not yield the complete mycelium. Nevertheless, artificial growth media like sand
or glass bead supplied with nutrient solution are frequently used as substrates for
AMF growth (Chen et al. 2001). These nutrient solutions allow the extraction and
analysis of AM mycelium samples. However, but it cannot be neglected that mor-
phology and activities of AM hyphae in nutrient solution differ considerably from
those in the soil. Studies of extraradical AMF development on employed axenic
cultures of AM fungi with Ri T-DNA-transformed roots have been done (de Souza
and Declerck 2003). A substrate consisting of 40 μm wet sieved soil and glass
beads, which allows for the complete extraction of intact AM mycelium, was pro-
posed as a growth substrate for AM mycelia (Neumann and George 2005). This
substrate may reproduce soil conditions better than previously used artificial
growth substrates.
The AM mycelia are made of morphologically different hyphal types. The coarse
and thick-walled hyphae with a diameter between 5 and 20 μm that appear to func-
tion mainly in nutrient transport and extension of the fungal colony at rates of
1–2 mm per day (Olsson and Wilhelmsson 2000) are called “runner-hyphae” that
elongate and form hyphopodia parallel to the plant root axis (Friese and Allen
1991). The runner-hyphae extend radially around the root and can reach distance up
to 24 cm (Drew et al. 2006). They may rapidly spread AM infection over non-
colonized neighboring roots of the same or different host plants (Drew et al. 2006;
Voets et al. 2009).
The AM hyphae bridges between neighboring roots and may be formed by

hyphal anastomosis of the same species (Croll et al. 2009). The coarse extraradical
AM hyphae form clusters of fine branched, thin-walled structures with a diameter
below 4 μm (Bago 2000). When AMF infection is watched on Petri plates, it resem-
bled in their morphology with 5–7 days old intraradical arbuscules (Cano 2005),
which show high metabolic activity (Bago et al. 2002). Since intra-radical arbus-
cules are presumably the major sites of AMF nutrient uptake from the soil, therefore
called branched absorbing structures (BAS).
13.4 ffect of Soil Salinity on Plants and Their Self-Defense

E
Mechanisms
Salinity represents the concentration of dissolved mineral salts present in the

soil and water. The dissolved mineral salts consist of the electrolytes of several
types of cations and anions. The establishment, growth, and development of
plants are affected by soil salinity that causes a huge loss in the productivity of
the plants (Evelin et al. 2009; Yadav et al. 2017a, b). Due to the soil salinity,
plants undergo some major physiological stresses that affect the plant system
very badly. The toxic effect of ions, mostly Na+ and Cl−, damage the structure of
enzymes and other macromolecules as well as cell organelles, disrupt photosyn-
thesis and respiration, inhibit protein synthesis and induce ion deficiencies
(Juniper and Abbott 1993). The high salt concentration induces physiological
drought condition in plants. The plant root cells are osmotically sensitive and
under normal conditions they must maintain lower internal osmotic potentials to
prevent water from moving into the soil. In addition, soil salinity creates a nutri-
ent imbalance in the plant caused by decreased nutrient transport to the shoot
(Adiku et al. 2001). Consequently, it affects all the systemic process of plants
such as growth, photosynthesis, protein synthesis, energy, and lipid metabolism
(Ramoliya et al. 2004).
The plant cell membranes have receptors for perceiving stress signal to activate
production of few secondary signal molecules such as Ca2+, inositol phosphates,
reactive oxygen species (ROS), and abscisic acid (ABA). The stress signals are
transduced inside the nucleus where they induce multiple stress-responsive genes
and the products of genes to induce plant adaptation to salinity. Plants start
expressing early genes within minutes of stress perception, and their products
(e.g., various transcription factors) can activate the expression of delayed genes
(e.g., RD gene [responsive to dehydration], KIN gene [cold induced], COR gene
[cold responsive]). Such gene products are either directly involved in cellular pro-
tection against the stress (e.g., late embryogenesis abundant proteins, antifreeze
proteins, antioxidants, chaperons, and detoxification enzymes) or indirectly (e.g.,
transcription factor and enzymes of phosphatidylinositol metabolism) (Tuteja
2007) protect the plant.
326 D. Kumar et al.
13.5 Effect of Soil Salinity on AMF
Soil salinity negatively impacts the host plant and associated AMF by inhibiting
colonization capacity, spore germination, and growth of fungal hyphae. It was
reported that AMF colonization on plant roots is reduced in the presence of NaCl
(Ojala et al. 1983; Poss et al. 1985; Duke et al. 1986; Rozema et al. 1986; Menconi
et al. 1995; Juniper and Abbott 2006; Giri et al. 2007; Sheng et al. 2008). The higher
NaCl concentrations directly affect fungal growth and metabolism (Juniper and
Abbott 2006) and could suppress the formation of AM (Tian et al. 2004; Sheng et al.
2008). The variable behavior of AM fungi in a similar ecosystem could be associ-
ated with soil salinity (Kilironomos et al. 1993) and some other environmental fac-
tors (Carvalho et al. 2001).
In the presence of higher concentrations NaCl, the germination of spores is
delayed (Cantrell and Linderman 2001). The rate of germination and maximum
germination of AMF spores may also depend on the salt type. According to Juniper
and Abbott (1993), the sodium salts like NaNO3 and Na2SO4 with osmotic potentials
similar to NaCl impart differential effects on the rate and germination of spores.
Jahromi et al. (2008) studied in vitro effects of salinity on the AM fungus, Glomus
intraradices and observed that there was no significant difference in hyphal length
and BAS between control (no salt) and 50 mM NaCl, but a significant decrease in
hyphal length and the number of BAS at 100 mM NaCl was observed. All these
results indicate that soil salinity directly affects fungal development by reducing
fungal mycelia formation and host root colonization.
However, contrary to the mentioned reports, increased AMF sporulation and
colonization under salt stress were reported by Aliasgharzadeh et al. (2001). Yamato
et al. (2008) reported that AM colonization rates were not reduced in coastal vegeta-
tions of Okinawa Island, Japan even when treated with salinity as high as 200 mM
of NaCl. The variation in the results invites researchers to look out for salt-tolerant
AMF species and to test mycorrhizal isolates that could maintain colonization
capacity and symbiosis efficiency under salinity.
13.6 Effect of AMF on Plant Biomass and Nutrient Uptake
For remediating the harmful effect of salinity, the AMF colonization plays a very
crucial role in plants. The AMF protects the symbiotic plants against salt stress. It
was demonstrated that the symbiosis results in increased uptake of nutrient, accu-
mulation of osmoregulator compounds, increase in photosynthetic rate, and water
use efficiency (Andrea et al. 2016). It was suggested that salt stress remediation by
AMF results from a combination of nutritional, biochemical, physiological, and
molecular effects (Atakan et al. 2018). However, remediation effect on plant devel-
opment depends on the AMF species (Marulanda et al. 2003, 2007; Wu et al. 2007).
Several workers reported that AMF-inoculated plants grow better than
non-inoculated ones under salt stress (Al-Karaki 2000; Giri et al. 2007; Sannazzaro
et al. 2007; Zuccarini and Okurowska 2008). Hajiboland et al. (2010) reported that
although high salinity reduces dry matter production of tomato cultivars; however,
in all treatments, mycorrhizal plants grow better by improving plant nutrient uptake,
especially P (Smith and Read 1997). Many other workers related improved growth
of mycorrhizal plants in saline conditions with the mycorrhiza-mediated enhance-
ment of P nutrition in plants (Hirrel and Gerdemann 1980; Ojala et al. 1983; Pond
et al. 1984; Poss et al. 1985; Al-Karaki 2000; Verma et al. 2017; Yadav et al.
2018; Kour et al. 2019; Rana et al. 2019a, b).
13.6.1 I ncreased Nutrient Uptake and Transport by AM

Hyphae
The enhanced growth of AM plants with improved P uptake is a common observa-

tion in greenhouse studies, particularly when plants are grown on soil with low
nutrient availability. Investigations on the uptake, transport, and transfer of mineral
elements by AMF mycelia to host plants were done under in vivo or in vitro micro-
cosms. Wang et al. (2002) studied the uptake, transport, and delivery of P and N of
plant-AM fungal combinations under varied environmental conditions. In many
studies, uptake and transport of Zn (Jansa et al. 2003), S (Allen and Shachar-Hill
2009), Fe (Caris et al. 1998), and Cu (Lee and George 2005) were verified via AM
pathway. However, the transport of nutrients other than P, N, Zn, Fe, and S
through AM mycelium has not been unequivocally verified.
The AM symbiosis shows increased tolerance to biotic and abiotic stress. Several
studies of plant-AM symbiosis confer host tolerance to drought (Ruiz-Lozano 2003;
Miransari 2010), heat (Compant et al. 2010), salinity (Evelin et al. 2009; Miransari
2010), or osmotic stress (Ruiz-Lozano 2003). Nevertheless, the early works on
biotic stress of mycorrhizae were mostly descriptive (Linderman 2000). The AM
symbiosis occurs in almost all the habitats including disturbed soils contaminated
with heavy metals and plays an important role in improving plant metal tolerance.
Several AM-mediated mechanisms of plant protection include dilution of plant tis-
sue toxins, sequestration of the toxic metals in the fungus, and tolerance develop-
ment by the fungi (Hildebrandt et al. 2007; Gamalero et al. 2009). Thus, it can be
concluded that some plants are unable to endure habitat-imposed abiotic and biotic
stresses in the absence of fungal endophytes.
The AM colonization of various plants improve salt tolerance and growth.
However, relatively few mechanisms demonstrate the increased tolerance of AM
plants to salt stress (Cho et al. 2006). The improved growth of AMF-colonized
plants was shown to enhance nutrient uptake, particularly N and P (Jeffries et al.
2003). In one study, salt tolerance was not found correlated with P concentration
(Ruiz-Lozano and Azcon 2000). According to Auge (2001), the contribution of AM
symbiosis to the salinity remediation of host plants can explain the salt tolerance
328 D. Kumar et al.
mechanisms such as enhanced osmotic adjustment, leaf hydration, increased water

use efficiency, reduced oxidative stress, and improved nutritional status. Due to soil
salinity, the water uptake ability of plants is inhibited leading to the slower growth,
causing osmotic or water-deficit effect (Munns 2005). This effect was reported in
wild and cultivated Phaseolus sp. where leaf water, osmotic and turgor potentials of
the plants decreased due to the salinity (Jimenez et al. 2003). Rosendahl and
Rosendahl (1991) reported improved water uptake by AM plants under salinity.
Kumar et al. (2009) reported that relative water content in the leaves of Jatropha sp.
was significantly higher in AM-inoculated plants growing under salinity. Under
salinity stress, the beneficial effects of AMF on plant growth may occur due to
improved water absorption, nutrient uptake and enhanced photosynthesis (Miransari
et al. 2008).
Decreased K+ concentration and K+/Na+ ratios at high salinity is another harmful
effect of salinity that disturbs the K+-specific plant functions (Tabatabaei 2006).
Generally, K+ is a preferred ion of cytoplasm as it provides a reactive environment
for cellular enzymes. In addition, K+ is also an osmotic equivalent under water stress
and salinity. However, under high salinity, the Na+ spontaneously enters plant cells
via nonselective K+ channels (Hammer et al. 2010). The spontaneous accumulation
of Na+ can be prevented and K+ uptake could be improved by AMF inoculation on
plants. Many studies show decreased Na+ accumulation under salt stress in Sesbania
spp. (Giri and Mukerji 2004) and Lotus glaber (Sannazzaro et al. 2006) due to AM
association. In addition, K+ uptake is also enhanced by the influence of mycorrhiza
(Rabie and Almadini 2005).
Interactions of Na+, Cl−, and many mineral nutrients affect the plant growth, caus-
ing an imbalance in nutrient availability, uptake, or distribution within plants, thus
increasing plant’s requirement for essential elements (Grattan and Grieve 1992).
Many reports show lowered Na+ concentrations in AMF plants growing under salin-
ity (Ashraf et al. 2004; Ghazi and Al-Karaki 2006; Sharifi et al. 2007; Kaya et al.
2009; Porras-Soriano et al. 2009). The growth and metabolism of plants are affected
by soil salinity, N availability, and other environmental factors (Shenker et al. 2003).
The P availability and activity also reduces in saline soil (Grattan and Grieve 1999).
However, plants colonized by Glomus sp. show higher N and P contents in shoots of
lettuce at the highest salt level than non-colonized ones at the lowest salt level (Ruiz-
Lozano and Azcon 2000). It was reported that the efficiency of P uptake by an AMF
is strongly affected by the spatial distribution of its hyphae in the soil and possibly
also by the differences in the capacity for uptake per unit length of hyphae (Jakobsen
et al. 1992). Mycorrhizal inoculation improves P nutrition of plants under salinity
stress and reduces the negative effects of Na+ by maintaining vacuolar membrane
integrity, which prevents this ion from interfering in growth metabolic pathways
(Rinaldelli and Mancuso 1996). It has been observed that the chief mechanism for
enhanced salinity tolerance in the mycorrhizal plant is actioned by improving P
availability (Copeman et al. 1996). The AMF plants increase P uptake in compensa-
tion of salt stress (Tian et al. 2004). A symbiotic association of specific mycorrhizal
fungi with plant species, and also the changes made by the AMF in plants for the
amelioration of soil salinity are shown in Table 13.1.
Table 13.1 Showing the effect of different AMF on enhancement tolerance of several plants
growing under salt stress
Plant species AMF species Effect of AMF on plant References
Glycine max Glomus etunicatum Increase the uptake of P, K, Ca, Sharifi et al.
and Zn (2007)
Gossypium Glomus mosseae Increase the uptake of P, Na, and Tian et al. (2004)
arboretum Cl
Lycoperssicon Glomus mosseae Increase the uptake of P and Na Al-Karaki (2000)
Esculentum and decreases the uptake of Cu
and Zn
Acacia nilotica Glomus fasciculatum Increase the uptake of Cu, Na, Giri et al. (2007)
and P
Acacia Glomus fasciculatum Increase the uptake of P and Na Giri et al. (2003)
auriculiformis and Glomus
macrocarpum
Citrus karma Mixed inoculum of Increase the uptake of N and P Murkute et al.
Glomus sp. and (2006)
Gigaspora sp.
Zea mays Glomus mosseae Increase the uptake of P and Feng et al.
increases soluble sugar and (2002)
electrolyte concentrations
Sesbania Glomus macrocarpum Increase the uptake of N and Mg Giri and Mukerji
aegyptiaca (2004)
Ocimum Glomus macrocarpum Increase the uptake of N and Mg Zuccarini and
basilicum Okurowska
(2008)
Musa sp. Glomus intraradices Increases the uptake of K and Cl Yano-Melo et al.
(1999)
Lactuca sativa Glomus clarum Increase the uptake of K Ruız-Lozano
et al. (1996)
Lactuca sativa Glomus sp. Increases transpiration, stomatal Ruız-Lozano
conductance, and water use et al. (1996)
efficiency
13.7 Mechanism of Remediation of Salinity Stress by AMF
13.7.1 Biochemical Change
The strategies adopted by the plant to remediate soil salinity include (1) synthesis
and accumulation of compatible solutes, (2) control of ion uptake by roots and
transport to plant tissues for ion homeostasis, (3) fine regulation of water uptake and
distribution to plant tissues by the action of aquaporins, and (4) reduction of oxida-
tive damage through improved antioxidant capacity. Additional plant responses
include selective build-up or exclusion of salt ions, maintenance of photosynthesis
at values adequate for plant growth, changes in membrane structure and synthesis of
phytohormones (Turkan and Demiral 2009). Many studies were done to understand
330 D. Kumar et al.
mechanisms for enhanced salt tolerance of AMF plants (Al-Garni 2006). The AMF
facilitate plants by enhancing nutrient mobility, improving water uptake through
extended fungal hyphae that act as root hairs, boosting root hydraulic conductivity,
and by maintaining osmotic balance by enhancing K+/Na+ ratios and lowering accu-
mulation of Na+ in the host plant shoots. Among the mechanisms, the best charac-
terized biochemical response of plant cells is osmotic stress in which accumulated
inorganic ions like Na+ and compatible organic solutes like proline, glycine betaine,
and soluble sugars were studied (Flowers and Colmer 2008). The compatible sol-
utes can accumulate to high levels in the cells without disturbing intracellular bio-
chemistry (Bohnert and Jensen 1996) and, consequently, protect subcellular
structures, mitigate oxidative damage caused by free radicals, and maintain the
enzyme activities under salt stress (Yokoi et al. 2002).
13.7.1.1 smotic Adjustment with the Help of AMF for Remediation

O
of Soil Salinity
The salt-accumulated soil has negative water potential, which implies less avail-
ability of soil water to the plant leading to the dehydration of the plant cells.
Therefore, plants must respond accordingly by decreasing their water potential to
maintain a favorable concentration gradient. Osmotic adjustment or osmoregulation
is the most important mechanism adopted by plants to reduce osmotic potential in
which some inorganic ions like Na+ and K+ and compatible organic solutes accumu-
late in the cells (Morgan 1984; Hoekstra et al. 2001). Among them, proline, beta-
ines, sugars (fructose, sucrose, and glucose), and complex sugars (trehalose,
raffinose, and fructans) are some compatible solutes that chiefly accomplish this
function in halophytes. The two major osmoprotectant osmolytes include proline
and glycine betaine (N, N, N-trimethylglycine betaine) which are synthesized by
plants in response to stress. These osmoprotectants help in maintaining the osmotic
status of the cell to ameliorate abiotic stress. Proline plays a role in scavenging free
radicals, stabilizing subcellular structures, and buffering cellular redox potential
under stresses. The salinity stress-responsive genes whose promoters contain
proline-responsive elements (ACTCAT) are also induced by proline (Chinnusamy
et al. 2005). Proline is an important osmoprotectant that plays a vital role in stabiliz-
ing subcellular structures, buffering cellular redox potential under stress and in
scavenging free radicals (Chen and Dickman 2005). In higher plants, proline is
synthesized from glutamic acid by the actions of two enzymes, pyrroline-5-
carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR).
Overexpression of P5CS gene in transgenic tobacco increases the production of
proline that helps in salinity tolerance (Kishor et al. 1995). Proline concentration
also increases during AMF colonization on plants. Several authors reported higher
proline concentration in AMF plants than in non-AMF plants at different salinity
levels (Sharifi et al. 2007; Talaat and Shawky 2011). In addition, another osmoregu-
lator named betaines also stabilize the structures and activities of enzymes, protein
complexes and maintain the integrity of membranes against the damaging effects of
excessive salt (Gorham 1995). The AMF plants increase the accumulation of beta-
ines under salt stress (Al-Garni 2006).
The increase in sugar content is found to be positively correlated with the mycor-
rhization of the host plant as reported by Thomson et al. (1990). Porcel and Ruiz-
Lozano (2004) reported increased sugar concentrations in soybean roots colonized
with Glomus intraradices subjected to drought stress. The positive correlation
between sugar content and mycorrhization is due to the sink effect of the fungus
demanding sugars from the shoot tissues (Augé 2000). The increased sugar accu-
mulation in plants may also be due to AMF-facilitated starch hydrolysis. However,
some authors reported negative correlations between AMF colonization and sugar
accumulation in host plants. Pearson and Schweiger (1993) reported a reduction in
carbohydrate content with an increase in the percentage of root colonization. Sharifi
et al. (2007) observed no role of soluble carbohydrates in the responses of AM
(colonized by Glomus etunicatum) soybean plants to salinity.
13.7.1.1.1 Polyamines
The polyamines are small organic cations necessary for eukaryotic cell growth. The
three main polyamine types existing in plants are putrescine (Put), spermidine
(Spd), and spermine (Spm). The plant-secreted polyamines play a major role against
a wide array of environmental stresses like salinity (Delauney and Verma 1993),
high osmolarity (Besford et al. 1993), and antioxidative stress (Kurepa et al. 1998).
They are also candidates for the regulation of root development under saline situa-
tions (Couee et al. 2004). Under salinity stress plants reduce the concentration of
free polyamine. The polyamine increases membrane stability and show a significant
effect on H+/ATPase and Ca2+/ATPase transporters during salinity stress (Pottosin
and Shabala 2014).
13.7.1.2 hytohormone Content Regulation by AMF for Remediation

P
of Salinity
Many phytohormones regulate cell division, plant growth, fruit ripening, stem elon-
gation, and other biochemical function. Out of these, ABA responds against abiotic
stress, and its concentration rise when plants experience stress. The ABA promotes
stomatal closure to reduce water loss. It also induces the expression of stress-related
genes and diminishes the environmental damage (Evelin et al. 2009). A report
showed that AMF plants change the ABA concentration under the saline condition
(Estrada-Luna and Davies 2003). It was observed that ABA concentration in AMF
plants decreased in lettuce plants colonized by G. intraradices (Jahromi et al. 2008).
The AMF-infected plants experience less environmental stress and, as a result,
accumulate less ABA. Evelin et al. (2009) observed the effect of AMF species on
ABA content varies with the host plants. As AM fungi help in remediating the salin-
ity effect, therefore could be used as agents for salinity-induced remediation.
332 D. Kumar et al.
13.7.1.3 Antioxidant System
A number of biotic, abiotic, and xenobiotic stresses can be linked to some degenera-
tive reactions that produce the ROS such as superoxide (O2−), hydroxyl radicals
(OH−), hydrogen peroxide (H2O2), and singlet oxygen in plants. These are produced
as reaction by-products of electron transport chains of chloroplast and mitochondria
where oxygen and electrons react to form ROS (Scandalios 1993). The plant ROS
production must be minimized because hydroxyl radical and singlet oxygen are
highly reactive and damage life-giving biomolecules (Jakob and Heber 1996). The
O2− and H2O2 are especially toxic ROS compounds that initiate cascade reactions,
resulting in the production of the hydroxyl radicals. According to Bowler et al.
(1992), free radicals and their derivatives are dangerous and reactive and could
damage biomolecules through lipid peroxidation, denaturation of proteins, and
DNA mutation.
Plant cells contain an array of protective and repair mechanisms that mini-
mize oxidative damage. According to Smirnoff (1993), the repair mechanisms
can be divided into two categories: (a) systems that scavenge activated oxygen
species which include carotenoids, glutathione, tocopherols, ascorbic acid, fla-
vonoids, and anthocyanins (Wu and Xia 2006) and (b) systems which possess
ROS-scavenging antioxidative enzymes like superoxide dismutase (SOD), cat-
alase (CAT), ascorbate peroxidase (APOX), glutathione reductase, dehydro-
ascorbate reductase, monodehydroascorbate reductase, guaiacol peroxidase,
oxidized glutathione, glutathione peroxidase, and the enzymes involved in the
ascorbate-glutathione cycle (Alguacil et al. 2003). Like other abiotic stresses,
salinity also induces oxidative stress in plants (Hajiboland and Joudmand
2009).
A correlation between antioxidant capacity and salinity tolerance was
reported in several plant species (Nunez et al. 2003; Turkan and Demiral 2009).
The reports suggest that AMF symbiosis helps plants to remediate salt stress by
enhancing the activities of antioxidant enzymes like superoxide dismutase
(SOD), catalase (CAT), ascorbate peroxidase (APOX), glutathione reductase,
dehydroascorbate reductase, monodehydroascorbate reductase, and guaiacol
peroxidase (Porcel et al. 2003; Garg and Manchanda 2009; Talaat and Shawky
2011).
13.7.2 Role of AMF in Physiological Changes
Salt stress disrupts plant physiological mechanisms by decreasing photosynthetic

efficiency, gas exchangeability, water status, and membrane integrity (Aroca et al.
2006; Porcel et al. 2006).
Several pieces of evidence demonstrate that AMF symbiosis can alleviate salt-
stress effects by employing several mechanisms that are discussed below.
13.7.2.1 Chlorophyll Content
Salt stress inhibits photosynthetic ability and induces physiological drought in

plants, causing decreased crop production. The soil salinity was reported to reduce
plant chlorophyll content (Sheng et al. 2008). The salinity-induced reduction in
chlorophyll content could be due to suppression of enzymes responsible for the
synthesis of photosynthetic pigments (Murkute et al., 2006). Under salinity the
plant uptake of Mg2+ decreases, which is needed for chlorophyll biosynthesis. The
reduction in Mg2+ concentration lowers leaf chlorophyll concentration (El-Desouky
and Atawia 1998). However, many studies report higher chlorophyll content in
leaves of AM plants facing saline stress (Sannazzaro et al. 2006; Zuccarini 2007;
Colla et al. 2008; Sheng et al. 2008). This suggests lower salt interference in chlo-
rophyll biosynthesis in mycorrhizal than in non-mycorrhizal plants (Giri and
Mukerji 2004). According to Giri et al. (2003), the antagonistic effect of Na+ on
Mg2+ uptake is counterbalanced and suppressed by the presence of mycorrhiza.
By chlorophyll content measurement, it was estimated that the photosynthetic
capacity of salt-stress AMF plant is superior to the non-stressed plants, which
implies that mycorrhization of the plants can fully counterbalance the salt stress
(Zuccarini 2007).
13.7.2.2 Relative Permeability
The AMF inoculation of host plants enable them to maintain higher electrolyte con-
centration that improves the integrity and stability of the cell membrane.
Consequently, the electrical conductivity of mycorrhizal roots was found to be
higher than the non-mycorrhizal roots (Garg and Manchanda 2008). The mycor-
rhizal roots of Cajanus cajan showed higher relative permeability than non-
mycorrhizal plants at different levels of soil salinity (Garg and Manchanda 2008).
Kaya et al. (2009) reported the electrolyte leakage of 31.66 and 42.45, respectively,
in leaves of Capsicum annum treated with 50 mM and 100 mM NaCl, while the
AMF-inoculated plants had a relatively lower electrolyte leakage of 26.87 and
30.98, respectively. This suggests that AM plants have lower root plasma membrane
electrolyte permeability than the non-mycorrhizal plants. The increased membrane
stability has been attributed to mycorrhiza-mediated enhanced P uptake and
increased antioxidant production (Feng et al. 2002).
13.7.2.3 Water Status
The water is essentially needed to mobilize plant nutrients. In saline soils, Na+
and Cl− ions bind with water and create a physiological drought-like condition
for plants (Fuzy et al. 2008). Several studies indicate that AMF colonization
can help plants to remediate the physiological drought. It has been reported
that plants inoculated with AMF maintain relatively higher water content
334 D. Kumar et al.
(Colla et al. 2008; Jahromi et al. 2008; Sheng et al. 2008). This is facilitated
by the improved hydraulic conductivity of the root at low water potential
(Kapoor et al. 2008). The improved root conductance is associated with a lon-
ger root and an altered root system morphology induced by AMF (Kothari
et al. 1990).
The AMF plants exhibit a higher stomatal conductance to increased leaf transpi-
ration (Jahromi et al. 2008; Sheng et al. 2008). Mycorrhizal plants hold lower
osmotic potential that is maintained by fungal accumulating solutes, resulting in
improved plant osmotic adjustment. Lower water saturation deficit and higher tur-
gor potential of AMF plants also improve their water status (Al-Garni 2006). All the
improved parameters facilitated by mycorrhizal colonization enable the host to uti-
lize water more efficiently and allow them to maintain a lower intercellular carbon
dioxide concentration. As a consequence, the gas exchange capacity increases in the
mycorrhizal plants.
13.7.2.4 Nodulation and Nitrogen Fixation
Nitrogen-fixing bacteria-mediated nodule formation in plants is considered a

target for salt stress, and its occurrence decreases due to salt accumulation
(Harisnaut et al. 2003). This is likely due to premature nodule senescence trig-
gered by salt stress (Gonzalez et al. 1998), which causes accelerated lytic activi-
ties, formation of green pigments from leghemoglobin, and loss of nitrogen
fixation (Delgado et al. 1994). Application of AMF in legumes could counteract
the harmful effects of salinity on nodulation and nitrogen fixation. The AMF
symbiosis could alleviate drought stress-induced premature nodule senescence
(Ruiz-Lozano et al. 2001). Giri and Mukerji (2004) reported a positive effect of
mycorrhizal inoculation on nodule formation under salt stress. The legume col-
onization by AMF could increase the number of nodules (Garg and Manchanda
2008). This indicates a positive influence of AMF on legume–nitrogen-fixing
bacteria symbiosis due to enhanced leghemoglobin content, which is deter-
mined by observing the nodule color change from pink to brownish pink due to
the synthesis of green pigments by leghemoglobin. The greening of the nodule
was observed much earlier in AMF-colonized legume plants (Garg and
Manchanda 2008). In addition, mycorrhizal plants hold higher nitrogenase
activity. All these parameters contribute to the higher nitrogen-fixing ability of
AMF plants. The increased nitrogenase activity and nitrogen fixation in AMF-
colonized legume plants is beneficial for nitrogenase enzyme functioning pos-
sibly through enhanced uptake of essential micronutrients, resulting in improved
plant growth (Founoune et al. 2002). Therefore, it may be concluded that mycor-
rhizal and nodule symbioses often act synergistically on mineral nutrition, and
plant growth to fulfill the need of N and P and to increase tolerance of plants to
salinity (Rabie and Almadini 2005).
13.7.3 Molecular Changes in Plants
The beneficial effects of the AMF symbiosis on plant salinity tolerance are assessed
by measuring plant growth, water status, or the expression of plant-specific stress-
related genes. The effects of AMF on plant responses to salinity stress are difficult
to measure due to the complex nature of the association. The heterokaryotic and
obligate nature of AMF also creates difficulty in understanding plant molecular
changes. However, molecular studies on salt tolerance by AMF are speeding up for
holistically understanding the mechanism of salt stress remediation by AM symbio-
sis. The expression and overexpression studies of proteins like Δ1-pyrroline-5-
carboxylate synthetase (P5CS), aquaporins, cation channels and transporters, late
embryogenesis abundant protein (LEA), and ABA synthesis in AMF plants treated
with salinity stress are underway (Hanin et al. 2011).
13.7.3.1 1-Pyrroline-5-Carboxylate Synthetase (P5CS)
The PC5S enzyme catalyzes the rate-limiting step in the biosynthesis of proline.
Proline is the widespread osmoprotectant in plants. The PC5S has two catalytic
sites: (1) γ-glutamyl-kinase and (2) glutamic-γ-semialdehyde-dehydrogenase. The
P5CS catalyzes the first two steps of proline biosynthesis from glutamate using
γ-glutamyl-kinase and glutamic-γ-semialdehyde-dehydrogenase activities. The last
step of proline biosynthesis is catalyzed by the Δ1-pyrroline-5-carboxylate reduc-
tase (P5CR) that reduces the Δ1-pyrroline-5-carboxylate to proline (Hu et al. 1992).
Yoshiba et al. (1995) observed that the P5CS-encoding gene is induced by drought
stress, salinity, and by ABA treatment in Arabidopsis thaliana. The P5CS-encoding
gene is of key importance for the biosynthesis of proline in plants (Abraham et al.
2003). The overexpression of the P5CS-encoding gene in transgenic tobacco plants
increase proline production, which confers plant tolerance to osmotic stress (Kishor
et al. 1995). Jahromi et al. (2008) reported the higher expression of Lactuca sativa
P5CS gene in non-AMF plants at 50 mM NaCl and 100 mM.
13.7.3.2 Aquaporins
Aquaporins belong to the major intrinsic protein (MIP) family of transmembrane

channels that facilitate and regulate the passive movement of water molecules follow-
ing a water potential gradient (Hill et al. 2004). In plants, aquaporins are subdivided
into five evolutionarily distinct subfamilies: (1) the plasma membrane intrinsic pro-
teins (PIPs), (2) the tonoplast intrinsic proteins (TIPs), (3) the small basic intrinsic
proteins (SIPs), (4) the nodulin-like intrinsic proteins (NIPs), and (5) the uncharacter-
ized X intrinsic proteins (XIPs) (Gupta and Sankararamakrishnan 2009), which were
shown to transport a variety of uncharged substrates (Bienert et al. 2011). The role of
aquaporin in water uptake was confirmed by inhibiting root water transport by the
336 D. Kumar et al.
general aquaporin blocker mercury ions (Maggio and Joly 1995). In general, AMF
differentially exerts control over the expression of different members of the large fam-
ily of aquaporins (Ouziad et al. 2006). The AM-plant symbiosis enhances the expres-
sion of PIP genes, and its product contributes in regulating root water permeability to
tolerate salinity-induced osmotic stress (Aroca et al. 2007). Such differences could be
a consequence of the mode of salt stress in plants. Sarda et al. (1999) observed com-
plex and differential expression pattern members of the large family of aquaporins in
tested plant species. This highlights the complex regulation of aquaporin genes in
response to the AMF symbiosis under abiotic stress with osmotic component.
13.7.3.3 Late Embryogenesis Abundant Proteins
Close (1996) reported the accumulation of proteins groups called late embryogenesis
abundant (LEA) proteins in mature plant seeds, responsible for desiccation tolerance.
During cellular dehydration, the LEA proteins maintain the structure of other pro-
teins, vesicles, or endomembrane constituents required for the sequestration. In addi-
tion, the LEA proteins help in absorbing water and also function as molecular
chaperones (Koag et al. 2003). The reports show that overexpression of LEA proteins
in plants and yeasts confer tolerance to osmotic stress (Imai et al. 1996; Babu et al.
2004). Dehydrins belong to LEA group 2 and represent the most noticeable soluble
proteins induced by dehydration stress. Biochemically, the activities of dehydrins are
diverse because they have multiple existences on the target sites (euchromatin, cyto-
sol, and cytoskeleton). Studies show increased gene expression of the LEA genes
during AMF symbiosis under salt stress. However, LEA gene expression and its effect
on AMF symbiosis under salt stress is not fully understood. Porcel et al. (2005)
cloned two dehydrin encoding genes from Glycine max (gmlea8 and gmlea10) and
one from L. sativa (lslea1) to analyze their response against drought stress of AMF-
infected soybean and lettuce plants. Results demonstrate that the levels of LEA tran-
script accumulation in soybean and lettuce plants colonized by G. mosseae or G.
intraradices were considerably lower than corresponding non-AMF plants.
It is suggested that the accumulation of LEA proteins is not a mechanism by
which the AMF symbiosis protects their host plant. The results suggest that mycor-
rhizal plants were less strained by drought stress through primary drought-avoidance
mechanisms. Jahromi et al. (2008) reported that LEA is expressed under salt stress
and is induced at lower rates in AMF plants.
13.7.4 Cation Channels and Transporters
13.7.4.1 Na+/H+ Antiporters
In soil the sodium uptake and distribution within the plant is responsible for their
sensitiveness to salts. Plants survive high salinity by restricting root entry of Na+
and by allocating Na+ within the leaf to sequester into the vacuole. The Na+/H+
antiporters mediate Na+ transfer out of cytoplasm into vacuole or apoplast.

Transgenic plants were reported to be more salt tolerant than the controls as
shown for Arabidopsis sp. (Sottosanto et al. 2004) and rice (Fukuda et al. 1999)
due to overexpression of Na+/H+ antiporters. In Arabidopsis sp., the Na+/H+ anti-
porters are found at various locations. The transporters contributing to Na+
homeostasis include plasma membrane (SOS1) antiporter, vacuolar Na+/H+ anti-
porters (e.g., NHX1), and the plasma membrane uniporter HKT1 (Zhu 2003).
The loss-of-function mutations in AtHKT1 render plants Na+ hypersensitive and
disturb the distribution of Na+ between roots and shoot. Ouziad et al. (2006)
analyzed the expression of two Na+/H+ antiporter genes in relation to salt and
mycorrhizal colonization. They observed no significant mycorrhization altera-
tions through the expression of LeNHX1 and LeNHX2, the latter of which had
previously been shown as K+/H+ antiporter (Venema et al. 2003). It is clear from
the presented studies that AMF colonization does not assist Na+/H+ antiporters
for balancing Na+ concentration.
13.7.4.2 Cyclic Nucleotide-Gated Channels
Ion influx is essential for signal transduction. Cyclic nucleotide-gated ion chan-
nels (CNGCs) are one of the potential pathways for ion uptake (Talke et al.
2003). Cyclic nucleotides monophosphate (CNMP) has only recently been
accepted as an important secondary messenger that might be involved in plant
response to biotic and abiotic stress. Donaldson et al. (2004) demonstrated that
rapid increase in cyclic guanosine monophosphate (cGMP) levels in Arabidopsis
thaliana are related to salt and osmotic stress, and these findings are consistent
with another finding according to which cAMP and cGMP improve tolerance to
salt stress (Maathuis and Sanders 2001). Interestingly, improved salt tolerance is
correlated with the cNMP-dependent decrease of channel opening probability
and the reduced influx of Na+ (Rubio et al. 2003). Members of the CNGC family
belong to the group of nonselective cation channels which enable the uptake of
Na+, K+, and Ca2+ (Kaplan et al. 2007). Plants hold a structure where CNGC and
cyclic nucleotide-binding domains occur in the cytosolic C-terminus of overlap-
ping regions. The Arabidopsis CNGC gene family is made of 20 members (Maser
et al. 2001). The ability of CNGCs to transport cations play a role in mediating
various environmental stresses, plant defense responses, and development
(Borsics et al. 2007). Kugler et al. (2009) reported upregulation of AtCNGC19
and AtCNGC20 in Arabidopsis thaliana shoots toward elevated NaCl. Salt
induction of CNGC20 was also observed in the shoot. No differences in K+ and
Na+ contents of the shoots were measured in homozygous T-DNA insertion lines
for CNGC19 and CNGC20, respectively, which developed a growth phenotype
in the presence of up to 75 mM NaCl similar to that of the wild type.
All these results suggest that CNGC19 and CNGC20 channels are involved
in salinity response of different cell types in the shoot. Both the channels
assist the plant to survive toxic effects caused by salt stress probably by real-
338 D. Kumar et al.
locating sodium within the plant. For obtaining a wider picture these studies
should be accomplished in combination with measurements of sodium and
potassium content in different plant tissues. This will allow us to understand
the effect of AMF symbiosis on Na+ and K+ uptake, distribution, and compart-
mentalization within the plant cell and may shed light on the mechanisms
involved.
13.8 Conclusion and Perspectives for Future Studies
It is well-recognized fact that halophytes conveniently grow in saline soil.

Now it is known that AM-inoculated plants can also grow under salinity. The
AM fungi do not completely remove the salt from the local environment.
However, it has the potential to alleviate the effect of soil salinity on plants.
Several counter-balancing processes of AMF symbiosis help in alleviating the
effect of soil salinity. In the saline soil, AMF-mediated mycoremediation of
the soil salinity causes accumulation of different compatible solutes and
higher uptake of water as well as nutrients. The symbiotic association of AMF
regulates the plant physiological and biochemical processes like water poten-
tial, ionic balance, stomatal conductance, maintenance of photosynthesis,
reduction of oxidative damage through antioxidant production, and hormone-
mediated signal transduction. Molecular basis of regulation of ionic homeo-
stasis, cation to proton antiporter and cyclic nucleotide-gated channels under
AMF symbiosis are less known and therefore should be the thrust area for
future research.
In Arabidopsis, the 20-member CNGC-encoding genes have overall
sequence similarities between 55% and 83% (Maser et al. 2001). However, in
mammalian genomes only six CNGC-encoding genes are present. The reason
for the large number and diversity of CNGCs in plants is because of important
and specialized physiological role there. For AM symbiosis no major CNGCs
specific studies were done. Only a few investigations were carried out on cat-
ion and proton antiporters, and therefore partial knowledge about the participa-
tion of cation/proton antiporters and cyclic nucleotide-gated channels is
available. In future research, the efforts should be made to understand the
effect of AM symbiosis on cation/proton antiporters, cyclic nucleotide-gated
channels, and AMF-mediated changes on plants. Mycoremediation-based
research is a promising field to shed light on mechanisms involved in the
enhanced tolerance of AM plants to salt stress. Further, transcriptomic analysis
of AMF is a promising route to furnish information about fungal genes partici-
pating in AMF-plant symbiosis. Overall, these investigations may open new
research lines aimed at harnessing maximum benefit from the AM symbiosis
under salinity or other osmotic stress.
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Chapter 14
Fungal Enzymes for Bioconversion
of Lignocellulosic Biomass
Subhadeep Mondal, Suman Kumar Halder, and Keshab Chandra Mondal
14.1 Introduction
Plant’s lignocellulosic material is the principal renewable biopolymer of our planet.

