8132227719

Springer Proceedings in Energy
Sachin Kumar
Samir Kumar Khanal
Y.K. Yadav Editors
Proceedings of the
First International
Conference on Recent
Advances in Bioenergy
Research
More information about this series at http://www.springer.com/series/13370
Sachin Kumar Samir Kumar Khanal
•
Y.K. Yadav
Editors
Proceedings of the First

International Conference
on Recent Advances
in Bioenergy Research
123
Editors
Sachin Kumar Y.K. Yadav
Sardar Swaran Singh National Institute Sardar Swaran Singh National Institute
of Bio-Energy of Bio-Energy
Kapurthala, Punjab Kapurthala, Punjab
India India
Samir Kumar Khanal

Department of Molecular Biosciences
University of Hawai’i at Mānoa (UHM)
Honolulu, HI
USA
ISSN 2352-2534 ISSN 2352-2542 (electronic)

ISBN 978-81-322-2771-7 ISBN 978-81-322-2773-1 (eBook)
DOI 10.1007/978-81-322-2773-1
Library of Congress Control Number: 2016935576
© Springer India 2016

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Preface
The increasing prices of crude oil, the depleting nonrenewable energy resources,
and the growing concern for climate change have led to a significant interest in
renewable energy, including bioenergy. There is a need to shift the current energy
spectrum to green energy by creating advanced technologies to utilize renewable
resources (such as agro-forestry biomass) for bioenergy production to keep pace
with the growing demand for energy. The research in bioenergy is receiving
unprecedented attention from all over the world due to the potential of biofuels as a
replacement for fossil fuels.
The First International Conference on “Recent Advances in Bio-energy
Research” (ICRABR-2015) organized by the Sardar Swaran Singh National
Institute of Bio-Energy as an attempt to bring together researchers to form a
meaningful conversation on directions of bioenergy. This proceedings volume
comprises the top 26 papers contributed by authors who participated and presented
their findings at ICRABR 2015. These chapters are peer-reviewed by experts in the
field.
We hope that the contents of this volume will help researchers, students, and
policy makers involved in this research field.
Kapurthala, India Sachin Kumar

March 2015 Samir Kumar Khanal
Y.K. Yadav
v
Acknowledgements
First, we would like to thank all the participants of ICRABR-2015, who contributed
their findings to prepare these proceedings in a comprehensive nature. Apart from
the authors, we would also like to acknowledge the reviewers for their constructive
and valuable comments and suggestions to improve the chapters that significantly
improved the quality of the proceedings:
Prof. Pranab Goswami, IIT Guwahati, India

Prof. Toshinori Kojima, Seikei University, Japan
Prof. B.S. Pathak, SPRERI Vallabh Vidyanagar, India
Dr. S. Dasappa, IISc Bangalore, India
Dr. D.K. Adhikari, CSIR-IIP Dehradun, India
Prof. B. Viswanathan, IIT Madras, India
Prof. M.M. Ghangrekar, IIT Kharagpur, India
Dr. Venkat Mohan, IICT Hyderabad, India
Dr. Dilip Ranade, ARI Pune, India
Prof. R.K. Behl, HAU Hisar, India
Prof. A.K. Jain, CUP Bathinda, India
Prof. I.M. Mishra, ISM Dhanbad, India
Dr. Rupam Kataki, Tezpur University, India
Prof. K. Mohanty, IIT Guwahati, India
Dr. Urmila Phutela, PAU Ludhiana, India
Prof. Rajesh Sani, South Dakota School of Mines and Technology, USA
Dr. D.K. Sahoo, CSIR-IMTech Chandigarh, India
Dr. Anuj K. Chandel, CTC, Brazil
Prof. Anil Kumar, Devi Ahilya University Indore, India
Dr. Pratibha Dheeran, CPUT, South Africa
Dr. R.C. Mathur, DBT-IOC Center Faridabad, India
Dr. P.V. Bhale, S.V. National Institute of Technology, Surat, India
Dr. Biplab K. Debnath, NIT Meghalaya, India
Prof. D.C. Deka, Gauhati University, India
vii
viii Acknowledgements
Dr. V. Sivasubramanian, PERC Chennai, India