Globally the estimated production of lignocellulosic materials is around 1 × 1010 ton
annually (Hekkert and Negro 2009). Lignocellulosic biomass obtained from plant
cell wall (PCW) is chiefly composed of cellulose, lignin, hemicellulose, and pectin
(Guerriero et al. 2016). Lignin is the copious biopolymer on earth ranked after cel-
lulose. A. P. de Candolle, a Swiss botanist, in 1813 coined the term “lignine” origi-
nated from the Latin word “lignum,” which means wood (Sjostrom 1993). In 1838,
French Chemist Anselme Payen discovered that the fibrous component of all higher
plant cells had an identical chemical structure, which he named “cellulose” (Payen
1938). Schulze first ever coined the term “hemicellulose” to represent the remnants
extracted from plant materials using a dilute alkali (Schulze 1891). Henri Braconnot
in 1825 first extracted and narrated pectin (Braconnot 1825).
Despite the huge morphological diversity of PCW, it is basically divided into
three strata: primary cell wall, secondary cell wall, and middle lamella (Buchanan
et al. 2016). Primary cell wall (PCW) (CWp) is defined as wall synthesized during
cell growth. The essential components of CWp are pectin (35–45%), cellulose (25–
45%), and hemicellulose (20–30%). The cellulosic microfibrils are associated with
the hemicellulosic chain to constitute the cellulose-hemicellulose network. The
principal hemicellulose in the CWp is xyloglucan (Fry 1989). When the plant cell
enlargement stops, secondary cell wall (SCW) (CWs) synthesis started. The compo-
nents of CWs are deposited between plasma membrane (PM) and the CWp. The
chief components of CWs are cellulose (35–50%), hemicellulose (20–35%), and
lignin (10–25%). Lignin is located by excluding water and provides rigidity to the
cell wall. The middle lamella layer is flourishing with pectin.
S. Mondal · S. K. Halder (*) · K. C. Mondal

Department of Microbiology, Vidyasagar University, Midnapore, West Bengal, India
e-mail: sumanmic@mail.vidyasagar.ac.in

350 S. Mondal et al.
Cellulose is a polysaccharide of β (1–4)-linked d-glucose and the chain length

varied as minimum of hundreds to few thousands (Klemm et al. 2005). At the PM
of vascular plants, the cellulose biosynthesis is done by the hexameric terminal,
rosette terminal complex (RTC). Cellulose microfibrils are synthesized by cellulose
synthase which form individual glucans, contained within each unit of the RTC
(Kimura et al. 1999). Hemicellulose is a heteropolymer that consists of mainly
xylan, arabinoxylan, glucuronoxylan, xyloglucan, and glucomannan.
In majority of cases, xylose is present in large amount as a sugar monomer, but
in the case of softwoods, mannose is present as the principal sugar. Along with regu-
lar sugars, the acidified form of hemicellulose, for example, galacturonic acid and
glucuronic acid, can be found (Chen 2014). Proteins synthesized on the ribosome of
the endoplasmic reticulum of the plant cell can be transferred to the Golgi complex
and form glycosides; the hemicellulose produced is contained in the Golgi vesicles
and moved to the cell membrane. In the cell membrane, the Golgi vesicles inoscu-
late to the continuous plasma membrane, further causing the hemicellulose to be
stuck to the cell wall. The Golgi apparatus can produce hemicellulose because it can
produce the enzymes needed for its synthesis. Lignin is extremely recalcitrant to
degradation, synthesized in plants from the phenylpropanoid precursors such as
coniferyl, synapyl, and p-coumaryl alcohols (Niladevi 2009). The phenylpropanoid
units analogous to lignin polymer are designated as p-hydroxyphenyl (H), syringyl
(S), and guaiacyl (G) units, respectively, depending on the methoxy group substitu-
tion on the aromatic rings. Usually softwood (gymnosperms, conifers such as hem-
lock, spruce, and cedar) lignin consists of principally G units and very minute level
of H units. Hardwood (leafy deciduous trees, viz. willow, poplar, alder, and birch,
angiosperms) lignin is rich in G and S units with minute level of H units. Grasses
(monocots) have all three types of units. Monolignol biosynthesis is conducted
through the phenylpropanoid pathway. In the shikimate pathway, glucose generated
from the carbon dioxide by photosynthesis first is converted to the final intermedi-
ate: shikimic acid. Then, through the prephenic acid, the shikimic acid is converted
into the final products of the shikimate pathway: phenylalanine and tyrosine. These
two amino acids are widely present in plants and serve as starting materials for the
cinnamic acid pathway. Under the effects of various enzymes, three monomers of
lignin are finally synthesized after a set of reactions, such as deamination, hydrox-
ylation, methylation, reduction, and so on (Geng et al. 2003). Pectin principally
contains d-galacturonic acid units (Sriamornsak 2003), tethered in chains via
α-(1,4)-glycosidic linkage. Mostly four kinds of pectic substances are found in
PCW: pectic acid, pectinic acids, protopectin, and pectin (Alkorta et al. 1998).
Highest amount of pectin is found in the middle lamella of PCW, with the continu-
ous lessening from the primary wall end route to the PM. Glycosyltransferase (GT)
accountable for pectin biosynthesis required specific nucleotide-sugar substrates
and acceptors for their activities. The recently accepted model for pectin biosynthe-
sis depends on an active site of Golgi luminal GT transmembrane protein with a
nucleotide-sugar substrate which is thought to reach into the Golgi lumen through
membrane traversing protein transporters or otherwise be synthesized within the
Golgi lumen.
14 Fungal Enzymes for Bioconversion of Lignocellulosic Biomass 351
14.2 Enzymes Attacking Lignocellulosic Biomass
In the PCW, lignin surrounds cellulose along with mainly hemicellulose and pectin
forming a matrix, its degradation leading to the major loss of utilizable wood and
concern for plant pathogenesis (Bholay et al. 2012). In Fig. 14.1, the structure and
composition of primary and secondary cell wall of plant was depicted with the
enzymes responsible for the degradation of the constituents. In the subsequent sec-
tion, a brief synopsis of those enzymes is discussed.
14.2.1 Ligninase
Ligninase is an array of oxidoreductive class of enzyme that mainly consists of

lignin peroxidase (LiP; EC 1.11.1.14), manganese peroxidase (MnP; EC 1.11.1.13),
and laccase (EC 1.10.3.2). Apart from these, the degradation of highly intractable
form of lignin requires the synergistic activities of several accessory enzymes,
which may include pyranose 2-oxidase/glucose-1-oxidase (EC 1.1.3.4), aryl
Fig. 14.1 Organization and composition of primary and secondary cell wall of plant with the
arrangement of cellulose, hemicellulose, pectin, and lignin moieties, along with respective degrad-
ing enzymes
alcohol oxidase/veratryl alcohol oxidase (EC 1.1.3.7), cellobiose dehydrogenase

(EC 1.1.99.18), glyoxal oxidase (EC 1.2.3.5), and cellobiose quinone oxidoreduc-
tase (EC 1.1.5.1) (Wong 2009). Ligninase production is regulated by nutrient star-
vation, especially in nitrogen- and carbon-limited cultures (Ntwampe et al. 2010).
MnP and LiP are pertaining to the heme proteins since they contain protoporphy-
rin IX prosthetic group. Related to diverse heme peroxidases, such as horseradish
peroxidase and cytochrome c peroxidase, the enzymatic reactions catalyzed by
MnP and LiP have three consecutive steps (Plácido and Capareda 2015).
Hydrogen peroxide causes the enzyme oxidation to produce compound I and
water:
Reduced peroxidase + H 2 O 2 → Compound I + H 2 O
The first altered enzyme (compound I) catalyzes the production of free radical
(S•) and the second altered form of the enzyme (compound II) with an electron shift
from the substrate (SH: reduced substrate):
Compound I + SH → Compound II + S •
Ultimately, compound II reacts with a second substrate molecule to synthesize

an additional free radical and water. In the meantime, the enzyme reduced to its
previous actual form.
Compound II + SH → Reduced peroxidase + S • + H 2 O
Ligninin Peroxidase This enzyme was first isolated from fungi Phanerochaete
chrysosporium in 1983 (Tien and Kirk 1988). LiPs are chemically oligomannose-
type glycoprotein with a MW that varies from 38 kDa to 43 kDa (Schmidt et al.
1990). LiP is having low optimum pH 3–4.5 and comparatively elevated redox
potential. This particular character of LiP makes it an important part of ligninolytic
system. LiP oxidizes both phenolics and nonphenolics. The catalytic reactions car-
ried out by LiP are involving cleavage of the propyl side chains, oxidation of benzyl
alcohols, hydroxylation of benzylic methylene groups, phenol oxidation, as well as
cleavage of aromatic ring in nonphenolic lignin compounds (Renganathan et al.
1985; Umezawa and Higuchi 1987).
Manganese Peroxidase Kuwahara first discovered manganese peroxidase in
1984 from Phanerochaete chrysosporium (Plácido and Capareda 2015). They are
glycoproteins with a MW ranging from 38 to 62.5 kDa. MnP especially favored
Mn(II) as its reducing substrate. MnP oxidizes Mn2+ to Mn3+, with the help of
organic acid chelators, viz., oxalate, malonate, glyoxylate, etc., and in turn oxi-
dizes several compounds randomly via hydrogen and electron abstraction (Glenn
and Gold 1985). The organic acids also assist the relief of Mn3+ from the catalytic
site. The one-electron oxidation of Mn2+ to Mn3+ is a multistep reaction as follows
(Niladevi 2009):
MnP + H 2 O2 → MnP compound I + H 2 O
MnP compound I + Mn ( II ) → MnP compound II + Mn ( III )
MnP compound II + Mn ( II ) → MnP + Mn ( III ) + H 2 O
Laccase Although laccases originated in plants, bacteria, and insects, the leading
producers of these enzymes are fungi. In 1983, laccase was first time isolated from
Rhus vernicifer (Japanese lacquer tree). Later, both Bertrand and Labrode in 1896
individually isolated laccase from fungi. In a holoenzymatic monomeric form of
fungal laccase (MW 60–85 kDa), generally it has four copper atoms (Coll et al.
1993) arranged in three groups: T1, T2, and T3 (Leontievsky et al. 1997). Laccase
catalysis involves (i) reduction of the T1 copper by reducing substrate; (ii) internal
electron shifting from T1 to T2 and T3 copper; and (iii) reduction of oxygen to
water at T2 and T3 (Archibald et al. 1997). Laccase certainly attacks the phenolics
of lignin, followed by Cα oxidation, aryl-alkyl cleavage, and Cα–Cβ cleavage. The
spectrum of substrate accessibility of laccase expanded toward the nonphenolic
constituents of lignin with the help of mediator such as ABTS and HBT. ABTS-
mediated oxidation of nonphenolic substrate is associated with an electron transfer
mechanism (Fabbrini et al. 2002; Baiocco et al. 2003). ABTS is first oxidized to the
radical cation (ABTS.+) and subsequently to dication (ABTS2+) with redox poten-
tials of 4.72 V (ABTS/ABTS.+) and 8.85 V (ABTS.+/ABTS2+), respectively
(Bourbonnais et al. 1998). The dicationic active intermediate is predominantly lia-
ble for the oxidation of the nonphenolic compounds. Fungal laccases are mono-
meric or homodimeric glycosylated protein having fewer monosaccharides
(10–25%), such as mannose, arabinose, fucose, galactose, and glucose. Glycosylation
is mainly subjected to secretion, proteolytic susceptibility, copper retention, activity,
and thermostability (Xu 1997).
14.2.2 Cellulase
Chemically, cellulase comprised a family of at least three groups of enzymes: endo-

(1, 4)-β-d-glucanase (EC 3.2.1.4), exo-(1, 4)-β-d-glucanase (EC 3.2.1.91), and
β-glucosidase (EC 3.2.1.21), which act cooperatively to break down cellulose.
Endoglucanase Endoglucanase or endo-(1, 4)-β-d-glucanase randomly digests the
internal O-glycosidic bond, resulting in glucan chains of various lengths.
Exoglucanase Exoglucanase or exo-(1, 4)-β-d-glucanase or cellobiohydrolase
(CBH) releases β-cellobiose from the reducing (CBH I) and nonreducing (CBH II)
ends of cellulose chain. Cellobiohydrolase is inhibited by its hydrolysis product, the
glucose.
β-Glucosidase It causes the degradation of cellobiose to form glucose and ingests

glucose from oligosaccharides by randomly hydrolyzing the β-1, 4 glycosidic bonds
in cellulose. β-Glucosidase is feedback inhibited by its end product glucose (Singh
1999; de Castro et al. 2010; Yadav et al. 2016, 2017a)
The cellulase complex from Trichoderma reesei comprised an endoglucanase of
about 52 kDa, an exoglucanase of about 61 kDa, and a β-glucosidase of about
76 kDa. Two catalytic models have been hypothesized for the reactions catalyzed by
cellulases:
The Inverting Mechanism In this mode of catalytic mechanism, the hydrolysis of
a glycosidic bond with anomeric configuration inversion is usually achieved in a
single-step, single-displacement reaction mechanism including transition states.
The catalytic reaction is typically performed with general acid or base support from
two specific amino acid side chains, usually glutamate or aspartate that are gener-
ally positioned 6–11 Å apart (McCarter and Withers 1994).
The Retaining Mechanism Hydrolysis with configuration retention is accom-
plished by two steps, double-displacement reaction mechanism including a covalent
glycosyl-enzyme transition state. Reaction is carried out with acid/base and nucleo-
philic support assisted by side chain of glutamate or aspartate, situated 5.5 Å apart.
In the initial step, one residue acts as a nucleophile, which attacks the anomeric
center to make a glycosyl-enzyme intermediate by displacing the aglycone. At this
situation, the other residue acts as acid catalyst and donates protons to the glyco-
sidic oxygen upon breakdown of the bond. In the next (deglycosylation) step, water
molecules cause the hydrolysis of glycosyl enzyme with the help of other residue
which being a base catalyst deprotonates the water molecules (Koshland Jr 1953).
14.2.3 Pectinase
According to their mode of action, the pectinase enzyme is basically divided into
three categories: pectin esterase, hydrolases, and lyases (Sharma et al. 2013; Garg
et al. 2016) (Kour et al. 2019b).
Pectin Esterase Pectin esterase (PE, EC 3.1.1.11) carried out de-esterification
reaction of the methoxy group of pectin resulting in pectic acid. The MW of major-
ity of microbial PEs ranges between 30 and 50 kDa, and the optimum pH for enzyme
activity ranges between 4.0 and 7.0 (Hadj-Taieb et al. 2002).
Hydrolases Hydrolases, namely, polygalacturonases and polymethylgalacturo-
nases, hydrolyze the α-(1,4)-glycosidic bonds in pectic acid and pectin, respec-
tively. There are three types of polygalacturonase: endopolygalacturonase
(endo-PG, EC 3.2.1.15) cleaves pectic acid randomly and produces oligogalactu-
ronates, exopolygalacturonase 1 (exo-PG1, EC 3.2.1.67) catalyzes the terminal
cleavage of polygalacturonic acid from the nonreducing termini and produces
monogalacturonates, and exopolygalacturonase 2 (exo-PG2, EC 3.2.1.82) catalyzes

the penultimate cleavage of pectic acid and produces digalacturonates. The MW of
endo-PG lies between 30 and 80 kDa, and in the case of exo-PGs, the MW lies
between 30 and 50 kDa. There are two types of polymethylgalacturonase: endo-
polymethylgalacturonase (endo-PMG) that cleaves pectin in random manner, pro-
ducing oligo-methyl-galacturonates, and exopolymethylgalacturonase (exo-PMG)
that catalyzes the terminal cleavage of pectin from the nonreducing termini, produc-
ing methyl monogalacturonates.
Lyases Lyases (polygalacturonate lyase and polymethylgalacturonate lyase) cleave
the α-(1,4)-glycosidic bond in pectin and pectic acid, respectively, through trans-
elimination reaction, thereby forming unsaturated methyl galacturonates and galac-
turonates, respectively. The MW of polygalacturonate lyase lies between 30 and
50 kDa, and the optimum pH of enzymatic activity is 8.0 and 10.0. There are three
types of polygalacturonate lyase: (i) endopolygalacturonate lyase (endo-PGL, EC
4.2.2.2) that randomly cleaves pectic acid to generate unsaturated oligogalacturo-
nates, (ii) exopolygalacturonate lyase (exo-PGL, EC 4.2.2.9) that cleaves pectic
acid from the nonreducing termini at the penultimate site to generate unsaturated
digalacturonates, and (iii) oligopolygalacturonate lyase (oligo-PGL, EC 4.2.2.6)
that cleaves the oligogalacturonate to generate unsaturated monogalacturonates.
The MW of PL lies between 30 and 40 kDa, and its optimum pH for enzymatic
activity ranges between 4.0 and 7.0. There are two types of polymethylgalacturo-
nate lyase: endopolymethylgalacturonate lyase (endo-PMGL EC 4.2.2.10) that
cleaves pectin randomly, producing unsaturated methyl oligogalacturonates, and
exopolymethylgalacturonate (exo-PMGL) that catalyzes the terminal cleavage of
pectin, producing unsaturated methyl monogalacturonates.
14.2.4 Hemicellulases
Hemicellulase is an array of enzymes mainly comprising xylanase, galactanse,

xyloglucanse, and mannanase (Bhat 2000). In an industrial scale, xylanase is mainly
beneficial because greater fraction of agricultural biomass utilized is composed of
mainly xylan as a hemicellulosic material.
Xylanase The substrate for xylanase, xylan, is the third most plentiful biopolymer
on earth (Mellerowicz and Gorshkova 2011), considering up to one third of the
renewable organic carbon on this planet (Collins et al. 2005). Xylans are polysac-
charides that consist of β-1,4-linked xylose (pentose sugar) residues with side chains
of α-arabinofuranose and α-glucuronic acids and assists in cross-linking of lignin
and cellulose microfibrils by ferulic acid residues (Balakshin et al. 2011). According
to substituted groups, xylan is of three types: (i) glucuronoxylan, (ii) neutral arabi-
noxylan, and (iii) glucuronoarabinoxylan (Faik 2010). Generally, the amount of
xylan in hardwoods and softwoods of hemicelluloses is 10–35% and 10–15%,
respectively. The major xylan ingredients in hardwoods and softwoods are O-acetyl-
4-O-methylglucuronoxylan and arabino-4-O-methylglucuronoxylans, respectively.

Basically, xylans of softwood varies from hardwood by the absence of acetyl groups
as well as the presence of arabinose units tethered by α-(1,3)-glycosidic linkages to
the backbone (Sixta 2006). The xylanolytic enzyme system that performed xylan
hydrolysis normally comprised a reservoir of hydrolytic enzymes, involving endox-
ylanase (endo-1,4-β-xylanase, E.C.3.2.1.8), β-xylosidase (xylan-1,4-β-xylosidase,
E.C.3.2.1.37), acetylxylan esterase (E.C.3.1.1.72), α-arabinofuranosidase (α-l-
arabinofuranosidase, E.C.3.2.1.55), and α-glucuronidase (α-glucosiduronase,
E.C.3.2.1.139) (Juturu and Wu 2012). These enzymes act synergistically to trans-
form xylan into its organizing sugars (Belancic et al. 1995). In xylanase family,
endoxylanases are the most efficient as they are directly involved in the cleavage of
glycosidic bonds, excluding short xylooligosaccharides (Verma and Satyanarayana
2012). Xylanase carried out the formation of xylan to xylooligosaccharides by
random hydrolysis, whereas β-xylosidase liberates xylose subunits from the nonre-
ducing terminus of xylooligosaccharides. However, the entire breakdown necessi-
tates the cooperation of acetyl esterase to liberate the acetyl units from the
β-1,4-linked d-xylose backbone of xylan (Wong and Saddler 1992; Coughlan and
Hazlewood 1993).
14.3 Biodiversity of Fungi-Producing Enzymes
Microorganisms are considered as a better source of valuable enzymes because of

its higher rate of multiplication and synthesizing biologically active products. From
the end of the twentieth century, remarkable level of increment in the utilization of
enzymes for industrial purposes was realized. These enzymes overcome the disad-
vantages of traditional chemical agents for various reasons, such as high catalytic
activity and substrate specificity, high production rate, biodegradability, economi-
cal, and environment friendly (Motta et al. 2013; Yadav et al. 2016, 2017b, 2018).
Fungi are the best lignin-degrading enzyme producer, and thereby, industrial-
scale production of the same is usually carried out with mainly fungal sources.
Within fungi, the white rots are the most potent producers of these enzymes, suc-
ceeded by the brown rots and the soft rots. White-rot fungi cause decaying of wood
that leads to pale color because of oxidative bleaching and loss of lignin and often
preserves a fibrous texture. Preferentially some white-rot fungi attack lignin more
easily than cellulose and hemicellulose of the woody tissue (Blanchette 1984;
Mester et al. 2004). This selective delignification procedure leaves enriched cellu-
lose that is detectable in the white portions of a mottled rot and in the pockets of a
white pocket rot. The lignin degradation system of Phanerochaete chrysosporium
was mostly studied. Lignin breakdown by P. chrysosporium is an orthodox second-
ary metabolic activity initiated particularly during nitrogen starvation. This organ-
ism secretes LiP, MnP, and laccase for lignin degradation (Tien and Kirk 1983;
Glenn and Gold 1985; Srinivasan et al. 1995). Different genera of white-rot fungi,
Phlebia ostreatus, P. radiata, Trametes hirsuta, T. versicolor, and T. ochracea, have
been found to be secreting LiP and MnP along with laccase. In disparity to white-rot
fungi, which are mainly found on deciduous (angiosperms) wood, brown-rot fungi
primarily grow on conifers (gymnosperms) (Gilbertson 1980). Brown-rot fungi
cause the breakdown of wood carbohydrates leading to brown-colored rot due to the
remaining oxidized lignin (Cowling 1961). Due to the cellulose breakdown, the
decayed wood represents a cross-checking pattern with cracks and clefts; this phe-
nomenon is known as “cubical” brown rot. These include Gloeophyllum trabeum,
Postia placenta, Fomitopsis lilacinogilva, Laetiporus portentosus, Piptoporus betu-
linus, and Serpula lacrymans. The roles of ligninolytic enzymes of fungal origin
include lignin degradation, breakdown of plant’s toxic products, and spore/fruiting
body formation (Dos Santos et al. 2007).
Fungi contributes lion’s share in the worldwide cellulose breakdown. This is
particularly important in forest ecosystem, where fungi are the chief decomposers
of cellulose (Alexopoulos et al. 1996). Many fungal strains synthesized cellulases
as inducible enzymes at the time of their growth in the presence of cellulosic com-
ponents. For cellulase production, fungi are more preferable than bacteria because
(a) bacterial cellulase usually lacks one of the three cellulolytic activities, i.e., FPase
activity, (b) downstream processing of fungal cellulase is much easier than bacterial
cellulases, and (c) the activity of fungal cellulase is far greater than that of the bacte-
rial cellulase (Sajith et al. 2016; Tomme et al. 1995). Most extensively studied cel-
lulolytic fungal genera are classified as soft-rot fungi like Aspergillus niger (Ong
et al. 2004), A. nidulans (Kwon et al. 1992), A. oryzae (Takashima et al. 1998), A.
fumigatus (Ximenes et al. 1996), Fusarium solani (Wood and McCrae 1977),
Humicola grisea (Takashima et al. 1996), Melanocarpus albomyces (Miettinen-
Oinonen et al. 2004), Penicillium brasilianum (Jørgensen and Olsson 2006),
Trichoderma reesei (Saloheimo et al. 1988), T. viride (Khokhar et al. 2012), T. har-
zianum (Galante et al. 1998), Chaetomium cellulyticum (Fähnrich and Irrgang
1981), Neurospora crassa (Romero et al. 1999), C. thermophilum (Li et al. 2009),
Thermoascus aurantiacus (Kalogeris et al. 2003), Mucor circinelloides (Saha
2004), and Paecilomyces inflatus (Kluczek-Turpeinen et al. 2007); brown-rot fungi
like Coniophora puteana (Schmidhalter and Canevascini 1992), Poria placenta
(Highley 1977), Tyromyces palustris (Yoon and Kim 2005), and Fomitopsis sp.
(Deswal et al. 2011); and white-rot fungi, viz., Phanerochaete chrysosporium (Jäger
et al. 1985), Sporotrichum thermophile (Kaur et al. 2004), Agaricus arvensis (Jeya
et al. 2010), Pleurotus ostreatus (Khalil et al. 2011), and Phlebia gigantea (Niranjane
et al. 2007).
Microbial sources of pectinolytic enzymes include filamentous and nonfilamen-
tous bacteria, yeasts, and filamentous fungi. Most extensively studied cellulolytic
fungal genera are Aspergillus niger, A. fumigatus, A. flavus, A. oryzae, A. sojae,
Penicillium viridicatum, P. chrysogenum, P. atrovenetum, Rhizomucor pusillus,
Trichoderma viride, Phytophthora infestans, etc. (Couri et al. 2000; Phutela et al.
2005; Demir et al. 2012; Silva et al. 2002; Banu et al. 2010; Siddiqui et al. 2013;
Irshad et al. 2014).
The xylanolytic enzymes are widely distributed among fungi. Industrially impor-
tant xylanases are preferentially obtained from thermophilic organism. Some fungal
genera show elevated thermal stability such as Thermoascus aurantiacus and

Paecilomyces themophila have been found to be stable at high temperature (Yang
et al. 2006). Thermostable Thermomyces lanuginosus exhibited optimum activity at
65–75°C, at pH 5–6.5 (Cesar and Mrša 1996). Except these, other important xyla-
nase producers of fungal origin are Aspergillus awamori, A. foetidus, Fusarium
oxysporum, Geotrichum candidum 3C, and T. reesei (Christakopoulos et al. 1996;
Rodionova et al. 2000).
14.4 Production of Fungal Enzymes
Solid-state fermentation (SSF) is the current trend for industrial-scale production of

most of the enzymes in comparison to the submerged fermentation (SmF). The ben-
efits of SSF over SmF are economically lower fermentation cost, minor risk of con-
tamination (Beg et al. 2001), simple recovery of the enzymes, compatible with the
natural environment of the fungus, production of a protein-augmented by-product,
production of enzymes with intensified enzyme activities, and higher specific activi-
ties (Coughlan 1989). SSF is especially compatible for the fungal growth because
their growth sustained at comparatively low-water activities at which the growth of
bacteria and yeast is not favored (Corral and Villaseñor-Ortega 2006). The selection
of fermentable substrate(s) is gaining special attention for the production of ligno-
cellulosic biomass-degrading enzymes to reach the expected level.
The lignocellulosic waste from agricultural-based industries has shown poten-
tiality to act as substrate for the high titers of laccase production. Laccase biosyn-
thesis by Pleurotus florida PF05 was reported by utilizing corncob oil seed cakes
as substrate. Marques De Souza et al. (2002) reported laccase production by uti-
lizing both wheat bran and straw as substrates in 1:1 ratio by Penicillium pulmo-
narius. Shinners-Carnelley et al. (2002) studied the production of laccase from
sugarcane bagasse by Penicillium chrysosporium. Rosales et al. (2007) performed
SSF using orange peel by Trametes hirsuta. Trametes versicolor efficiently uti-
lizes barley bran for maximum laccase production. Karim and Annuar (2009)
used coconut husk for laccase synthesis by Pycnoporus sanguineus. During SSF,
Coriolus versicolor produced laccase when rice bran was used as substrate
(Chawachart et al. 2004).
Agricultural wastes such as brans and straws of wheat and rice, sugarcane
bagasse, corn stover, sawdust, etc. are principal substrates used for the synthesis of
cellulase. For instance, Liu et al. (2011) investigated the cellulose preparation on
different lignocellulosic substrates such as straws of rice, wheat, and cotton, corn
stover, and corncob using Aspergillus fumigatus; of them, the corncob supported the
maximum synthesis of endoglucanase. Penicillium echinulatum showed the maxi-
mum biosynthesis of cellulolytic enzymes on the medium containing a mixture of
pretreated wheat bran and sugarcane bagasse (Camassola and Dillon 2007). Dutta
et al. (2008) studied the production of cellulases from Penicillium citrinum using
brans of wheat and rice and rice straw as substrate; all these substrates supported the
cellulases production at high magnitude. Aspergillus niger efficiently utilized the

lignocellulosic substrates like grass, bagasse, and corncob with variable cellulase
activities (Sohail et al. 2009). Aspergillus flavus competently utilized different lig-
nocellulosic substrates and supported higher levels of cellulase activity (Sajith et al.
2014). Aspergillus flavus produces cellulose when grown on various natural sub-
strates like bagasse, sawdust, and corncob (Ojumu et al. 2003).
Manimaran et al. (2009) reported higher-level synthesis of xylanase by
Thermoascus aurantiacus during growth on bagasse pulp under optimized solid-
state fermentation. Kang et al. (2004) reported xylanase biosynthesis by A. niger
KK2 using rice straw as substrate. Mohana et al. (2008) studied the production of
xylanase by Burkholderia sp. by employing distillery spent wash pretreated anaero-
bically. Yang et al. (2006) synthesized higher amount of xylanase under optimized
conditions by Paecilomyces thermophila J18 by utilizing wheat straw as substrate.
Thangaratham and Manimegalai (2014) carried out a comparative study by
involving Aspergillus oryzae, Aspergillus flavus, and Rhizopus oryzae for pectinase
production by applying different agro-industrial wastes, including sawdust, pine-
apple peel, cassava waste, lemon peel, and wheat bran. Jahan et al. (2017) reported
higher production of polygalacturonase using Bacillus licheniformis using wheat
bran as SSF substrate. A combination of wheat bran and orange bagasse as sub-
strates in the ratio 1:1 allows the higher production of pectinase using the filamen-
tous fungus Penicillium viridicatum RFC3 (Silva et al. 2005). Biz et al. (2016)
construct an identical process to synthesize pectinases using sugarcane bagasse and
citrus peel as SSF substrates.
14.5 Biotechnological Applications
The aforementioned enzymes have versatile fields of application (Fig. 14.2) in dif-
ferent sectors either in purified or in cocktail form. In the following section, the
prime applications are stated.
14.5.1 In Biofuel Industry
The combustion of fossil fuel has a serious impact on atmospheric CO2 levels and is
an important agent to man-made global climate change (Topakas et al. 2013).
Production of biofuels overcomes this issue, but the major problem in producing
biofuels from lignocellulosic biomass is the processing cost rather than availability
and cost of feedstock (Lynd et al. 2008). To reach the sufficient level of biofuels
production to compete with fossil fuels, the ability of transforming lignocellulosic
biomass to monomeric sugars must be enhanced by producing enzymes of cost-
effective and greater specific activities (Galbe et al. 2007; Kour et al. 2019a;
Rastegari et al. 2019)
Fig. 14.2 Different fields of application of enzymes associated with lignocellulosic biomass
degradation
Laccases assist the pretreatment of lignocellulosic materials by excluding their

phenolic compounds for the subsequent yield of bioethanol (Fang et al. 2015).
White-rot fungal genus Phlebia sp. MG-60 selectively converts lignin under SSF
and from this delignified biomass ethanol is synthesized under semi-aerobic sub-
merged fermentation (Kamei et al. 2012a). Phlebia sp. MG-60 is unique in that it
degrades lignin and subsequently produces ethanol from cellulose (Kamei et al.
2012b). After removing lignin and hemicellulose contents from lignocellulosic bio-
mass, cellulase is treated to hydrolyze cellulose residue to generate fermentable sug-
ars, and finally a fermentable microbe is applied for biofuel production by utilizing
those sugars (Sukumaran et al. 2005). Xylanase along with ligninase and cellulase
facilitates the biofuel production from lignocellulosic residues (Dominguez 1998;
Olsson and Hahn-Hägerdal 1996). The plants which are rich in pectin source like
apple pomace, citrus, and sugar beet have been recommended as possible sources of
hydrocarbon for bioethanol production (Edwards and Doran-Peterson 2012).
14.5.2 In Paper and Pulp Industry
The mechanical pulping process involving refining and grinding of the woody raw
material assists pulps with fine particles of greater amount of bulkiness and stiffness
(Bhat 2000). Wood fiber has a multilayered structure consisting primarily of cellu-
lose, hemicellulose, and lignin. In pulp and paper industry, enzymes related to lig-
nocellulosic biomass breakdown aid biomechanical pulping for prospering the
rough mechanical pulp (Bedford et al. 1997), hand-sheet strength characteristics
(Akhtar 1994), deinking of reprocessed papers (Pere 1995), and elevating drainage
waste effluent from paper mills (Prasad et al. 1992).
Lignin is an insoluble complex biopolymer of phenolic compounds. Conventional
methods for delignifying paper pulp include either chlorinated or oxygenated
agents (Bajpai and Bajpai 1992). Albeit effective, these methods have severe dis-
advantages such as environmental pollution, weakening the cellulose fiber strength,
etc. Enzymatic delignification procedure overcomes these problems and has the
potential as an alternative to the conventional methods. During delignification,

laccases are found to act on small phenolic lignin parts that subsequently reacted
with the lignin polymer, leading to its degradation (Madhavi and Lele 2009; Yadav
et al. 2019a, b).
Application of cellulase for pulping enhances the energy efficiency of the pro-
cess and also improves the physical properties, such as interfiber bonding and
mechanical strength of the ultimate paper product (Chen et al. 2012). Cellulase was
also used for deinking of waste papers. During deinking, the ink attached to the
surface of recycled cellulose fibers was released by the enzymatic hydrolysis of
carbohydrates, leading to the peeling of individual fibers or bundles (Kuhad et al.
1997). Recycling of used papers reduces the solid wastes and also decreases the
burden of deforestation for wood fibers (Lee et al. 2011; Singh et al. 2012).
Enzymatic deinking of waste papers, especially employing the mixtures of cellulase
and hemicellulase, enhances the quality and brightness of the recycled paper (Lee
et al. 2007; Ibarra et al. 2012). Cellulases are also used to elevate the drainage of
several paper mills by dissolving clogged fiber residues (Kuhad et al. 2011).
Moreover, the cellulase preparations are also used to make easily biodegradable
cardboards, tissue papers, and sanitary papers (Buchert et al. 1998; Bajpai 1999).
During the Kraft process, i.e., the chemical transformation of wood into wood
pulp (lignocellulosic fibrous components), the lignin-carbohydrate complex is
hydrolyzed. Thus, endo-1,4-β-xylanase can be used in this process to increase the
extraction of lignin. In that way it becomes more accessible for bleaching. By this
approach the consumption of chlorine chemicals for bleaching is reduced. In addi-
tion to being environment friendly, enzymatically treated pulp is brighter and has
improved fiber quality (Kalim et al. 2015). It was also reported that pulps and papers
treated with hemicellulases lead to the reduction in necessity of chlorine bleaching
(Viikari et al. 2001).
Pectins found in papers impair dewatering during sheet production because of
their high cationic requirement and give yellowness of paper. Depolymerization of
polygalacturonic acids by pectinases lowers the cationic requirement in the hydro-
gen peroxide-bleached thermomechanical pulp filtrate (Viikari et al. 2001).
Pectinases exclusively or with other enzymes synthesized by same or other micro-
organisms have been effectively utilized for biobleaching of bamboo kraft pulps and
mixed hardwood (Dhiman et al. 2009).
14.5.3 In Food Processing
Along with progressive health-conscious civilization, fruit and vegetable juice prep-
arations gain huge impact on both the human health and industrial sector. Fruit and
vegetable juice production at industrial level needs detailed knowledge about the
methods of extraction, clarification, and stabilization. During the early 1930s, when
fruit industries were just blooming to produce juice, many difficulties were encoun-
tered, such as the yields were low and clarity of fruit juices was not to a receivable
grade (Uhlig 1998). At present, a mixture of pectinases, laccase, cellulases, and

hemicellulases, collectively acquainted as macerating enzymes, are utilized in the
juice extraction and clarification from fruits and vegetables (Galante et al. 1998).
Stutz (1993) prepared a clear and stable apple juice with the help of laccase and
ultrafiltration. Laccase treatment at suitable pH and consequent ultrafiltration ame-
liorated the juice quality by means of flavor and durability in contrast to the conven-
tional treatments with ascorbic acid and sulfites.
Cellulases performed a leading role in macerating enzymes complex (mainly
cellulases, pectinase, and xylanases); they are used to ameliorate texture and cloud
stability and reduce viscosity of the purees and nectars of the juice obtained from
tropical fruits like, mango, papaya, peach, apricot, pear, and plum (Sukumaran et al.
2005; Bhat 2000; De Carvalho et al. 2008). Improving sensory qualities of fruits
and vegetables juices like texture, aroma, and flavor can be achieved by reducing the
extra bitterness of citrus fruits through the addition of enzymes such as β-glucosidases
and pectinases (Baker and Wicker 1996; Youn et al. 2004; Rai et al. 2007). The same
enzyme mixture is also helpful for improving the extraction procedure of olive oil.
Cellulase along with pectinase is utilized in the extraction of pigments (e.g., carot-
enoid) which are used as food coloring agents (Choudhari and Ananthanarayan
2007). A mixture of pectin methylesterase with calcium is used as softening agent
in pickle industries (Baker and Wicker 1996).
Pectinase treatment improved tea fermentation by degrading pectin that is pres-
ent in the tea leaves and improves the quality of instant tea powder. A characteristic
aroma of tea is associated with alteration in tea color during the fermentation
(Praveen and Suneetha 2015). Marimuthu et al. (1997) documented that a mixture
of cellulase, pectinase, and xylanase upgrades the quality of tea by increasing
thearubigin (TR), theaflavin (TF), high polymerized substances (HPS), and total
soluble solids (TSS) content. The robusta coffee obtained from the bean is sur-
rounded by the gelatinous and viscous mucilage layer that comprised sugars (4.0%),
pectin (2.8%), moisture (84.0%) with protein (8.9%), and ash (0.9%). Pectinases
eliminate the layer of pectic substances from the coffee bean. Pectinase treatment
minimizes demucilization time associated with sugar content increment and lower-
ing of pH (Murthy and Naidu 2011).
14.5.4 In Wine and Brewery Industry
One of the vital applications of laccase in alcoholic beverage industry is wine stabi-
lization. Wine aroma depends on the alcohol and organic compounds, whereas phe-
nolic compounds determine the color and taste of the wine. Availability of metal ions
(like copper and iron), enzymes, amino acids, proteins, aldehydes and polyphenols
in wine causes oxidative reactions, resulting in turbidity, aroma, color intensification,
and flavor changes. This oxidative phenomenon is called maderization. In compari-
son to the conventional chemical treatment of Riesling wine with sulfur dioxide and
polyvinylpyrrolidone (PVP), the polyphenol content, sensorial quality, color, and
haze stability were uplifted with laccase treatment (Madhavi and Lele 2009).
In wine production, a combination of enzymes such as glucanases, pectinases,

and hemicellulases in well-balanced conditions performs a major role by ameliorat-
ing color extraction, must clarification, skin maceration, filtration, and lastly the
wine stability and quality (Singh et al. 2007; Galante et al. 1998). β-Glucosidases
are responsible for developing aroma in wines by modifying glycosylated precur-
sors. Macerating enzymes also maintain the pressability, settling, and juice produc-
tion from grapes utilized in wine fermentation. Oksanen et al. (1985) reported that
enzymatic preparations of exoglucanase II and endoglucanase II from Trichoderma
cause maximum amount of reduction in polymerization and wort viscosity level.
Galante et al. (1998) conducted an experiment for wine production by treating three
qualities of white grapes (namely, Soave, Chardonnay, and Sauvignon) by a cocktail
of cellulase, pectinase, and xylanase, commercially known as Cytolase 219, and
found that improvement in wine must extraction, wine must filtration rate, and sta-
bility was realized with minimization of pressing time and must viscosity.
14.5.5 In Textile Industry
Laccases play an essential role in preventing back staining of printed or dyed tex-
tiles. Laccases in washing solution thoroughly bleach released dyestuff, leading to
the reduction in energy, processing time, and water needed to obtain textile up to
satisfactory level. Laccase-based bleaching system replaced the traditional chemical
oxidation step and showed its potentiality to bleach the indigo dye in denim and
acquire various bleached outlooks on the fabric (Madhavi and Lele 2009).
Conventional stonewashing of jeans included amylase-mediated dispel of starch
coating followed by pumice stone-mediated machine washing of jeans. In commer-
cial sector, cellulases find its role by biopolishing the cotton and various other
cellulose-based fabrics and biostoning of jeans. During the biostoning procedure,
cellulases attack the cotton fabrics and split the tiny fiber endings on the yarn sur-
face which leads to dye slacking causing easy removal during washing by mechani-
cal friction. The benefits associated with cellulose-based treatment include less
harm to the cotton fibers, less intensive work, and improved production efficiency
of the machines in an environmentally protected manner (Sukumaran et al. 2005;
Uhlig 1998). Acidic cellulases are particularly helpful in improving softness and
water-absorbing properties of fibers, sharply reduce the propensity for pill forma-
tion, and give a bright and cleaner surface feature with reduced fuzz (Sreenath et al.
1996). Endoglucanases-rich cellulase preparations are efficient in biopolishing by
improving look, color, and feel without any chemical anointment of fibers (Galante
et al. 1998). In regular washing, the cotton clothes became fluffy and dull which is
chiefly due to the partially loosed microfibrils on the clothes surface. Application of
cellulase causes removal of those microfibrils, thereby retaining a smooth surface,
original color, and appearance to the clothes (Hebeish and Ibrahim 2007; Ibrahim
et al. 2011). Infliction of cellulase also helps in softening the clothes by removing
dirt particles entrapped within microfibril network.
Pectinases in combination with amylases, cellulases, and hemicellulases are used

to dispel sizing agents from cotton in such a safe and ecofriendly way that displaces
traditional process of using toxic sodium hydroxide. Pectinases are employed for
bioscouring (noncellulosic impurities removal from the fiber by applying specific
enzymes) (Jayani et al. 2005).
14.5.6 In Bakery Industry
The application of enzyme preparations in bread making to produce fluffier bread

has been reported long ago (Blagoveschenski and Yurgenson 1935). It was reported
that the incorporation of laccase in baked products in bakery industries causes oxi-
dation of dough materials and strengthens gluten structures of wheat as well as
increased dough volume, smoothness of final baked product, a better crumb struc-
ture, increased strength, reduced stickiness, and stability, thus overall ameliorating
the machinability of the dough.
Xylans play a major role in bread making because of their interaction with gluten
and high water absorption potentiality. Endoxylanase attacks arabinoxylan back-
bone to reduce the degree of polymerization and consequently its structure and
function (Courtin and Delcour 2002). Xylanase heightens dough stability, dough
machinability, lump volume, crumb structure, oven spring, and shelf life when
applied in optimum level (Hamer 1995; Poutanen 1997).
14.5.7 In Biomedical Sector
Laccase covalently conjugated to bioadhesive molecules (antibody, antigen, DNA,