Dr. D.K. Bora, AEC Guwahati, India
Dr. Neeraj Atrey, CSIR-IIP Dehradun, India
Dr. Nasreen Munshi, Nirma University Ahmadabad, India
Dr. S.K. Tyagi, SSS-NIBE, Kapurthala, India
Dr. Sasikumar Elumalai, CIAB Mohali, India
Dr. T. Bhaskar, CSIR-IIP Dehradun, India
Dr. S.K. Soni, PAU Ludhiana, India
Dr. A.K. Sarma, SSS-NIBE, Kapurthala, India
Dr. V.P. Sethi, PAU Ludhiana, India
Dr. Surender Singh, IARI, New Delhi, India
Dr. Neha Mittal, University of North Carolina at Charlotte, USA
Dr. Madhulika Shukla, SSS-NIBE, Kapurthala, India
Kapurthala, India Sachin Kumar

October 2015 Samir Kumar Khanal
Y.K. Yadav
Organizing Committees
International Advisory Committee

Dr. Robert Dekker, Lakehead University, Canada
Prof. Mohammed Farid, University of Auckland, New Zealand
Prof. S.C. Bhattacharya, International Energy Initiative, India
Prof. B.S. Pathak, SPRERI, India
Prof. Rajesh Sani, South Dakota School of Mines and Technology, USA
Prof. Toshinori Kojima, Seikei University, Tokyo, Japan
Prof. Rakesh Bajpai, University of Louisiana, USA
Prof. S.K. Sharma, PU Chandigarh, India
Dr. Samir K. Khanal, University of Hawaii, USA
Prof. Mohammad J. Taherzadeh, University of Boras, Sweden
Dr. Giuliano Grassi, European Biomass Industry Association
Prof. Giuliano C. Premier, University of South Wales, UK
Prof. I.M. Mishra, IIT Roorkee, India
Dr. Badal C. Saha, US Department of Agriculture, USA
Dr. Lalini Reddy, CPUT, South Africa
National Advisory Committee

Prof. Subhash Chand, IIT Delhi
Prof. S. Dasappa, IISc Bangalore
Prof. A.K. Jain, CUP, Bathinda
Prof. U.C. Banerjee, NIPER, Mohali
Prof. V. Sivasubramanian, Director-Tech, PERC, Chennai
Dr. D.K. Adhikari, IIP, Dehradun
Dr. Dilip Ranade, ARI, Pune
Prof. D. Das, IIT Kharagpur
Prof. Pranab Goswami, IIT Guwahati
Prof. V.K. Vijay, IIT Delhi
ix
x Organizing Committees
Prof. M.M. Ghangrekar, IIT Kharagpur

Prof. M.K. Jha, NIT Jalandhar
Dr. Ashok Pandey, NIIST, Trivandrum
Dr. D.K. Sahoo, IMTech, Chandigarh
Dr. R.K. Behl, HAU, Hisar
Dr. S.S. Sooch, Director, SES, PAU, Ludhiana
Dr. Kaustubha Mohanty, IIT Guwahati
Contents
Part I Biochemical Conversion

Comparison Between Separate Hydrolysis and Fermentation
and Simultaneous Saccharification and Fermentation Using Dilute
Acid Pretreated Lignocellulosic Biomass. . . . . . . . . . . . . . . . . . . . . . . . 3
Madhuri Narra, Jisha P. James and Velmurugan Balasubramanian
Liquid Hot Water Pretreatment of Paddy Straw for Enhanced
Biomethanation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Abhinav Trivedi, Virendra Kumar Vijay and Ram Chandra
Improving Yeast Strains for Pentose Hexose Co-fermentation:
Successes and Hurdles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Shalley Sharma, Sonia Sharma, Surender Singh, Lata and Anju Arora
Pretreatment of Paddy Straw to Improve Biogas Yield . . . . . . . . . . . . . 43
Rupali Mahajan, Harmanjot Kaur, Raman Rao and Sachin Kumar
Bioethanol Production from Fermented Cashew Apple Juice
by Solar Concentrator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Y.P. Khandetod, A.G. Mohod and H.Y. Shrirame
Potential Role of Xylose Transporters in Industrial Yeast for
Bioethanol Production: A Perspective Review. . . . . . . . . . . . . . . . . . . . 81
Nilesh Kumar Sharma, Shuvashish Behera, Richa Arora and Sachin Kumar
Part II Chemical Conversion

Transesterification of Neem Oil Using Na Ion-Doped
Waste Fishbone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Himadri Sahu and Kaustubha Mohanty
xi
xii Contents
Assessment of the Seed Oils of Persea americana and Melia dubua

for Their Potentialities in the Production of Biodiesel and Possible
Industrial Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Kariyappa S. Katagi, Ravindra S. Munnolli, Sangeeta D. Benni
and Sneha S. Kulkarni
Investigation of CI Engine Fueled with Ethanol Nano Additives
Blended Diesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
V. Karthickeyan, P. Balamurugan and R. Senthil
Designer Biodiesel: An Optimization of Fuel Quality by Blending
Multiple Oils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Anuchaya Devi, Renuka Barman and Dhanapati Deka
Biodiesel Production from Moringa oleifera Oil and Its Characteristics
as Fuel in a Diesel Engine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Mohd. Zeeshan, Mohit Vasudeva and A.K. Sarma
Growth and Lipid Production from Scenedesmus sp. Under
Mixotrophic Condition for Bioenergy Application. . . . . . . . . . . . . . . . . 159
Monika Prakash Rai and Shivani Gupta
Experimental Evaluation of Combustion Parameters of a DI
Diesel Engine Operating with Biodiesel Blend at Varying
Injection Timings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Abhishek Sharma and S. Murugan
Performance and Emission Characteristics of CI Engine Fueled
with Alternative Fuel with Special Reference to Modification
for Combustion: A Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . 179
Himansh Kumar, Y.K. Yadav and A.K. Sarma
Part III Thermochemical Conversion

Upgradation of Bio-oil Derived from Lignocellulose Biomass—A
Numerical Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Anjani R.K. Gollakota, Malladi D. Subramanyam, Nanda Kishore
and Sai Gu
Sustainability Assessment of Biomass Gasification Based
Distributed Power Generation in India. . . . . . . . . . . . . . . . . . . . . . . . . 213
Amit Kumar Singh Parihar, Virendra Sethi and Rangan Banerjee
Overview of Biogas Reforming Technologies for Hydrogen
Production: Advantages and Challenges. . . . . . . . . . . . . . . . . . . . . . . . 227
Priyanshu Verma and Sujoy Kumar Samanta
Modifications in Improved Cookstove for Efficient Design. . . . . . . . . . . 245
Amit Ranjan Verma, Rajendra Prasad, Virendra Kumar Vijay
and Ratnesh Tiwari
Contents xiii
Part IV Biomass Management

Thermochemical Characterization of Pine Needles
as a Potential Source of Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Anil Kumar Varma and Prasenjit Mondal
Biomass Supply Chain Management: Perspectives and Challenges . . . . 267
Yogender Singh Yadav and Y.K. Yadav
Part V Electrochemical Processes

Enhanced Power Generation in Microbial Fuel Cell Using
MnO2-Catalyzed Cathode Treating Fish Market Wastewater . . . . . . . . 285
Md.T. Noori, M.M. Ghangrekar, A. Mitra and C.K. Mukherjee
Bioelectricity Generation from Marine Algae Chaetoceros
Using Microbial Fuel Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
P.P. Rajesh and M.M. Ghangrekar
Selective Enrichment of Electrochemically Active Bacteria
in Microbial Fuel Cell By Pre-treatment of Mixed Anaerobic
Sludge to Be Used as Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
B.R. Tiwari and M.M. Ghangrekar
Performance Analysis of Separators in Dual-Chambered Microbial
Fuel Cell and Treatment of Combined Industrial Effluent of South
Gujarat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Purvi Zaveri, Tanvi Modi, Lipi Parekh, Aditi Patel, Sameer Kureshi
and Nasreen Munshi
Part VI Integrated Systems