RNA, biotin, and streptavidin) can be employed as a marker enzyme in the immu-
nological, histological, cytological, or molecular biological assay. In such proto-
cols, the binding moiety attached to the target, and subsequently the laccase signals
the binding incident (Schmid and Urlacher 2007). Laccases have shown its ability
to cure aceruloplasminemia. The disease is associated with the lack of ceruloplas-
min, a multicopper serum oxidase whose ferroxidase activity controls iron homeo-
stasis (Harris et al. 2004). Poison ivy dermatitis is caused mainly by urushiol
(catechol-derived toxin). The application of laccase causes oxidation, polymeriza-
tion, and detoxification of urushiol (Madhavi and Lele 2009). The first significant
pharmaceutical chemical that has been made by employing laccase is actinocin,
produced from 4-methyl-3-hydroxyanthranilic acid. Actinocin is an anticancer
compound which restricts the tumorous growth by blocking DNA transcrip-
tion (Burton 2003). Another example of the anticancer drug is vinblastine which is
useful in curing leukemia. The plant Catharanthus roseus naturally produces vin-
blastine in small amount. This compound is synthesized in plant from the precur-
sors—katarantine and vindoline. Laccases find its role by 40% transformation of
these precursors to the end product (Yaropolov et al. 1994). Catechins found in tea,
vegetables, and herbs consist of small amount of tannins. The antioxidative property
of catechin makes them beneficial for healing several diseases including cancer,
cardiovascular diseases, and inflammatory diseases. The antioxidative property of
catechin can be upgraded by the oxidation of laccase and producing catechins in
numerous goods with amplified antioxidative capability (Kurisawa et al. 2003).
Erb-Downward et al. (2008) reported that laccase was involved in the creation of
immunomodulatory prostaglandins. Laccase of oyster mushroom can obstruct hep-
atitis C virus penetration into hepatoma and peripheral blood cells (El-Fakharany
et al. 2010). Laccase of edible mushroom Agrocybe cylindracea was reported to
inhibit HIV-1 reverse transcription (Hu et al. 2011).
Cellulases are applied to synthesize chitosan with antibacterial, immunomodula-
tory, and antitumor activities (Qin et al. 2004; Wu and Tsai 2004). Cellulases are
directly applied to human for the treatment of phytobezoars. A phytobezoar is a
calculus formed in the gastrointestinal tract that consists of swallowed foods of
plant origin, such as indigestible vegetables and/or fruit fibers. Few medical compli-
cations are healed by using cellulases alone (Pinos et al. 2015). Cellulases cause
breakdown of biofilm by attacking cellulose which is one of the vital constituents in
the matrix of biofilm (Flemming and Wingender 2010; McCrate et al. 2013).
Pathogenic microbes construct biofilms, allows spreading and protecting them-
selves. It is a matter of concern because maximum drugs are incapable to penetrate
the biofilm structure. Several studies and patents suggested that the direct insertion
of cellulases acts as antibiofilm agent for medical implants (Loiselle and Anderson
2003), diverse prosthetic materials (Rajasekharan and Ramesh 2013), treatment of
cystic fibrosis (Ma et al. 2009; Rajasekharan and Ramesh 2013), treatment of noso-
comial infections (Huertas et al. 2014), among others.
Endoxylanase-catalyzed xylan hydrolysis results in xylooligosaccharides (XOs).
Xylan results in the formation of xylose, arabinose, and methyl-d-glucuronic acid-
bearing XOs (Goswami and Rawat 2015). XOs have numerous impacts in various
fields like agricultural, food pharmaceuticals purposes, and feed formulations
(Vazquez et al. 2000). XOs have prebiotic action as food additives by improving the
intestinal health by flourishing the growth of health-beneficial Bifidobacteria (Fooks
and Gibson 2002). XOs are applied as dietary supplements due to their instrumental
effect on gastrointestinal tract and may decrease the threat of colon cancer
(Whitehead and Cotta 2001). Acceptance of XOs is due to the allowable odor and
noncarcinogenic nature (Kazumitsu et al. 1987; Kazumitsu et al. 1997). XOs have
low calorific value and therefore are applied as antiobesity diet products (Taeko
et al. 1998; Toshio et al. 1990).
Non-digestibility of pectin in the gut of human makes it as a dietary fiber by
increasing the viscosity in the intestinal tract. Enhancement of the viscosity of the
intestinal tract results in reduced cholesterol absorption by increasing the excretion
of neutral sterols and fecal bile acids, by intervening with the formation of micelles,
and/or by reducing the diffusion rate of bile acid and cholesterol-carrying micelles
through the bolus, therefore curtailing the incorporation of cholesterol and bile
acids. High amount of negative charges on demethylated pectin makes them as a
weak cation exchanger, and therefore, at favorable pH, they chelate toxic ions.
Intestinal bacteria easily ferment demethylated pectins, subsequently producing
short-chain fatty acids, propionate, butyrate, and acetate. These products protect the
bowel against inflammatory diseases, and also regulate the insulin release and appe-
tite by modulating the release of gut hormones (Tolhurst et al. 2012).
14.5.8 In Animal Feed Sector
Zearalenone (produced by Fusarium sp.) is globally present in an array of cereal

crops, such as maize, barley, oats, wheat, rye, rice, millet, and sorghum. Zearalenone
is the main toxin responsible for infertility, abortion, and breeding-associated prob-
lems, especially in pigs. The symptoms are particularly complicated in prepubertal
gilts involving enlarged breast, atrophy of the ovaries, and swelling of uterus and
vulva. Humans come in contact with zearalenone by consuming animal meat, by
swallowing contaminated grain, and also from bread baked with contaminated
wheat. Vikso-Nielsen and Sorensen (2015) reported that laccase treatment causes
the conversion of zearalenone into nontoxic substances. Treatment of cereal straw
with enzyme alone or in conjunction with other treatments improved their degrad-
ability by the rumen microbes (Rodrigues et al. 2008). Feeding the sheep with
24 hours lignolytic enzymes-treated straw (Eleusine coracana) causes not only
higher lignin degradation but also higher digestibility in comparison to consuming
the immediate enzyme-treated straw (Sridhar et al. 2014).
The cereal-centered food of poultry and pigs are far less complicated than forage
food of ruminants comprising cellulose, pectin, hemicellulose, and lignin. An enzy-
matic complex having higher amounts of cellulase, hemicellulase, and pectinase
increases the nutritive value of forages (Lewis et al. 1996). Together, cellulases and
hemicellulases improve the nutritional status of animal feed by semihydrolysis of
lignocelluloses, hydrolysis of β-glucans, better emulsification and pliability, and
dehulling of cereal grains (Galante et al. 1998; Cowan 1996). Furthermore, these
enzymes are involved in incomplete hydrolysis of PCW at the time of silage or fodder
preservation. Propionic acid, a bacteriostatic agent produced during cellulases-based
cereal fermentation, has a positive impact on ruminant animals by decreasing the
colonization of pathogenic bacteria (Fortun-Lamothe et al. 2001). Supplementation of
fibrolytic enzymes like xylanases and cellulases significantly raised the average milk
production per day in Murrah buffaloes (Shekhar et al. 2010). Similar results were
also noticed for goats (Bala et al. 2009). Wheat, triticale, and rye are rich in arabinox-
ylans (Bonnin et al. 1998). Endo-(1,4)-d-xylanase ease the mobility of the gut materi-
als leads to the improved nutrient absorption and diffusion of the enzymes secreted
from pancreas. Consumption of xylanase-treated rye-based feed to broiler chickens
leads to the reduction in intestinal viscosity, improved feed conversion efficiency, and
weight gain (Bedford and Classen 1993). Incorporation of pectinases to the animal
feed lessens feed viscosity which in turn improves nutrients absorption and decreases
the amount of fecal matter (Jayani et al. 2005).
14.5.9 In Agricultural Sector
Mixture of cellulases, hemicellulases, and pectinases produced by fungal strains

of Trichoderma and Penicillium are utilized for the synthesis of protoplast of plant
and fungi which are fused to yield mutant or hybrid strains with anticipated char-
acteristics (Beguin and Aubert 1994; Bhat and Bhat 1997; Yadav et al. 2015,
2017b). The pathogenic fungal genera Fusarium sp. and Rhizopus solani are lia-
ble for leakage of cytoplasm, hyphal tip swelling, formation of many septae, etc.
Such pathogen-inducing plant diseases are limited by β-1,3-glucanase isolated
from T. harzianum CECT 2413 (Kuhad and Singh 2013; Kubicek and Harman
1998). Cucumber seedlings infected by the plant pathogen Pythium sp. can be
limited by the inhibition of its growth by hypercellulolytic mutant of T. longibra-
chiatum, which synthesize high amount of β-1,4-endoglucanase compared to the
wild type (Kubicek and Harman 1998). Thus, cellulase performed beneficial role
as biocontrol agents to defend the plants and its seeds from plant pathogens (Bhat
2000). Application of exogenous cellulase leads to improvement in soil fertility
by decomposition of cellulose (straw) in soil and thus minimizes reliance on min-
eral fertilizers (Han and He 2010; Fontaine et al. 2004). Paenibacillus ehimensis
KWN38 synthesizes antifungal compounds along with the lytic enzymes, mainly
cellulases and β-glucanases, which defend plants against pathogenic oomycetes,
such as strains Phytophthora (Naing et al. 2014). Moreover, cellulases synthe-
sized by this Paenibacillus species may be responsible for rhizospheric soil
decomposition; thus more nutrients are now accessible to the plant (Han and He
2010; Kour et al. 2019b; Yadav et al. 2017a; Yadav 2018).
Xylanase treatment of plant cells can induce glycosylation and fatty acylation of
phytosterols. Treatment with purified endoxylanase of Trichoderma viride of
tobacco suspension cells leads to a 13-fold rise in the intensities of acylated sterol
glycosides which in turn causes large amount of phytoalexins synthesis (Moreau
et al. 1994). Xylanases from the germinating plant seed during ripening convert
reserve food to the suitable end product. Xylanases are also reported to take active
part in softening of fruits, e.g., papaya. During ripening when the fruits become
soften, endoxylanases modify the polysaccharide content in the matrix of cell wall
(Manenoi and Paull 2007).
The fiber which is obtained from fiber crop holds gum. Ramie fiber is an
excellent natural textile, but it contains 20–35% ramie gum that mostly contains
pectin and hemicellulose and is therefore detached by further treatment for tex-
tile processing. Conventionally degumming can be done by chemical treatment,
12–20% NaOH in solution form is used to remove gum of decorticated fibers
(Cao et al. 1992). But chemical treatment is not much efficient, and this type of
degumming is toxic, contaminating, and nonbiodegradable. Pectinases with
xylanase can be employed for degumming in a sustainable manner (Jayani et al.
2005). A mixture of xylanase with pectinase can be employed for the degum-
ming of bast fibers such as flax, ramie hamp, and jute (Sreenath et al. 1996). The
same enzymatic mixture can also be involved in the debarking process, the early
step in wood handling (Bajpai 1999). Alkaline pectinases and cellulases are
employed for the extraction of viruses in pure form when the viral particles are
present in phloem (Jayani et al. 2005).
14.5.10 In Waste Management
A leading problem of our present world is contamination of air, soil, and water by
harmful chemicals which may have catastrophic influence not only on human health
but also their surrounding environment. Unrelenting legislation have been executed
on industries for treating their waste effluents before ultimate discharge into the
ecosystem.
Fungal laccases are employed for the detoxification and decolorization of
industrial effluents as well as wastewater treatment (Chandra and Chowdhary
2015). Major environmental role of laccases is the remediation of toxic soils
involving the oxidation of toxic organic chemicals, such as polycyclic aromatic
hydrocarbons, chlorophenols, organophosphorus compounds, lignin-related
structures, phenols, and azo dyes in free or immobilized state (Saratale et al.
2011; Khan et al. 2013). Gaitan et al. (2011) found that laccase of Trametes
pubescens can degrade mixture of pentachlorophenol, 2,4-dichlorophenol, and
2,4,6-trichlorophenol. Saparrat et al. (2010) reported that the water-soluble
fraction of “alpeorujo,” a by-product pulled out during the olive oil extraction,
can be detoxified by laccase. Zhao et al. (2010) studied that the application of
laccase in soil causes the degradation of dichlorodiphenyltrichloroethane. Along
with the biodegradation of 2,4-dichlorophenol by laccase, treatment of the same
can weaken the bisphenol-A-mediated cancer cell propagation (Bolli et al.
2008). Pozdnyakova et al. (2006) reported the biodegradation of polycyclic aro-
matic hydrocarbons like fluoranthene, anthracene, fluorene, phenanthrene,
pyrene, and perylene using synthetic mediator-based laccase production by
Pleurotus ostreatus.
The wastes from agricultural fields, agroindustries, and forests contain a huge
mass of unprocessed or underprocessed cellulose, resulting in environmental pollu-
tion (Milala et al. 2005). At present these biowastes are judiciously applied to pro-
duce valued products, viz., enzymes, biofuels, sugars, nutrients, biochemicals, as
well as cheap substrate for fermentation and improved animal feeds (Kuhad et al.
2010; Kuhad et al. 1997; Karmakar and Ray 2011; Humpf and Schreier 1991; Gupta
et al. 2009).
Hemicelluloses (xylan)-rich agrowastes are treated with xylanase to convert
xylan into xylose which are subsequently employed for the production of biogas,
enzyme (xylanase), and other value-added products (Stalin et al. 2012; Fang
et al. 2010).
The wastewater discharged from the citrus fruit processing industries contains
large amount of pectinaceous substances which are not fully decomposed during the
activated sludge treatment process (Tanabe et al. 1986). Vegetable food processing
industries also discharged pectin substances as waste effluent. Treatment of this

waste effluent with pectinases makes it suitable for decomposition by activated
sludge treatment (Jayani et al. 2005).
14.6 Conclusion and Future Perspectives
Irresistible demand for environment-friendly products elevated the value of indus-

trial enzymes, among which lignocellulose-degrading enzymes gained pivotal role.
Ample supply of lignocellulosic biomass makes it a potential source in agricultural,
biofuel, textile, biomedical, food processing, and paper and pulp industries, as well
as in research and development. Most significant lignocellulose biomass attacking
enzymes such as laccase, cellulase, xylanase, and pectinase are produced by fungi
belonging to diverse genera. These enzymes also availed worldwide attention in an
expected level regarding the waste management, and their utilization overcome the
drawbacks in various conventional chemical-based treatments in different indus-
tries. Present scenario for the fermentative production of these enzymes from ligno-
cellulosic biomass in an industrial scale is still not reached to the expectation. The
major aims for future research regarding the synthesis of these enzymes would be
cost-effective production and increment in the potentiality of the enzymes. Attaining
such goals required tremendous level of research for searching new microbes of
better enzyme production both quantitatively and qualitatively, their growth optimi-
zation, and genetic engineering to develop the properties of these enzymes.
Acknowledgement The authors are grateful to the Department of Science and Technology and
Biotechnology, Govt. of West Bengal, India for financial assistance (Memo No: 532/(Sanc.)\ST/P/
S&T/2G-48/2018 dated: 27/03/2019).
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Molecules 15(11):7961–7970
Chapter 15
Bioconversion of Biomass to Biofuel Using
Fungal Consortium
Pavana Jyothi Cherukuri and Rajani Chowdary Akkina
15.1 Introduction
The global economy is highly influenced by fuel energy. Global pollution and
energy consumption expanded tremendously during the last two decades which
led to exhaustion of fossil fuels, resulting in emerging of energy crisis.
Therefore, there is a need to search for alternative new energy sources and
technologies which have increased logistically in recent years. The entire globe
depends on petroleum as a sole energy source. The high usage of petroleum has
led to adverse impact on environmental issues such as catastrophic emission of
greenhouse gases (Hill et al. 2006). In India, the required petroleum is imported
from Middle East; these high imports of petroleum significantly influence the
Indian economy. Many attempts are being done to search for alternative fuel
sources in transportation sector, such as diesel, gasoline and natural gas
(Tabassum Ansari and Choube 2012). But no fuel exhibits unique feature like
petroleum such as high energy density, compatibility with vehicles, and being
in liquid state. Developed countries like the United States, Europe, Brazil, and
China invented new technologies such as solar, hydro, and wind energy usage
as an alternative to petroleum or alternative liquid fuels such as butanol, etha-
nol, methane, and CNG. Among all the liquid fuels, ethanol occupies the first
place by its unique properties.
P. J. Cherukuri (*) · R. C. Akkina

Department of Microbiology & Food Science and Technology, Institute of Science, GITAM
(Deed to be University), Visakhapatnam, Andhra Pradesh, India

382 P. J. Cherukuri and R. C. Akkina
15.2 Ethanol Production
Ethanol exhibits almost similar properties with petroleum. It can mix with gasoline
and reduce emission of smog by unburned hydrocarbons and CO (carbon monox-
ide) from vehicles. Ethanol blending with gasoline enhances octane number and
oxygen content of fuel (Limayem and Ricke 2012). The specific properties of etha-
nol like high octane number, water free, low flame temperature, high gas volume
change, high heat of vaporization, and total combustibility make it convenient to use
as automotive fuel (Balan et al. 2013). Ethanol is a transparent liquid, with mild
odor boiling temperature at 78 °C and freezing temperature at −112 °C. Energy
Policy Act of 2005 in the United States, demands the blending of 7.5 billion gallons
of alternative fuels by 2012. Global rapid depletion of fossil fuels has increased the
demand for alternate fuels. The usage and production of ethanol have to be increased
markedly (Haghighi et al. 2013).
The production of ethanol is not sufficient to meet today’s demand. Industrial
ethanol production is obtained from coarse grains (56%), cane (32%), molasses
(4%), wheat (3%), nonagricultural substrates (3%), and sugar beet (2%) (Tabassum
Ansari and Choube 2012). Economically feasible ethanol production from cellu-
losic wastes is one of the best ways (Rastegari et al. 2019). Nowadays, integrated
biomass system is being used for production of biofuel through biotechnological
process. Lignocellulosic biomass provides competitive renewable resource for gen-
eration of ethanol in an eco-friendly manner. This technology ensures increased
energy security and economic progress and eliminates problems of solid waste
management (Das and Singh 2004). According to the studies by Sanchez and
Cardona (2008), 73.9 Tg of dry crop waste and 1 × 1010 MT weed biomass is pro-
duced annually.
Numerous edible crops like cane, corn, beet, and sorghum are used in ethanol com-
mercial production. In this technology, the raw material cost is very expensive. This
leads to food insecurity and very high cost of food products. Therefore, plant biomass
(lignocelluloses waste) has become an inexpensive carbon source for the production
of ethanol. The lignocellulosic wastes such as agricultural residues like corn stover,
corncobs, rice straw, wheat straw, sugarcane bagasse, rice husk, wheat husk, and weed
biomass like Lantana camara, Prosopis juliflora, Saccharum spontaneum, Eichhornia
crassipes, etc. are promising substrates for the production of ethanol.
15.2.1 Demand for Ethanol Production
It was estimated that there was 90% increase from the existing levels on the India’s
dependency on oil imports by 2020 AD. In order to reduce the oil imports, India
proposed an EBP (ethanol blending program report 2014–2015) during the year
2002 with 5% blending of ethanol in petrol which is mandatory in nine major sugar-
producing states (Tamil Nadu, Gujarat, Andhra Pradesh, Karnataka, Uttar Pradesh,
15 Bioconversion of Biomass to Biofuel Using Fungal Consortium 383
Maharashtra, and Union Territories). In the year 2008, Indian Government intro-
duced a “National Biofuel Policy” for blending of petrol with 20% ethanol
(Tabassum Ansari and Choube 2012). To fulfill the supply of bioethanol, it is highly
essential to search for new cost-effective raw materials like agricultural residues and
weeds. Global ethanol production is around 30 billion liters per year. Global ethnaol
production is around 30 billion litters per year, in this, the United States occupies a
major share (53%), followed by Brazil (21%), Europe (6%), China (7%) and India
(3%). In India, the main source of ethanol production is by molasses (80%) and
grains (20%). The production of bioethanol in India is around 2.4 billion liters
per annum.
The industrial production of bioethanol by biotechnological process using micro-
organisms and gross productivity ranges from 75% to 90%, and the remaining small
fraction of production is from chemical technology by ethylene hydration reactions
(Mc. Millan 1997). Currently, ethanol production is by sugarcane and corn in devel-
oped countries like the United States and Brazil (Limayem and Ricke 2012). The
first-generation biofuels are the results of bioconversion of edible food crops (sug-
arcane, corn), the second-generation biofuels are from nonedible sources like agri-
cultural and nonagricultural residues, and third-generation biofuels are from algal
biomass (Bacovsky et al. 2010; Yewale et al. 2016; Kim and Dale 2004; Chen et al.
2010).
15.3 Biomass for Biofuels
Hard wood angiosperms such as a poplar, eucalyptus, and beech wood are rich in
cellulose, hemicellulose, and lignin. Efficient conversion of ethanol production
from hard wood spent sulfite liquor (HSSL) using Pichia stipitis, Candida shehatae,
and Pachysolen tannophilus was reported (Jeffries et al. 2007). HSSL contains high
amount of microbial inhibitors. Biofuel production from renewable plant sources is
an attractive and alternative process in many countries. Mainly, three substrates like
sugars, starches, and cellulose are used for ethanol production in a cost-effective
manner. Among these, cellulose materials are renewable and plenty. The agro-
residues such as sugarcane bagasse, corncobs, corn fiber, corn stover, wheat straw,
rice straw, forestry, paper pulp, weed plants, sawdust, sorghum straw, cotton seeds,
sunflower seed coats, kitchen waste, and fruit and vegetable waste are collectively
known as organic biomass (Lin and Tanaka 2006). The plant biomass is an attractive
feedstock for ethanol production consisting of rich carbohydrate composition in
various polymeric complex forms (lignin, cellulose, and hemicellulose).
Pretreatment process is necessary to use available carbohydrates in biomass. The
current technologies are not cost-effective process for commercialization of bio-
ethanol. Elaborate investigations have been done from the past two decades by
many researchers for value addition of lignocellulosic biomass (Zhang et al. 2014;
Yewale et al. 2016). Weed, starch, and by-products of paper industry were used for
ethanol production which includes spent sulfite (Pereira et al. 2013).
15.4 Composition of Biomass
In the present chapter, focus is on biofuel production from lignocellulosic biomass.

According to the plant taxonomy, lignocelluloses are classified into soft wood
(belongs to gymnosperms), hard wood (belongs to woody angiosperms), and annual
plants (herbaceous angiosperms, crops). Biomass of lignocelluloses is a heteroge-
nous mixture consisting of hemicellulose, cellulose, and lignin, and these composi-
tions vary from plant to plant (Yadav et al. 2019a, b). Generally, cellulosic fractions
of biomass comprise 40–60% by weight, and long-chain polymers of glucose are
bonded together which appear as fiber bundles. Hemicellulose comprises 20–40%
by weight and contains short-chain polymers of heterogenous sugars (glucose,
galactose, mannose, arabinose, and xylose) and co-jointly binds the cellulose fibers
(Bobleter 1994). Lignin consists of 10–30% by weight and contains three-
dimensional propyl-phenol polymers and contributes rigidity to the entire structure.
Lignin is derived from the dehydrogenated products of lignin monomers (p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol). These main aromatic phenol rings
of monomer residues of p-hydroxy phenyl, guaiacyl, and syringyl and their compo-
sition vary from plant to plant (Campbell and Laherrere 1998).
Lignocellulosic biomass shows resistance toward degradation and hydrolysis
because of structural firmness. The presence of cross-linkages exists between the
cellulose, hemicellulose, and lignin components in biomass. Hemicellulose and lig-
nin are compactly packed cellulosic fraction. For hydrolysis of cellulose, initially
digest the hemicellulose and lignin fractions. Cellulosic fraction is protected and
occupies the central position in the three-dimensional structure of lignin and hemi-
cellulosic fraction (Balan et al. 2013). For exposure of cellulose by hydrolysis, it is
essential to remove the lignin seal. The degradation of cellulosic and hemicellulosic
fractions of plant biomass are carried by acid hydrolysis or enzymatic hydrolysis or
enzyme producing microrganisms. From these methods, acid hydrolysis requires
high temperatures and energy, and this process releases various non-eco-friendly
inhibitors (furfurals, hydroxyl furfurals, acids) during the hydrolysis process
(Sreenivas et al. 2006). Secondly, enzymes used for the hydrolysis of biomass are
eco-friendly and expensive. Among these, bioconversion of biomass using fungal
consortium is cost-effective and eco-friendly process (Dirk et al. 2003).
The bioconversion of biomass to ethanol requires more processing steps, i.e.,
pretreatment of lignocellulosic biomass, hydrolysis, and conversion (fermentation/
transformation) of sugars into (biofuel) ethanol. An extensive work has been carried
out by utilization of microorganisms (yeast, bacteria, and fungi) for ethanol produc-
tion. Commercial production of ethanol by fermentation process is highly influ-
enced by fungal and yeast strains. In pretreatment process, initially digest the
hemicellulose and lignin crystalline structure and then expose it to cellulose. In the
pretreatment process of lignocellulosic matrix, major sugar released from hemicel-
lulosic fraction is xylose, and small amounts of arabinose, galactose, and glucose
are also released by the laccase and xylanase enzymes using fungal consortium.
Then, cellulose is broken down to monomeric sugars by cellulase-producing fungal
strains and release of hexose (glucose) sugars. These sugars are converted to ethanol
by fermentation process using yeast strains such as Saccharomyces and Candida sp.
The yeast Saccharomyces cerevisiae is used for ethanol production from glucose as
carbon source. This strain is unable to utilize xylose as carbon source. The Candida
and Pichia sp. of yeast are used to convert pentose sugars to ethanol by biotechno-
logical process (Steven and Lee 1990).
15.5 Pretreatment of Lignocellulose Materials
The lignocellulosic biomass derived from plant biomass is initially treated by chem-
ical or enzymatic or by both methods. By this degradation, polymeric forms of
biomass such as lignin, cellulose, and hemicelluloses are digested to release mono-
meric sugars. These monomeric sugars are further fermented by microorganisms to
produce bioethanol (Claudio et al. 2011).
The pretreatment methods are classified into three types:
1. Physical process: Physical process such as steam explosion or auto-hydrolysis,
carbon dioxide explosion, ammonium fiber/freeze explosion, and liquid hot
water.
2. Chemical process: Chemical process consists of wet oxidation ozonolysis, acid
pretreatment, alkaline pretreatment, and organosolv.
3. Biological process employed by microorganism or their enzymes (Buruiana et al.
2013).
The pretreatment of starch substrates includes digestion by gasification of ligno-
cellulosic materials by acid or enzymatic hydrolysis for solubilization of cellulose.
Novel pretreatment methods such as ultra-sonication, nano-technological methods,
and microwave digestion processes are used for pretreatment of lignocellulosic bio-
mass in order to improve the bioethanol production and reduce inhibitors. After this
treatment process, the obtained slurry contains two different types of fractions: one
is liquid fraction consisting xylose and small amounts of glucose, galactose, and
arabinose, and another is solid fraction containing lignin and cellulose material. The
potential utilization of these two (hemicellulosic and cellulosic) fractions of bio-
mass is one of the cost-effective method for ethanol production (Agbogbo and
Wenger 2007).
15.5.1 Parameters Affecting Pretreatment of Biomass
Optimization by screening the efficient strains and maintaining the culture condi-
tions can make this process more effective and reduce the treatment time. The gov-
erning process parameters include biomass type, nature, and composition, and
physical parameters include incubation temperatures, pH, incubation time, moisture
content, and aeration rate. Biological parameters such as type of microorganism,

culture conditions, growth characteristics, optimum conditions for growth, and
enzyme production are the key factors for this process.
Lignocellulosic biomass is available abundantly and consists of various propor-
tions of lignin, cellulose, and hemicelluloses. The selection of microorganism for
biological treatment is based on the nature and composition of the biomass. The
optimum temperature varies with fungal strain such as ascomycetes with optimum
temperature at 39 °C while basidiomycetes at 25–30 °C. Incubation time also varies
depending on strain and nature of biomass. Long incubation time is required for
pretreatment and delignification process. Moisture content plays a key role in solid-
state fermentations and is essential for growth establishment of microorganisms.
White-rot fungi or brown-rot fungi are used for efficient enzymatic saccharification.
Pretreatment with fungal strains could enhance enzymatic hydrolysis of lignin.
Several studies have revealed that fungal consortium has faster degradation ability
of lignocellulosic biomass when compared with single strain. Lignin degradation is
an oxidative process, and aeration is a critical factor for production of lignolytic
enzymes such as lignin peroxidase and manganese peroxidase (Millati et al. 2011).
pH is another important factor for microbial growth; majority of fungal strains grow
tremendously in acidic pH range between 4.0 and 5.0. Inoculum level and cell bio-
mass are also critical factors and influence the treatment time of biomass. In SSF
(solid-state fermentation), particle size plays a crucial role in biological treatment.
Penetration limitations are large with particle size, when compared to small particle
size (Kuijk et al. 2015).
15.6 Limitations by Chemical Pretreatment
Inhibitors were produced during the digestion of hemicellulosic fraction. These

inhibitors are classified into three types: (a) organic acids (acetic acid, formic acid,
levulinic acid, ferulic acid, and p-coumaric acid), (b) furan derivatives (furfural and
5-hydroxy furfural), and (c) phenolic compounds (4-hydroxybenzoic acid and feru-
lic acid) (Jonsson and Martin 2016). All these inhibitors influence the growth and
ethanol production efficiency of microorganism (Cragg et al. 2015). Advanced
treatment methods (ultrasonication, microwave digestion) are used to reduce the
inhibitor formation and increase the fermentation efficiency of organism. Therefore,
detoxification steps are required for removal of inhibitory compounds from hydro-
lysate before the fermentation process. Detoxification strategies include active char-
coal treatment, ion exchange resins, alkali treatment, overliming using calcium
hydroxide, change in the fermentation methodologies, and treatment with soft-rot
fungi Trichoderma reesei to degrade inhibitors (Yu et al. 2011).
The need for efficient pretreatment or hydrolysis process for the recovery of
maximum amount of fermentable sugars with minimum toxic chemicals is a major
challenge. WRF (white-rot fungi) is one of the promising organisms to convert bio-
mass to bioethanol production with eco-friendly and cost-effective manner. This
process has ample advantages over chemical process such as simpler technique, less
energy utilization, less wastage, and absence of inhibitors. In this process, various
strains of WRF that include Pleurotus ostreatus, Cyathus stercoreus, Ceriporiopsis
subvermispora, Trametes versicolor, and Phanerochaete chrysosporium are used
for conversion of biomass to ethanol production (Nigam and Pandey 2009).
15.7 ugal Consortium Used for Biofuel Production

F
from Plant Biomass
Several studies have proved that co-culture studies are promising processing meth-
ods for production of ethanol from lignocellulosic biomass. Development of fungal
consortium has played a significant role for the conversion of polymeric fractions of
lignin, cellulose, and hemicellulose into sugars. This consortium includes lignin-,
cellulose-, hemicellulose-degrading white-rot fungi and ethanol producing efficient
strains. Examples including P. chrysosporium, Pleurotus ostreatus, Pycnoporus cin-
nabarinus and Cyathus stercoreus are able to produce lignin-degrading enzymes.
Laccases are involved in degradation of lignin and show activity with lignin peroxi-
dase and manganese peroxidase (Binod et al. 2010). Cellulose-degrading enzymes
endoglucanases, cellobiohydrolase, and β-glucosidease are produced by a number
of fungal species. Hemicellulose-degrading enzymes are xylanases and β-xylosidases
and produced by Aspergillus niger, Trichoderma reesei fungal strains (Zhang et al.
2012; Kour et al. 2019; Rana et al. 2019a, b).
Currently, genetic engineering techniques are widely used for bioconversion of
value-added products from lignocelluloses biomass. Transfer of genes encodes
xylose reductase and xylitol dehydrogenase from Pichia stipitis to S. cerevisiae
(wild strain) for utilization of xylose for enhanced production of ethanol (Agbogbo
and Wenger 2007). In industrial scale, it is expensive to maintain the biochemical
and fermentative characters of recombinant strains. Due to this disadvantage, a co-
culture process (both glucose- and xylose-utilizing strains) is cost-effective method
for ethanol production along with enzyme-producing white-rot fungi (Cheng et al.
2010). Various factors influencing the production efficiency of the strain include
oxygen, aeration, agitation, pH, temperature, concentration of carbon source, inhib-
itor presence in hydrolysate, and medium components (Sreenivas Rao et al. 2006).
15.8 Enzymes Produced by White-Rot Fungi
White-rot fungi produce various extracellular oxidases such as laccase, Mn peroxi-

dase, and lignin peroxidase (LiP) including lignin-modifying enzymes (LME).
These enzymes effectively degrade the lignin content in lignocellulosic biomass.
Lignin degradation is a key process for biofuel production from lignocellulosic bio-
mass. White-rot fungi (WRF) Phanerochaete chrysosporium produce multiple
isoenzymes (LiP, MnP) but not laccase. Other WRF were able to produce laccases.
Based on the enzyme production, WRF are categorized into three main groups: (1)
lignin-manganese peroxidase (P. chrysosporium, Phlebia radiata), (2) manganese
peroxidase (Dichomitus squalens, Rigidoporus lignosus), and (3) lignin peroxidase
(Phlebia ochraceofulva and Junghuhnia separabilima). These enzymes are able to
degrade various types of plant polymers (Heinzkill 1998; Yadav et al. 2016)
(Table 15.1).
Table 15.1 Enzymes released by WRF

Biomass Microorganism Enzyme Reference
Corn stover Gloeophyllum Cellulase Gao et al. (2012)
trabeum KU-41
Cotton stalks Phanerochaete Cellulase Jian et al. (2008)
chrysosporium
Milled tree leaves, Pleurotus spp., Laccase Songulashvili et al.
banana peel, apple Lentinus edodes (2005)
peel, mandarin peel
Sugarcane trash Aspergillus Cellulases Singh et al. (2008)
terreus
Sorghum husk Phanerochaete Lignin peroxidase and Pankajkumar et al.
chrysosporium manganese peroxidase (2018)
Young plant leaves Fusarium Endopolygalacturonases Mikan and Castellanos
(from Aster genus), oxysporum galactosidase (2004)
lamella from oats
and maize plants
Sugarcane bagasse Aspergillus Xylanases, cellulases Park et al. (2002)
niger
Clavel leaves, young Fusarium Endo-xylanase, cellulases, Fernández-Martín et al.
plant leaves (from merismoides arabinofuranosidase, (2007)
Aster genus), acetylesterase
lamella from oats
and maize plants
Sugi wood Strobilurus Lignin peroxidase and Homma et al. (2007)
ohshimae manganese peroxidase
Bagasse of cane Pleurotus Xylanases, cellulases, Márquez et al. (2007),
maize straw ostreatus laccase, manganese Okamoto et al. (2002)
peroxidase
Grape seeds, barley Phanerochaete Lignin peroxidase and Rodríguez et al. (1997),
bran, and wood chrysosporium manganese peroxidase Srinivasan et al. (1995),
shavings Kersten and Cullen
(2007), Quintero et al.
(2006)
Clavel leaves, young Clonostachys Endopolygalacturonases Mikan and Castellanos
plant leaves (from rosea galactosidase endo-xylanase, (2004), Rezácová et al.
Aster genus), cellulases, (2006)
lamella from oats arabinofuranosidase, acetyl
and maize plants
(continued)

Biomass Microorganism Enzyme Reference
Coffee pulp, used P. ostreatus, P. Endoglucanase, Marnyye et al. (2002),
nappy, grass pulmonarius cellobiohydrolase, laccases, Delfin and Duran de
residues, cleaned manganese peroxidase bazúa (2003), Okamoto
coffee (substrates et al. (2002)
analyzed separately
and in mixture),
wheat straw,
industrial cotton
fiber
Clavel leaves, young Streptomyces Cellulases, xylanases, Mikan and Castellanos
plant leaves (from arabinofuranosidase (2004), Benimelia et al.
Aster genus), xylosidase, acetylesterase (2007)
lamella from oats
and maize plants
Wood shaving, Trametes Laccases Moredo et al. (2003),
carozo maize, and versicolor Márquez et al. (2007),
compost of Dumonceaux et al.
gardening wheat (2001), Villagran and
straw Renan (1991), Cabuk
et al. (2006), Tong et al.
(2007)
Oat husk Cerrena Laccases, manganese Moilanel et al. (2015)
unicolor peroxidase
Saw dust Coriolopsis Laccases Daassi et al. (2016)
gallica
15.9 Ligninolytic Enzymes from White-Rot Fungi
Ligninolytic enzymes from WRF are used in industrial biotechnological process.