Role of Rhamnolipid: A Biosurfactant in Methane
Gas Hydrate Formation Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Amit Arora, Swaranjit Singh Cameotra, Rajnish Kumar,
Anil Kumar Singh, Pushpendra Kumar, Chandrajit Balomajumder
and Sukumar Laik
Economic Analysis of Solar PV/Wind/Diesel Generator/Battery
Connected Integrated Renewable Energy Systems for Residential
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Suresh Muthusamy and Meenakumari Ramachandran
Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

About the Editors
Dr. Sachin Kumar is Deputy Director in the

Biochemical Conversion Division at the Sardar Swaran
Singh National Institute of Bio-Energy, Kapurthala,
India. Dr. Sachin Kumar obtained his Ph.D. in
Chemical Engineering from Indian Institute of
Technology Roorkee, India in 2011. He has more than
11 years of research experience in biochemical con-
version of biomass to biofuels. His areas of research
interest include biofuels including bioethanol, biogas,
biobutanol, and biohydrogen, algal biomass, biopro-
cess engineering, enzyme technology, and metabolic
engineering.
Dr. Samir Kumar Khanal is Associate Professor of

Biological Engineering at the University of Hawai’i at
Mānoa and Collaborating Professor at Iowa State
University. Dr. Khanal obtained a Ph.D. in Civil
Engineering with focus in Environmental Biotechnology
from the Hong Kong University of Science and
Technology (HKUST), Hong Kong in 2002. Dr. Khanal
is an internationally leading researcher in the bioenergy
field and has over ten active research projects in the field
of bioenergy/bio-based products and environmental
biotechnology.
xv
xvi About the Editors
Prof. Dr. Y.K. Yadav received his Ph.D. in Energy

Studies from Indian Institute of Technology, Delhi and
at present he is the Director General, Sardar Swaran
Singh National Institute of Bio-Energy (Formerly
Sardar Swaran Singh National Institute of Renewable
Energy), Kapurthala, India. Professor Yadav has been
actively engaged in research, teaching, consultancy,
administration, and management in the field of
renewable energy for more than two decades. He
developed and taught renewable energy courses to
undergraduate and postgraduate students at CCS
Haryana Agricultural University, Hisar, India and
established a Renewable Energy Laboratory there. He also contributed in the
establishment the Department of Energy at Tezpur University, Assam, India.
About the Conference
The First International Conference on “Recent Advances in Bio-energy Research”

ICRABR-2015 was organized by Sardar Swaran Singh National Institute of
Bio-Energy, Kapurthala, India during March 14–17, 2015. The conference was
inaugurated by Shri Upendra Tripathy, Secretary, MNRE. Dr. B.S. Dhillon,
Vice-Chancellor, Punjab Agricultural University, Ludhiana, Dr. N.P. Singh,
Adviser, MNRE, Prof. B.S. Pathak, Former Director, SPRERI, Vallabh
Vidhyanagar, and Shri Balour Singh, Director, PEDA, Chandigarh, and Prof. Y.K.
Yadav, Director General, SSS-NIBE were also present during the inaugural func-
tion. The prominent personalities among delegates from overseas were Dr. Lalini
Reddy, Cape Peninsula University of Technology, South Africa, Dr. Hanna
Tashyreva, Zabolotny Institute of Microbiology and Virology of National Academy
of Sciences of Ukraine, and Dr. Baba Shehu Ibn Abubakar, University of
Maiduguri, Nigeria.
Eminent speakers including Prof. B.S. Pathak, Dr. Lalini Reddy, Dr. Abubakar,
Dr. Hanna Tashyreva, Prof. A.K. Jain, Dr. D.K. Adhikari, Mr. Vasudeo Joshi,
Dr. A.S. Mathur, Dr. R.C. Ray, Dr. D.K. Sahoo, Prof. I.M. Mishra, and
Mr. Parikshit Dhingra delivered the plenary speeches. Two plenary speeches were
conducted through video conferencing by Prof. Lee R. Lynd, Thayer School of
Engineering, Dartmouth College, USA and Prof. Ram B. Gupta, School of
Engineering, Virginia Commonwealth University, USA.
Fifteen technical sessions were conducted in three parallel slots each day with
different themes such as Biomass & Energy Management; Thermo-chemical
Conversion; Biochemical Conversion; Chemical Conversion; Electrochemical
Processes; and Integrated/Waste to Energy. About ten invited speakers delivered
their research findings and review papers. More than 30 participants presented their
work through oral presentation, whereas 38 participants presented their work
through posters. About 218 abstracts were published in the souvenir under the
different sections including plenary speakers, invited speakers, biochemical con-
version, chemical conversion, biomass and energy management, thermochemical
conversion, electrochemical processes, waste to energy, and integrated systems.
xvii
xviii About the Conference
On the last day of event, one session was conducted for the formation of a
“Bio-Energy Alliance.” During the session, the representatives of different orga-
nizations including academic and research institutions, universities, industry,
and international groups were present and agreed on fully supporting the
Bio-Energy Alliance.
Part I
Biochemical Conversion
Comparison Between Separate Hydrolysis
and Fermentation and Simultaneous
Saccharification and Fermentation Using
Dilute Acid Pretreated Lignocellulosic
Biomass
Madhuri Narra, Jisha P. James and Velmurugan Balasubramanian
Abstract In the present study, two different processes, separate hydrolysis and
fermentation (SHF), and simultaneous saccharification and fermentation (SSF) were
compared. Three different lignocellulosic biomass viz. rice straw (RS), wheat straw
(WS), and sugarcane bagasse (SB) were pretreated with dilute acid at two different
concentrations (2 and 4 % H2SO4 w/v) and at two different time intervals, i.e., 30
and 60 min. RS, WS, and SB with 4 % H2SO4 at 121 °C for 30 min yielded
maximum reducing sugars (110, 90, and 95 g l−1). Delignification of the solid
residues were carried out with 0.5 % NaOH, at 121 °C for 30 min. In-house
cellulase produced by Aspergillus terreus was used for separate hydrolysis studies
at 10 % solid loading and 9 FPU g−1 substrate enzyme loading for 0–48 h at 42 °C.
Maximum yield of reducing sugars from RS, WS, and SB were 266, 242, and
254 mg g−1 substrate, respectively. Acid and enzymatic hydrolysates from RS, WS,
and SB produced 5.1, 4.9, 5.2 g l−l, and 14.0, 13.9, 12.9 g l−1 of ethanol with Pichia
stipitis and Saccharomyces cerevisiae in 24 and 36 h, respectively. Whereas SSF at
10 % solid loading and 9 FPU g−1 substrate enzyme loading for different time
intervals 0–72 h at 42 °C was carried out using in-house thermotolerant yeast strain
Kluyveromyces sp. RS, WS, and SB yielded maximum ethanol of 23.23, 18.29, and
17.91 g l−1, respectively. Ethanol yield was enhanced by addition of Tween 80 1 %
(v/v) by 8.39, 9.26, and 8.14 % in RS, WS and SB, respectively.
Keywords Lignocellulosic biomass Delignification Thermotolerant yeast

strain SHF SSF Ethanol
Madhuri Narra (&) J.P. James Velmurugan Balasubramanian

Sardar Patel Renewable Energy Research Institute, P. Box No. 2, Vallabh Vidyanagar
388120, Gujarat, India
e-mail: madhuri68@gmail.com
© Springer India 2016 3