Free enzyme applications were limited in industrial scale due to instability and lack
of reusability. Immobilization techniques were used to improve stability and can be
reusable. Voberkova et al. (2018) have reported that immobilization methods are
desirable, operational stability and cost-effective process.
The delignification is a key task for proper utilization of lignocelluloses bio-
mass. The first demonstration of commercial plant for the ethanol production
from lignocellulosic biomass is in operation in Canada since 2004 (Tampier
et al. 2004). White-rot fungi is a filamentous fungi, which can produce ligni-
nase, cellulase enzymes for degradation of lignocellulosic plant material.
Pleurotus cystidiosus, P. ostreatus, Phlebia, Ganoderma lucidum, and
Flammulina velutipes are able to produce ethanol from biomass. Various WRF
are used for production of enzymes, which degrade the lignocellulosic material.
Integrated production of ethanol by WRF is influenced by factors like moisture
content, temperature, pH, and chemical nature. Various reports of biological
pretreatment of lignin are presented in Table 15.2.
Table 15.2 Lignin degradation by fungal strains

Microorganism Biomass Effect Reference
Merulius tremellosus Aspen wood 0.26–0.37 mg/ml as compared to Bradley et al.
0.15–0.16 mg/ml of control (1989)
Phanerochaete Cotton stalks 0.027 g/g Jian et al. (2008)
chrysosporium
Trichoderma viride Rice straw 56% of lignin reduction Ghorbani et al.
(2015)
Trichoderma reesei Wheat straw Sugar Yield -270 Reduction (mg/g Barakat and
dry substrate) Rouau (2014)
Ceriporiopsis Corn stove 2- to 3-fold increase in reducing Wan and Li
subvermispora sugar yield (2011)
Irpex lacteus Corn stalks 82% of hydrolysis yield Du et al. (2011)
Phanerochaete Corn stover Improved degradation Liu et al. (2014)
chrysosporium silage of substrate cell wall components
39% lignin removal of initial
substrate
Pleurotus ostreatus Wheat straw 35% of lignin reduction (1983)
Pleurotus ostreatus Rice straw 33% lignin removal Mustafa et al.
(2016)
Ceriporiopsis Corn stover 31.59% lignin loss Wan and Li
subvermispora (2010)
Fusarium spp. Paddy straw 17.1% decrease in lignin content, Phutela and Sahni
10.8% decrease in silica content (2012)
compared with controls
15.10 Biochemical Pathway of Ethanol by Fungi
The yeast cells have the sense to identify the sugar-rich environment. This intensity
by the yeast can affect the enzyme activity during biochemical processes, change of
translation by mRNA, stability of protein degradation, and concentration of metab-
olites (Yadav et al. 2017, 2018). After glucose uptake then enters the glycolytic
pathway and is converted to pyruvate and produces ATP and then coupled to inter-
mediate products and reducing power through NADH for biosynthetic pathway.
Pyruvate in glycolysis enters TCA cycle or fermentative pathway. In alcoholic fer-
mentation, decarboxylation of the pyruvate gives acetaldehyde by pyruvate decar-
boxylase enzyme. This enzyme converts acetaldehyde to ethanol by reduction of
NADH to NAD+, from one molecule of glucose to two CO2 molecules and ethanol
is formed.
The second abundant sugar, from hemicellulosic fraction of biomass, is xylose.
Xylose is a five-carbon sugar molecule, utilized through pentose phosphate pathway
in fungi. The xylose transportation in fungi is followed by two different mecha-
nisms. Pichia stipitis and Candida spp. follow proton symport (PS) mechanism,
whereas Saccharomyces cerevisiae follows facilitated diffusion system (FDS) for
transport of xylose. The PS transport is for pentose sugars and FDS transport for
both hexose and pentose sugars. The medium consisting of low quantities of hex-
Lignocellulosic
biomass
Pretreatment
Cellulose Hemicellulose
Cellulases
Cellobiose Xylose Acetate
Cellodextrin
transporter Xylose Acetate
XR ACS
Cellobiose Xylitol Acetyl-CoA
β-glucosidase XDH AADH

Xylulose
XK Acetaldehyde
PPP ADH
Glucose Glycolysis Ethanol
Ethanol
Fig. 15.1 Biochemical pathway of ethanol production by fungi
oses will inhibit xylose transport by FDS mechanism. In fungi xylose metabolism,
xylose is converted to xylulose through xylitol by xylitol dehydrogenase, and then
xylulose is phosphorylated and enters to pentose phosphate cycle. Conversion of
xylose to xylitol in the presence of xylose reductase utilizes NADH or NADPH as a
cofactor. In anaerobic or microaerophilic conditions, yeast utilizes NADH for con-
version. High expression of xylose reductase and xylitol dehydrogenase will tend to
enhance the ethanol production. Co-current isomerization of xylose and co-fermen-
tation of xylose and glucose increase the production of ethanol (Fig. 15.1).
Overcoming the challenges of fossil fuels through the biotechnological route by

fungal consortium is one of the promising approaches to reach global demand. The
lignocellulosic biomass is a significant substrate for bioethanol production using
fungal consortium for commercial production of ethanol throughout the year in a
cost-effective process. This biotechnological approach of ethanol production is an
eco-friendly process through enhancing the yield and reduction of the greenhouse
gas emission. Saccharifications of lignocellulosic biomass by white-rot fungi have
overcome the challenges with chemical digestive methods such as gasification and
acidification. The chemical process using high temperatures, acids, and high-energy
input lead to release of inhibitors and pollutants. The development of fungal consor-
tium consists of lignin-, cellulose-, and hemicellulose-degrading strains in combi-
nation with ethanol-producing strains (from hexoses and pentoses) which can
achieve the global demand for ethanol production. The enzymes like laccases, cel-
lulases, and xylanases play a significant role for the digestion of lignocellulosic
plant biomass by eco-friendly manner. Several advantages were reported by fungal
consortia which include high adaptability, productivity, and efficiency of the pro-
duction. This process is also considered as inexpensive and eco-friendly. The future
prospect for ethanol production using fungal consortium is a need to development
of unique fungal consortia with noncompetitive synergistic fungal strains for pro-
duction of lignocellulosic digestive enzymes along with efficient ethanol-producing
strains. Standardization of co-culture studies for optimization of digestive enzymes
and ethanol production, optimized conditions for microbial growth, and metabolite
production (enzymes) were varying from one strain to another. In co-culture studies
(development of fungal consortium), physical factors (temperature, pH, agitation,
moisture levels, surface area, and SSF) and chemical factors such as carbon source,
nitrogen sources, minerals, salts, and their concentrations need to standardize for
commercial production of ethanol. Simultaneous saccharification and fermentation
have enhanced the yield.
Acknowledgments Authors sincerely acknowledge University Grant Commission (UGC),

Government of India, for providing financial support for major research project entitled
“Overcoming fossil fuel challenges: Co-culture fermentations for biofuel production using agro-
industrial waste materials.”
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Chapter 16
Role of Fungi in the Removal of Heavy
Metals and Dyes from Wastewater
by Biosorption Processes
Ajay Kumar, Vineet Kumar, and Joginder Singh
16.1 Introduction
World population is increasing day by day; hence, to meet the demand of the growing
population, clean water is the major concern. Water is being polluted by human acti-
vates and industrial discharges. These pollutants are categorized into three major
groups: organic, inorganic, and biological particles. Heavy metals and dyes as waste
from various industries including textile, pharmaceutical, leather, etc. are the major
pollutant present in water (Burakov et al. 2018). Heavy metal ions are elements from
the fourth period of the periodic table, mostly chromium (Cr), cobalt (Co), nickel (Ni),
copper (Cu), zinc (Zn), arsenic (As), lead (Pb), and mercury (Hg). Removing heavy
metals is necessary because they are toxic substances with carcinogenic nature that
should not to be discharged directly into the environment. Conventional techniques like
membrane separation, precipitation, coagulation, and flocculation are widely used for
removal of heavy metals (Azimi et al. 2017; Marzougui et al. 2017). Biosorption is
preferred over these conventional techniques due to high affinity, capacity, and selectiv-
ity of the materials from the solution. There are different mechanisms involved in bio-
sorption phenomenon (Fig. 16.1). There are different kinds of adsorbent available for
wastewater treatment. Adsorbents are broadly classified into conventional and noncon-
ventional. Biosorbents are nonconventional adsorbents and have several advantages
over other conventional and nonconventional methods (Fig. 16.2).
Microorganism is one type of biosorbents that has been used for wastewater
treatment. Many living or dead microorganisms such as bacteria, fungus, and
A. Kumar (*) · V. Kumar

School of Bioengineering and Biosciences, Lovely Professional University,
Phagwara, Punjab, India
J. Singh

398 A. Kumar et al.
Fig. 16.1 Different mechanisms involved in biosorption phenomenon (Asgher 2012)
algae are widely used for heavy metal removal because of high adsorption capacity,
low cost, and its availability in large quantities (Kour et al. 2019a; Rastegari et al.
2019). However, it suffers from some of the drawbacks like waste may be con-
verted into more potential toxic compounds. Most of the microorganism-based
methods deal with discoloration of dye instead of removal of dye or other wastes.
Dyes are mostly used in textile, leather, and pharmaceutical industries. Discharge
of dyes directly into the water bodies causes threats to aquatic fauna and flora as
it interferes with gas solubility. Higher concentration of dyes resulted in carcino-
genicity and toxicity. Use of fungi for wastewater treatment is one of the promis-
ing alterative to physical and chemical process (Couto 2009; Yadav et al. 2016,
2019b). Classification of dyes according to the chemical structure is depicted in
Fig. 16.3.
However, nanomaterial-based approaches of wastewater treatment are consid-
ered more useful as they allow comparatively better removal of wastewater (Masoudi
et al. 2018). With advances in nanotechnology, more and more useful nanomaterials
such as graphene, carbon nanotubes, and fullerenes are being produced for waste-
water treatment. Along with wastewater treatment, these nanomaterials are also use-
ful for various other environmental applications (Hegab et al. 2018; Nyairo et al.
2018; Park et al. 2017). Figure 16.4 shows nanomaterials used for heavy metal treat-
ment of water. Synthesis of bionanocomposite is now in practice for wastewater
treatment. Wang et al. (2018) reported the removal of methylene blue by adsorption
on yeast composite assisted with Fe2O3 nanoparticles. Figure 16.5 shows the scheme
diagram for the synthesis of bio-nanocomposites.
16 Role of Fungi in the Removal of Heavy Metals and Dyes from Wastewater… 399
Fig. 16.2 Various methods for wastewater treatment methods (Crini et al. 2018)
16.2 Fungi as Biosorbent
Fungi are osmo-heterotrophic eukaryotes placed in the kingdom Fungi. The fungal
cell wall is composed of acid polysaccharides such as chitin (a polymer of acetyl-
glucosamine unit), and chitosan, which is characterized by phosphate, amine, and
400 A. Kumar et al.
Fig. 16.3 Classification of dyes according to the chemical structure (Yagub et al. 2014)
Fig. 16.4 Nanomaterials for heavy metal treatment of water (Lu and Astruc 2018)
hydroxyl groups, is involved in biosorption of heavy metals, dyes, and phenolic

compounds (Zhu et al. 2019). They lack chlorophyll and their vegetative structure
may be filamentous or unicellular. They reproduce through spore formation
(Raghukumar 2017). Figure 16.6 shows the structure of chitin (Fig 16.6a) and chi-
tosan (Fig 16.6b) and their binding with metal ions. Table 16.1 shows the character-
istics of major fungal divisions.
Fig. 16.5 The scheme diagram for the synthesis of bio-nanocomposites
Fig. 16.6 (a) The units of a chitin polymer molecule. (b) Chitosan is deacetylated chitin. (c) Binding
of metal anions on chitin or chitosan (Kotrba 2011)
402 A. Kumar et al.
Table 16.1 Characteristics of major fungal divisions (Stajich et al. 2009)

Division Characteristics
Chytridiomycota The fungi produce zoospores capable of moving on their own through a
liquid medium by simple flagella
Zygomycota The hyphae do not have one nucleus per cell but rather have long
multinucleate, haploid hyphae that comprise their mycelia. Asexual
reproduction is by spores produced in stalked sporangia
Ascomycota They contain more than 30,000 species of unicellular (yeasts) to
multicellular fungi. Yeasts reproduce asexually by budding and sexually by
forming a sac/ascus
Basidiomycota Mushrooms, toadstools, and puffballs are commonly encountered
basidiomycetes. These conspicuous features of the fungi are the
reproductive structures. Sexual reproduction involves the formation of
basidiospores on club-shaped cells known as basidia
Deuteromycota A group of fungi that either lack the perfect stage (i.e., sexual
reproduction) or whose perfect stage is as yet undiscovered. They
reproduce most frequently by conidia or conidia-like spores. Many forms
of deuteromycota are pathogenic, affecting man, animals, or plants
For removal of dyes from wastewater, different forms of fungal sorbents are used
such as fungal pellets, mycelium, or dead fungus by many researchers (Yagub et al.
2014). Many molds and filamentous microorganisms such as Aspergillus niger,
Penicillium simplicissimum, Aspergillus fumigatus, Termitomyces clypeatus,
Penicillium brevicompactum, Saccharomyces cerevisiae, Trichoderma, etc. are used
for removal of heavy metals and dyes (Rana et al. 2019a, b; Yadav et al. 2019a, b).
Fungus can survive in the presence of high metal concentration. So it can be used
for heavy metal removal from wastewater. Heavy metal adsorption by fungi through
ion exchange and coordination is due to the presence of chitin–chitosan, glucuronic
acid, phosphate, and polysaccharides present in/on the cells of fungi. Different
kinds of functional groups such as amine, carboxyl, hydroxyl, phosphate, and sulf-
hydryl pay a vital role in the adsorption of heavy metals and dyes by fungal stains
(Yin et al. 2018). Fungi especially white-rot fungi and their enzymes (laccase, lignin
peroxidase, and Mn peroxidase) can be used to bioremediate various xenobiotics
and wastewaters (Kour et al. 2019b; Yadav et al. 2017a, b; 2018). Figure 16.7 shows
the schematic diagram of adsorption mechanism model.
16.3 Growth Models for Filamentous Organisms
Fungal biomass can be easily cultivated, or it can be available as industrial waste

product such as Aspergillus niger (waste from citric acid production) and
Saccharomyces cerevisiae (brewery industry waste) (Dhankhar and Hooda 2011).
At high cell density, filamentous organisms such as molds often form microbial pel-
lets in suspension culture. During the growth process, filamentous organism
Fig. 16.7 (a) Schematic diagram of adsorption mechanism model. (b) SEM images of fungus
mycelia and (c) dyes adsorbed onto fungus mycelia (Li et al. 2019)
increases in their size and mass. Thus, in the absence of mass transfer limitations,
the radius of the microbial pellet increases linearly with time.
dR
= k=
p cons tan t (16.1)
dt
The growth rate of mold colony can be expressed as follows:
dM dR
= ρ 4π R 2 = kp 4π R 2 ρ (16.2)
dT dt
dM
= γ M 2/3 (16.3)
dT
where γ = kp(36πρ)1/3
The mass of spherical pellet as a function of time is given as follows:
3 3
 γt  γt 
M =  M 01/ 3 +  ≈   (16.4)
 3 3
404 A. Kumar et al.
where M0 is the initial mass which is very small as compared with the M and there-
fore M varies with cubic power with time.
16.4 Surface Modification of Fungal Biomass
Surface modification of biomass is one of the strategies used for adsorption of heavy
metals and dyes. Various pretreatment methods such as acid, base, and thermal
treatment are used for surface modification of biomass to enhance the adsorption
capacity of biomass (Yin et al. 2018) as shown in Fig. 16.8.
16.4.1 Acid Pretreatment
Biomass treated with acid improved the positive charge density on the surface which
provide strong electrostatic attraction for negatively charged heavy metal ions.
16.4.2 Base Pretreatment
Biomass treated with alkali may increase negative charge on the surface of biomass
to enhance the electrostatic attraction for positively charged heavy metal ions.
Fig. 16.8 Enhancing heavy metal removing efficiency of biomass through surface modification
(Yin et al. 2018)
16.4.3 Thermal Treatment
The porosity and surface area of biomass can be enhanced by thermal treatment for
adsorption capacity of biomass. The thermal treatment can also increase the surface
functional groups by adding metal-binding groups.
16.5 Biosorption Models and Isotherms
Biosorption is defined as the removal of substance from biological materials whether

it is living or dead which involves the phenomenon of the mass transfer. Biosorption
involves both the adsorption and absorption processes.
Sorption mechanism can be divided into four consecutive steps:
(i) Transport of solute in the bulk solution
(ii) Diffusion of solute through the liquid film surrounding the adsorbent particles
(iii) Diffusion of solute in the pores of the sorbent (intraparticle diffusion)
(iv) Chemical reaction as adsorption and desorption on the solid surface
16.5.1 Adsorption Thermodynamics
Thermodynamic behavior of heavy metals and dyes on biosorbent is cited as exo-

thermic or endothermic sorption processes. Free energy gives the information about
the physical sorption or chemical sorption (Yao et al. 2010; Madala et al. 2017).
∆G  = − RT ln K (16.5)
∆G  = ∆H  − T ∆S  (16.6)
 
∆S ∆H
ln K = − (16.7)
R RT
where ∆G0 (J/mol) is Gibb’s energy, R is the ideal gas constant (8.314 J/mol K), T
is temperature in Kalvin (K), ∆S0 (J/mol K) is adsorption entropy, and ∆H (J/mol)
is adsorption enthalpy.
K can be obtained from qe/Ce, while the values of ΔH0 and ΔS0 were determined
from the slope and intercept of the van’t Hoff plot of ln K versus 1/T.
The negative values of ΔGo suggested the spontaneous behavior of adsorption
process. The positive values of ΔHo indicate the endothermic process for the adsorp-
tion of metals and dye. The positive value of ΔSo suggested the increasing random-
ness between the solid and solution interface during the adsorption process.
406 A. Kumar et al.
16.5.2 Adsorption Isotherm
Several mathematical models have been developed by the researchers to validate the
process of adsorption (Lei et al. 2018). Kumari and Abraham (2007) describe bio-
sorption of anionic textile dyes by nonviable biomass of fungi and yeast.
The Freundlich model assumes that the adsorbent surface is heterogeneous and
sorption on its surface is multilayer.
qe = K f c1/e n (16.8)
1
ln qe = ln K f + ln ce (16.9)
n
where Ce (mg/L) is the equilibrium concentration in solution, qe (mg/g) is the lead

adsorbed at equilibrium, n is Freundlich constant related to adsorption intensity, and
Kf is adsorption constant for Freundlich model.
Thermodynamic parameters of adsorption of silver onto biochar were calculated
from Langmuir isotherm as documented by Antunes et al. (2017).
bqm ce
qe = (16.10)
1 + bce
The linear form of Langmuir isotherm model equation
Ce Ce 1
= + (16.11)
qe qm ( qm .b )
where ce (mg/L) is the equilibrium concentration of Cu(II), qe (mg/g) is the adsorp-

tion capacity, qm (mg/g) is the theoretical maximum sorption capacity, and b (L/mg)
is the Langmuir constant related to adsorption energy.
The plot of Ce/qe against Ce gives a straight line with a slope and intercept of 1/qm
and 1/qmb, respectively.
The separation factor, RL, can be determined from Langmuir plot as per the fol-
lowing relation:
1
RL = (16.12)
(1 + bC0 )
where RL values indicate the type of adsorption to be irreversible (RL = 0), favorable
(0 < RL < 1), linear (RL = 1), or unfavorable (RL > 1), and C0 is the initial metal or
dye concentration (ppm).
The Dubinin–Radushkevich (D-R) equation is described for adsorption nonpo-

rous, macroporous, and mesoporous adsorbents. The linear D-R isotherm model
equation
2
  1 
ln qe = ln qD − BD  RT ln  1 +   (6.13)
  C e 

where BD is related to the free energy of adsorption and qD is the D-R isotherm con-
stant related to the degree of adsorption by the adsorbent.
The Temkin isotherm is based on the assumption that heat of adsorption would
decrease linearly with increase of coverage of adsorbent due to adsorbate/adsorbent
interactions.
The linearized Temkin isotherm equation
qe = QT ln K T + QT ln Ce (16.14)
where QT = RT/bT, bT is the Temkin constant related to the heat of adsorption (kJ/
mol), KT is the Temkin isotherm constant (l/g), R is the gas constant (8.314 J/
mol · K), and T is the Kelvin temperature (K).
16.5.3 Adsorption Kinetics
Adsorption kinetics was studied by Bayramoglu and Yilmaz (2018). Aljeboree et al.
(2017) have described the pseudo-first-order and pseudo-second-order kinetics and
equilibrium study for the adsorption of textile dyes on coconut shell activated
carbon.
k1t
log ( qe − qt ) = log qe − (16.15)
2.303
t 1 t
= + (16.16)
qt k2 qe2 qe
where qt and qe (mg/g) are adsorbed lead amount at time t (h) and equilibrium and
k1 (1/h) and k2 (g/(mgh)) are the rate constant for the pseudo-first-order and pseudo-
second-order adsorption kinetics, respectively.
Elovich equations
1 1
qt =   ln (αβ ) +   ln t (16.17)
β  β 
408 A. Kumar et al.
where qe (mg/g) is the experimental amount of dye adsorbed at equilibrium and qt

(mg/g) is the amount of dye adsorbed at time t. For Elovich equations, α is the initial
adsorption rate (mg/g/min), and the parameter β is related to the extent of surface
coverage and activation energy for adsorption (g/mg).
16.5.4 I ntraparticle Diffusion Model on Metal or Dye

Adsorption
For most adsorption process, the amount of adsorption varies almost proportional
with t1/2 .
qt = K diff t 1/ 2 + C (16.18)
where qt is the adsorption capacity at time t, t1/2 is the half-life time in second, and
Kdiff (mg/g min1/2) is the rate constant of intraparticle diffusion.
To find out the rate constants, plot qt versus t1/2 gives a linear relationship, and
Kdiff can be determined from the slope of the plot.
Arrhenius equation is to determine the adsorption activation energy using the
kinetic data. The kinetic constants (k) at each temperature are derived from the
intraparticle diffusion models.
 Ea 
− 
k = Ae  RT 
(16.19)
Ea
ln k = ln A − (16.20)
RT
The adsorption activation energy is calculated by depicting ln(k) versus 1/T .

A is the Arrhenius constant, R is the gas constant (8.314 J/mol K), Ea is the
adsorption activation energy (J/mol), and T is temperature (K).
16.6 Characterization of Fungal Biosorbent
Several techniques are used for the characterization of fungal biosorbent, namely,
ultraviolet (UV) spectroscopy, scanning electron microscopy (SEM), transmission
electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX),
Brunauer–Emmett–Teller (BET), Fourier-transform infrared spectroscopy (FTIR),
Zeta potential analyzer, particle size analysis, and differential scanning calorimetry
(DSC). Figure 16.9 shows the determination of the basic properties of an adsorbent
by multifarious techniques.
Fig. 16.9 Determination of the basic properties of an adsorbent by multifarious techniques

(Unuabonah et al. 2019)
16.7 Adsorption Study
Adsorption studies are mainly done through batch and column study such as packed
bed study.
16.7.1 Batch Adsorption Study
Adsorption of the heavy metals on fungal biomass is carried out in batch mode until
equilibrium is established. The adsorption capacity of each metal ion adsorbed by
the fungal biomass is determined (qe, mmol g−1) as the difference between their
initial and final concentrations as given by Karunanayake et al. (2018) and Liu et al.
(2018). Schematic diagram of batch biosorption equilibrium experimental proce-
dure is represented in Fig. 16.10.
410 A. Kumar et al.
Biosorbent
After
Equilibrium
Filter
Solute Agitation
C0 initial
Filtrate analyzed
Cf Final
Fig. 16.10 Schematic diagram of batch biosorption equilibrium experimental procedure

(Vijayaraghavan and Yun 2008)
V
Qe = (C0 − Ce ) × (16.21)
m
C0 − Ce
Decolourization Rate ( % ) = × 100% (16.22)
C0
where Co and Ce (mg L−1) are the initial and equilibrium concentrations after adsorp-
tion and Qe (mg g−1) is the equilibrium adsorption capacity. V (L) is the volume of
acid dye solution, and m (g) is the mass of the adsorbents.
16.7.2 Column Adsorption Study
The fixed-bed column is made of a glass tube and packed with fungal biosorbent.
The effluent samples are collected periodically from the bottom of the column dur-
ing the experiment to determine the concentration of each collected sample
(Fig. 16.11). A 45 mL of sample is placed in the Teflon liner with 4 mL of 15.9M
HNO3 and 1 mL of 12.1M HCl. The final extract was filtered through 0.45 μm
syringe filter and analyzed using inductively coupled plasma mass spectrophotom-
etry (ICP-MS) (Östman et al. 2017).
Packed-bed
Diatomite embedded with

Wastewater PEI-PY nanoparticles
source
Syringe
pump
sampling
Fig. 16.11 Schematic representation of the fixed-bed experimental setup (Hethnawi et al. 2018)
16.7.3 Breakthrough Analysis
In the fixed-bed adsorption system, the breakthrough curve (BTC) behavior is

affected by the operational parameters (i.e., Q, Co, Z, and % nps) and the designed
parameters (length over diameter) of the column as well as the characteristic of the
adsorbent (size and shape) (Fig. 16.12).
16.7.4 Packed Bed Column Model
Packed bed column is an effective process for cyclic sorption/desorption, as it makes

the best use of the concentration difference known to be a driving force for pollutant
sorption and results in a better quality of the effluent. Two frequently used models,
i.e., Thomas and Bed Depth Service Time (BDST), were used to analyze the com-
patibility of experimental data of the tested metals.
16.7.4.1 Thomas Model
The Thomas model can be implemented to analyze the breakthrough curves and
adsorption capacity for sorbent.
412 A. Kumar et al.
Adjustable plunger To collection

Outlet
concentration Outlet concentration
Biosorbent
Inlet concentration
level
Breakthrough Elution
curve curve
Breakthrough
point
Time Glass beads Time

Two way valve
Metal/dye Elutant
solution solution
Fig. 16.12 Schematic diagram of packed column arrangement with biosorption breakthrough and
elution curves (Vijayaraghavan and Yun 2008)
C  k qm
ln  0 − 1  = Th 0 − kTh C0 t (16.23)
 C  Q
where C0 and C are the inlet and the effluent solute concentrations at any time
t (m); kTh is the Thomas model constant (mL m−1 mg−1); q0 is the maximum
solid-phase concentration of solute (mg g−1); and M is the total mass of the
adsorbent (g).
The model constants kTh and q0 can be determined from slope and intercept of a
plot of ln[(C0/C) – 1] against t, respectively.
16.7.4.2 BDST Model
The BDST model is used to predict the column performance of any bed length, if
data for some depths are known.
N0 Z 1 C 
t= − ln  0 − 1  (16.24)
C0ϑ K a C0  Cb 
where t is the service time (h), N0 is the adsorption capacity (mg cm−3), Z is the
height of column (cm), Cb is the breakthrough sorbate concentration (mg L−1), ϑ is
the linear velocity (cm h−1), and Ka is the rate constant (L mg−1 h−1) at time t.
16.8 Validation of Adsorption Kinetics Models
The kinetic data was fit using the nonlinear form of the PFO and PSO models,
where the best-fit was estimated by coefficient of determination (R2) as validated by
Jawad et al. (2019).
n =1
∑ ( qt .meas − qt .cal )
2
R = 1−
2 n
(16.25)
( )
N =1 2
∑ qt .cal − qt .cal
N
qt.meas and qt.cal are the measured and calculated adsorption capacity at time t, and n
is the number of observations.
Chi-square and the normalized standard deviation are used to validate the kinetic
models as cited by Inyinbor et al. (2016).
(q − qcal )
2
i =1
exp
χ 2
=∑ (16.26)
n qcal
∆qe ( % ) = 100
(q exp − qcal ) / qexp
(16.27)
N −1
where N is the number of data points, while qexp and qcal are experimentally deter-
mined quantity adsorbed at equilibrium and calculated quantity adsorbed at equilib-
rium, respectively.
16.9 Factors Affecting Fungal Biosorption
Several factors affect the process of biosorption (Dhankhar and Hooda 2011; Arief
et al. 2008; Bankar and Nagaraja 2018). Common factors are as follows:
(i) Type and nature of biomass
(ii) Initial solute concentration
(iii) Biomass concentrations (biosorbent dose/solution volume) in solution
(iv) Physicochemical factors like temperature, pH, and ionic strength
16.10 Factors Affecting Desorption
Numerous factors such as effect of desorption reagent, desorption temperature, and

desorption time have been investigated by several researchers (Zhang and Wang
2015; Mahfoudhi and Boufi 2017). Desorption or recovery is an essential concept,
414 A. Kumar et al.
especially if the pH has an effect in the sustainable manner of adsorption study.

But the regeneration process should not damage the adsorbent inside the fixed-bed
column; otherwise, their reuse will be inefficient; 0.05 mM of HNO3 at pH 5 can be
used for desorption study.
qde
Desorption Efficiency ( % ) = × 100 (16.28)
qad
where qde is the quantity desorbed by each of the eluent and qad is the adsorbed
quantity during loading.
16.11 Fungal Bioreactor for Wastewater Treatment
For the growth of fungus, different bioreactor configurations such as stirred tank
reactor (STR), bubble column, airlift, and fluidized bed reactors are used. These
reactors are used for wastewater treatment. Figure 16.13 shows fungal pellet reactor
for removal of pollutants in wastewater, although batch reactors are also used for
wastewater treatment (Espinosa-Ortiz et al. 2016).
(i) STR: Stirred tank reactor is widely used for culturing fungal pellets and com-
monly used for removal of heavy metals and dyes during wastewater treatment.
(ii) Bubble column bioreactor: Bubble column reactor belongs to the category of
multiphase reactors, and it is advantageous for the use of fungal pellets to treat
pollutants from wastewater.
(iii) Airlift bioreactor: Airlift bioreactor is similar to bubble column bioreactor but
contains draft tube which is always an internal or an external tube to improve
circulation and oxygen transfer. It is used for wastewater treatment.
(iv) Fluidized bed reactor: The fluidized bed reactor is characterized by its plug
flow nature of fluid movement inside the reactor. The fluidization occurs when
solid material (i.e., biomass) is suspended in an upward-flowing stream of
fluid, which can be either liquid or gas. These reactors are used in wastewater
treatment.
Methods exist for wastewater treatment, but they have some disadvantages like
chemical methods used chemicals that are threat to environment. Biological meth-
ods have advantages over chemical methods. Fungal biomass can be a novel inex-
pensive biosorbent for removal of heavy metals and dyes from aqueous solution.
The structural arrangements of various functional groups collectively make fungal
biomass a good biosorbent. Actually, the sorption (adsorption/absorption) is affected
by interaction of waste material with functional groups of fungal biomass with various
waste materials.
Fig. 16.13 Fungal pellet reactor for removal of pollutants in wastewater (Espinosa-Ortiz
et al. 2016)
Acknowledgments The authors are thankful to the School of Bioengineering and Biosciences,
Lovely Professional University, India, for providing library facilities.
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Chapter 17
Impact of Arbuscular Mycorrhizal Fungi
(AMF) in Global Sustainable
Environments
Sanjeev Kumar and Joginder Singh
17.1 Introduction
The arbuscular mycorrhizal (AM) association is a symbiosis between soil-borne fungi

with more than 80% of the terrestrial land plants. Colonization of AM fungi with host
roots resulted in increased growth and development of plants. This association further
improved nutrient uptake and increased protection against soil-borne pathogenic fun-
gus (Smith and Read 1997; St-Arnaud and Vujanovic 2007). Arbuscular mycorrhizal
fungi are important components of soil microbiota and form mutual interactions with
other microorganisms of rhizospheric soil (Bowen and Rovira 1999; Mathur et al.
2011; Yadav et al. 2019a). These interactions are mainly confined to the mycorrhizo-
sphere region of plant that leads to changes in the physical and chemical structures of
the soil. Furthermore, associations change in root exudation pattern (composition and
quantity) and fungal exudates, and mycorrhizal formation can affect the microbial
population present in rhizosphere. This also affects the colonization pattern by soil
microorganisms, and it is known as mycorrhizal effect (Gryndler 2000).
Mycorrhizosphere is known as a zone which influences the mycorrhiza (fungus roots)
in the soil (Oswald and Ferchau 1968). Moreover, the mycorrhizosphere consists of
two components as shown in Fig. 17.1. One is a thin layer of soil that surrounds the
root, known as rhizosphere. The other is a zone of AM hypha–soil interaction
(Marschner 1995) called hydrosphere. Hydrospheric zone consists of AM soil myce-
lium, and this is not affected by plant roots. Therefore, mycorrhizosphere region affects
soil fertility and quality. Current application of chemical fertilizers and pesticides with
S. Kumar (*)
Department of Genetics and Plant Breeding, Lovely Professional University, Jalandhar, India
J. Singh

420 S. Kumar and J. Singh
Fig. 17.1 Arbuscular mycorrhizal fungi interacted with plant growth-promoting bacteria
(Beneficial microorganism) in the mycorrhizosphere affecting soil properties and quality.
(Modified from Jeffries et al. (2003))
continuous husbandry of single crop may affect the community composition pattern of
AM fungi in different soil types.
Describing the diversity of AM communities at sites becomes, therefore, an
important step in determining the effect of different agricultural practices. Mycorrhizal
communities are site-specific, and each species can be affected in several ways by
different agricultural management practices. Agricultural practices such as tillage,
fertilization, and crop rotation affect the structure of AMF communities. There are a
number of reports that have described AM community composition in differently
managed ecosystems, as briefly shown in Table 17.1. In recent years, organic farming
practices have gained importance in many industrialized countries for the conserva-
tion of natural resources and reduction of environmental degradation (Maider et al.
2002; Yadav et al. 2017, 2018, 2019b). Several reports concluded significantly
greater AMF colonization in organically managed soil which further enhances
microbial activity and biodiversity (Maider et al. 2002; Kumar and Adholeya 2016).
In contrast, very few studies have dealt with structural and functional differences
among AM fungi in different land-use systems. The screening and identification, and
multiplication of diverse AMF species occurring over a broad range of land-use
intensity will contribute to meeting the future need for sustainable development.
17.2 Extraction and Propagation of AMF
The obligate symbiotic nature of AM fungi has greatly hindered their use in agricul-
ture, agroforestry, and the commercialization of inocula. Additionally, biodiversity
of AMF species is measured mainly by extracting, counting, and identifying their
field-collected asexual spores containing limited taxonomic characters. To over-
come this limitation, new methodologies have been required that allow studies of
different aspects of life cycle of AM fungi. For this purpose, in the last few decades,
17 Impact of Arbuscular Mycorrhizal Fungi (AMF) in Global Sustainable Environments 421
Table 17.1 A summary of number of AMF species reported in different land-use system
Number of AM Trap culture
Habitats species used References
Bamboo Forest, Taiwan 14 – Wu and Chen (1986)
Old meadow, Quebec 13 – Hamel et al. (1994)
Grassland, North Carolina, USA 23 + Bever et al. (1996)
Tallgrass prairie, Kanas, USA 14 – Bentivenga and Hetrick
(1992)
Sonoran deserts crub, Arizona, 7–-9 + Stutz and Morton
USA (1996)
Sand dune, Rhode Island, USA 6 – Koske and Halvorson
(1981)
Sao Paulo, Brazil 19 – Trufem (1995)
Santa Catarina, Brazil 12 – Stürmer and Bellei
(1994)
Sandy soil, Hel Peninsula, Poland 34 – Blaszkowski (1994)
Native vegetation, Cultivated Soil, 46 – Blaszkowski (1993)
Poland
Native woodland, Florida, USA 10 + Schenck and Kinloch
(1980)
Malus domestica orchards, USA 3–6 + Miller et al. (1995)
Wheat monoculture, Cultivated 24 – Schalamuk et al. (2006)
Amazon forest, Colombia 18 – Peňa-Venega et al.
(2007)
Western Brazilian, Amazon 54 – Stümer and Siqueira
(2011)
Changins, Canton Vaud, 26 + Mathimaran et al.
Switzerland (2005)
Native forest and grassland in 08 Xu et al. (2017)
southeast Tibet
Cr contaminated site of Jajmau, 07 + Akhtar et al. (2017)
Kanpur, India
Modified from Douds and Millner (1999)
a number of reports are available (Schenck 1982; Brundrett et al. 1994; Clapp et al.
1996; Verma and Adholeya 1996) that deal with isolation and enumeration of AM
fungal propagules, propagation of AM propagules in greenhouse pot culture, and
finally, storage of fungal material for germplasm collection, which required thor-
ough understanding of AM fungal life cycle and growth pattern.
17.2.1 Pot-Culture Inoculum
The most intensively used pot-culture technique consisted of growing bait plants in
field-collected soil. It allows the sporulation and multiplication of AM fungal propa-
gules (spores and colonized roots) present in the collected soil sample. The
collected soil sample was frequently diluted with a variety of inert substrates
(Feldman and Idczak 1994; Lilly and Santhanakrishnan 1999), sterilized sand
(Bragaloni et al. 1998; Gaur and Adholeya 2002), vermiculite, or Terra green
(Baltruschat 1987). This propagation approach multiplies AM species of unknown
composition.
17.2.2 Trap Culturing
Field-collected AM spores are found to be low in numbers, parasitized with microbes

and fungi, lacking suitable information for taxonomic characters, and hindering a
more accurate identification. Trapping is necessary to obtain healthy AM spores for
identification as well for using as inoculum to establish monospecific culture. Trap
cultures are helpful in unveiling AM community that is undetected in initial extrac-
tion of spores from field soil (Morton et al. 1995). Beaver et al. (1996) and Koske
et al. (1997) observed that trap cultures tended to encourage preferential sporulation
of some species when different conditions are applied. Miller et al. (1985) recov-
ered 14 AMF species in Sorghum and Coleus trap cultures that were not previously
present in field sampling of apple orchards. Trap culture has been extensively used
to investigate AM biodiversity and isolates of indigenous fungi (Morton et al. 1995).
Beaver et al. (1996) established Sorghum trap cultures and also transplanted intact
plants from the field to microcosms in order to complement their diversity estimates
based on field-collected spores from mown grassland. Successive generation of trap
cultures using the same soil sample often allows the initiation of nonsporulating
dormant species (Stutz and Morton 1996). Establishment of trap cultures greatly
improves the assessment of species composition in an ecosystem, and in some
cases, it can promote the sporulation of cryptic AMF species that were not sporulat-
ing in field conditions (Stürmer 2004). AMF biodiversity studies from 30 sites in
Arizona, USA, were compared to determine the impact of urbanization on AMF
communities by Bills and Stutz (2009). In this study, they used trap cultures for AM
spores propagation collected from the field soil of nonindigenous and indigenous
plants at urban sites and from indigenous plants at desert sites.
17.2.3 Single-Spore Culture
It is well known that pot culture established from multiple spores setup from field
soil may contain more than one species/isolate of AM fungi. Establishment of a
single-spore culture is the same like pot culture but, as their name implies, it is initi-
ated using single spore. Single-spore culture of AM fungi constitutes valuable
resource, not only for plant growth experiment but also for taxonomic and bio-
chemical studies. Several techniques were reported for establishing monosporic
culture from germinated and ungerminated spores by Hepper (1984) and Brundrett
and Juniper (1995). Later, more improved methods for hypha and single-spore
propagation were described by Fracchia et al. (2001). Single-spore culture using
healthy-looking AM spores was collected from pot culture and originated from field
soil usually with high success rate (80%) particularly for ‘aggressive’ species of
Glomeromycota (Walker 1999). However, the success rate for the development of
monosporic culture using field-collected spore is only 1%. This may be due to con-
tamination of (1) fragment of hyphae, (2) other species sporulating inside dead
spores, and (3) production of a culture of a species other than the one thought to
have been used for inoculation.
17.3 Biodiversity of AMF
17.3.1 Subtropical Agricultural Soil
Many soils of tropics are low in soil biodiversity and prone to degradation because
of soil moisture stress, low nutrient capital, high erosion, low pH, high P fixation,
and low amount of soil organic matter (Sanchez et al. 2003; Kour et al. 2019; Rana
et al. 2019a, b). In the last decade, intensive use of inorganic fertilizers and pesti-
cides along with introduction of new high-yielding cultivar had overcome these
constraints (Dalgaard et al. 2003). However, at the same time, decline in quality and
fertility of soils leads to gradual decline in household food production in tropic and
subtropic ecosystems. Several recent studies have demonstrated that AM fungi are
common and ecologically important in tropical ecosystems; and they co-occur with
mixture of plant species; maintain soil fertility; guard against erosion; and fully
utilize soil resources (Alttieri 2004).
Almost all tropical plants have mycorrhizal association. There are 102 AM
fungi reported in diverse tropical habitats from India (Ragupathy and
Mahadevan 1993; Manoharachary et al. 2005). The occurrence of AM fungi in
a forest and also coastal regions of Andhra Pradesh was reported by
Manoharachary and Rao (1991); distribution and identification of AM fungi in
the rhizosphere soils of the tropical plains were done in Tamil Nadu, India, by
Ragupathy and Mahadevan (1993); and from natural forest regions in the Old
Delhi Ridge, Saraswati Range of Haryana, by Thapar and Uniyal (1996).
Diversity of AM fungi has also been studied in the coastal sand dunes of the
west coast of India (Beena et al. 2000); in deciduous forests in Dharwad dis-
trict of Karnataka (Lakshman et al. 2001); in the Western Ghats of Goa (Khade
and Rodrigues 2003); and in coastal saline soils of Kerala, South India,
(Karthikeyan and Selvaraj 2009).
The AM biodiversity in rhizospheric soil of plant Leucaena leucocephala
was studied from the agricultural field of Bangalore and reported by Nalini
et al. (1987). Diversity of AM fungi was studied in the pot-culture setup from
the agricultural field soil of different wheat-growing regions of India by Singh
and Adholeya (2002). A subsequent study by Sunil Kumar and Garampalli
(2010) reported AM fungal diversity in agricultural soils of maize, wheat,