S. Kumar et al. (eds.), Proceedings of the First International Conference on Recent
Advances in Bioenergy Research, Springer Proceedings in Energy,
DOI 10.1007/978-81-322-2773-1_1
4 Madhuri Narra et al.
1 Introduction
Bioethanol can be produced from any lignocellulosic biomass such as RS, WS, and
SB as they are readily available renewable resources of carbohydrates for biological
conversion to fuels and chemicals (Borbala et al. 2013). One of the most abundant
lignocellulosic biomass in the world is RS. About 731 million tones of RS is
produced annually which are distributed in Africa (20.9 million tones), Asia
(667.6 million tones), Europe (3.9 million tones), America (37.2 million tones), and
Oceania (1.7 million tones). Around 205 billion liters bioethanol per year can be
potentially produced from this quantity of RS, which is the largest amount from a
single biomass (Faveri et al. 2004). Sugarcane industries also generate huge amount
of bagasse annually and some of this residue is currently used for energy cogen-
eration in sugar mills while the surplus being stockpiled.
Lignocellulosic biomass primarily consists of cellulose, hemicellulose, and lig-
nin and its composition varies with different feed materials used. They are very
complex materials; hence a single pretreatment method cannot be applied to all
lignocellulosic biomass. Various pretreatment methods have been developed
including chemical, physical, physico-chemical, and biological for lignocellulosic
biomass and commonly used methods are steam, dilute acid, alkaline, and oxidative
pretreatment methods. At current scenario, an up-to-date-technology like dilute acid
pretreatment is used for pretreating any lignocellulosic biomass. In acid hydrolysis,
removal of hemicellulosic content with small fraction of lignin takes place and the
remaining part of lignin remains fixed to the cellulosic content (Kaya et al. 2000).
Delignification of lignocellulosic solid biomass is essential to achieve maximum
cellulosics hydrolysis due to the greater affinity of cellulase components, β-glu-
cosidase, and endoglucanase towards lignin than to the carbohydrates, resulting in
lower saccharification efficiency during enzymatic hydrolysis. Agricultural residues
such as RS and herbaceous crops are very effectively pretreated using alkali agents.
(Chen et al. 2007). Advantages of alkali pretreatment over other pretreatment
technologies include lower temperature, pressure, and time requirement. Sodium
hydroxide (alkali) has been widely studied for many years to increase the ease of
access of cellulases towards cellulose and hemicelluloses by disrupting the lignin
structure of the lignocellulosic biomass.
During enzymatic hydrolysis, addition of cellulases to pretreated material con-
taining holocellulosic (cellulose and hemicelluloses) material converts into mono-
meric sugars and subsequent addition of yeast ferment these sugars to ethanol. SHF
is a two step process where enzymatic hydrolysis is followed by fermentation.
However, in SSF using single reactor both the process steps can be carried out
simultaneously. SSF is more advantageous compared to SHF as it reduces the cost
of reactors by performing saccharification and fermentation in single vessel and
better ethanol yields by reducing the product inhibition exerted by saccharification
products (Scordia et al. 2013).
The need of the hour is to develop a suitable technology for bioethanol pro-
duction as a partial replacement of gasoline from lignocellulosic biomass as most of
Comparison Between Separate Hydrolysis and Fermentation … 5
these materials are found in surplus and burnt in the open fields, thereby creating
environmental pollution. In order to have an economically viable ethanol plant, the
primary focus should be on low cost pretreatments, novel enzymes with higher
activities, innovations on fermentation technologies for complete sugar utilization,
bioreactors design as well as strains for SSF to provide higher ethanol productivity.
Hence, it is essential to develop an indigenous technology with low capital cost and
operational expenditure.
In this study, two different processes, SHF and SSF were compared with respect
to production of ethanol from dilute acid pretreated lignocellulosic biomass.
Both SHF and SSF were carried out at 10 % solid loading and 9 FPU g−1 substrate
enzyme loading at 42 °C by the in-house cellulase produced by Aspergillus terreus
for different time intervals 0–48 h and using thermotolerant in-house yeast strain
Kluyveromyces sp. for different time intervals 0–72 h, respectively. Comparative
performance of both the processes was reported in this paper.
2 Materials and Methods
2.1 Lignocellulosic Biomass, Media and Chemicals
Raw materials such as RS and WS were collected from local farmers, Anand,
Gujarat, India. SB was procured from a local sugar factory. After collection, the raw
materials were exposed to physical pretreatment, i.e., passing through 5 mm mesh
in hammer mill prior to chemical pretreatment (Finex, India). The physically pre-
treated lignocellulosic biomass were washed thoroughly with tap water, air dried,
and stored at room temperature in air tight containers. All the chemicals, reagents,
and media of analytical grade were purchased from local vendors.
2.2 Acid Pretreatment
One fifty grams of pre-sized RS, WS, and SB were mixed with 750, 1500, and
3000 ml of 2 and 4 % H2SO4. Substrate to acid ratio (w/v) maintained were 1:5,
1:10, and 1:20, respectively. Pretreatment of RS, WS, and SB were conducted at
temperature 121 °C for time period 30 and 60 min as described earlier (Narra et al.
2015). Double layered muslin cloth was used to filter the acid hydrolysate. The acid
hydrolysate was detoxified with calcium hydroxide as described by Kuhad (2010)
and analyzed for sugars, phenolics, and furans. The leftover solid residues after acid
pretreatment were thoroughly washed with continuous flow of water till neutral pH
and dried under sunlight. The dried acid pretreated solid residues were further
subjected to delignification using 0.5 % NaOH at 121 °C for 30 min of 1:20
substrate to alkali ratio. The cellulosic material separated from lignin portion were
filtered using double layered muslin cloth and washed thoroughly with tap water till
neutral pH and sun dried. These dried solid residues were further used for SHF and
SSF studies or stored at 4 °C in air tight bags.
2.3 Microorganisms and Culture Conditions
Standard cultures for SHF were viz. Saccharomyces cerevisiae 3570 and Pichia
stipitis NCIM 3499 were procured from National Chemical Laboratory (NCL),
Pune, India whereas, newly isolated thermotolerant yeast strain isolated from fruit
waste was used for SSF. Partial sequencing of purified strain was carried out at
National Collection for Industrial Microorganisms (NCIM), Pune, India. The
hexose fermenting yeast strains were maintained on agar slants containing (g−1 l):
glucose, 30.0; yeast extract, 3.0; peptone, 5.0; agar, 20.0 at pH 6.0 ± 0.2, and
Pichia stipitis was maintained on agar slants containing (g−1 l) xylose, 20.0; yeast
extract, 4.0; peptone, 5.0; KH2PO4, 1.5; MgSO4, 7H2O, 0.5; agar, 20.0 at pH
5.0 ± 0.2 and temperature 30 °C, respectively. The cultures were stored at 4 °C.
2.4 Yeast Inoculum Preparation
The Saccharomyces cerevisiae inoculum was grown for 12 h at 30 ± 2 °C in a