Pigeon pea, and chickpea plants; and soil samples were collected from Gwalior
and Hassan districts, India. AM fungi were collected from the tannery effluent-
polluted soil of Tamil Nadu, India, by Sambandan et al. (1991). Raman et al.
(1993) described and identified Glomus and Gigaspora spp. in the mycorrhizo-
spheres of 14 plant species collected from a magnesite mine spoil in India.
Later studies by Raman and Sambandan (1998), and in soils of Kanpur, Uttar
Pradesh, by Khade and Adholeya (2009), described AM community from tan-
nery-contaminated soils.
17.3.2 Inorganically Managed Agricultural Soil
Arbuscular mycorrhiza is an association between plants and fungus and must be

studied as a dynamic system, not as an individual organism. This dynamic sys-
tem leads to hypotheses for a number of investigations, and also edaphic proper-
ties influence both plants and mycorrhiza diversity. An earlier study by Hayman
(1975) found that crop rotation and fertilizer’s treatment caused changes in spore
population in soil. Abbott and Robson (1991) observed that more of AM com-
munity is in the top 8 cm of zero-tilled soil as compared with tilled soil. Later
studies by McGonigle and Miller (1993) observed that less tillage of soil is better
for mycorrhizal population, and that reduced disturbance of soil with ridge till-
age resulted in more induced mycorrhizal population. Subsequent studies by
Johansen et al. (1993) observed that application of inorganic fertilizers increased
the abundance of specific mycorrhizal fungi, for example, Rhizophagus intrara-
dices and Funneliformis mosseae, whereas, other species like Gigaspora gigan-
tea, Gigaspora margarita, Scutellospora calospora, or Paraglomus occultum
significantly decreased in abundance. In a subsequent report, Treseder et al.
(2004) show that conventional agricultural practices such as application of inor-
ganic fertilizers and tillage tend to decrease AMF spore abundance and alter
community composition. Similar findings by Hijri and Sanders (2005) observed
AMF species diversity to be low in agricultural field and also found that low-
input agricultural land with crop rotation practices induces relatively high AMF
species richness. Jasper et al. (1989) observed that soil disturbance decreases
AM colonization of plants via destruction of AM fungal network. Tillage prac-
tices reduce AMF spore density and species richness (Hendrix et al. 1990; Altieri
1999). Galvez et al. (2001) observed negative impact on AMF spore numbers and
on the density of AMF hyphae in tilled soil. Higher AMF infection potential, as
well as faster development of AMF colonization, was observed in soils under
reduced tillage conditions by McGonigle and Miller (1996). By contrast, Jansa
et al. (2002) found no significant effect of tillage on diversity indices of the AMF
community structure. Jansa et al. (2002, 2003) found that Rhizophagus species is
generally predominantly present in tilled soil. On the contrary, Scutellospora sp.

was observed in minimum-tilled soil.
17.3.3 Organically Managed Agricultural Soil
Arbuscular mycorrhizal fungi are important members of soil microbial community

and can exert beneficial effect by application of low-input organic agricultural prac-
tices. Many agricultural practices can negatively affect AM fungal population while
organic agricultural practices may be the sustainable practices and increase the
number of AM spores in soil. This may be due to increased pore volume of soil
which has a beneficial effect on AM colonization, the mycorrhizal growth response,
and AM spore density (Giovanetti and Avio 1985). The addition of organic matter
also decreases the mechanical soil resistance to the growth of AM hyphae (Joner
and Jakobsen 1995). AM sporulation was found to be enhanced when soil rich in
organic amendments was investigated by Douds et al. (1997) and Johnson and
McGraw (1988). Application of farmyard manure overall reduced AM density in
rhizospheric soils of corn, millet, and sunflowers as reported by Harinikumar and
Bagyaraj (1989); however, other field studies have observed negative effects of
farmyard manure on mycorrhizal colonization (Jensen and Jokobsen 1980).
Subsequent studies by Muthukumar and Udaiyan (2000) found that organically
managed soil improves colonization of roots with mycorrhizal fungus. Additionally,
application of organic matter can have a beneficial effect on the growth of indige-
nous AM fungi in nutrient-limited soils (Caravaca et al. 2002; Gaur and Adholeya
2002). Overall, higher biomass and diversity of soil animals and a higher microbial
activity were noted in organically managed soil (Maider et al. 2002). Recent study
by Gosling et al. (2010) examined 11 sites to test the hypothesis that organic man-
agement increases AM fungal number and colonization potential of tilled agricul-
tural soil. They concluded overall spore number to be significantly higher in
organically managed soil with no overall difference in soil physicochemical proper-
ties. However, it was also found in the study of Eason et al. (1999) that organic
farming not necessarily resulted in large numbers of AM spores in absolute terms.
17.3.4 Industrial Wasteland Soil
Wastewater discharged from textile and dye industries is the major cause of serious
environmental hazards. Many physical and chemical methods have been used to
detoxify the wastewater, but unfortunately, due to high operating costs and complex
operational process, a suitable alternative will be required; also, these methods do
not take away dyes completely. In contrast, introduction of biological organism,
arbuscular mycorrhizal fungi (AMF), has enormous potential to enhance phytoac-
cumulation process of heavy metal by naturally grown plant species in
Identification and
multiplication Field inoculation
under in-Vitro with AM fungi
Mycorrhizal Technology
Functional
diversity of
AMF using
different AM fungal
concentration spore
of heavy
metal Trap
culture/
Single
spore
culture
Fig. 17.2 Mycorrhizal-based technology use as a bioremediation of polluted soil
contaminated soils as shown in Fig. 17.2. However, very limited information is

available about community composition of tolerant mycorrhizal species/strains
associated with heavy metal accumulator plant grown naturally under tropical soil.
Study suggested that consortia of Rhizophagus species from metal polluted soil
contribute to higher uptake and transportation of heavy metals, as well as tolerance
of heavy metals toxicity than single AMF species. This study also suggested differ-
ent AM fungi to differ in their susceptibility and tolerance to heavy metals, and
being responsible for uptake of specific metals only constitutes some limitation. It
was also suggested that roots of some plant are species-specific and sporulate differ-
ently in the presence specific AMF species/strains. Many reports described that the
presence of AMF increases the efficiency of plants for the removal of heavy metals
from toxic environment (Regvar et al. 2003; Turnau and Mesjasz-Przybylowicz
2003). The study by Kumar and Adholeya (2016, 2018) recorded that Rhizophagus
fasciculatus obtained from trap culture originated from sludge-contaminated field
used for the clean-up of multi–metal-contaminated tannery sludge. More recent
report by Nazir and Bareen, (2011) investigated the synergistic effect of Rhizophagus
fasciculatus and Trichoderma pseudokoningii on Heliathus annuus for decontami-
nating toxic metals from tannery sludge. They showed that consortia of AM fungi
could be exploited for decontamination of heavy metals from tannery sludge.
17.4 ife Cycle of AMF, and Economic and Ecological

L
Significance
Arbuscular mycorrhizas are mainly symbiotic with herbaceous plants in temper-

ate areas, but are also found frequently in tropical trees (Smith and Read 2008).
80–90% of terrestrial plant species form association with AM (Smith and Read
2008). In a recent survey of 3617 plant species, Wang and Qiu (2006) showed
that 92% of the families (80% of the species) potentially form at least one mycor-
rhiza type. A very detailed review about mycorrhizal associations was most
recently published (by Brundrett 2009). AM fungi enhance mobilization and
transportation of nutrients to roots (Smith and Read 1997). AM fungi improved
nutrient uptake, especially nitrogen and phosphorus, by increasing the abilities
of the host plants to explore a larger volume of soil than plant roots alone would
have been able to cover, and to mobilize phosphate from a greater surface area
(Joner et al. 2000). It also reduces water stress and improves soil aggregation in
eroded soils (Caravaca et al. 2002). There are three different phases of AM fun-
gal interaction: (1) the asymbiotic phase, when AM fungal spores germinate and
hypha grow in the absence of plant signals; (2) the presymbiotic phase, during
which AM fungal hyphal growth and differentiation occur in the presence of
signal exuded by host roots; and (3) the symbiotic phase, following colonization
of roots, during which there is formation of intraradical structures and an
exchange of nutrients between host and fungus. The formation of AM symbiosis
is a complex developmental event leading to the coordination of gene expression
of both partners. The fungus life cycle starts with germination of hyphae from
resting spores. The spores are able to germinate in the absence of host plants, but
the growth of the hyphae is restricted after days or weeks, depending on the spe-
cies/isolate of AM fungi (Tamasloukht et al. 2003). Germination of AM spores
need not necessarily be in the presence of plant host root, although the percent-
age of germination is sometimes increased in their presence. Gigaspora spores
germinated directly through the spore wall, Acaulospora and Scutellospora
through germination shields, and Glomus (Siqueira et al. 1985) through the
hyphal attachment. On the other hand, germinating spores produce diffusible fac-
tors that lead to an expression of specific genes in the host plant root cells even
in the absence of direct physical contact (Kosuta et al. 2003). Furthermore, sev-
eral reports confirm plant–microbe interaction and show that root exudates also
produce primary plant signal that triggers a complex cascade of signals from
both the plant and the AM fungus (Morandi 1996; Vierheilig et al. 1998). Root
exudates of AM host plants stimulate spore germination (Gianinazzi et al. 1989;
Schreiner and Koide 1993) and hyphal growth (Mosse and Hepper 1975; Graham
1982; Balaji et al. 1995; Pinior et al. 1999). It is known that in the absence of
roots or root exudates, the hyphae have very slow metabolic rates, and all attempts
at long-term culture have failed (Azcón-Aguilar et al. 1999; Giovannetti 2000;
Bécard et al. 2004). The presence of a root or root exudates stimulates growth
and branching of the mycelium and apparently converts it into an ‘infection-

ready’ state.
The chemical nature of diffusible factors of plants and of fungi is not yet
known. First observation by Akiyama et al. (2005) suggested that strigolactones
are known to stimulate the germination of seeds of root parasite in the genera
Striga and Orobanche and their role as general signaling molecule for establish-
ment of AM symbiosis. Furthermore, Besserer et al. (2006) investigated and
observed that strigolactones are responsible not only for spore germination but
also for stimulating the growth of hypha of AMF. Several other phenolic com-
pounds produced by roots or seeds are known to influence symbiotic develop-
ment between Rhizobium and Agrobacterium and their hosts. Ex Flavonoids have
stimulatory effects on the growth and branching of germ tubes of Gigaspora
margarita and some Glomus species (Gianinazzi-Pearson et al. 1989; Tsai and
Phillips 1991; Bécard et al. 1992; Buee et al. 2000) and can also lead to increased
colonization of roots by the fungi (Nair et al. 1991; Siqueira et al. 1991; Akiyama
et al. 2002). Oldroyd and Downie (2004) confirmed rhizobial symbioses, and
AM fungi utilize the same factor (at least seven proteins) of a common signaling
(Sym) pathway in legumes. Therefore, it is likely that different rhizobial and
mycorrhizal signals (Nod factors and Myc factors) result in a common signaling
pathway, whereas the output is unique in both symbioses (Oldroyd and Downie
2006; Kosuta et al. 2008). In AM, Myc factors are thought to induce calcium
oscillations in root epidermal cells (Kosuta et al. 2008) and activate plant symbi-
osis-related genes (Kosuta et al. 2003).
The second step in the root–AM fungus interaction prior to colonization is the
formation of aspersorium on the root surface. Genre et al. (2005) observed that
AM fungi on the root surface involve changes in the plant cells, encompassing
wall alterations, nuclear movements, alterations in cytoskeletal activity, and
membrane proliferation and modification, including the formation of a complex
prepenetration apparatus (PPA). Using a plant mutant of Lotus japonicus affected
in the symbiosis genes SYM15 or SYMRK, Demchenko et al. (2004) found that
the plant actively allows the fungus to penetrate the rhizodermis. They observed
three steps in the interaction that were differentially impaired in the mutants: (1)
the surface opening, where the anticlinal cell walls of two adjacent epidermal
cells separate from each other in the vicinity of fungal hyphae: (2) the intracel-
lular passage of hyphae through an exodermal cell and an adjacent cell of the
outermost cortical layer; and (3) the arbuscule formation in cells of the two
innermost cortical layers. Gallaud (1905) identified two different morphological
growth patterns of AMF on the root surface. First, Arum type is characterized by
fast-growing hyphae spreading through intercellular air spaces and penetrating
cortical cells by side branches, in which arbuscules are formed. Smith and Read
(2008) show that Arum type in particular is typical for fast-growing crops.
Second, Paris type is characterized by hyphae g rowing intracellular from cell to
cell in which coils are formed. This type of growth pattern is found in Gentianaceae
(Sýkorová et al. 2007).
Subsequently, AM fungus forms tree-like structures, called arbuscules, inside

inner cortical cells of host roots. On the other hand, genera belonging to Paraglomus,
Scutellospora, and Gigaspora form intra-and intercellular storage organs called
vesicles in the late stage of the symbiosis (Smith and Read 1997; Morton and
Redecker 2001). Sander et al. (1977) reported that arbuscules in AM fungi are the
central place for nutrient exchange. This structure is degraded by plant cells after 4
to 10 days, and then plant regains its original morphology (Jacquelinet-Jeanmougin
et al. 1987). Consequently, plant cells form new colonization. The life cycle is com-
pleted by the formation of new spores by the AM fungus.
17.5 Role of AMF for Global Sustainable Environments
Climate change and food security have now become major problems mainly in
developing countries. Therefore, there is need to fulfill demand of growing popula-
tion of developing countries by increasing food production through high-input agri-
cultural practices and at the same time by minimizing negative environmental
impact (Foley et al. 2011; Rillig et al. 2016). Holistic use of several beneficial
microorganisms may lead to minimization of environmental pollution and conser-
vation of soil ecosystem. Moreover, many microbes symbiotically associated in soil
life play a role as major pillars in conservation agriculture. Among these symbioses,
a well-known player is mycorrhiza, the extensive symbiotic association of fungi
with roots of higher plant (Smith and Read 2008). AM fungi offer several benefits
to the plants: (1) improved nutrient uptake, (2) faster growth, (3) greater drought
resistance, (4) protection from pathogens, (5) increased seedling survival, (6)
improved soil structure, and (6) greater resistance to invasion by weeds. Colonization
of the root system by AM fungi confers benefits directly to the host plant growth and
development, through the acquisition of phosphate and other mineral nutrients from
the soil. In addition, colonization may also enhance the plant’s resistance to biotic
and abiotic stresses (Newsham et al. 1995). AM fungi also develop an extensive
hyphal network out with the plant root system, which makes a significant contribu-
tion to the improvement of soil texture and water relationship (Bethlenfalvay and
Schuepp 1994). Mycosorption using AM fungi in heavy metal-contaminated soil
showed significantly greater accumulation as compared with plant noncolonized
with AM fungi (Utomo et al. 2014). Therefore, AM fungi constitute an integral and
important component of ecosystems and may have significant applications in sus-
tainable agricultural system (Schreiner and Bethlenfalvay 1995). Current produc-
tion technique of mycorrhizal inocula presents with certain limitations with regard
to purity and quality. Moreover, Cardoso and Kuyper (2006) suggested that side by
side with mycorrhizal technology, mycorrhizal management practices may increase
crop production through intensive use of agroforestry. They also concluded that
sustainable conservation of soil ecosystem may be possible through mycorrhiza-
tion, multi cropping, and crop rotation practices.
17.6 Conclusions and Future Prospect
This chapter suggests that the presence of AMF increases the efficiency of plants for
the removal of heavy metals from toxic environment. Study revealed that plant species
diversity would increase the diversity of AMF in soil and contribute to the efforts to
restore degraded lands. Review dealt with selection and multiplication of AMF from
wasteland sites and the manner in which we can use them for future revegetation pro-
grams. The characterization and identification of AMF adapted from harsh conditions
of soils/area affected by industrial effluents can be put to use for future utilization of
the reclaimed areas for the successful management of revegetation programs.
Acknowledgments The authors wish to thank financial contributions from Department of

Biotechnology, Government of India and The Energy and Resource Institute (TERI), New Delhi,
India. We thank Dr. R K Pachauri, Director General, TERI, for providing the necessary infrastruc-
ture for carrying out the research work.
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Cham
Chapter 18
Fungal Phytoremediation of Heavy Metal-
Contaminated Resources: Current
Scenario and Future Prospects
Amit Kumar, Ashish K. Chaturvedi, Kritika Yadav, K. P. Arunkumar,

Sandeep K. Malyan, P. Raja, Ram Kumar, Shakeel Ahmad Khan,
Krishna Kumar Yadav, Kusam Lata Rana, Divjot Kour, Neelam Yadav,
and Ajar Nath Yadav
18.1 Introduction
Phytoremediation is the technique in which living plants are used for remedia-
tion of the contaminated soils, water, sediment, and ecosystem (Cunningham and
Ow 1996). The utilization of fungus for remediation of the contaminated
A. Kumar
Host Plant Section, Central Muga Eri Research & Training Institute, Central Silk Board,
Lahdoigarh, Jorhat, Assam, India
K. P. Arunkumar
Central Muga Eri Research and Training Institute, Central Silk Board, Jorhat, Assam, India
A. K. Chaturvedi
Water Management (Agriculture) Division, Centre for Water Resources Development and
Management, Kozhikode, Kerala, India
K. Yadav
Department of Botany, Dayalbagh Educational Institute, Agra, Uttar Pradesh, India
S. K. Malyan
Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization
(ARO), The Volcani Center, Rishon LeZion, Israel
P. Raja
ICAR-IISWC, Regional Centre, Ooty, Tamil Nadu, India
R. Kumar · S. A. Khan
Centre for Environment Science and Climate Resilient Agriculture, ICAR-Indian Agricultural
Research Institute, New Delhi, India
K. K. Yadav
Institute of Environment and Development Studies, Bundelkhand University,
Jhansi, Uttar Pradesh, India
K. L. Rana · D. Kour · N. Yadav · A. N. Yadav (*)
Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib,
Sirmour, Himachal Pradesh, India

438 A. Kumar et al.
resources is fungal phytoremediation. Fungi survive about 5300 years (Gams and
Stalpers 1994). Armillaria bulbosa is the longest and largest living fungal spe-
cies in the world (Smith et al. 1992). Fungi play vital role in all ecosystems and
are capable of regulating the nutrient as well as energy flow through their myce-
lial networks, and hence, they are considered as natural and true ecosystem engi-
neers (Lawton and Jones 1995). The ecological and biochemical capacity of
fungi to degrade environmental chemicals and decrease the risk associated with
metals and metalloids through chemical modification or its bioavailability makes
them as a potent bioremediation agent. However, to date, the ecological demands
and ecophysiological strengths of fungi in bioremediation have not been poten-
tially explored. Unlike bacteria, the fungal phytoremediation does not require
absolute water phase as fungus can grow in the air-water interface. However, the
water phase acts as a carrier for nutrient transport for hydrophobic organic
contaminants.
Interaction of fungi with metals includes mobilization and immobilization in
the mycosphere, sorption to cell walls, and uptake into fungal cell. Thereafter,
chemical transformation, translocation, and metabolization along with reactions
of pollutants on fungal enzymes such as extracellular oxidoreductases/cell-bound
enzymes allow fungi to act on various metal pollutants (Harms et al. 2011;
Prakash 2017). Hence, the role of filamentous fungi becomes important where
translocation of essential factors necessitates for the transformation or detoxifica-
tion of environmental chemicals. Conversely, requirement of fungal degradation
is needed for pollutant classes, i.e., dioxins, 2, 4, 6-trinitrotoluene, synthetic
drugs, or endocrine-disrupting chemicals found in medium as these are ineffi-
ciently degraded by bacteria (Harms et al. 2011; Macellaro et al. 2014; Mnif et al.
2011). Fungi can be used in the treatment of contaminated soil surface with
organic/metal contaminants, water streams with trace organic contaminants and
removal of metals from water stream, VOCs from air, and organic pollutants using
isolated extracellular enzymes instead of whole fungi (Nguyen 2015; Pinedo-
Rivilla et al. 2009).
Conversely, an increasing trend toward energy- and cost-efficient passive phy-
toremediation methods for the reclamation of contaminated natural resources,
i.e., land, water, and air is the need of hour. The low degree of mechanical inter-
vention in natural attenuation of natural resources especially soils favors the
importance of filamentous fungi in sustainable fungal phytoremediation (Harms
et al. 2011). Another aspect involves arbuscular mycorrhizal (AM) fungal asso-
ciation with plants, as these are integral, functioning parts in plant roots and
enhance plant growth even under highly contaminated soils with heavy metals.
AM fungi play an important role in metal tolerance and accumulation of heavy
metals in the plants root growing on heavy metal-contaminated soils. Hence, iso-
lation of stress-adapted indigenous AM fungi could be targeted as a potential
biotechnological tool for inoculation of plants for degraded ecosystems. Major
role of AM fungi attributed to the secretion of glomalin (a glycoprotein), stabiliz-
18 Fungal Phytoremediation of Heavy Metal-Contaminated Resources: Current… 439
ing the aluminum in soil and in the roots of Gmelina plants, has been reported
(Dudhane et al. 2012). There are several fungal species such as A. niger, A. pul-
lulans, C. resinae, F. trogii, G. lucidum, Penicillium sp. (Loukidou et al. 2003;
Say et al. 2003), R. arrhizus, and T. versicolor, which efficiently recover heavy
metals from the contaminated environment. Heavy metal bioaccumulation poten-
tial of A. versicolor was observed 6 for 50 mg/L Cr (VI) and Ni (II) and 5 for Cu
(II) ions with the 99.89, 30.05, and 29.06% removal yield, respectively at optimal
pH by Taştan et al. (2010). Kumar Ramasamy et al. (2011) found that Aspergillus
fumigates is very suitable for removal of Pb (II) ions from the electronic waste
aqueous solution (containing Pb 100 mg/L) through batch sorption with adsorp-
tion capacity of 85.41%. El Zeftawy and Mulligan (2011) found that micellar-
enhanced ultrafiltration MEUF could treat phosphorous-rich heavy metal
wastewater with a transmembrane pressure of 69 kPa, at 25 °C and pH 6.9.
Häyrynen et al. (2012) observed that pressure and cross-flow velocity signifi-
cantly affects the flux of Cd and Cu in MEUF purification methods, while P was
not retained (Landaburu-Aguirre et al. 2012). Thus, potential application of
MEUF for heavy metal decontamination of nutrient-rich wastewaters has been
recently justified (Mani and Kumar 2014).
18.2 otential Sources of Heavy Metal Contamination

P
and Associated Risks
18.2.1 Anthropogenic
Based on the relative higher densities (3.5–7.0 g/cm3), atomic weights, or atomic
numbers (>20), metals are termed as heavy metals. Some heavy metals are essen-
tial nutrients (Fe, Co, Zn), relatively harmless (Ru, Ag, and Id), but potentially can
be toxic in larger amounts or certain forms. Conversely, heavy metals, such as Cd,
Hg, and Pb, are highly poisonous. The common source of heavy metals is antisep-
tics, fertilizers, sedimentation, cars, golf clubs, mobile phones, plastics, self-
cleaning ovens, solar panels, and particle accelerators (Gupta et al. 2018; Singh
et al. 2013; Hübner et al. 2010; Singh et al. 2017; Yadav et al. 2018b, c)
(Table 18.1). The potential sources are atmospheric deposition; automobile
exhausts, metal industries, mine spoils, river dredging and urban refuse disposal,
pyrometallurgical industries, and fossil fuel combustion are also the main sources
of heavy metals (Lottermoser 2010a, b; Matta et al. 2018; Prasad 2001)
(Table 18.1). Industries such as microelectronics, plastics, refinery textiles, wood
preservatives, agrochemicals (fertilizers and pesticides), sugar-based industries
and waste disposal sewage sludge, landfill leachate, and fly ash disposal are also
some of the chief sources of the heavy metals (Bhatia et al. 2015; Gupta et al.
2018; Singh et al. 2013a; Kumar et al. 2016; Singh and Kumar 2006; Yadav et al.
2018b, c) (Fig. 18.1).
440 A. Kumar et al.
Table 18.1 Sources of heavy metals and respective anthropogenic activities (adapted and modified
from Yadav et al. 2017)
Heavy metals Anthropogenic activities
Antimony Alloys, Britannia metal, electrical applications, flame-proof pigments and glass,
(Sb) pewter, medicines for parasitic diseases, queen’s metal, semiconductors
Arsenic (As) Geogenic processes, fuel, smelting operations, thermal power plants
Beryllium Alloy, electrical insulators in power transistors, moderator, nuclear power plants
(Be)
Cadmium e-waste, incinerations and fuel combustion, paint sludge, waste batteries, Zn
(Cd) smelting
Chromium Mining, industrial coolants, chromium salt manufacturing, leather tanning
(Cr)
Cobalt (Co) Ceramics, glass industry, metallurgy (in super alloys), paints
Copper (Cu) Mining, electroplating, smelting
Iron (Fe) Alloys, cast iron, construction, machine manufacturing, steel, transportation,
wrought iron
Lead (Pb) Alloys, antiknock agents, cable sheathings, ceramics, glassware, lead-acid
batteries, plastic, ordinance, pigments, solder, tetramethyl lead, pipes
Manganese Alloys, antiknock agents, batteries, catalysts, coating welding rods,
(Mn) ferromanganese steels production, fungicides, pigments, dryers, wood
preservatives
Mercury(Hg) Catalysts, dental fillings, fungicides, electrodes, electrical and thermal
measuring apparatus, metals extraction by amalgamation, mobile cathode
production, mercury vapor lamps, pharmaceuticals, oscillators, scientific
instruments, solders, rectifiers, X-ray tubes
Molybdenum Alloys, cast irons, catalysts, corrosion inhibitors, dyes, electroplating, flame
(Mo) retardants, lubricants, nonferrous metals, smoke
Nickel (Ni) Alloys, arc-welding, catalysts, computer components, electroplating, Ni/Cd
batteries, paint pigments and ceramics, rods, surgical and dental instruments,
ceramic molds, and glass containers
Selenium (Se) Dandruff treatment glass industry, inorganic pigments, lubricants, photoelectric
and photo cells, rubber production, semiconductors, stainless steel, thermo-
elements, and xerographic materials
Stannum (Sn) Brasses, bronzes, catalysts, dental amalgam, pesticides, pewter, stabilizers,
tin-plated steel
Titanium (Ti) Ti as alloy in aeronautics nucleation, catalyst, deep temperature thermometers,
electronics industry, glass ceramics, infrared optical systems, low melting
glasses, semiconductors, supraconductors, UV-filtering agents, white pigments
Vanadium (V) Alloys, catalyst, steel production
Zinc (Zn) Electroplating, smelting
18.2.2 Water Resources
Water contamination due to heavy metals is a known threat and has been attributed
to anthropogenic sources involving untreated domestic and industrial wastewater
discharges, chemical spills, and agricultural residues (Malyan et al. 2014;
Fig. 18.1 Overview of sources of heavy metal pollution and its agroecological consequences.
(Source: Srivastava et al. (2017))
Tchounwou et al. 2012). The outcome is poor water quality, degradation, and water
borne-human health risks even at lower doses of heavy metals (Kumar et al. 2014;
Micó et al. 2006; Wongsasuluk et al. 2014). Major heavy metals such as lead, mer-
cury, chromium, cadmium, copper, and aluminum for water contaminations are
originated through anthropogenic activates and natural incidents like seepage from
rocks, volcanoes, and forest fires. Over a time period, heavy metals enter in the
food chain through water, and there chronic effects could be manifested for many
years and may exert several threats such as mental disorders, pain in joints, gastric
disorders, and even cancer. Human population living near industries are more sus-
ceptible to heavy metal toxicity. Along with that, pregnant women and malnutri-
tioned children are more vulnerable to heavy metal toxicity. Freshwater bodies are
heavily affected by pathogens from untreated wastewater and heavy metals from
mining and industrial release (Caravanos et al. 2016). It has been reported that
more than 80% of the world’s wastewater is released to the environment without
treatment, which is the major cause of nearly 58% diarrheal disease (major cause
of child mortality) (Connor et al. 2017). Hence, it is of utmost importance in the
coming future to mitigate this global threat of water toxicity with proper remedia-
tion measure, and techniques are required for the treatment of water. In that con-
text, fungal phytoremediation serves as an environment-friendly, pocket-friendly,
and reliable technique.
442 A. Kumar et al.
18.3 Role of Heavy Metals in Living Beings
Heavy metals such as chromium (glucose metabolism), cobalt (metabolism), copper

and iron (oxygen and electron transport), zinc (hydroxylation reactions) (Nieboer
and Richardson 1978), manganese and vanadium (enzyme regulation), nickel (cell
growth), and selenium (antioxidant and hormone production) (Emsley 2011) are
important for certain biological processes. Molybdenum (catalysis of redox reac-
tions), cadmium (in marine diatoms), tin (growth in a few species), and tungsten
(metabolic processes of archaea and bacteria) may be required for growth of differ-
ent species (Emsley 2011). A deficiency and excess of any of these above-discussed
heavy metals may impart heavy metal poisoning of living beings (Venugopal and
Luckey 1978). Hence, excess amount of heavy metals could dysfunction various
physiological and biological effects in the human beings which have been elabo-
rated in next sections.
18.4 Possible Impacts of Heavy Metal Contaminations
18.4.1 On Humans
Non-essential metals can escape control mechanisms such as binding to specified

cell constituents, cellular processes malfunctioning, compartmentalization, homeo-
stasis, oxidative deterioration, and transport, and therefore, they have toxic and fur-
ther lethal effects (Gupta et al. 2018). The important health symptoms of heavy
metal toxicity in human are central nervous system disorders, dementia in adults,
emotional instability, insomnia, intellectual disability in children, kidney diseases,
liver diseases, depression, vision disturbances, and increased morbidity and mortal-
ity rate (Jain et al. 2015; Yadav et al. 2018b, c). The metal toxicity depends on the
generation of oxidative stress (increased reactive oxygen species (ROS) and reactive
nitrogen species (RNS) production; depletion of intracellular antioxidant stores and
free radical scavengers) (Jan et al. 2015). Heavy metals toxicity due to occupational
exposure mainly responsible for multiple organ systems and toxicity levels mainly
depends on the form and type of the heavy element, on route and duration of the
exposure, and, to a greater extent, on a person’s individual susceptibility (Jan et al.
2015) (Fig. 18.2).
18.4.2 On Plants
Heavy metal contamination in soil and water resources affects growth and yield per-
formance as well as nutritional quality of plants to a great extent. For the plants
which are grown in close vicinity to the contaminated soil and water or at the con-
taminated site, metals cause physiological dysfunctioning and biochemical
Fig. 18.2 Trophic transfer of toxic HMs from soil to plants to humans and organism’s food to
humans and their toxicity. (Adapted with permission from Saxena et al. (2019))
alterations (Sharma et al. 2012a; 2012b). In case of vegetables requiring high mois-
ture percentage, the use of heavy metal-contaminated irrigation water is one of the
major causes for high metal toxicity in plants. Some of the heavy metals at a lower
concentration are required for optimum performance of plants; however, excess
amount may cause toxicity, e.g., chromium (Yadav et al. 2018b, c). Common fea-
tures pertaining to metal toxicity are reduced biomass reduction, leaf chlorosis, and
root growth and seed germination inhibition (Ghani 2011). Cr toxicity considerably
affects the physio-biochemical processes in barley, cauliflower, citrullus maize,
wheat, and vegetables (Ghani 2011). ROS signalling and oxidative damage affect
enzymes like catalase; cytochrome oxidase and peroxidase with iron as their compo-
nent are affected by chromium toxicity. The catalase activity stimulated with an
excess supply of chromium-inducing toxicity has been studied, concerning nitrate
reductase activity, photosynthesis, photosynthetic pigments, and protein content in
algae (Nath et al. 2008). Pb and Cd also affect the gas exchange attributes, ROS sys-
tem, cause chlorophyll deterioration, and ultimately the overall performance of major
agricultural crop worldwide (Anjum et al. 2015; Mobin and Khan 2007; Pinho and
Ladeiro 2012; Zhu et al. 2007). The microbes are ubiquitous in nature and have been
reported from diverse sources including extreme habitats (Yadav et al. 2015a, b, c,
2017b) and as plant microbiomes (Kour et al. 2019b; Yadav 2018; Yadav et al. 2016).
These microbes have potential applications in agriculture, industry, pharmaceutical,
and environment (Kour et al. 2019a; Yadav et al. 2017a, 2018a, 2019a, b).
444 A. Kumar et al.
18.5 hytoremediation of Contaminated Soils/Water

P
Resources
In general, phytoremediation is the process of bioremediation using plant species

called hyperaccumulators to reduce the toxic contaminants in the environment. This
is a novel advanced technology, considered as eco-friendly having lesser investment
cost. Current scenario explains the feasibility and accountability of this technique.
Many plant species are being used as hyperaccumulators, and new species are being
explored (Ali et al. 2013). Eventually, phytoremediation is an interdisciplinary
branch that requires knowledge for soil composition, soil microbiology and envi-
ronment engineering, plant physiological processes, and in recent development use
of lower plant groups as a sustainable system for the bioremediations of toxic heavy
metals (Pisani et al. 2011). Some of the species of the plants used in phytoremedia-
tion are Robinia pseudoacacia and Sesbania drummondii for Pb, Stanleya pinnata
for Se, etc. (Yang et al. 2016).
18.6 Fungal Phytoremediation
As its name explains, fungal phytoremediation or mycoremediation is a form of

bioremediation where the degradative abilities of fungi are utilized to remove or
neutralize the harmful contaminants present in soil and water. It is a relatively new
form of bioremediation where its use only spans a few decades, beginning as early
as 1966 (Matsumura and Boush 1966), but it is known or being practiced to a lesser
extent. Malathion (an insecticide and neurotoxin) breakdown was successfully done
using Trichoderma viride and Pseudomonas (Matsumura and Boush 1966). There
are several mushroom species identified till date to remove the heavy metals from
the contaminated resources. The important species are Galerina vittiformis (Cu, Cd,
Cr, Pb, and Zn), Hypholoma capnoides (Ti, Sr, and Mn), and Marasmius oreades
(bismuth and titanium). The other important fungal species which are having high
fungal phytoremediation potentials are Agaricus bisporus, Lentinus squarrosulus,
Phanerochaete chrysosporium, Pleurotus ostreatus, Pleurotus tuber-regium, P.
ostreatus, P. pulmonarius, and Trametes versicolor (Adenipekun and Lawal 2012;
D’Annibale et al. 2005).
In this chapter, the sources of different heavy metals (HMs) with adverse effects
in major countries on human health along with the permissible limits of HMs has
been highlighted to have the understanding on the current scenario of fungal
phytoremediation works (Table 18.2). Similarly, the different groups of fungus hav-
ing remediation potential for the most potent heavy metals have been highlighted in
Table 18.3. Further, the categorical classification of different fungus and their
importance in particular metal have been worked out with extensive literature sur-
vey in order to target potential fungal phytoremediation techniques for the metal
Table 18.2 Sources of heavy metals (HMs) and their target organs in human with adverse effects in major countries along with the permissible limits of HMs
18
HMs limitation
Major world mine in (ppm)
HMs Sources Target organs Harmful effects countries EPA WHO
As Pesticides and wood Pulmonary, As (especially as arsenate) is a phosphate analog China, Chile, Morocco, 0.10 –
preservatives nervous system, which interferes with oxidative phosphorylation and Russian Federation
skin ATP synthesis
Cd Paints and pigments, plastic Renal, skeletal Cd is carcinogenic, mutagenic, teratogenic, and China, Korea, Japan, 5.0 0.05
stabilizers, electroplating, pulmonary endocrine disruptor; Cd interferes with calcium Mexico, Canada
incineration of Cd-containing regulation in livings; renal failure and chronic
plastics, phosphate fertilizers anemia
Cr Tanneries, steel industries, fly Pulmonary Cr causes hair loss, nephritis, cancer, and ulceration South Africa, – 0.02
ash in humans Kazakhstan, India,
Turkey, Russian
Federation
Cu Pesticides, fertilizers Liver, kidney, Elevated Cu levels may cause brain and kidney Chile, China, Peru, 1.3 2.0
blood damage, liver cirrhosis and chronic anemia, stomach Australia, the United
and intestinal irritation States
Hg Release from Au-Ag mining Nervous system, Anxiety, autoimmune diseases, depression, China, Kyrgyzstan, 2.0 –
and coal combustion, medical renal balancing difficulty, drowsiness, fatigue, hair loss, Chile, Russian
waste insomnia, irritability, memory loss, recurrent Federation
infections, restlessness, vision disturbances,
tremors, temper outbursts, ulcers and damage to
brain, kidney and lungs
Ni Industrial effluents, kitchen Pulmonary, skin Allergic dermatitis known as nickel itch; inhalation Philippines, Russia, – 0.2
appliances, surgical can cause cancer of the lungs, nose, and sinuses; Brazil, Indonesia,
instruments, steel alloys, cancers of the throat and stomach have also been Canada, Russia
Fungal Phytoremediation of Heavy Metal-Contaminated Resources: Current…
automobile batteries attributed to its inhalation; hematotoxic,

immunotoxic, neurotoxic, genotoxic, reproductive
toxic, pulmonary toxic, nephrotoxic, and
hepatotoxic; causes hair loss
445
(continued)
446
HMs limitation
Major world mine in (ppm)
HMs Sources Target organs Harmful effects countries EPA WHO
Pb Aerial emission from Nervous system, Impaired development in children, reduced China, Australia, the 15 0.01
combustion of leaded petrol, hema-topoietic intelligence, loss of short-term memory, learning United States, Peru,
battery manufacture, system, renal disabilities and coordination problems; causes renal Mexico
herbicides and insecticides failure; increased risk for development of
cardiovascular disease
Zn Fertilizers Brain, respiratory Over dosage can cause dizziness and fatigue China, Australia, Peru, 0.5 –
tract India, the United States
Mn Industrial dust and fumes Nervous system Central and peripheral neuropathies South Africa, Australia, – –
China
Sources: Ali et al. (2013); Mahurpawar (2015); Yadav et al., 2017; Gupta et al. 2018; Rajendran et al., 2003; USGS, 2012
A. Kumar et al.
Table 18.3 Categorical classification of fungal species targeting metal remediates for fungal
phytoremediation
Species Metals remediate References
Agaricus bisporus Ni, Cu, Pb, Mn, Cd, Nagy et al. (2014)
Zn, Hg, Fe
Agaricus bitorquis Cu, Zn, Fe, Cd, Pb, Ni, Lamrood and Ralegankar
(2013)
Alternaria alternata Cd, Cr, Cu, Ni Seshikala and Charya (2012)
Armillaria mellea Ni, Cu, Pb, Mn, Cd, Zn Ita et al. (2008)
Ascochyta betae Cr Seshikala and Charya (2012)
Aspergillus fumigatus Cu, Cd, Ni, Co, Pb Rao et al. (2005)
Aspergillus flavus Zn, Cu, Ni, Pb Thippeswamy et al. (2012a)
Aspergillus foetidus Cr Prasenjit and Sumathi (2005)
Aspergillus fumigates Pb Kumar Ramasamy et al. (2011)
Aspergillus niger Cd, Pb, Zn, Cu, Ni, Cr, Pal et al. (2010)
Aspergillus ochraceus Cr Seshikala and Charya (2012)
Aspergillus oryzae Cr Nasseri et al. (2002)
Aspergillus terreus Pb, Cu, Ni, Cr Seshikala and Charya (2012)
Aspergillus versicolor Cr, Ni, Cu Taştan et al. (2010)
Aspergillus versicolor Pb Çabuk et al. (2005)
Candida tropicalis Zn Akhtar et al. (2008)
Candida utilis Cr Pattanapipitpaisal et al. (2001)
Circinella sp. Ni Alpat et al. (2010)
Cladonia rangiformis (lichen) Pb Ekmekyapar et al. (2012)
Cladosporium resinae Cu Gadd and de Rome (1988)
Cunninghamella echinulata Pb, Ni, Zn Shouaib et al. (2011)
Curvularia lunata Cu, Cr, Cd Seshikala and Charya (2012)
Drechslera rostrata Cr Seshikala and Charya (2012)
Fusarium oxysporum Cr Amatussalam et al. (2011)
Fusarium solani Cr, Zn, Ni Sen and Dastidar (2011)
Ganoderma lucidum Cu Muraleedharan et al. (1995)
Ganoderma lucidum, Penicillium sp. Ar Loukidou et al. (2003)
Gliocladium sp. Cu Tahir (2012)
Lactarius piperatus Cd Nagy et al. (2014)
Lentinus edodes Cd, Pb, Cr Tu and Huang (2005)
Metarhizium anisopliae Pb Çabuk et al. (2005)
Mucor hiemalis Cd, Cu Srivastava and Hasan (2011)
Mucor rouxii Pb, Cd, Ni, Zn Majumdar et al. (2010)
Mucor sp. Cu Tahir (2012)
Neurospora crassa Pb, Cu Kiran et al. (2005)
Penicillium canescens Cr Say et al. (2003)
Penicillium canescens As, Pb, Cd, Hg Say et al. (2003)
Penicillium chrysogenum Cu, Ni, U, Cr, Th, Zn, Tan and Cheng (2003)
Cd, Pb
(continued)
448 A. Kumar et al.