culture medium containing (g−1 l); glucose, 30.0; yeast extract, 3.0; peptone, 5.0;
(NH4)2HPO4, 0.25 at pH 6.0 ± 0.2 (Chen et al. 2007). Kluyveromyces inoculum
was prepared at 42 ± 2 °C for 12 h. After incubation, the flask contents were
aseptically collected, centrifuged, and used for SHF and SSF studies. An optical
density of 0.6–0.8 at 620 nm was used for cell culture. Inoculum of Pichia stipitis
was prepared as described by Nigam (2001) using (g−1 l); xylose, 50.0; yeast
extract, 3.0; malt extract, 3.0; peptone, 5.0 at pH ± 0.2, and temperature 30 °C.
A 12 h seed culture of Pichia stipitis with 1 % v/v were inoculated in separate
flasks.
2.5 SHF of Cellulosic Residue
2.5.1 Cellulase Preparation
Crude cellulase used for separate hydrolysis and SSF was indigenously produced
by Aspergillus terreus under solid state fermentation as described earlier (Narra
et al. 2012). RS was used as a substrate for cellulase production and the crude
enzyme contained FP activity, β-glucosidase, and endoglucanase, of 0.98 ± 0.13,
5.2 ± 0.30, and 14.2 ± 0.40 U ml−1, respectively.
2.5.2 Enzymatic Hydrolysis of Delignified Cellulosic Residue
Enzymatic hydrolysis was carried out with cellulosic solid residues of RS, WS, and
SB at 42 °C in 50 ml capacity oak ridge wide mouth bottles at 16 rpm for 4–40 h
with an enzyme load of 9 FPU g−1 substrate. Total volume of the system was 20 ml
(0.05 M citrate buffer, pH 4.8). Other conditions were kept constant as described
earlier (Narra et al. 2012). At regular intervals, the supernatant samples were
analyzed for total reducing sugars by DNSA method (Miller 1959) after centrifu-
gation at 10,000 g for 15 min. Saccharification efficiency was calculated as men-
tioned previously (Narra et al. 2012).
2.5.3 Ethanol Fermentation
The enzymatic and acid hydrolysate were fermented with Saccharomyces cere-
visiae and Pichia stipitis, respectively, in a 100 ml stoppered flask at 30 ± 2 °C for
40 h. Nutrients containing NH4Cl, 0.5, KH2PO4, 0.15, yeast extract, 3.0 were added
to the acid hydrolyse (20 ml) containing (20.0 g l−1) sugars and the pH was adjusted
to 5.5 ± 0.2. While the cellulosic hydrolysate having 40.0 g l−1 sugar was sup-
plemented with yeast extract 3.0 g l−1 and (NH4)2HPO4, 0.25 g l−1. The flasks were
inoculated with 10 % (v/v) inoculum and incubated at 30 °C for 60 h at 150 rpm.
2.6 SSF
SSF was performed with cellulosic solid residues of RS, WS, and SB in 50 ml
capacity oak ridge wide mouth bottles at 42 °C. The total volume of the system
maintained was 20 ml (0.05 M citrate buffer, pH 4.8). Crude cellulases were used to
hydrolyze the cellulosic substrates at 10 % solid loading and 9 FPU g−1 substrate
enzyme loads. After 6 h of hydrolysis at 42 °C, an in-house yeast strain
Kluyveromyces sp. was added under sterile conditions for better conversion of
cellulosic material. The experimental flasks placed in a rotating assembly and were
rotated at 16 rpm for 60 h as described earlier (Narra et al. 2015).
2.7 Analytical Methods
Endoglucanase activity was assayed using 2 % carboxymethyl cellulose (CMC,