Species Metals remediate References
Penicillium cyclopium Cu Ianis et al. (2006)
Penicillium decumbens Cd, Ni, Cr Levinskaite (2001)
Penicillium digitatum Cd, Cu, Pb Galun et al. (1987)
Penicillium notatum Cr Seshikala and Charya (2012)
Penicillium purpurogenum Cr Say et al. (2003)
Penicillium verrucosum Pb Çabuk et al. (2005)
Phanerochaete chrysosporium Cu, Ni, Cd, Pb, Zn, Mamun et al. (2011)
Mn, Fe
Pleurotus florida Cu, Zn, Ni, Pb Prasad et al. (2013)
Pleurotus floridianus Cu, Zn, Pb, Cd, Fe, Ni Lamrood and Ralegankar
(2013)
Pleurotus ostreatus Pb, Ni, Cu, Zn, Cu, Cr, Arbanah et al. (2012)
Mn
Pleurotus sajorcaju Pb, Cd, Cu, Hg, Zn, Fe Arıca et al. (2003)
Pleurotus sapidus Ni, Cu, Pb, Cd, Mn, Zn Ita et al. (2008)
Polyporus frondosus Ni, Cu, Pb, Cd, Mn, Zn Ita et al. (2008)
Polyporus sulphureus Ni, Cu, Pb, Cd, Mn, Zn Ita et al. (2008)
Pyrenochaeta cajani Cr Seshikala and Charya (2012)
Rhizoctonia solani Cr Seshikala and Charya (2012)
Rhizopus arrhizus Ni, Zn, Cd, Pb Fourest and Roux (1992)
Rhizopus arrhizus Pb, Cr, Cd, Cu, Zn, Ni Prakasham et al. (1999)
Rhizopus cohnii Cd Luo and Xiao (2010)
Rhizopus nigricans Cr, Pb, Zn Bai and Abraham (2001)
Rhizopus sp. Cu, Cd Tahir (2012)
Saccharomyces cerevisiae Cd, Ni, Pb, Cr, Zn, Cu Thippeswamy et al. (2012b)
Serpula himantioides As Adeyemi (2009)
Species of Aspergillus, Mucor, Cd, Cu, Fe Fulekar et al. (2012)
Penicillium, and Rhizopus
Trichosporon cutaneum Cr Bajgai et al. (2012)
Volvariella diplasia Cu, Cd, Pb, Ni Lamrood and Ralegankar
(2013)
Volvariella volvacea Cu, Zn, Pb, Cd, Ni, Fe Lamrood and Ralegankar
(2013)
Sources: Adapted and modified from Archana and Jaitly (2015)
contamination in soil. In addition to that for the mechanistic understanding on

growth conditions, enzyme production, type of compound degradation has been
explored (Table 18.4). The bioconversion efficiency of wastes by some fungal species
has been reported worldwide (Table 18.5).
Table 18.4 Some commonly used fungal species, for mechanistic understanding on growth
conditions, enzyme production, type of compound degradation, and key references
Growth condition
Fungal species required Enzymes produced Compound degraded Reference
Phanerochaete Lignin peroxidases Xenobiotic Paszczynski
chrysosporium and manganese compounds and Crawford
peroxidases (1995)
Aspergillus Grows best in Laccase Removing Ghosh and
flavus cereals nuts surfactants and dyes Ghosh (2018)
legumes
Bjerkandera Commonly grows Lignin peroxidases Xenobiotic Rhodes (2014)
adusta on dead wood compounds
Fusarium Grows in desert, Endoglucanase Degrades silver Danesh et al.
oxysporum temperate, and (2013)
tropical, soils of
tundra
Rhizopus Arises from Lipases Heavy metals like Fourest and
arrhizus nodes where Ni, Zn, Cd, Pb Roux (1992)
rhizoids are borne Also remediated
uranium- and
thorium-affected soil
Table 18.5 Bioconversion of waste by fungal species

Bioconversion of
Fungal species waste Remarks References
Pleurotus Handmade paper and Successfully cultivated. Kulshreshtha et al.
citrinopileatus cardboard industrial Basidiocarps possessed good (2013)
waste nutrient content and no
genotoxicity
Aspergillus niger Waste office paper to Used turbine blade reactor Ikeda et al. (2006)
gluconic acid and production increased to
four times in the presence of
oxygen than air
Lentinus edodes Biodecoloration Decoloring reactive dye Vinciguerra et al.
(1995)
Beauveria bassiana Production of Utilized prawn chitinous Suresh and
chitinase enzyme waste Chandrasekaran
(1998)
Phlebia radiata Production of ethanol Successfully produced Mäkinen et al.
biocompound and biofuels at (2018)
low cost
Aspergillus flavus Production of laccase Potential candidate for the Ghosh and Ghosh
enzyme production of lactase, used in (2018)
bioremediation and bleaching
of dyes, etc.
Monascus purpureus Production of Successfully produced by Kantifedaki et al.
and Penicillium biobased pigments semisolid fermentation and (2018)
purpurogenum submerged fermentation
technique
450 A. Kumar et al.
18.6.1 Mechanistic Approach of Fungal Phytoremediation
In fungal phytoremediation, mechanism of fungal partner is very important to

understand. Fungal phytoremediation has got several mechanistic pathways for bio-
remediation process. In general, fungus increases the ability of roots to absorb more
heavy metals. Its mechanism could be devised as (i) avoidance and (ii) sequestration
mechanisms. Avoidance ameliorates the metal toxicity though decreasing the con-
centration of metal by biosorption, precipitation, and uptake or efflux. Conversely,
sequestration involves the formation of compounds for intracellular chelation (−)
and further dilution in plant tissues due to plant growth, exclusion from uptake
through precipitation, and chelation in the rhizosphere (Danesh et al. 2013). Both of
these mechanisms may play part or even could counteract. Overall, the reduction in
absorption owing to retention and immobilization takes part in fungal structures or
mycorrhizal roots. The activation of specific/nonspecific transporters and pores play
the part in the plasma membrane in plants and fungi, chelation in the cytosol and the
sequestration into the vacuoles of plants as well as in fungal cells. Further, transpor-
tation and exportation occur through the fungal hyphae, involving both active and
passive transportation into the mycorrhizae (Fig. 18.3).
Fungal phytoremediation is proven to be efficient, where the abilities of hyperac-
cumulators diminished. One of the limitations of hyperaccumulators is to accumulate
Fig. 18.3 Mechanisms involved in remediation of HM-contaminated soil by HMT-PGP microbes-

plant interaction. (Sources: Mishra et al. (2017))
less concentration of contaminants due to their small biomass while fungi can accumu-
late more due to their some molecular mechanisms. Hence, intervening the interaction
of hyperaccumulator plant with fungi and other legume plant and herbs could help us
to use it as a potent strategy for phytoremediation (Yang et al. 2016). Therefore, further
exercise is required for explaining the molecular mechanisms underlying.
18.6.2 Factors Influencing the Fungal Phytoremediation
Several factors influencing the fungal phytoremediation include species of plant and
fungi, their association strength, plant-soil interaction, physical and chemical proper-
ties of soil, and biophysical aspects such as temperature, pH, salinity, soil microbes,
and metal characteristics (Fig. 18.4).
18.6.2.1 Temperature
The fungi are having their different temperature range for growth based on different
habitat, such as mesophilic (5–35 °C), psychrophilic (below °C), thermophilic
(above 40 °C), etc. With the change in the temperature, the bioavailability of the
Plant type, Translocation Resistance to

Plant Metal and Growth rate
species and and disease and
characteristics salt tolerance and biomass
varieties accumulation pests
Climate
characteristics Humidity Temperature Air Rainfall Sunlight
Microbial
Rootzone Root Redox activity
Exudation pH
characteristics depth potential
Medium Soil Soil Composition Organic

Salinity
characteristics type pH and texture matter
Amount Cation
Chelator Chelation Time of
of Toxicity exchange
characteristics application
chelator capacity
Concentration
Pollutant Nature of
of metal Bioavailability
characteristics pollutants
pollutants
Speciation
Chemical
Phytoremediation and
structure
toxicity
Fig. 18.4 Relationships among the factors affecting phytoremediation efficiency. (Adapted with
permission from Saxena et al. (2019))
452 A. Kumar et al.
heavy metals is also changed. An increase in soil temperature tends to speed up the
concentration of metals in the soil due to increase rate of organic matter degrada-
tion. It was observed that high temperature is favorable for the absorption of heavy
metals. However, the temperature also affects the growth of fungi. So, fungi with
high temperature tolerance will be beneficial for the bioremediation process (Yadav
et al. 2018b, c). Fe and Mn are mobile in alternating in dry and wet conditions
(Boisselet 2012).
18.6.2.2 pH
pH is an important parameter which controls the availability of heavy metals to get

remediated. Heavy metals are present in a dissolved state if the pH of the solution is
at 2–3. However, the bioavailability, dissolution, and precipitation of each metal
have its own intrinsic capacity along with the pH range.
18.6.2.3 Redox Potential
The redox potential affects the state of oxidation of the metals, as different forms
show different behaviors in solubility. Anaerobic conditions in deeper parts of the
soil for oxidoreductive reactions of microorganisms can accelerate the heavy metal
degradation. Redox potential along with pH affects the fungal-phyto interactions
with the soil components by altering the sorption capacity and influencing stability
of complexes.
18.6.2.4 Heavy Metals Bound with Hydrocarbon
Some of the heavy metals are present in the bound form of the other compounds
such as polycyclic aromatic hydrocarbons (PAH). The remediation of such metals
can be achieved only after degradation of the host compound. Some fungal species
such as Agaricus bisporus, Pleurotus ostreatus, and Ganoderma lucidum are
observed to degrade the hydrocarbons in petroleum. Pleurotus ostreatus is benefi-
cial in degrading the PAH (García-Delgado et al. 2015).
18.6.2.5 Other Growth Requirements
Apart from the temperature, other factors such as moisture percentage, sugar and
other organic materials, oxygen, amino acids, vitamins, fatty acids, etc. are also
important for fungal growth. The change in these requirements can also enhance/
limit the fungal phytoremediation potential.
18.6.2.6 Fungal Species
Different fungal species are having different capacity to remediate the heavy met-
als from the soil and water based on their internal genetic constitutes and external
growth and environmental factors. To check the any new/existing species reme-
diation potential, the arsenic test (preliminary assessment) will serve as good
choice. Later on, the heavy metals-based potential check can be made and com-
pared with the existing data. However, some of the fungal species can serve as a
bioindicator of particular heavy metals. In this case, these species serve as the
reference species for the remediation potential. For example, Lycoperdon perla-
tum may be employed as a bioindicator of heavy metals and selenium in soil pol-
lution (Quinche 1990).
Filamentous fungi are known to possess higher adsorption capacities for heavy
metal removal (Singh and Gauba 2014). Trichoderma and Mortierella species iso-
lated from the soil and Aspergillus and Penicillium species isolated from marine
and terrestrial environments, respectively, have the high ability to remediate con-
taminated environment (Thenmozhi et al. 2013). Arbuscular mycorrhizal fungus
Glomus mosseae formed a symbiotic associate of P. vittata L. and possessed sub-
stantial resistance to arsenic toxicity by increasing the plant biomass, and this
mycorrhiza can enhance the arsenic sink. Mycorrhiza can be a potential tool for
fungal phytoremediation by choosing the native species of fungi/host and altera-
tion in the a ssociation by changing any of the fungi/host or controlling factors or
inoculation of the new fungal strains. This can be achieved through re-vegetation
on the contaminated sites such as mine areas.
18.7 Precaution Prerequisite
Some prerequisite precautions are needed for successful achievement of fungal phy-
toremediation which involves selection of correct fungal species for targeted metal
contamination for developing a screening protocol (Matsubara et al. 2006). Among
these precautions, major points have been prescribed in general which should be
considered. These involve as follows:
• The catabolic activity and capacity of organisms involved to transform the target
compound(s) and bring the concentrations to levels that meet regulatory
standards
• The rate of bioremediation
• The possible production of toxic by-products at dangerous levels during the
remediation process
• Adaptability of the process to site conditions (environmental and
anthropogenic)
• Economic viability of the process
454 A. Kumar et al.
As explained above, fungal phytoremediation is a very potent technology for sus-

tainable bioremediation of contaminated soils and water. In general, it is still in
infancy in laboratory conditions and greenhouse, which limits the outcome to the
actual field condition pertaining to multiple factors. Hence, the assessment of the
efficiency rate of fungal phytoremediation must be tested in field condition in order
to commercialize this green technology by evaluating the different plant for targeted
heavy metal. Similarly, there are few lags in this eco-friendly remediation technol-
ogy such as to increase the growth rate of plants, increase the biomass of such plants
for maximum absorption of heavy metals, and take a look on possible hazards on
food chain. Field experiments should be devised to explore the hyperaccumulators
from where these metals can be harvested easily and feasible techniques to harvest
these metals without exerting a negative impact on environment. Also, the key to
mycoremediation is determining the right fungal species to target a specific pollut-
ant. Desirable traits should be identified from the hyperaccumulator and fungi
genome. Such gene can be selected by the conventional techniques or new technolo-
gies of hybridization such as protoplast fusion. Identification of genes coding for
different toxicants from different hyperaccumulators and their transformation in
same plant can develop SUPERBUG plant for phytoremediation. Besides the sev-
eral constraints and limitations, fungal phytoremediation appears to be the most
potent, eco-friendly, economical, and environmentally attractive option of bioreme-
diation in heavy metal-contaminated soils and water resources. Many fungal species
can grow under various contaminated conditions, thus enabling remediation in the
contaminated environment that may not be suitable for other organisms. Based on
our review of the subject and key questions raised on the concerned topic, we do not
conclude that it could solve the issues of metal or hydrocarbon contamination com-
pletely. Conversely, a synergistic approach involving proactive policy designing in
the field of fungal phytoremediation ranging from lab-based desirable trait based
targeted metal contaminations with a particular fungal species, after testing it in
green houses it has a potential to be replicated in the field environment for the future
safety of soil, plant, water resources and rising human population prone to future
heavy metal contaminations.
Acknowledgments The authors are grateful to Prof. Harcharan Singh Dhaliwal, Vice Chancellor,
Eternal University, Baru Sahib, Himachal Pradesh, India, for providing infrastructural facilities
and constant encouragement.
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Chapter 19
Fungal Enzymes for Bioremediation
of Xenobiotic Compounds
Peter Baker, Araven Tiroumalechetty, and Rajinikanth Mohan
19.1 Introduction
Xenobiotics are natural or synthetic organic compounds foreign to an organism that

are potentially toxic and entail negative ecological or physiological consequences,
in the form of pollution and disease, respectively (Olicon-Hernandez et al. 2017).
Industry and agriculture are two major sources of environmentally prevalent xeno-
biotics which include fertilizers, insecticides, pesticides, dyes, plastics, and hydro-
carbon derivatives (Sharma et al. 2018). Chemically, the vast majority of these
compounds are aromatic with one or more frequently substituted phenyl functional
groups. Many of these xenobiotic compounds are of major health and ecological
concern as they are frequently carcinogenic and teratogenic, thereby disrupting
development and reproductive capabilities in humans, fish, fish-eating birds, and
other animals. A serious environmental and health issue is the accumulation of per-
sistent, toxic chemical pollutants requiring new cost-effective and efficient ways to
tackle the growing threat of environmental toxicity in the modernized world.
The majority of xenobiotic compounds can be decomposed or modified by
microbes (Cameron et al. 2000). Bioremediation utilizes the metabolic potential of
biological organisms to degrade or transform hazardous compounds in the environ-
ment into less toxic or nontoxic forms (Watanabe 2001; Yadav et al. 2019a, b). In
particular, the use of fungi, referred to as mycoremediation, has attained widespread
Peter Baker and Araven Tiroumalechetty contributed equally with all other contributors.
P. Baker · A. Tiroumalechetty
Department of Biology, Colgate University, Hamilton, NY, USA
R. Mohan (*)
Department of Biology, Colgate University, Hamilton, NY, USA
Department of Biology, Mercyhurst University, Erie, PA, USA
e-mail: rmohan@colgate.edu

464 P. Baker et al.
attention. Although bacteria are also versatile decomposers of xenobiotic pollutants,

certain fungal strains are able to tolerate higher levels of pollutants. In some cases,
such as the degradation of polyaromatic hydrocarbons (PAHs), bacteria can metab-
olize them and utilize them as carbon and energy sources, but they cannot mineral-
ize them completely the way fungi can (Mougin et al. 2009). Fungi are capable of
metabolizing a wider range of pollutants than their prokaryotic counterparts due to
both extra- and intracellular degradation mechanisms and the powerful, nonspe-
cific nature of the enzymes involved in both processes (Christian et al. 2005a).
Additionally, the enzymes in question are tolerant of an active under diverse condi-
tions including broad pH ranges, making fungi and their enzymes desirable for
bioremediation (Verma and Madamwar 2002) (Rastegari et al. 2019; Yadav et al.
2017, 2018).
Filamentous fungi, particularly from the group called white rot fungi and mainly
from Basidiomycetes, demonstrate a striking ability for oxidative decomposition of
lignin, a recalcitrant component of wood composed of polyphenols (Mougin et al.
2009). These enzymes were evolved by such fungi to assist in the breakdown and
detoxification of potentially hazardous by-products resulting from the decomposi-
tion of wood and other organic matter (Morel et al. 2013). The presence of these
enzymes in many species of white rot fungi has been confirmed using comparative
genomics to identify the so-called xenome, which consists of genes involved in
xenobiotic detoxification. In the last two decades, these enzymes have been exten-
sively adapted for bioremediation of pollutants in a low-cost, eco-friendly manner.
Bioremediation involves environments toxic to the survival of biological sys-
tems and despite fungal robustness and innate degradation infrastructure, there is a
limit to the organisms’ tolerance of xenobiotic-induced environmental toxicity.
Frequently, the polluted areas are too nutrient-poor to support microbial growth.
Moreover, the fungi capable of detoxifying a pollutant may be sensitive to other
pollutants in the environment (Mougin et al. 2009). To circumvent these problems,
one option is to utilize isolated fungal enzymes or enzyme mixtures. The use of
fungal enzymes rather than complete fungal populations is also a more eco-friendly
approach in contrast to using live microbes as it allows for the mitigation of any
adverse environmental impact resulting from the introduction of novel species
(Sharma et al. 2018; Kour et al. 2019; Rana et al. 2019a, b). This is especially
desirable as release of genetically modified fungi or other organisms is contingent
on both acceptance by regulatory bodies like EPA and the general public (Ang
et al. 2005).
The most prominent groups of enzymes utilized in xenobiotic bioremediation
transformations are oxidoreductases: peroxidases, laccases, and oxygenases
(Sharma et al. 2018). Oxidoreductases can detoxify compounds by catalyzing oxi-
dative coupling reactions using oxidizing agents to support the reactions. Laccases
(LACs) and CYP monooxygenases (P450s) use molecular oxygen as the electron
acceptor, while peroxidases use hydrogen peroxide to oxidize the substrates; both
reactions result in the formation of water as a by-product. Peroxidases are heme-
containing proteins, and the major types of peroxidases involved in detoxification
processes in fungi are manganese peroxidase (MnP), lignin peroxidase (LiP), and
versatile peroxidases (VP) (Doddapaneni et al. 2005). Xenobiotic detoxification
19 Fungal Enzymes for Bioremediation of Xenobiotic Compounds 465
enzymes are produced by a great diversity of fungi, but many are often produced by
a large physiological group called the white rot fungi. These fungi actively degrade
lignin – a large, complex, aromatic-containing networked polymer, creating a
bleached appearance in the host, hence the name white rot fungi (Pointing 2001).
Given the complexity of lignin and the structural motifs common to both lignin and
various xenobiotics, these enzymes are well suited for xenobiotic detoxification
(Fig. 19.1). A few genera of fungi are used extensively in bioremediation processes;
these include Trametes, Pleurotus, Phanerochaete spp., etc.
In this chapter, we will review the utility of fungal enzymes in bioremediation
processes. In particular, we focus on the detoxification of organic xenobiotics by
oxidoreductase enzymes including the extracellular peroxidases and laccases as
well as the intracellular CYP450.
19.2 Sources of Xenobiotic Pollutants
Synthetic dyes released from paper, textile, plastic, cosmetic, food, and drug indus-
tries can be toxic and carcinogenic (Asgher et al. 2008; Levin et al. 2005). Polycyclic
aromatic hydrocarbons (PAHs) like anthracene, pyrene, benzopyrene, and naphtha-
lene are toxic, potentially carcinogenic xenobiotics that are produced from fuel
combustion, gas plants, and industrial applications. Endocrine disrupting chemicals
(EDCs) include alkylphenols such as nonylphenol and octylphenol as well as biphe-
nyls such as bisphenol A (BPA), stilbene, and genistein estrogens. These are phar-
maceutically active compounds that disrupt endocrine homeostasis in animals and
are of grave concern to human health (Asgher et al. 2008). Other sources of xenobi-
otics include bleach plant effluents from paper and pulp industry, which contain
toxic polychlorinated phenols (PCPs) and other organic compounds used for bleach-
ing, and include dyes like azo dye and crystal violet (D'Souza et al. 2006).
Chlorinated aromatic compounds like dichloro- and trichlorophenol and derivatives
such as DDT, chlordane, and lindane are used in pesticides.
19.3 Metabolism of Xenobiotic Pollutants by Fungi
Bioremediation through fungi may be achieved by direct metabolism, whereby a

fungus may completely degrade a xenobiotic compound to innocuous end products
like carbon dioxide and other simple inorganic compounds (Mougin et al. 2009).
This is particularly likely under nutrient-limiting conditions when the substrate
compound could serve the carbon and/or energy needs of the organism. While this
metabolic pathway is preferred as the toxic compound is eliminated or nearly so, the
more common method of detoxification is co-metabolism. In this process, a cosub-
strate is used to transform a xenobiotic compound without utilizing the compound
for growth or energy needs. Generally, this only results in minor changes in the
structure of the pollutant. For instance, substrate-free radicals may be generated
466 P. Baker et al.
Fig. 19.1 The relationship and commonalities between organically produced lignin and varied
xenobiotic chemical structures. (a) The structure of lignin according to Laurichesse and Averous
(Laurichesse and Avérous 2014) and the monomeric alcohols which polymerize to form the cross-
linked complex. (b) Common xenobiotic classes and associated structures as lignin model com-
pounds targeted by lignin-degrading enzymes. Alkylated phenolic structures are common to both
EDCs and lignin, as is the geminal phenyl motif of BPA. Nitrogen is not commonly present in
lignin structures, nor are fused polycyclic aromatics nor polychlorinated phenols making lignin-
degrading enzymatic degradation of these compounds less obvious
which subsequently cross-link the substrates with themselves or with environmental

structures such as soil components, thereby forming adducts with decreased
bioavailability and toxicity (Bollag 1992). Alternatively, these modified compounds
could be further metabolized by other organisms leading to further detoxification. A
third detoxification pathway involves conjugation or oligomerization of a pollutant.
In this process, the resulting products of enzymatic action have larger and more
complex structures than the parent compounds, but the structural modification or
aggregation reduces the bioavailability of the compound, thereby negating its bio-
logical potency. In conjugation, typically, the compound may be methylated, acety-
lated, alkylated, or conjugated with sugars and amino acids and subsequently
excreted or sequestered into storage structures. In oligomerization, a xenobiotic is
coupled with a second molecule of the same or different xenobiotic following oxi-
dation, aggregating into structures that become less bioactive.
19.4 Fungal Enzymes in Xenobiotic Remediation
The most commonly studied enzymes involved in mycoremediation all catalyze

oxidoreduction reactions in the transformation of xenobiotics (Sharma et al. 2018).
Among these enzymes are some active extracellularly and other which are intracel-
lularly involved in the transformation and subsequent detoxification of xenobiotics.
Within these oxidoreduction-catalyzing enzymes, some such as laccases (LACs)
and CYP monooxygenases (P450s) use molecular oxygen as an eventual electron
acceptor, while peroxidases use hydrogen peroxide as electron acceptors in their
respective reaction pathways. One specific class of enzyme common to many xeno-
biotic degradation mechanisms are the heme peroxidases of which the most exten-
sively involved and studied are manganese peroxidase (MnP), lignin peroxidase
(LiP), and versatile peroxidases (VP) (Doddapaneni et al. 2005). The relatively
lower substrate specificity of the extracellular enzymes in oxidative degradation
allows them to target a wider variety of xenobiotic compounds and is as such of
great interest in bioremediation processes (Harms et al. 2011). As with many
secreted proteins, these peroxidases and laccases are glycosylated, at times heavily;
for instance, certain laccases show 16% sugar content (Salony et al. 2006). Many
fungal metabolites bearing diverse functional groups can serve as redox mediators
for the oxidative function of these enzymes and can enhance their activity (Asgher
et al. 2008). These include veratryl alcohol, N-hydroxyacetanilide, and acetosyrin-
gone to name a few. Mediators are particularly important when the substrates are
too large to be accommodated into the active site of the enzyme (Mougin et al.
2009). The toxic xenobiotics or the products formed following their metabolism by
extracellular enzymes can be further detoxified by intracellular enzymes including
CYPs that are ubiquitously present in all organisms (Morel et al. 2013). These
enzymes can catalyze various reactions including dealkylation, hydroxylation, and
sulfoxidation and frequently function by inserting molecular oxygen into various
substrates.
468 P. Baker et al.
19.4.1 Laccase
LACs, originally identified as ligninolytic enzymes, are present in ascomycetes,

basidiomycetes, and deuteromycetes and are commonly found in gene families
(Harms et al. 2011). Laccases are secreted blue multicopper oxidases that catalyze
single-electron oxidation of phenolic and other aromatic substrates to create free
radicals with the concomitant reduction of molecular oxygen to water. The radical
products cross-link by self-coupling or cross-coupling to form less toxic polymers;
during this process they may undergo further decomposition reactions such as
decarboxylation, dechlorination, and demethoxylation. Following LAC-dependent
oxidation events, metabolites commonly exhibit oxidative coupling or radical
polymerization resulting in compounds of greater molecular masses than the parent
compound (Junghanns et al. 2005) which potentially may further negate the biologi-
cal effects of such foreign compounds (Tsutsumi et al. 2001). The use of abundantly
available oxygen as an oxidizing agent with the formation of water as a harmless
by-product makes laccases a desirable bioremediation enzyme. Compared to per-
oxidases, different laccases can tolerate a wider pH range (2–10) and have broader
substrate specificities (Xu 1996). The broad substrate range of laccases encom-
passes various xenobiotic compounds contaminating soil and water, i.e., PAHs,
organophosphorus insecticides, and toxic dyes which include aromatic compounds
like phenols, trichlorophenols, aminophenols (anilines), phenylenediamine deriva-
tives, and benzenethiols (Amitai et al. 1998; Kues 2015; Sharma et al. 2018; Xu
1996; Yadav et al. 2016).
The potential of LACs in xenobiotic remediation has long since been a point of
interest and investigation. Laccases from Trametes versicolor and Pleurotus ostrea-
tus have been used in the detoxification of PCBs (polychlorinated biphenyls) which
were used as insulators in electrical equipment until they were banned in 1979 for
their high toxicity (Keum and Li 2004). Laccases facilitate substrate oligomeriza-
tion and dechlorination, leading to detoxification of these compounds. LACs cata-
lyze the initial oxidation of polycyclic aromatic hydrocarbons (PAHs), beginning
their extracellular degradation (Pozdnyakova et al. 2018b). A LAC isolated from
Coriolopsis gallica oxidizes the PAHs carbazole, N-ethylcarbazole, and dibenzo-
thiophene in concert with proper mediators such as 1-hydroxybenzotriazole and
2.2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (Viswanath et al. 2014).
When free laccase was applied to PAH-contaminated soil, 15 PAHs including
anthracene and benzopyrene were degraded, demonstrating the potential of direct
enzyme application for bioremediation (Wu et al. 2008). Covalently immobilized
laccases from Trametes versicolor were shown to efficiently degrade the PAHs,
anthracene, naphthalene, and phenanthrene (Bautista et al. 2015). Multiple studies
revealed the ability of laccases from Pleurotus ostreatus, Trametes versicolor, and
other strains to effectively degrade BPA and other endocrine disruptors like alkyl-
phenols. The detoxification of these compounds by laccases may be brought about
by oligomerization or mineralization to carbon dioxide, and this action was
enhanced by supplementation with mediators (Macellaro and Pezzella 2014;
Margot et al. 2013; Zhang et al. 2015). Laccases from Pycnoporus sanguineus,
Myceliophthora thermophila, Trametes trogii, and Ceriporiopsis subvermispora
effectively metabolized the toxic dyes like bromophenol blue, methyl violet, and
malachite green and other dyes used in the textile industry (Antosova et al. 2018;
Chmelova and Ondrejovic 2016; Mougin et al. 2009). Synthetic laccases are also
used in the paper and pulp industries to bleach paper and clothes (Sharma et al.
2018). The addition of mediator compounds can dramatically enhance the detoxifi-
cation of substrates as witnessed in the transformation of halogenated pesticides by
laccases from the fungus, Coriolopsis gallica (Torres-Duarte et al. 2009); in this
study, acetosyringone and syringaldehyde proved to be the most effective media-
tors. Finally, laccases from Trametes versicolor have been used to oxidize the phar-
maceutical drugs, diclofenac and mefenamic acid in municipal wastewater (Margot
et al. 2013). Beyond naturally occurring LACs, some researchers have explored the
possibility of engineering LACs with the intention of improving transformational
efficiency and demonstrating transient expression by means of directed evolution
(Camarero et al. 2012; Gu et al. 2014; Mate and Alcalde 2015; Theerachat et al.
2012; Wong et al. 2013b).
19.4.2 Peroxidase
The ligninolytic peroxidases of interest in bioremediation carry high redox poten-

tials (>1.4 V) and catalyze the oxidative breakdown of lignin and other compounds
with aromatic ring structures using hydrogen peroxide as a cosubstrate and with the
help of certain mediator compounds like veratryl alcohol (Piontek et al. 2001;
Sharma et al. 2018). These peroxidases are glycosylated secreted proteins carrying
an iron protoporphyrin (heme) ring at the catalytic center. Peroxidases can oxidize
and generate phenolic radicals which may aggregate and precipitate. There are sev-
eral groups of secreted peroxidases in fungi:
19.4.2.1 Lignin Peroxidase
LiPs are heme-containing enzymes which can metabolize various aromatic com-
pounds many of which are generally refractory to breakdown, with pH optima in
the acidic range (2–5) (Shrivastava et al. 2005). Since the 1983 discovery of P.
chrysosporium, LiP in extracellular media, isozyme forms among various basidio-
mycetes have been isolated ranging in weight from 38 kDa to 43 kDa (Falade et al.
2017). LiPs have been shown to completely oxidize both methylated lignin model
and non-methylated lignin model compounds as well as PAHs (Kadri et al. 2017).
Through powerful, nonspecific catalytic transformative activity, the aptly named
LiP is capable of direct transformation of up to 90% of lignin structural compo-
nents (Falade et al. 2017).
470 P. Baker et al.
LiPs are commonly produced by Phanerochaete and Trametes spp. As

demonstrated by LiP isolated from P. chrysosporium which transforms the PAHs
benzo[a]pyrene, anthracene, 1-methylanthracene, 2-methylanthracene, 9-methyl-
anthracene, fluoranthene, acenaphthene, and dibenzothiophene, these enzymes
possess broad substrate ranges (Pozdnyakova 2012). LiPs also demonstrate
expanded substrate ranges in the presence of veratryl alcohol which increases oxi-
dation of weak or terminal LiP substrates. With the advantageous kinetics con-
ferred by mediators like veratryl alcohol, LiP transforms most aromatic compounds
with an ionization potential less than 8 eV. LiP also has the unique ability to cleave
esters in non-phenolic aromatics, thereby further demonstrating the significance of
LiPs to bioremediation efforts of diverse, aromatic-containing xenobiotics
(Pozdnyakova 2012).
19.4.2.2 Manganese Peroxidase
MnPs are also heme-containing secreted enzymes which function under relatively
less acidic conditions (pH 4–7) than those of LiP (Asgher et al. 2008). As a heme
peroxidase, MnPs share much of their characteristics and mechanism with LiPs
(Deshmukh et al. 2016). MnP appears not to occur in the same large gene families
characteristic of other ligninolytic enzymes such as LAC (Torres-Farrada et al.
2017) and is only found in Basidiomycota (Harms et al. 2011). MnP is capable of
aromatic ring cleavage within monoamino-dinitrotoluene and chlorophenol deriva-
tives (Harms et al. 2011). The oxidative potential of MnP relies on the oxidation of
Mn2+ to Mn3+ by the enzyme followed by the indirect, single-electron oxidation of
subsequent substrates as Mn3+ is reduced, thereby reverting to Mn2+.
Due to the indirect mechanism, the substrate range is broad, and the extent of
transformation of resulting metabolites is near complete. MnP oxidizes phenols,
aromatic amines, and dye compounds as well as mineralizes CO2 from various qui-
nones – common products of PAH radical polymerization – by ring fission transfor-
mations. MnPs isolated from Anthracophyllum discolor clearly demonstrate this
PAH-substrate promiscuity as they are able to oxidize pyrene, anthracene, fluoran-
thene, and phenanthrene as well as various derivatives of these compounds
(Pozdnyakova 2012). A MnP from the white rot fungus, Trametes, displayed a
strong ability to degrade azo and indigo dyes as well as PAHs (Zhang et al. 2016).
Another MnP from Peniophora incarnata not only displayed the potential to break
down the PAH anthracene, but this ability was transferable by heterologous expres-
sion in yeast, signifying a bioremediatory potential. MnP from Ganoderma lucidum
used as cross-linked enzyme aggregates efficiently degraded the endocrine disrupt-
ing nonylphenol and triclosan (Bilal et al. 2017a).
MnP is of great interest in bioremediation efforts as it has been shown to be sta-
ble under adverse conditions; however, a complicating factor of its use in remedia-
tion effort and applications is the mechanistic need for a suitable chelator (Bogan
et al. 1996). Such chelators are commonly organic acids such as oxalic or malonic
acid derivatives (Kadri et al. 2017). These compounds complex with Mn3+, enabling
the oxidation of substrate lignin model compounds. In addition to a mechanistic
dependence on chelators, MnP activity significantly increases in the presence of
redox mediators such as Tween 80. With the added effect of Tween 80, MnP has
been shown to transform compounds with ionization potentials of 8.2 eV. Despite
the powerful oxidative potential and resistance to adverse conditions of MnP, the
application of these enzymes in biotechnical pursuits is complicated by not only
chelator requirements and redox mediator reliance for elevated efficiency but also
by LAC-dependent initiation of Mn3+ complexing (Schlosser and Hofer 2002). No
Mn3+ complexing was observed in in vitro mixtures of semi-purified MnP, Mn2+,
and oxalate or malonate when H2O2 sources were excluded. However, the addition
of LAC stimulated Mn3+ complexing and ultimate MnP-stimulated substrate oxida-
tion. In response to PAH-polluted media, MnP secretion by Fomes is very high,
reaching concentrations of approximately 1299 U/L after 21 days of xenobiotic
exposure (Godoy et al. 2016).
19.4.2.3 Versatile Peroxidase
Versatile peroxidases (VP) are a hybrid between LiPs and MnPs as they contain a
heme group and oxidize Mn2+to Mn3+, inducing indirect oxidations, as well as oxi-
dize phenolic and non-phenolic substrates; VPs conjoin mechanisms and substrate
ranges between these two enzymes (Kues 2015; Pozdnyakova 2012). VPs have thus
far only been identified in Basidiomycetes (Harms et al. 2011). In the initial charac-
terization of the first identified VP – specifically, PS1 isolated from Pleurotus eryn-
gii – it was shown to possess both the Mn oxidation domain of MnPs and the
aromatic substrate oxidation center (AS) of LiPs (Camarero et al. 1999). Furthermore,
PS1 and subsequently characterized VPs have been shown to actually retain LiP- or
MnP-like enzymatic activity in conditions that would inactivate LiPs or MnPs,
respectively. Little is understood about the role of VPs in the mediation of xenobi-
otic transformation; however, it is known that VP production is induced by the pres-
ence of PAH pollutants (Pozdnyakova et al. 2018b). In addition to PAHs, VPs can
also degrade polyhalogenated aromatic pesticides containing diverse functional
groups as demonstrated by VP isolated from Bjerkandera adusta which success-
fully transformed dichlorophen (an antimicrobial polycyclic), bromoxynil (a nitrile
herbicide), and pentachlorophenol (PCP) – a potent pesticide (Davila-Vazquez et al.
2005). Given the hybridization of LiP and MnP substrate ranges, they have the
potential to be a major component of the subsequent remediation of many chemi-
cally diverse xenobiotics. Due to their broad substrate range and various transfor-
mation mechanisms, VPs warrant further characterization and investigation
regarding their potential application in bioremediation and biotechnical efforts.
Examples of fungal laccases and peroxidases employed for xenobiotic detoxifica-
tion are presented in Table 19.1.
472 P. Baker et al.
Table 19.1 Details of microbes producing laccase and peroxidase in the bioremediation of xenobiotics
Enzyme source Xenobiotic Compounds Mediator Reference
Laccases
Trametes PAH Anthracene, benzopyrene ABTS Dodor et al.
versicolor (2004)
T. versicolor, PCB Hydroxy PCBs TEMPO Keum and Li
Pleurotus (2004)
ostreatus
P. ostreatus Insecticides, VX, Russian VX, ABTS Amitai et al.
nerve agents diisopropyl-Amiton 1998)
P. pulmonarius Toxins Aflatoxin B1 ABTS, AS, Loi et al. (2016)
SA
T. sanguineus Endocrine Bisphenol A, benzopyrene, ABTS Balcazar-Lopez
disruptor, phenanthrene et al. (2016)
PAH
Echinodontium Azo dyes Brilliant Violet 5R, Direct Lignin Han et al. (2014)
taxodii Red 5B, Direct Black 38 derivatives
Clavariopsis Endocrine Nonylphenol ABTS Junghanns et al.
aquatic disruptor (2005)
Peroxidases
Trametes spp. Dyes, PAH Indigo, anthraquinone, azo, – Zhang et al.
triphenylmethane, fluorene, (2016)
fluoranthene, pyrene,
phenanthrene, anthracene
Peniophora PAH Anthracene – Lee et al. (2016)
incarnata
Irpex lacteus Dyes Azo, indigo dyes – Qin et al. (2014)
Phanerochaete PCB 2,4-dichlorophenol – Chen et al.
Penicillium Dyes Cotton blue – Shedbalkar et al.
chrochloron (2008)
ABTS 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid, AS acetosyringone, PAH polyaro-
matic hydrocarbons, PCB polychlorinated biphenyls, TEMPO 2,2,6,6-tetramethylpiperidine-
N-oxyl radical, SA syringaldehyde
19.4.3 Cytochrome P450 Monooxygenase
Many fungi also possess intracellular transformation enzymatic machinery capable

of further degradation of xenobiotics (Syed and Yadav 2012). They belong to the
larger group of oxygenases that are the principal intracellular enzymes involved in
the aerobic degradation of aromatic xenobiotics using oxygen (Sharma et al. 2018).
These are also heme-containing enzymes that add one or more oxygens to destabi-
lize aromatic rings to break down and even solubilize the compound. Oxygenases
could catalyze the addition of one oxygen molecule (monooxygenase) or two mol-
ecules (dioxygenase) to the substrate. Many halogenated herbicides, fungicides, and
pesticides are detoxified using oxygenases, and the best-studied enzyme belongs to
the cytochrome P450 enzyme family, which utilized NADPH as a cofactor to cata-
lyze redox reactions (Doddapaneni et al. 2005).
P450s are very distinct from other xenobiotic-degrading fungal enzymes as it is

active intracellularly (Deshmukh et al. 2016). Furthermore, CYP450 are found in
massive gene families with multiple subfamilies as discovered in the genome of P.
chrysosporium with at least 149 isozymes (Doddapaneni et al. 2005; Olicon-
Hernandez et al. 2017; Syed et al. 2011). Such gene families have been identified in
ascomycetes, basidiomycetes, mucoromycetes, and chytridiomycetes (Harms et al.
2011). As monooxygenases, P450 incorporates a single atom from molecular oxy-
gen into the substrate while reducing the remaining oxygen to water. Compared to
CYPs involved in primary metabolism, CYPs mediating detoxification processes are
generally less specific (Cresnar and Petric 2011). P450 monooxygenases are a major
component in the degradation of various xenobiotics as they are quite nonspecific
and demonstrate very powerful enzymatic activity in the transformations of such
diverse chemical motifs. However, as these are intracellularly active proteins, their
application in fungal enzyme isolation and immobilization is further complicated.
Within each class of P450s, multiple subfamilies exist sharing general function
and some sequence identity. Many class-II subfamilies are involved in biosynthetic
pathways and homeostatic processes. The subfamily CYP51 has been indicated in
maintaining cell wall integrity while CYP61 (a fungi-specific subfamily) is respon-
sible for spore outer wall formation. Various Class II P450s transform PAHs, alkyl
phenols (APs), alkanes, and polychlorinated dibenzo-p-dioxins (PCDDs) (Harms
et al. 2011; Kues 2015). CYPs from the basidiomycete Phanerochaete chrysospo-
rium have been shown to catabolize PAHs including anthracene and the endocrine
disrupting alkylphenols (Hirosue et al. 2011; Syed et al. 2011). Among Class II
P450 subfamilies, CYP57 has been shown to detoxify pisatin, a heterocyclic,
aromatic-containing fungus growth inhibitor produced in pea plants upon microbial
attack. Furthermore, CYP53 has been shown to degrade and detoxify benzoate
derivatives within multiple species from both the Ascomycota and Basidiomycota
phyla. Additionally, CYP504 is known to degrade phenylacetate derivatives. The
significance of Class II P450 activity is further emphasized in observed biodegrada-
tion by non-ligninolytic fungi such as Scopulariopsis brevicaulis which was shown
to completely transform anthracene (a tricyclic PAH), producing the same major
metabolite – 9,10-anthraquinone – as ligninolytic fungi such as the basidiomycete
Fomes, despite the absence of extracellular enzymes (LAC, LiP, and MnP) demon-
strating the versatility and potency of Class II P450-dependent intracellular trans-
formation pathways (Godoy et al. 2016). CYPs have been tapped to detoxify a
variety of pharmaceutical compounds, including antibiotics, anti-inflammatories,
and β-blockers (Olicon-Hernandez et al. 2017). Naproxen is an environmentally
prevalent pharmaceutical pollutant and has even invaded drinking water systems
due to overuse in treating human and animal diseases. CYPs were shown to detoxify
this drug through demethylation and hydroxylation (Aracagok et al. 2017). A fun-
damental limitation in utilizing CYP enzymes in cell-free systems is the fact that
CYP functions in intracellular networks that also involve other enzymes (Haroune
et al. 2017). Shotgun proteomics of proteins in response to the PAH, anthracene in
Penicillium oxalicum, revealed regulation of hundreds of proteins, and intracellular
metabolism of such xenobiotics typically follows two distinct phases with the CYPs
working in conjunction with other enzymes like epoxide hydrolases and transfer-
ases (Lucero Camacho-Morales et al. 2018).
474 P. Baker et al.
19.4.4 Unspecific Peroxygenase
UPOs are secreted hybrid enzymes which combine the functionalities of heme
peroxidases and P450 monooxygenases (Karich et al. 2017). Among this class of
peroxygenases, two common structural motifs have been observed: short UPOs are
approximately 29 kDa, while long UPOs are approximately 44 kDa. Short UPOs
are found across all fungal phyla, but long UPOs are exclusive to ascomycetes and
basidiomycetes. Both short and long UPOs have immense substrate ranges, acting
in association with hydrogen peroxide as a cofactor, a marketed improvement in
simplicity from the nuanced requirements of MnP. Furthermore, 41 of EPA-listed
priority pollutants have been shown to be transformed by UPOs, and more than 300
other aromatic, poly- and heterocyclic, and aliphatic substrates have been identified
thus far. Despite the recent findings regarding xenobiotic substrates, the physiologi-
cal role of UPOs remains to be identified (Olicon-Hernandez et al. 2017); however,
Karich et al. theorized that UPOs and P450s may work in harmony with UPOs
crudely transforming xenobiotics extracellularly to reduce negative biological
effects, while P450s “fine-tune” the resulting metabolites so that they may be fur-
ther transformed and rendered inert within the cell or even consumed as carbon
sources due to the incredibly diverse functionality of these enzymes. Thus far, pres-
ent understanding explains that UPOs’ substrate transformations are limited by ste-
ric hindrance, bioavailability and potential substrate solubility, or strong inactivation
of an aromatic ring by electron withdrawing groups (Karich et al. 2017). Despite
these regulatory factors in UPOs’ substrate specificity, UPO isolated from Agrocybe
aegerita demonstrated significant transformation of naphthalene, phenol, anisole,
toluene, ethylbenzene, acenaphthylene, acenaphthene, fluorene, phenanthrene,
anthracene, pyrene, benzo[a]anthracene, 1,2-diphenylhydrazine, benzidine, and
2,4-dimethylphenol as well as various phthalate esters, nitroarenes, and polychlori-
nated benzenes (Karich et al. 2017). Among these known substrates exist several
previously only thought to be transformed by P450s or MnPs and LiPs, again dem-
onstrating the remarkable potential of these enzymes. Such versatile enzymes in
extracellular degradation processes carry great implications for biotechnical devel-
opment of xenobiotic mitigation strategies.
19.5 echanisms of Xenobiotic Detoxification by Fungal

M
Enzymes
Fungal extracellular xenobiotic degradation occurs in two conserved steps (Rao

et al. 2010). Firstly, the hydrolytic system targets macromolecules for degradation
by hydrolase activity. The hydrolyzed substrates are then further transformed by the
ligninolytic system comprised of nonspecific oxidative enzymes. The enzymatic
mechanisms we focus on in this section are the oxidoreductases whose mechanisms
are well researched (Fig. 19.2).
Fig. 19.2 Simplified mechanism of fungal enzymes used in bioremediation. (a) Laccase enzy-
matic cycle. Mediators include veratry alcohol (VA), Tween 80, and other small organic molecules.
(b) Lignin peroxidase enzymatic cycle. Both oxidized states of LiP are able to oxidize substrates
and mediators. Mediator molecules are used when the substrates cannot access enzyme active site.
(c) Manganese peroxidase enzymatic cycle. MnP preferentially oxidizes Mn2+ in the first redox
reaction but can also oxidize other mediators. Mn2+ oxidation is highly specific for the second
redox reaction which restores the base state. (d) CytP450 enzymatic cycle. The reduction of heme-
bound molecular oxygen is either directly catalyzed by the reductase coenzyme or indirectly via
the use of cytochrome B5 as a redox mediator. (Adapted from Kues (2015) and Guengerich
(2001b))
19.5.1 Laccases
Laccases are multicopper oxidases with low substrate specificity. Within basidio-
mycetes these families range from 5 to 17 isozymes (Yang et al. 2017). Within
LAC isozymes, enzymatic copper involvement is conserved with four copper
atoms at +2 oxidation states in the resting enzyme. The four copper atoms are
characterized as T1, T2, and T3 – which is binuclear. The T1 copper facilitates
substrate oxidation, while T2 and T3 copper atoms store the resulting electrons
which then convert diatomic oxygen to water. They catalyze coupled redox reac-
tion between a substrate and molecular oxygen resulting in the formation of a
radical cation and water, respectively. The type I copper is found in a wide cavity
of the enzyme surface, which allows it to bind to many different types of substrates
(Su et al. 2018).
The significance of LACs in the remediation of xenobiotics is only amplified by
laccase-mediator systems (LMSs) which act as electron transfer chains and thereby
476 P. Baker et al.
further broaden the substrate range as well as increase kinetic favorability of trans-
formation (Wong 2013a). LMSs share the common mechanism of facilitating the
indirect oxidation of a substrate following the primary oxidation event of the media-
tor by LAC. The enzymatic cycle of laccases can be simplified as a four-electron
abstraction from the substrate by the oxidized enzyme followed by the reduction of
molecular oxygen to water to regenerate the active enzyme (Fig. 19.2a). The enzyme
primarily exists in the resting oxidized state (RO) with each of the copper atoms
oxidized. The catalytic reaction is initiated by the donation to the type I copper of
four electrons from suitable substrates (reductants). Three electrons are transferred
through a conserved His-Cys-His sequence of amino acids to the trinuclear cluster
(TNC) consisting of the type II copper and the two type III copper atoms, resulting
in the fully reduced enzyme. The electrons are used to reduce molecular oxygen
which binds to the TNC, forming a peroxide intermediate (PI). The reduction then
proceeds by donation of a hydrogen by a nearby glutamic acid residue resulting in
the cleavage of the oxygen-oxygen bond and the native intermediate (NI) state of
the enzyme. This state can directly proceed to the fully reduced state in the presence
of large amounts of suitable substrates, producing two water molecules or proceed
to the fully oxidized state with loss of a single water molecule in the absence of
significant levels of reductants (Jones and Solomon 2015).
Laccases tend to accommodate substrates with relatively low redox potentials
such as phenolic compounds which are oxidized to phenoxyl radicals which may
undergo coupling reactions or isomerization to form quinones. Laccases are gener-
ally unable to directly oxidize compounds with high redox potentials or steric hin-
drance. Instead, such compounds are oxidized via the laccase-mediator system
which involves oxidation of micromolecular organics which can then oxidize target
compounds through redox reactions leading to cleavage of bonds (Su et al. 2018).
19.5.2 Peroxidases
Of the various peroxidases discussed herein, only the general mechanism of lignin
peroxidase and manganese-dependent peroxidases has been elucidated. The shared
mechanism of these two types of peroxidases involves the oxidation of the enzyme
by two electron abstractions by the H2O2 cosubstrate followed by two one-electron
transfer steps where the oxidized enzyme abstracts an electron from the substrate
(Mougin et al. 2009).
19.5.2.1 Lignin Peroxidases
In LiPs, Fe(III) in the heme ring is coordinated with four heme tetrapyrrole nitro-
gens and to a histidine residue. LiP has been shown to fold into a globular profile
measuring about 50 × 40 × 40 ¯ ; this form is subdivided into proximal and distal
domains relative to the heme group. Two small molecular channels allow for heme
accessibility despite its fixed position within the protein’s tertiary structure. LiPs are
capable of cleaving Cα-Cβ bonds as well as bonds between aryl Cα (Kadri et al.
2017). LiP is further differentiated from other peroxidases by the optimal pH of
approximately 3.0 (Falade et al. 2017). Lignin peroxidases are activated by a two-
electron oxidation of the native enzyme by the cosubstrate H2O2 (Mougin et al.
2009). This results in compound I which is reduced by the substrate as part of a
one-electron redox reaction to compound II. Compound II then abstracts a second
electron from the substrate resulting in regeneration of the native enzyme. The acti-
vated forms of the enzyme have a high redox potential allowing it to oxidize com-
pounds such as lignin that other peroxidases are unable to transform. LiPs act
through three different mechanisms based on the availability of its heme cofactor
toward the substrate. First, LiPs act directly on certain phenolic and non-phenolic
compounds which can access the heme group. This can lead to breakage of carbon-
carbon bonds in substrates leading to conformational changes (Fig. 19.2b). The sec-
ond lignin peroxidase mechanism is indirect, acting through a redox mediator to
oxidize compounds that cannot access the heme group (Fig. 19.2b). Lignin peroxi-
dase oxidizes mediators such as veratryl alcohol (VA) to a cation radical (VA+)
which then oxidizes compounds through a redox reaction. The third mechanism
occurs through further reactions of VA. The cation radical oxidizes organic acids
into anion radicals which act as reductant. These radicals can also reduce molecular
oxygen to a dioxygen anion which acts as a reductant. Through the reduction of fer-
rous ions, this dioxygen anion can also reduce hydrogen peroxide to a hydroxyl
radical which can oxidize compounds through the non-enzymatic Fenton’s reaction
(Christian et al. 2005b).
19.5.2.2 Manganese Peroxidase
Manganese peroxidases, also known as manganese-dependent peroxidases, are oxi-

dized to a two-electron-deficient state (compound I) and restore its basal oxidation
state by two single-electron abstracting steps (Christian et al. 2005b). The first step
which reduces the compound I to a single-electron-deficient state (compound II) is
not very specific and can use either Mn2+ or small compounds such as VA as reduc-
tants. The second step which restores the basal oxidation state of the enzyme is
highly specific to Mn2+ (Fig. 19.2c). Both of the activated enzyme states oxidize
Mn2+ to Mn3+ which acts as a nonspecific small, diffusible redox mediator. Mn3+ are
chelated to carboxylic acids such as oxalic acid and are thus able to cause one-
electron abstraction oxidation of various compounds (Fig. 19.2c). They can also
react with carboxylic acids to produce radicals such as VA+ and superoxide.
Manganese peroxidases are also able to act indirectly as reductors. This is done
through oxidation of hydroquinone to semiquinone radicals which reduce highly
oxidized compounds. This generates a quinone which is then reduced back to
hydroquinone by quinone reductase enzyme.
478 P. Baker et al.
19.5.3 Cytochrome P450
Cytochrome P450 enzymes are part of a superfamily with remarkable variation.

Due to this variation within the CYP superfamily, the reactions associated with
P450 enzymes are classified based on reactionary motifs and various protein
involvements into ten distinct classes, three of which have been observed in fungi:
class II, class VIII, and class IX (Cresnar and Petric 2011). Most commonly,
CYP450 enzymes act as monooxygenases that incorporate an atom of oxygen into
substrates (van den Brink et al. 1998).
P450 enzymes therefore serve as versatile catalysts for region-specific and
stereospecific oxidation resulting in hydroxylation, heteroatom oxygenation,
dealkylation, and epoxidation of C=C bonds (Deshmukh et al. 2016; Olicon-
Hernandez et al. 2017). However, the basis of all these reactions can be sum-
marized as being
XH + NAD ( P ) H + H + + O2 → XOH + NAD ( P ) + H 2 O

+
with R-H as the substrate of cytochrome P450. Among these transformations,

NADH or NADPH frequently acts as an electron donor to the second oxygen as
it is reduced to water (Kues 2015). The reaction does not usually produce a phe-
nol but instead an oxide which isomerizes to the more favorable phenolic confor-
mation (Christian et al. 2005b). The phenolic form is activated toward further
enzyme- catalyzed reactions, resulting in trans-dihydrodiols (Tongpim and
Pickard 1999).
Much like the peroxidases, P450 enzymes are heme-based, with their catalytic
active site containing a heme-bound iron atom (Guengerich 2001a). The substrate
first binds to the heme iron Fe3+ which is reduced by an accessory enzyme, NADPH
reductase (Fig. 19.2d). This reduced iron Fe2+ then binds with molecular oxygen.
The accessory enzyme then reduces molecular oxygen which is then protonated.
This leads to cleavage of the O-O bond, causing the protonated oxygen to be released
as H2O. The resulting FeO3+complex radicalizes the substrate by proton (or electron
abstraction). The radical then accepts the hydroxyl group (or oxygen atom in case
of electron abstraction) before being released, simultaneously returning the iron to
its base state. There exists a shunt pathway in which a peroxide is used as cosubstrate
instead of molecular oxygen, skipping the need for the reductase coenzyme
(Fig. 19.2d).
The monooxygenation mechanism of P450 is not limited to carbons but can also
apply to heteroatoms such as nitrogen, sulfur, phosphorus, and iodine. In these
cases, the oxygen transfer is more complicated, with the oxygen being added to the
substrate after two successive electron transfer steps.
19.6 trategies and Considerations in Xenobiotic

S
Bioremediation
Either fungi or enzymes isolated from fungi could be used for the detoxification of
xenobiotic compounds. Some fungal phyla possess more versatile degradation sys-
tems relative to bacterial strains (Pozdnyakova et al. 2018a). The fundamental limi-
tations in using isolated fungal enzymes include productivity as well as stability and
retention of activity (Sharma et al. 2018). Laccases, for example, have an acidic pH
optimum that may not be available in effluent soils and waters, presenting a major
limitation to their use. Furthermore, cost of application, intolerance of enzymes to
high levels of cosubstrate hydrogen peroxide, and issues with enzyme reusability
have hampered the application of these enzymes in the field (Nicell and Wright
1997). Once applied to sites for remediation, the enzymes are also under threat of
denaturation and destruction by physical and chemical forces and by the action of
microbes and their enzymes. Besides, excessive exposure of heme-containing pro-
teins to oxidative species can lead to inactivation and subsequent degradation of the
protein (Valderrama et al. 2002). Indeed, these limitations are so critical that they
have delayed or limited large-scale application of these enzymes in bioremediation
processes (Ayala et al. 2008).
One approach to the identification of potential fungal strains with significant
remediation potential and biotechnical application is to isolate populations from
xenobiotic-polluted environments (Godoy et al. 2016). Doing so results, in part, in
fungi with innate tolerance to the environmentally present pollutants. The pollutant-
resistant detoxifying fungi often produce biodegradative enzymes that are too lim-
ited in amount to isolate and utilize. To scale up production of these enzymes,
genetic engineering could be used to overexpress fungal enzymes in fungi, plants,
or other organisms that could colonize the polluted substratum. Alternatively,
enzymes could be mass-extracted from such organisms by heterologous expression
and utilized for bioremediation. This is a cost-effective strategy that not only allows
purification of large amounts of enzyme with stability and activity, the purification
process of recombinant proteins is also simpler as they can be isolated using cleav-
able tags (Alcalde et al. 2006). Furthermore, the stability, activity, and other features
of an enzyme can also be enhanced using genetic and enzyme engineering
approaches. By introducing mutations using strategies like DNA shuffling, error-
prone PCR, and site-directed mutagenesis (Dua et al. 2002), enzyme engineering
could be accomplished, where a change in protein sequence from mutations results
in a possible change in enzyme structure or regulation with improved traits includ-
ing increased stability and activity; increased xenobiotic substrate specificity or
wider substrate range; tolerance to a wider range of pH, temperature, and stress
conditions; and decreased susceptibility to proteases in the natural environment
(Rayu et al. 2012). One such mutated laccase protein from Pleurotus ostreatus iden-
tified in a screen gained greater enzymatic activity and higher stability at acidic pH,
widening its pH tolerance in detoxifying toxic industrial dyes (Miele et al. 2010).
480 P. Baker et al.
Similarly, directed evolution of a laccase resulted in 3.5-fold increase in acetonitrile

catabolism while tolerating relatively high concentrations of it (Alcalde et al. 2005).
As enzymes are sensitive to physical and chemical environmental changes, vari-
ous strategies have been evolved to fortify and preserve their structure and function
while exposed to adverse conditions. Enzyme immobilization in carbohydrate-
based matrices has been shown to be effective in prolonging the half-life and
enhancing the stability and catalytic function of the remediative enzymes. Enzymes
can be immobilized to solid support systems for more effective use in bioremedia-
tion. Enzyme immobilization can be done by covalent linkage or adsorption of
enzymes onto solid surfaces like glass, affinity tag-bearing beads or nylon mem-
branes, suspension in polymeric gels like chitosan, gelatin and encapsulation in
solid matrices such as sodium alginate and could also accompany enzyme cross-
linking using glutaraldehyde (Diano et al. 2007; Koyani and Vazquez-Duhalt 2016;
Sirisha et al. 2016). Immobilization has been shown to improve the stability, cata-
lytic function, and longevity of enzymatic function of the fungal enzymes and addi-
tionally protects the enzymes from proteolytic degradation (Asgher et al. 2007;
Cheng et al. 2007; Jing and Kjellerup 2018). This is brought about in part by the
increased resistance to physical, chemical, and biological denaturing agents afforded
by the immobilization. Additionally, immobilized enzymes can be recovered and
reused, thus economizing the process. Laccases immobilized on glass beads from
Trametes retained 90% of their activity and showed greater resistance to proteases
(Bilal et al. 2017b; Dodor et al. 2004). Similar benefits have been observed for other
fungal enzymes like MnPs (Bilal and Asgher 2016). Enzymatic nanoreactors with
dendritic copolymers bearing glycosidic groups anchoring laccases not only showed
improved enzymatic activity but also increased thermostability (Gitsov et al. 2008).
Similarly, vault nanoparticles with hollow cores covalently anchoring multiple MnP
enzymes showed enhanced stability while displaying a threefold increase in phenol
degradation (Wang et al. 2015).
The addition of cofactors, cosubstrates, and mediators is important to drive the
action of the enzymes in bioremediation scenarios; this is particularly important for
recalcitrant pollutants. Additionally, the presence of mediator compounds can
enhance enzyme activity. The fungal metabolites, veratryl alcohol and 2-chloro-1,4-
dimethoxybenzene, can stimulate LiP enzyme activity, and this is particularly use-
ful when treating recalcitrant substrates (Asgher et al. 2008), while acetohydroxamic
acid serves as an excellent mediator for laccases (Minussi et al. 2007). In some
cases, the addition of glucose as a carbon source to fungal cultures also enhanced
the detoxification process (Asgher et al. 2008).
Marine fungi are adapted to much harsher conditions than terrestrial fungi. Their
enzymes are able to withstand high salinity and concentration of phenolic com-
pounds. Certain marine fungi have shown bioremediation potential for water-soluble
crude oil fractions between 0.01 and 0.25 mg/mL (Deshmukh et al. 2016). Other
extremophilic fungi such as Pestalotiopsis palmarum are able to survive in high
concentration of extra-heavy crude oil and salt while producing oxidative exoen-
zymes (laccases and lignin peroxidases) which degrade the maltene and asphaltene
fractions of oil for use as a carbon and energy source (Naranjo-Briceno et al. 2013).
The isolation of enzymes from marine organisms can select for traits such as heat
and cold tolerance and salt and pressure tolerance (Lima and Porto 2016).
Recently, non-protein enzyme mimics or next-generation artificial enzymes in
nanoparticles or nanocomposites have been used to replace enzymes in bioremedia-
tion processes (Gao and Yan 2016). Desired for their low cost and higher stability,
these structures display enzyme-like properties under physiological conditions.
Nanozymes, although lacking an active site, bind substrates specifically and cata-
lyze their transformation. The Fe3O4-based magnetic nanoparticles mimic peroxi-
dases and can degrade toxic dye compounds like methylene blue (Wu et al. 2015).
Carbon-based nanomaterials made of graphene oxide (GO) also display peroxidase-
like activity (Ma et al. 2017). Similarly, a guanosine monophosphate (GMP) coor-
dinated copper nanocomposite mimicked laccases in being able to degrade phenolic
compounds including hydroquinone and naphthol (Liang et al. 2017).
Although it is desirable to identify and isolate detoxification enzymes in polluted
environments, a basic handicap is the ability to culture these microbial species.
Only a handful of microbes are culturable, and these may not include the microbes
that make the enzyme of interest. This problem is circumvented by recent develop-
ments in metagenomics which can identify potential detoxification enzyme-coding
genes. These genes could be expressed in the heterologous system to mass-produce
the enzymes of interest for bioremediation. Clues to the functionalities of these
genes could be revealed by metatranscriptomic and metaproteomic approaches,
which could also reveal biochemical pathways and synthetic pathways for xenobi-
otic-transforming enzyme production.
In silico approaches can be employed to understand the evolution of xenobiotic
detoxification by phylogenetic analyses. More recently, bioinformatic analysis in
the form of molecular docking tools has been developed to predict pollutant sub-
strates of the detoxification enzymes. This approach has been particularly useful for
laccases which have broad substrate specificity; in one study, laccase enzyme struc-
tures were screened against a database of toxic compounds to identify putative sub-
strates (Suresh et al. 2008). The study found that 30% of the studied compounds that
were recognized as environmental pollutants could be potentially metabolized by
fungal laccases. Similar approaches could be employed for other enzymes with
known structures to test if they could be used to detoxify pollutants of interest.
19.7 Conclusions and Future Perspectives
Increased global industrialization has presented many challenges including the pro-
duction of ecotoxic industrial wastes that also present health threats as xenobiotic
compounds. Fungi have specialized enzymes that are highly efficient in detoxifying
these pollutants. Harnessing the power of these enzymes is proving to be an effec-
tive strategy for the bioremediation of the xenobiotic compounds. In particular, oxi-
doreductase enzymes including laccase, peroxidase, and oxygenases like CYP
from various fungal species are employed to detoxify a wide range of pollutants.
482 P. Baker et al.
In addition to bioremediation by detoxification, enzymes can also be employed for

the detection and quantification of xenobiotics in the environment. White rot fungi
(WRF) are a versatile group of microbes that are capable of oxidative detoxification
of a variety of chemical pollutants and xenobiotic compounds that are environmen-
tally harmful. These organisms and their enzymes have the ability to not only toler-
ate the xenobiotics but in many cases also metabolize or help sequester them. The
limitation in biodegradation of xenobiotic compounds is frequently the initial stages
of degradation. The oxidoreductase enzymes are all extracellular and specialized for
less specific activity in initial stages of xenobiotic metabolism and, as such, as ideal
candidates for detoxification of these compounds.
Development of heterologous expression systems and industrial scale expression
is still a limitation. Limitations of enzyme quantity and stability are being overcome
by the adoption of genetic engineering technology and enzyme immobilization
approaches. Fungal enzymes can also be modified for enhanced catalytic activity in
addition to thermostability to maximize the efficiency of the detoxification process.
Creation of enzymes with increased redox potentials, especially in those such as
laccases which have a lower redox potential, could widen the substrate range. The
utility of laccases and CYPs may be limited by the availability of molecular oxygen.
Development of peroxidases to target those substrates could be one possible
approach. Present bioengineering tactics are focused on developing superior
enzymes for the bioremediation of known pollutants, but such approaches may be
extended to target novel pollutants, not known to be biodegradable. Alternatives to
fungal enzymes in the form of nanozymes are also being explored.
An inherent limitation in the use of single enzymes in bioremediation systems is
the fact that detoxification processes are often multi-step pathways, requiring the
action of multiple enzymes in a sequence. The ability to use fungal enzymes in
synthetic pathways for biotransformation of toxic pollutants into economically
valuable compounds would be a great future direction. Since fungal-fungal and
fungal-bacterial consortia have been employed for remediation of xenobiotics like
PAHs, a similar strategy with enzymes could follow suit.
Structural studies of enzymes as well as directed evolution can facilitate changes
in structure that could substantially enhance activity and xenobiotic metabolism.
Better structural understanding of laccases as well as other enzymes could lead to
minimization of the use of redox mediators, especially in in situ bioremediation
scenarios in environmental settings, where the massive release of these eco-unfriendly
compounds may be undesirable. The solving of crystal structures of fungal detoxi-
fication enzymes enables molecular docking analyses to predict the ability to
metabolize various xenobiotic substrates. Similarly, the application of enzymes also
requires a fine understanding of soil structure and soil-water interactions as these
can significantly affect enzyme activity. Recent advances in this area are likely to
benefit soil modeling studies and assessment of enzyme function for future biore-
mediation projects. Adopting a bioprospecting-like approach to find new strains of
fungi with superior ligninolytic enzymes in environments like rainforests could be
a promising way forward in continued efforts to combat environmental pollutants of
industrial origin in cost-effective and environmentally sustainable tactics.
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Chapter 20
Fungal White Biotechnology: Conclusion
and Future Prospects
Ajar Nath Yadav
This book contains current knowledge about fungi; their biodiversity from diverse
sources including natural as well as extreme, associated with plants (epiphytic,
endophytic, and rhizospheric); and potential biotechnological applications of fungi
and fungal products for different processes in agriculture, industry, pharmaceutical,
food and feed processing, and environments for sustainable developments. Fungi
are prominent sources of pharmaceuticals and are used in many industrial fermenta-
tive processes such as the production of enzymes, vitamins, pigments, lipids, glyco-
lipids, polysaccharides, and polyhydric alcohols. In the past 50 years, several major
advancements in medicine came from lower organisms such as molds, yeasts, and
the other diverse fungi. Fungi are extremely useful in making high-value products
like mycoproteins and act as plant growth promoters and disease suppressors.
Fungal secondary metabolites are important to our health and nutrition and have
tremendous economic impact. In addition to this, fungi are extremely useful in car-
rying out biotransformation processes. Recombinant DNA technology, which
includes yeasts and other fungi as hosts, has markedly increased the market for
microbial enzymes (Fig. 20.1).
Today, fungal white biotechnology is a major participant in the global industry
due to its mind-blowing potential in medical with pharmaceutical importance. The
secondary metabolites with pharmaceutical importance of Aspergillus nidulans and
other fungi could be used as drug worldwide (Keller et al. 2005). Ergot alkaloids
(ergometrine and Ergotamine) and lovastatin, a popular cholesterol-lowering drug,
are the secondary metabolites (Beekman and Barrow 2014). Fungal metabolites
have antitumour, antiviral, antibacterial, and immunosuppressant activities. Fungi
are used as high-cost food because of its high protein and low calorific value (Siso
1996). Some of the edible fungi (mushrooms) are used such as Agaricus bisporus
(white button mushroom), Agaricus campestris, Morchella (temperate zone
A. N. Yadav (*)
Department of Biotechnology, Akal College of Agriculture, Eternal University,
Baru Sahib, Sirmour, Himachal Pradesh, India

492 A. N. Yadav
Fig. 20.1 Biotechnological applications of fungi and their value-added products in agriculture,
health, industry, and environments. (Adapted with permission from Kour et al. (2019a))
mushroom), Pleurotus sp. (oyster mushroom), and Volvariella (paddy straw

mushroom) (Mathur et al. 2011; Reddy 2015). Fungi are used as the rich sources of
single-cell proteins. Some of the fungi for SCP are given as Aspergillus niger,
Fusarium avenaceum, Neurospora sitophila, Penicillium chrysogenum, and
Saccharomyces cerevisiae (Kuhad et al. 1997; Ravindra 2000) (Fig. 20.2).
Fungi are widely used in fermentative industries for the production of ethanol,
organic acids, antibiotics, and enzymes like fungal cellulases, gluconase, and glyco-
sidase. Certain fungi like Penicillium notatum, P. chrysogenum, and Cenococcum
sp. are used in antibiotic production (Broadbent 1966; Mishra et al. 2019), whereas
Saccharomyces cerevisiae and Monilia sp. are used in ethanol production (Chiang
et al. 1982; Molaverdi et al. 2019). Fungi are also useful in ripening of cheese and
processing of other products.
With the expanding population, environment is changing greatly, and agriculture
is one of the most exposed sectors to these changes and faces a number of challenges
like pollution, pathogenic attack, salinity, drought, high temperature, low tempera-
ture, and so on. All these challenges ultimately affect the productivity (Fig. 20.3). To
overcome such issues, eco-friendly approaches are very vital. The use of fungi as
biofertilizers is one emerging area which is getting a greater attention, as it is proving
its importance by enhancing the plant growth and productivity by diverse plant
growth-promoting traits including the production of phytohormones, siderophores,
and hydrolytic enzymes, making the availability of different nutrients and protecting
plants against pathogens (Kour et al. 2019b; Yadav et al. 2019b). Fungi are very
important for the soil ecosystem and play a considerable role in the daily life of
human beings; additionally, they are important for agriculture, bioremediation, natu-
ral cycling, food industry, and as biofertilizers (Karthikeyan et al. 2014). Plant
20 Fungal White Biotechnology: Conclusion and Future Prospects 493
Fig. 20.2 White fungal biotechnology applications. (Adapted with permission from Challa et al.
(2019))
growth-promoting fungi are gaining a significant interest to be used as bioinoculants,

as they possess manifold benefits on quantity as well as the quality of the plants as
well as the positive relation they exhibit with the ecological environment. Vesicular-
arbuscular mycorrhizae are the mutualistic symbiosis between the roots of higher
plants and certain fungi. The mycorrhizae help in the phosphate nutrition of plants
and protect the roots by forming the mantle (Mathur et al. 2011; Yadav et al.
2019a). Furthermore, fungi have been demonstrated to produce phytohormones
including indole-3-acetic acid (IAA), gibberellins (Maor et al. 2004; Tudzynski
and Sharon 2002; Kumar et al. 2018), and siderophores (Kumar et al. 2018;
Milagres et al. 1999). Fungi are known for its quite specific and effective action
and have low residual effects in comparison with synthetic pesticides. Fungi are
used as bioherbicides such as Cercospora ageratinae (Pamakani weed),
Colletotrichum gloeosporioides (mistletoes), Leptosphaerulina trifolii (Passiflora),
Phyllosticta (Glycosmis), Puccinia chondrillina (rush weed), Septagloeum gillis
(mistletoes), and Wallrothiella arecuthobii (mistletoes) (Charudattan and Dinoor
2000; Poudel et al. 2019).
Employing live fungi or fungal enzymes for industrial applications is known as
fungal white biotechnology. However, fungal biotechnology, an essential technology,
494 A. N. Yadav
Fig. 20.3 A schematic representation of plant-associated rhizospheric microbiota. The

rhizosphere-associated fungi in relation with plants exhibit widespread applications in the field of
agriculture, pharmaceutical and biomedical industries, and industrial sectors. (Adapted with per-
mission from Pattnaik and Busi (2019))
uses renewable sources for sustainable growth of population. Fungi or fungal

enzymes play a role in food and feed industries. Fungal white biotechnology brings
down greenhouse emissions and is eco-friendly in nature. The applications of fungi
as food (edible fungi) and fodder and using fungi in processing food (bread, cheese,
and other bakery products) and fermenting food (alcohols, beverages) are indis-
pensable. Fungal white biotechnology enhances flavor in cheese, bread, and bever-
ages, protein quality and yield in SCPs, and stability and shelf-life of the products
with much efficacy. Though there are many advantages with white fungal biotech-
nology, tolerance to the extreme conditions during processing and enrichment of
products are the major challenges observed with white fungal biotechnology (Challa
et al. 2019) (Fig. 20.4).
Enzymes from microbes have gained great appreciation worldwide for their
extensive uses in a variety of sectors such as food, agriculture, chemical, and medi-
cine. In the field of medicine, these are used to treat health disorders associated with
deficiency of human enzymes caused by genetic problems (Anbu et al. 2015; Singh
et al. 2016). The processes mediated by enzymes are speeding up in food, pharma-
ceutical, textile, paper, and leather industries and are gaining interest because of
certain advantages such as reduced process time, intake of low energy input,
Fig. 20.4 Classification and biological activity of fungal natural products. (Adapted with permis-
sion from Gholami-Shabani et al. (2019))
cost-effectiveness, greater efficiency, nontoxicity, higher-quality products, and

eco-friendly characteristics (Gurung et al. 2013; Kamini et al. 1999; Singh et al.
2016). The fungi and fungal enzymes are used for biodegradation of azo dye and
hydrocarbons for sustainable environments. Peroxidase enzymes of Penicillium
chrysosporium and Streptomyces sp. have potential biodegradable activities that
degrade amaranth dye, Orange G, heterocyclic dyes like Azure B and Lip dye
(Fig. 20.5). The filamentous fungi are also playing a role in the degradation of toxic
hydrocarbons (Hasan 2018; Leahy and Colwell 1990). Another potential application
of fungal communities for sustainable environments is remediation of explosive con-
taminated soil by its lignin-degrading enzymes (Maqbool et al. 2016). The fungi
have eminent role in the removal and recovery of heavy metals from wastewater and
industrial effluents. Hg, Cu, Ni, Pb, and Cd are extracted at pH 2–5 by myceliar
beads of Penicillium.
496 A. N. Yadav
Fig. 20.5 Fungal enzymes and their biotechnological applications in diverse sectors. (Adapted
with permission from Kour et al. (2019a))
White biotechnology or industrial biotechnology uses enzymes and microorgan-

isms to make bio-based products in sectors such as chemicals, food and feed, deter-
gents, paper and pulp, textiles, and bioenergy. White biotechnology also employs
living organisms as cell factories preferably utilizing renewable natural resources
such as lignocellulose for production of a variety of materials and biocompounds
with energy efficiency, increased productivity, and environmentally sustainable
characteristics. Fungi are the organisms that play a potential role in degradation of
explosives. It is observed by repeated laboratory studies involving pure cultures of
white rot fungi. It also helps in degradation of hydrocarbons in the environment.
Fungi attract considerable attention due to their possible involvement in the
diverse applications. So far, large numbers of enzymes have been purified from
fungal cultures and characterized in terms of their biochemical and catalytic proper-
ties. It possesses antimicrobial activities and is used in bio-mineralization, as a food
for its high protein contents and as a biofertilizer.
Acknowledgments The authors are grateful to the Department of Biotechnology, Akal College of
Agriculture, Eternal University, Baru Sahib, and Department of Environment, Science and
Technology, Shimla, HP-funded project “Development of microbial consortium as bio-inoculants
for drought and low temperature growing crops for organic farming in Himachal Pradesh,” for
providing the facilities and financial support to undertake the investigations. There are no conflicts
of interest.
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Cham
Index
A Anthraquinone, 119, 120

Abiotic stress, 324, 327, 330–332, 336, 337 Anthropogenic activities, 17
Actinomycin, 122 Anthropogenic and natural sources, 196–197
Adsorption Antihyperglycemic, 101
metal ions, 397 Antihypocholesterolemic, 101
wastewater treatment, 397 Antioxidant system, 332
Adsorption activation energy, 408 Antioxidative property, 365
Adsorption effects, 165 AOX1 gene expression, 58
Adsorption mechanism model, 402, 403 Aquaporins, 335, 336
Adsorption method, 161 Arabidopsis, 337, 338
Adsorption process, 408 Arabinosidases, 264
Adsorption studies Arbuscular mycorrhizal (AM), 438
batch adsorption study, 409–410 Arbuscular mycorrhizal fungi (AMF), 30,
breakthrough analysis, 411 195, 425
capacity, 409 biochemical processes, 321
column adsorption study, 410–411 biodiversity, 423–427
equilibrium adsorption capacity, 410 biodiversity studies, 422
packed bed column, 411 cation antiporter (see Cation channels and
Agricultural practices, 420 transporters)
Agrowastes, 118 colonization, 420
Airlift bioreactor, 414 community composition, 420
Alcohol oxidase (AOX), 58, 65 counter-balancing processes, 338
Aliphatic hydrocarbons, 240 ecophysiology mechanisms
Alkyl glucosides, 266 extraradical AMF networks, 324, 325
AMF symbiosis, 327, 334 and plants, 324
Amino and alkylamino groups, 75 enhancement tolerance, plants, 329
Amylases, 134, 135, 310, 311 extraction and propagation, 420–423
Anaerobic conditions, 452 fungal symbiosis, 322, 323
Anaerobic rumen fungi (ARF) germination, 427
brown rot fungi, 6 life cycle, 323
plant biomass degradation, 3, 4 microbiota, 419
secretome, 12, 13 mobilization and transportation, 427
wood-decaying fungi, 8 morphology, 322, 323
wood-degrading fungi, 13 mycelia, 324
Analytical profile index (API), 302 mycoremediation, soil salinity, 321
Anthracene, 119, 217, 224, 228 mycoremediation-based research, 338

Fungi, Fungal Biology, https://doi.org/10.1007/978-3-030-25506-0
500 Index
Arbuscular mycorrhizal fungi (AMF) (cont.) classification, 258

nutrient uptake, 326, 327 co-culturing, 269
AM colonization, plants, 327 CRISPR/Cas9, 272
interactions, Na+ and Cl-, 328 deinking of waste paper, 266
K+, 328 flavour and nutrition enhancement, 264
Na+, 328 fungi, 260
P availability and activity, 328 genetic manipulation, 270
P uptake, 327 high-value bioproducts, 263
salinity stress, 328 hydrolyse flavonoid compounds, 265
physiological processes, 321 isoflavone glycosides, 264
plant biomass, 326, 327 lignocellulose, 262
plant growth-promoting bacteria, 420 lignocellulose bioconversion (see
plants, 321 Lignocellulose bioconversion)
pot-culture technique, 421 lignocellulose degradation, 271
propagation, 421, 422 metagenomics approach, 271
roots, 321 microbial sources, 261
saline soils, 321 mutagenesis, 270, 271
salinity-induced stress, 321 plant biomass, 263
salinity stress remediation sources, 259, 260
(see Salinity stress remediation) synthetic activity, 266
soil salinity (see Soil salinity) Betaines, 330
species, 420, 421 Bioaugmentation process, 225, 227–229, 243,
trap cultures, 422 248, 249
Arbuscular mycorrhizas, 427 advantages, 24
Arbuscules, 323 degradation properties, 24
Arctic environment, 201 DNA manipulation, 23
Aromatic peroxygenase (APO), 130, 192 GEMs
Arrhenius equation, 408 advantage, 24
Aryl alcohol oxidase (AAO), 106, 127, 128 biodegradation process, 24
applications, 129, 130 biotic and abiotic factors, 24
biochemical characteristics, 128 genetic engineering microbes
structure, 128, 129 strategies, 24
Ascomycota, 240, 241, 244, 245 rate-limiting steps, 24
2-Azino-bis-(3-ethylbenzothiazoline-6- recombinant DNA technology, 24
sulfonic acid (ABTS), 127 molecular biological techniques, 24
Azo dye (RB5) wastewater, 74, 89 recombinant DNA, 24
Bioconversion
edible food crops, 383
B fungal consortium, 384
Basidiomycota, 240, 241, 244, 245 value-added products, 387
Batch adsorption study, 409–410 Bio-decolourization, 84
Batch biosorption equilibrium, 410 Biodegradation process, 205
Benzene, toluene, ethylbenzene and xylene Biofertilizers, 492, 497
(BTEX), 248, 249 Biofuel
Benzoanthracene, 217 bioconversion, 383
Benzopyrene, 119, 217 biomass, 383
β-glucosidases, 7, 10, 354, 363 lignocellulose, 262, 263, 387
approaches, 268 production, 261, 272, 382, 384, 387
aryl-β-glucosidases, 259 Biofuels, 121
biotechnological applications, 261 Bioherbicides, 493
biotechnology-based approaches, 272 Biological oxygen demand (BOD), 299
cassava, 266 Biomass
cellobiose, 258 algal, 383
cellulose degradation, 258 biofuels, 383
Index 501
composition, 384, 385 Biosensors, 165

ethanol production, 387 Biosorbent, 397
hemicellulosic fraction, 390 chitin, 399
lignocellulosic, 382, 385, 387, 389, 391 chlorophyll, 400
plant, 387 Biosorption, 397, 405
pretreatment, 385, 386 heavy metals and dyes, 405
Biomedical industries, 121 thermodynamic behavior, 405
Bio-mineralization, 497 Biosorption phenomenon, 398
Bio-nanocomposites, 398, 401 Biostimulation, 227, 229, 243, 248, 249
Biopulping process, 307 Biostimulation/bioaugmentation, 23
Bioremediation, 64, 204, 444 Biotechnological applications, 259
advantages, 18 in agricultural sector, 367, 368
aerobic conditions, 21 in animal feed sector, 366
agents, 18 in bakery industry, 364
application, 238, 244 β-glucosidases, 261
atrazine, 249 in biofuel industry, 359, 360
biological processes, 19 in biomedical sector, 364–366
chemical pollutants, 22 in food processing, 361, 362
classifications, 20 paper industry, 360, 361
definition, 18 pulping process, 360, 361
extracellular enzymes, 18, 19 in textile industry, 363, 364
fungal enzymes, 19, 31–34, 239–240 in waste management, 368, 369
(see also Enzymes) in wine and brewery industry, 362, 363
fungus, 19 Biotechnology, 312
hazardous chemical by fungi, 243 Bisphenol A, 201
limitations, 25, 26 Branched absorbing structures (BAS), 325
metabolic potential, 21, 22 Breakthrough curve (BTC) behavior, 411
microbes, 20, 21 Brown rot fungi (BRF), 11, 12, 357, 386
microbial bioremediation, 19, 20 Brunauer–Emmett–Teller (BET), 408
microorganisms, 20 Bubble column reactor, 414
monooxygenases act, 246
nitroaromatic compounds, 247
oil contaminated soil, 244 C
organic pollutants, 18 C1-compounds, 53
persistent organic pollutants, 249 Carbohydrate esterases (CEs), 10, 11
phytoremediation, 19 Carbohydrate-active enzymes (CAZymes), 4,
pollutants, 238 6, 9, 10, 12, 13
types, 20, 22, 243 Carbon sequestration, 286, 287
bioattenuation, 23 Carboxymethyl cellulase (CMCase), 132,
bioaugmentation, 23 301, 303
biopiles, 25 Catalase, 244
biostimulation, 22 Catalase activity, 443
bioventing, 25 Catalytic mechanism, 156
mycoremediation/fungal remediation, 26 Cation channels and transporters
Bioremediation mechanism, 199 CNGCs, 337, 338
Bioremediation processes, 467 Na+/H+ antiporters, 336, 337
environmental toxicity, 464 Cellobiohydrolase, 7, 10, 11, 132, 257, 267,
fungal enzymes, 465 387
metabolic potential, 463 Cellobiose, 257, 258, 263, 267, 271
oxidoreductases, 464 Cellulase
pH ranges, 464 alkaline, 305
white rot fungi, 464 application, 361
xenobiotic pollutants, 464, 465, 467 biocatalysts, 303
xenome, 464 deinking photocopier papers, 306
502 Index
Cellulase (cont.) Deinking process

endoglucanase, 353 biological, 299
enzymatic deinking, 307 cellulase, 306
enzymatic pulping process, 305 chemical, 299
exoglucanase, 353 cutinase, 312
fungal, 303 enzymatic technologies, 300
fungi vs. bacteria, 306 enzymatic treatment, 299
and hemicellulase, 303 laccase, 310
inverting mechanism, 354 lipase, 312
isolated Bacillus strains, 306 microbial enzymes, 300
laccase mediator system, 307 xylanase, 309
methods, 306 Depolymerization, 2, 3, 6–9, 12
office wastepaper, deinking, 306 Desorption, 413
OMG, 306, 307 Deuteromycete fungus, 157
ONP, 306, 307 Deuteromycetes family, 155
retaining mechanism, 354 Differential scanning calorimetry (DSC), 408
Cellulose 6-Dimethoxyphenol (DMP), 125
in cotton fibres, 257 Dubinin–Radushkevich (D-R) equation, 407
enzymatic hydrolysis, 258 Dye-decolorizing peroxidases (DyPs), 39
microfibrils, 257 Dye degradation, 162
Cellulosome, 262 Dye industry, 76
Cephalosporins, 122 Dyeing process, 79
Chemical oxygen demand (COD), 299 CETP, 78
Chitin, 400 chemicals, 80
Chitin polymer molecule, 401 effluents, 80
Chlorophenols, 119, 240, 248 Dyes, 400
Chlorophyll, 333 Dyestuffs, 75
Chromium-inducing toxicity, 443
Chrysene, 217
Column adsorption study, 410–411 E
Common Effluent Treatment Plants Ecosystem’s equilibrium, 201
(CETP), 78 Ecosystems, 290
Coniferyl alcohol, 384 Effluents containing dyes, 93
Contaminants Elovich equations, 407, 408
HMs (see Heavy metals (HMs)) Endoamylases, 311
Conventional techniques, 397 Endocrine disrupting chemicals (EDCs), 465
Coralene Golden Yellow (CGY), 92 Endocrine disrupting compounds (EDCs), 165
Cosmetic industries, 121 Endoglucanase, 257, 258, 262, 269, 270,
Crude laccase, 92 353, 387
Cutinase, 312 Energy-and cost-efficient passive
Cyclic guanosine monophosphate (cGMP), 337 phytoremediation methods, 438
Cyclic nucleotide-gated ion channels Enterococcus pseudoavium, 311
(CNGCs), 337, 338 Environmental contamination
Cyclic nucleotides monophosphate metallic properties, 195
(CNMP), 337 soil and water, 194
Cyclosporin A, 122 toxic compounds, 194
CYP monooxygenases (P450s), 464 Enzymatic deinking, 307
Cytochrome P450 (CYP), 225, 241, 247 Enzymatic system, 221, 224
Enzyme commission (E.C.) number, 35
Enzyme immobilization methods, 161
D Enzymes
Decolouration, 84, 85, 88, 90–92, 120 active sites, 31
Dehalogenases, 248 advantages, 40, 41
Dehydrins, 336 apoenzyme, 35
Index 503
biological moieties, 31 polluted environments, 221

classification and types, 191 terrestrial and aquatic habitats, 221
disadvantages, 41 Fungal consortium
E.C. number, 35 bioethanol production, 391
holoenzyme, 35 biomass, 384
scope, 42 development, 392
types, 35 ethanol production, 392
catalase, 38 laccase and xylanase enzymes, 384
laccases, 36, 37 Fungal decolourization processes, 93
oxidoreductase, 36 Fungal divisions, 402
peroxidases, 38–40 Fungal enzymes
Epoxide hydrolases (EHs), 225 agricultural wastes, 358
Ergot alkaloids, 491 agro-industrial wastes, 359
Ethanol application, 479
production, 382, 383, 391 biodegradation, 495
Ethanol blending program (EBP), 382 bioremediation, 239–240, 465, 475
Ex situ bioremediation technique, 20, 21 catalytic reaction activity, 191
Exoglucanases, 257, 258, 262, 269, EDCs, 190
270, 353 food and feed industries, 494
Extracellular oxidative enzymes, 193 fungal extracellular enzymes, 191
geophysical area, 190
high-cost food, 491
F hydrocarbons, 191
Facilitated diffusion system (FDS), 390 immobilization, 480
Ferulic acid, 386 laccase biosynthesis, 358
Fibre-reactive azo dye, 75 limitations, 479, 482
Filamentous fungi, 453 marine fungi, 480, 481
Filamentous organisms mediators, 480
growth process, 402 medicine, 494
industrial waste product, 402 multicellular fungal mycelium, 189
Fixed-bed experimental setup, 411 non-protein enzyme mimics, 481
Fluidized bed reactor, 414 oxidoreduction reactions, 192
Fluoresomidearyl glucoside, 242 remediation, 243, 244, 250
Fluoroquinolones, 242 secondary metabolites, 190, 491
Food industry, 120 size and shape, 189
Free radicals, 468 SSF, 358
Freundlich model, 406 synthesis of xylanase, 359
Fungal biomass value-added products, 492
acid pretreatment, 404 xenobiotic detoxification (see Xenobiotic
biomass, 404 detoxification)
thermal treatment, 405 xenobiotic pollutants, 465, 467
Fungal bioreactor, 414 xenobiotic remediation (see Xenobiotic
Fungal biosorbent, 408 remediation)
Fungal biosorption, 413 Fungal metabolites, 491
Fungal biotechnology, see White Fungal mycelium, 26
biotechnology Fungal pellet reactor, 415
Fungal cellulase, 303 Fungal peroxidase
Fungal colonization, 26 distal histidine, 168
Fungal communities mechanisms, 168–169
bacterial and archaeal, 229 Fungal phytoremediation
contaminated soils, 228 bioremediation, 444, 454
indispensable tool, 222 categorical classification, 444
PAH-contaminated soil, 222 eco-friendly, 454
PAH degradation, 226, 227 factors, 451
504 Index
Fungal phytoremediation (cont.) play, 290

fungal species, 453 secondary metabolites, 491
growth requirements, 452 soft rots, 357
HMs (see Heavy metals (HMs)) sustainable fungal phytoremediation, 438
hydrocarbon, 452 usage, 438
mechanisms, 450, 451 wastewater treatment, 398
metal contamination, soil, 448 white rots, 356
pH, 452 xylanolytic enzymes, 357, 358
precautions, 453 Fungus, 286, 402
prerequisite precautions, 453 2,5-Furandicarboxylic acid (FDCA), 129
redox potential, 452
species, 444
temperature, 451, 452 G
Fungal species Galacto-oligosaccharides, 266
AMF, 30 Generally Regarded As Safe (GRAS), 137
dye decolorization, 27 Genetic diversity, methylotrophic yeast
ectomycorrhizal fungi, 30 evolutionary analysis, 58
electrostatic interactions with metals, 27 expression system, 58
extremophilic fungi, 29, 30 gene regulation study, 54
laccases, 26, 27 H. polymorpha, 57, 58
marine fungi, 28, 29 LSU rRNA, 59
white-rot fungi, 26–28 methanol utilisation, 59
wood degraders, 27 O. Polymorpha, 59
yeasts, 27 phylogenetic analysis, 58
Fungal strains, 390 Pichia pastoris, 58
Fungal unspecific peroxygenases, 246 Pichia strains, 57
Fungi SSU rRNA, 59
AM, 438 strains, 60
bioherbicides, 493 woody materials, 56
biotransformation processes, 491 Genetically engineered microbes (GEMs), 23
brown rots, 357 Global forest soils efflux, 288
and carbon, 285 Global sustainable environments, 429
detoxification, environmental Glucanases, 10
chemicals, 438 Glucomannans, 1
ecological and biochemical capacity, 438 Glucosidic isoflavones, 264
ecosystems, 438 Glycoside hydrolases (GHs), 9–11
factor, soil, 288 Glycosyltransferase (GT), 4, 350
fermentative industries, 492 (see also Glyoxal oxidase (GLOX), 106
Fungal enzymes) Golgi apparatus, 350
growth Greenhouse gases (GHGs), 283
ecosystem services, 289 Guaiacyl, 104
fertilizer, 289–290
pesticides, 290
physical disturbance, 289 H
soil moisture, 290 Haloalkaliphilic bacteria, 301
soil pH, 289 Hansenula polymorpha, 65, 66
temperature, 288 Hard wood spent sulfite liquor (HSSL), 383
high-value products, 491 Hazardous chemical
lignin-degrading enzyme producer, 356 bioremediation, 243
literature, 291 definition, 237
and metals, 438 degradation, 241
pectinolytic enzymes, 357 environmental, 237
pharmaceuticals, 491 fungal degradation, 248, 249
plant growth promoters, 493 Heavy metal removing efficiency, 404
Index 505
Heavy metals (HMs) Indian dyestuff industry, 76

alkaline desorption solution, 198 Industrial applications
AM fungi, 438 lignocellulose process, 104–106
anthropogenic in VP, 127
essential nutrients, 439 Industrial biotechnology, 496
sources, 439 Industrial dye effluents, 92–93
water resources, 440, 441 Industrial Wasteland Soil, 425–427
binding, 197 Inorganically Managed Agricultural Soil,
bioaccumulation, 439 424–425
living beings, 442 Intergovernmental Panel on Climate Change
MEUF, 439 (IPCC), 283
on humans, 442 Intra-radical arbuscules, 325
Ni and Cu, 198 Ion influx, 337
nutrients availability, 197 Isobutyl-galactosides, 266
on plants, 442, 443 Isoflavone glycosides, 264
phytoremediation, 444
Heme-thiolate peroxidase (HTP)
applications, 131 K
biochemical characteristics, 130, 131 Konzo syndrome, 266
catalytic properties, 130 Kraft lignin, 105
classification, 130 Kraft process, 361
CPO, 130
Hemicellulase, 266, 312
xylan, 355 L
xylanase, 355 Laccase-based bioremediation, 158
xylanolytic enzyme system, 356 Laccase catalytic cycle, 85
xylooligosaccharides, 356 Laccase enzyme, 160
Hemicelluloses, 1, 2, 7, 9, 10, 262, 264, 267, 268 Laccase-mediator systems (LMSs), 307,
Hemicellulosic fraction, 386 313, 475
Hidroxywarfarin, 242 Laccase producing fungi, 155
High molecular weight (HMW), 202 Laccases (LACs), 83, 84, 107, 114, 116, 244,
High molecular weight PAHs 309, 310, 467
(HMW-PAHs), 247 agriculture and industrialization, 158
Histidines, 114 applications, 118
Homopolysaccharide, 267 biological functions, 158
Humic substances (HS), 173 bioprocessing, 116, 117
Hydrolases, 193–194 bioremediation, 154
Hydrolytic enzymes catalytic domain, 157
cellulose/hemicellulose, 121 covalent attachment, 161
class, 137 cross-linked method, 162
complex lignocellulosic materials, 102 detoxification
and non-hydrolytic, 107 copper involvement, 475
Hydrophobic cluster analysis (HCA), 258 LMSs, 475
Hydroxyalkylation, 105 multicopper oxidases, 475
4-Hydroxybenzoic acid, 386 redox potentials, 476
2-Hydroxycarbamazepine, 242 TNC, 476
Hydroxyflumequine, 242 type I copper, 475
Hydroxylation, 204 type II copper, 476
type III copper, 476
encapsulation, 161
I enzyme-supportive matrix, 161
In situ bioremediation technique, 20, 21 extracellular and intracellular enzymes, 154
Indian Agro and Recycled Paper Mills functions, 156
Association (IARPMA), 299 fungal laccases, 156
506 Index
Laccases (LACs) (cont.) Lignocellulose bioconversion

fungi and plants, 156 challenges, 268
glycosylation, 156 end-product inhibition, 267
growing fungi, 159 plant biomass, 267
immobilization, 159 pre-treatment methods, 267
mechanism, 157 Lignocellulose degradation, 263, 267, 271
paper and pulp industry effluents, 154 Lignocellulosic biomass
polyphenol oxidase, 154 applications (see Biotechnological
remediation applications)
applications, 468, 469 cellulase, 353, 354
aromatic compounds, 468 fungi, 356
detoxification of PCBs, 468 hemicellulase, 355, 356
free radicals, 468 hemicellulose, 350, 351
ligninolytic enzymes, 468 lignin polymer, 350
PAHs, 468 ligninase, 351–353
pH range, 468 monolignol biosynthesis, 350
toxic dyes, 469 morphological diversity of PCW, 349
structure, 156–157 PCW, 349
toxic environment, 159 pectinase enzyme, 354, 355
types, 156 RTC, 350
Langmuir isotherm model equation, 406 Lignolytic fungi, 26
Late embryogenesis abundant (LEA) Lignosulfonate lignin, 105
proteins, 336 Lignosulfonates, 106
Leghemoglobin, 334 LiP and MnP enzymes, 172
Legume colonization, 334 Lipases, 137, 311, 312
Leucine pNA (LPNA), 135 Lovastatin, 491
Lichens, 287 Lytic polysaccharide monooxygenases
Lignin, 298 (LPMOs), 7, 10
polymer monomeric units, 103
Lignin degradation, 390
Lignin-degrading enzymes actions, 107 M
Lignin-modifying enzymes (LME), 192, 387 Manganese peroxidase (MnP), 40, 81–84, 88,
Lignin peroxidase–graphite electrode 106, 122, 170, 243, 245, 249, 464
biosensor systems, 171 application, 124
Lignin peroxidase (LiP), 39, 40, 81, 83, characterization, 122, 123
106, 125, 169–170, 243, 245, 249, mechanism of action, 123, 124
387, 464 Manganese peroxidases, 40, 170
Lignin vs. xenobiotic chemical Mannans, 1
structures, 466 Mediators, 467
Ligninase Meiosporic ascomycetes, 240
enzymatic reactions, 352 MEROPS database, 135
heme peroxidases, 352 Metagenomics, 271
laccase, 353 Metal pollutants, 438
LiPs, 352 Metal tolerance, 438
MnP, 352 Metal toxicity, 442, 443
oxidoreductive class, 351, 352 Metaproteomics, 222
Ligninolytic extracellular enzymes, 171 Methane cycle, 54
Ligninolytic fungi, 123 Methanol, 54
Ligninolytic oxidoreductases, 106 Methanol oxidising genes, 54
Lignins, 102 Methylotrophic yeast
Lignocellulose, 382, 384, 387, 389 biotechnological application, 64
biorefineries, 127 environmental impact
cellulose and hemicellulose, 129 bioremediation, 64
and industrial applications, 104–106 bioremediation, soil, 66
Index 507
biotechnological applications, 63 Penicillium citrinum, 302

heavy-load wastewater treatment photocopier printers, 299
process, 65 pulp, 298
lactate-selective microbial biosensor, 65 pulp and paper industry, 297
oil-contaminated soil, 65 SSF, 303
Pichia pastoris, 65 sustainability issues, 299
plant growth promotion, 63 thermophilic, novel, 301
pollutants, 65 wastepaper (see Wastepaper)
genetic diversity, 55–56 (see also Genetic whole-cell hydrolysates, 301
diversity, methylotrophic yeast) xylanase, 303 (see also Xylanase)
genetic regulation, 60, 62, 63 Microeukaryotes, 220
glycerol metabolism, 54 Microorganism, 238, 240, 241, 243, 245, 249
metabolism and physiology, 57 autochthonous, 227
methane cycle, 54 PAH-degrading, 219
methanol Microwaves, 105
inducible gene expression, 61 Middle lamella layer, 349
metabolism, 62 Mitomycin, 122
oxidising genes, 54 Mixed office wastepaper (MOW), 312
methanol utilisation, 53 Molecular biological techniques, 24
recycling, carbon, 54 Monolignol biosynthesis, 350
Micellar-enhanced ultrafiltration (MEUF), 439 Monooxygenases, 246
Microbial enzymes Mucoromycotina, 241, 242, 244
alkaline-tolerant fungi, 301 Multicopper oxidases, 83
amylases, 310, 311 Multifarious techniques, 409
API, 302 Municipal office waste (MOW), 299
avicelase, 303 Mushroom enzymes, 137
biological deinking, 299–300 Mutagenesis, 270
biological process, 299 Mycoremediation, 189, 195, 444, 454
biotechnical applications, 298 biotransformation, 200
cellulases (see Cellulases) exclusion, 199
CMCase, 303 extrusion, 199
Crude cellulose, 302 fixation, 200
cutinase, 312 stress condition, 199
deinking process, 299 Mycoremediation/fungal remediation, 26
deinking wastepapers, 297 Mycorrhiza, 429, 453
EndoA, 302 Mycorrhizal-based technology, 426
EndoB, 302 Mycorrhizal fungi hyphae, 286
enzymatic technologies, 300 Mycorrhization, 429
enzymatic treatment, 299 Mycorrhizosphere, 419
haloalkaliphilic bacteria, 301 Mycosorption, 429
Heleococcum alkalinum sp., 301
hemicellulase, 312
IARPMA, 299 N
laccase Na+/H+ antiporters, 321, 337
alkaliphilic laccase activity, 310 Nanomaterial-based approaches, 398
bleaching, kraft pulp, 310 Naphthalene, 204–205, 217, 228
ink removal capability, 310 National Oceanic and Atmospheric
plants and fungi, 310 Administration (NOAA), 284
lignin, 298 Natural Biomass Utilization Systems
lipases, 311, 312 (NBUS), 3
MOW, 299 Nitrogen fixation, 334
natural polymers, 297 Nitrophenols, 119
novel alkalothermophilic actinomycete, 301 Nitroreductases, 247
papermaking, 298 Nodulation, 334
508 Index
Nucleotide sequence identity (NCI), 258 remediations

Nutrient uptake, 320, 325–328 LiP, 469, 470
MnPs, 470, 471
P450s, 472, 473
O UPOs, 474
Ogataea polymorpha, 59 VP, 471
Ogataea thermomethanolica TBRC656, 62, 63 types, 166
Old magazine (OMG), 306 versatile, 40
Old newspaper (ONP), 306 Persistent organic pollutants (POPs), 203
Organic biomass, 383 Pesticides, 194, 195, 200–205, 240, 242, 248,
Organic compounds, 200 249, 290
Organically Managed Agricultural Soil, 425 pH, 452
Organosolv lignin, 105 Phanerochaete chrysosporium, 9, 10
Organosolv process, 105 Pharmaceutical industries, 121, 398
Osmoregulation, 330 Pharmaceutical personal care products
Osmotic stress, 336 (PPCPs), 190
Oxidoreductases, 192–193, 464 Pharmaceutical protein production system, 66
Oxyalkylation, 105 Phenanthrene, 217, 224, 225, 228, 229
Oyster mushroom, 101, 102 Phenol 2-monooxygenases, 246
Phenol oxidases, 192
Phenylpropane lignin, 106
P Phenylpropene monomers, 103
P5CS-encoding gene, 335 Phosphorus(P)-based fertilizers, 289
Packed Bed Column Model, 411–412 Photocopier printers, 299
BDST model, 412 Photocopier wastepaper, 306
kinetic data, 413 Photosynthates, 286
Thomas model, 411 p-Hydroxyphenyl, 104
Paper and pulp industry, 173 Phylogeny, 55, 59
Paper industries, 121, 162–164 Phytoremediation, 19, 437, 444
P-coumaryl alcohol, 384 Pichia pastoris, 58, 61, 63
Pectin esterase, 354 Pichia strains, 57
Pectinase, 133, 134 Plant cell wall (PCW), 349
hydrolases, 354, 355 Plant growth promoters, 491–493
lyases, 355 Pleurotus ostreatus
pectin esterase, 354 amylases, 134, 135
Pectinolytic enzymes, 357 cellulases, 132
Pectinylase (PL), 134 different enzymes, 108–113
Peroxidase, 245 laccase isoenzymes, 116
Peroxidase catalytic activity, 169 lipases, 137
Peroxidase–catalase superfamily manganese peroxidase IV, 123
(PCATS), 167 pectinase, 133, 134
Peroxidase-cyclooxygenase superfamily proteases, 135, 136
(PCOXS), 166 PUF cubes, 118
Peroxidases, 166, 241, 243, 245–247 xylanases, 131–133
detoxification Polluted soils
cytochrome P450, 478 metatranscriptomics, 222
LiPs, 476, 477 microbial diversity, 229
MnPs, 477 PAH, 220–223, 228
heme peroxidases, 38, 39 Polyamines, 331
lignin, 39, 40 Polyaromatic hydrocarbons (PAHs), 18, 119,
LiP, 166 464, 465, 468
manganese, 40 biodegradation, 202–203
non-heme peroxidases, 39 contamination, 202
PCOXS, 166 oxidation, 202
Index 509
Polychlorinated biphenyls (PCB), 164, 248 Reactors, 414

laccase-mediated degradation rate, 164 Recalcitrant, 221, 238, 248, 249
Polychlorinated dibenzofurans (PCDFs), 248 Recombinant DNA technology, 24, 491
Polychlorinated dibenzo-p-dioxins Redox mediators, 157
(PCDDs), 248 Redox potentials, 168, 452
Polycyclic aromatic compounds (PAC), 217 Remazol Brilliant Blue R (RBBR), 119, 120
Polycyclic aromatic hydrocarbon (PAH), 164, Remazol Brilliant Blue SN4R, 120
174, 240–242, 247–249, 452 Response surface methodology (RSM), 85
atmospheric and aquatic transport, 219 Resting oxidized state (RO), 476
barcode-primer pair, 223 Rhizo-deposited carbon, 287
biological sciences, 230 Rhizo-deposition, 287
contaminated soils, 227–229 Rhizo-deposition material, 290
culture-dependent techniques, 222 Rhizospheric soil, 423
culture-independent techniques, 222, 223 Rosette terminal complex (RTC), 350
environments, 217, 220
extracellular mechanism, 224, 225
fungal communities, 226, 227 S
fungal mechanisms, 223, 224 Saline soil
genetic engineers, 230 AM fungi, 321
heterocyclic, 218–219 and irrigation, 320
intracellular enzymatic pathways, 225, 226 salt concentration, 319
microbial communities, 230 Salinity stress remediation
phenanthrene degradation pathway, 225 biochemical change
photodecompositions, 219 antioxidant system, 332
physicochemical properties, 217 osmotic adjustment, 330, 331
polluted soils, 220–222 osmotic stress, 330
Polysaccharide lyases (PL), 4 phytohormones regulation, 331
Polyurethane foam (PUF), 134 polyamines, 331
Polyvinylpyrrolidone (PVP), 362 molecular changes, plants
Pot-culture technique, 421 aquaporins, 335, 336
Powdered activated carbon (PAC), 18 heterokaryotic and obligate
Prerequisite precautions, 453 nature, 335
Primary cell wall (PCW), 349 LEA proteins, 336
Proline, 330, 335 P5CS, 335
Proteases, 135, 136 physiological changes
Proton symport (PS) mechanism, 390 chlorophyll content, 333
Proxidases nitrogen fixation, 334
classification, 167 nodulation, 334
Pulping process, 360 relative permeability, 333
Pyrroline-5-carboxylate reductase (P5CR), 330 water, 333, 334
Pyrroline-5-carboxylate synthetase (P5CS), Salt-affected soils, 319
330, 335 Salt-stressed soil, 321
Secondary cell wall (SCW), 349
Secretome
Q fungal, 4, 7
Quinone reductases, 247 wood-degrading fungi, 2
Sexual basidiospores, 241
Sinapyl alcohols, 2, 384
R Single-spore culture, 422–423
Reactive Black 5 (RB5), 85 Soil, 319
Reactive dyes, 75 Soil bioremediation, 119, 205–206
Reactive nitrogen species (RNS), 442 Soil fungi, 285
Reactive oxygen species (ROS), 38, 221, 244, Soil moisture, 290
325, 442 Soil organic matter (SOC), 284–285
510 Index
Soil salinity 1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane

AMF, 326 (DDT), 248
development, salt-tolerant crop Trinitrotoluene (TNT), 18, 240, 242,
varieties, 321 247–249
dissolved mineral salts, 325 Trinuclear cluster (TNC), 476
nutrient imbalance, 325 Triphenylmethane, 119
nutritional disorders, 320 2,4-Dichlorophenol (2,4-DCP), 127
physiological stresses, 325 Tyrosinases, 244
plant cell membranes, 325
reduced growth, 320
salt concentrations, 320 U
significance, 320 Ultrasonication, 386
stress signals, 325 United Nations Environment Program
Soil salinization, 319, 320 (UNEP), 319
Solid–liquid operations, 168 Unspecific peroxygenase (UPOs),
Solid-state fermentation (SSF), 132, 303, 39, 474
358, 386
Sorption mechanism, 405
Starch and cellulose, 287 V
Stirred tank reactor (STR), 414 Veratryl alcohol, 245
Stress tolerance, 324 Versatile peroxidases (VP), 40, 170, 172, 193,
Subphylum mucoromycotina, 240 245, 471
Subtropical agricultural soil, 423–424 applications, 127
Symbiosis, 427–429 biochemical characteristics, 125
Syringaldazine, 116 CIP, 125
Syringyl, 104 lignin manganese peroxidases, 124
ligninolytic enzyme, 125
in Pleurotus sp., 124
T structural characteristics, 125, 127
Temkin isotherm, 407 Vesicular-arbuscular mycorrhizae, 493
Temperature, 451, 452 Vinblastine, 122
Textile dyes Volcanic eruption, 217
azo groups, 74
colourants, 74
fabrics, 74 W
Textile effluent treatment, 162 Wastepapers
Textile industries, 73, 76, 121 cutinase, 312
dye, 171 deinking
Theaflavin (TF), 362 biological process, 299
Thearubigin (TR), 362 chemical process, 299, 300
Thermochemical hydrolysis, 104 conventional methods, 297
Thermodynamic behavior, 405 enzymatic pretreatment, 311
Thermotolerance, 270 laser-printed office papers, 299
Thermotolerant yeast, 57 microbial enzymes, 299
Thomas model, 411 industrial utilization, 297, 313
Thyroid peroxidase (TPO), 166 raw materials, 298
Toxic agrochemicals, 174 Wastewater treatment, 119, 120, 398, 399
Trametes hirsute Water, 333, 334
fungus laccase, 115 Water toxication, 440, 441
Transcription factors, 62 White biotechnology
Transcriptome, 7–9, 13 advantages, 494
Transglycosylation, 261, 266 bio-based products, 496
Trap culturing, 422 eco-friendly, 494
Trichoderma viride, 302 greenhouse emissions, 494
Index 511
in medical, 491 X
renewable natural resources, 496 Xenobiotic compounds, 463
White rot fungi (WRF), 3, 6, 9, 10, 73, 153, Xenobiotic detoxification
173, 203, 224, 225, 356, 386–389, LACs, 475, 476
391, 464, 482 peroxidases, 476–478
colour reduction levels, 89 silico approaches, 481
decolouration, 90 Xenobiotic pollutants, 465
dye degradation, 86–88 Xenobiotic remediation
En3, 90 bioremediation processes, 467
enzymes, 89 fungal strains, 479
laccases, 80, 81, 83 heme peroxidases, 467, 479
ligninolytic enzymes, 73 heterologous expression, 479, 482
lignolytic systems, 85 LACs, 468, 469
LiP, 81 mediators, 467
manganese peroxide and lignin oxidoreduction-catalyzing enzymes, 467
peroxide, 84 peroxidases (see Peroxidaes)
MnP, 81 Xenobiotics, 242
nitrogen, 85 Xenome, 464
nitrogen-limited glucose ammonium Xylan, 307
media, 88 Xylanase, 9, 12, 132, 133, 307–309
oyster mushroom, 74 Bacillus pumilus SV-205, 308
recalcitrant nature, 93 biological treatment, 309
toxicity and xenobiotic nature, 92 biopulping process, 307
types, 81 deinked ONP pulp, 309
xenobiotic degradation capacity, 73 hydrolysis, hemicellulose, 307
Wood optimizing submerged fermentation
biopulping, 310 conditions, 308
cheap source, paper pulp, 312 pollution free environment, 308
chipping, 307, 310 SSF, 308, 309
debarking, 307 xylano pectinolytic, 309
lignin biodegradation, 297 Xylanolytic enzyme system, 356–358
pulp and paper industry, 297, 313 Xyloglucans, 1
screening, 307 Xylulose, 391
triglycerides, 312
Wood-degrading fungi, 2, 4, 7–9
Wood-rotting fungi, 106 Z
Woollen industry, 76 Zygospores, 241

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Fungal Biology

Ajar Nath Yadav

More information about this series at http://www.springer.com/series/11224

ISSN 2198-7777 ISSN 2198-7785 (electronic)

© Springer Nature Switzerland AG 2019

White biotechnology is industrial biotechnology dealing with the potential applica-

enzymes used for bioremediation for environmental cleaning. Chapter 3 by Kumar

Vice Chancellor Dr. H. S. Dhaliwal

Dr. H. S. Dhaliwal is presently the Vice Chancellor of

and molecular breeding. Dr. Dhaliwal made a signifi-

Perspective for environmental sustainability is to main-

White biotechnology is drawing much attention as a solution to producing enzymes

Baru Sahib, Himachal Pradesh, India Ajar Nath Yadav

1 Secretomics of Wood-Degrading Fungi and Anaerobic

8 Bioremediation of Polycyclic Aromatic Hydrocarbons (PAHs)

19 Fungal Enzymes for Bioremediation of Xenobiotic Compounds�������� 463

Farid Agouillal Research Unit on Analysis and Technological Development in

Ulises Conejo-Saucedo Department of Microbiology, Institute of Water Research,

Jagdeesh Kumar Department of Hydrology, Indian Institute of Technology

Priyanka Priyanka Department of Biochemistry, University of Lucknow,

Neha Vishnoi Department of Environmental Sciences, Babasaheb Bhimrao

Ajar Nath Yadav is an Assistant Professor in the

Biotechnology. Dr. Yadav is currently handling two proj-

Sangram Singh is an Associate Professor in the

Shashank Mishra is presently working as Scientist

Scientific Reports and Archive of Phytopathology and

Arti Gupta is an Assistant Professor in the

(MSET); and Dr. V.P. Agarwal Gold Medal by D.A.V.

Nasib Singh and Joginder Singh

Lignocellulose is a widely available recalcitrant plant biopolymer composed of

© Springer Nature Switzerland AG 2019 1

Hemicelluloses are considered as the second most abundant polysaccharide in

1.2 Degradation and Depolymerization of Plant Biomass

1.3  ecretomics and Mechanism of Lignocellulose

hemicellulose components. This activity is achieved in white rot fungi by extracel-

of cellobiohydrolases (GH families GH6 and GH7), β-glucosidases (GH1 and

1.4 Wood-Degrading Fungi

mechanistic details of lignocellulose degradation by various species of bacteria and

Wood-degrading fungi mostly belong to the class Agaricomycetes of phylum

1.4.1 Secretomes of White Rot Fungi (WRF)

species mediated by peroxidases which facilitate physical disruption of crystalline

1.4.2 Secretome of Brown Rot Fungi (BRF)

from Wolfiporia cocos growing on different media containing glucose, purified

1.5 Secretome of Anaerobic Rumen Fungi (ARF)

ARF have indispensable role in digestion of recalcitrant lignocellulosic feed materi-

In spite of the availability of enormous amount of plant matter, woody substances,

Reina R, Kellner H, Hess J, Jehmlich N, García-Romera I, Aranda E, Hofrichter M, Liers C (2019)

Neha Vishnoi and Sonal Dixit

© Springer Nature Switzerland AG 2019 17

Bioremediation is considered as one of the safer, cleaner, cost-effective, and eco-­

MICROBIAL BIOREMEDIATION PHYTOREMEDIATION

Oxidoreductase Lyases Peroxidase Hydrolase Dehalogenase

Fig. 2.1 An overview of types of bioremediation

Fig. 2.2 An outline of

Solid Phase Slurry Phase

are known to act as agents of bioremediation under laboratory conditions. Microbes

pollutant-­degrading characteristics) from various contaminated environments, or it

2.2.1 Types of Bioremediation

It is also known as natural attenuation which means elimination of varying concen-

Baru Sahib, Himachal Pradesh, India Ajar Nath Yadav

1 Secretomics of Wood-Degrading Fungi and Anaerobic

8 Bioremediation of Polycyclic Aromatic Hydrocarbons (PAHs)

19 Fungal Enzymes for Bioremediation of Xenobiotic Compounds�� 463

1.2 Degradation and Depolymerization of Plant Biomass

1.3 ecretomics and Mechanism of Lignocellulose

1.4 Wood-Degrading Fungi

1.4.1 Secretomes of White Rot Fungi (WRF)

1.4.2 Secretome of Brown Rot Fungi (BRF)

1.5 Secretome of Anaerobic Rumen Fungi (ARF)

Bioremediation is considered as one of the safer, cleaner, cost-effective, and eco-

pollutant-degrading characteristics) from various contaminated environments, or it

2.2.1 Types of Bioremediation

2.2.2 Limitations of Bioremediation

2.3 Fungi as Bioremediator

p ollutants under different environmental conditions. Fungal enzymes have also

2.3.1 White-Rot Fungi

2.3.2 Marine Fungi

2.3.3 Extremophilic Fungi

2.3.4 Symbiotic Fungi with Plants

2.4 Fungal Enzymes in Bioremediation

2.4.1 Enzymological Background

2.4.2 Advantages of Enzymatic Bioremediation

2.4.3 Disadvantages of Enzymes

2.4.4 Scope of Enzymatic Bioremediation

2.5 Conclusion and Future Prospects

3.2 Genetic Diversity of Methylotrophic Yeast

3.3 Genetic Regulation in Yeast Methylotrophy

3.4 Methylotrophic Yeast and Impact to the Environments

3.5 Conclusion and Future Prospects

4.2 Textile Dyes

4.3 Dyes Used in India

4.4 escription of Dyeing Process and Sources of Effluent