Sigma Chemical Co.) in 0.05 M sodium acetate buffer, pH 4.8 as substrate. The
release of reducing sugars in 20 min at 50 °C was determined by DNSA (Miller,
1959). β-glucosidase assay was carried out using p-nitrophenyl—β-D-glucopy-
ranoside (PNPG, Sigma Chemical Co.) as substrate at 50 °C for 30 min. The
reaction was terminated by addition of 4 ml NaOH-glycine buffer (0.2 M, pH 10.6).
FP activity was measured as describer earlier (Narra et al. 2012). Lignin, cellulose,
and hemicellulose contents of the untreated and pretreated RS, WS, and SB were
analyzed according to Goering and Vansoest (1975).
The samples from SHF and SSF were withdrawn at regular intervals, centrifuged
at 10,000 × g for 15 min, and the supernatant was analyzed for residual sugars by
DNSA method as described earlier. Ethanol was estimated using high performance
liquid chromatography (Shimadzu, Japan) equipped with a refractive index detector
(RID) and packed with an Aminex-HPX-87 column (Biorad, Hercules, USA, CA)
with dimension of 300 mm × 7.8 mm. Samples were eluted using 5 mM H2SO4
with the flow rate of 0.6 ml min−1. Column temperature was maintained at 65 °C.
The saccharification efficiency and the theoretical yield of ethanol were calculated
as described by Narra et al. (2012, 2015).
3 Results and Discussion
3.1 Biomass Composition Analysis
RS, WS, and SB contained cellulose content of 41.02 ± 1.45 %, 38.50 ± 1.07 %,
39.00 ± 1.83 %; hemicellulose content of 28.47 ± 1.91 %, 27.00 ± 1.36 %,
25.00 ± 1.44 %; lignin content of 9.20 ± 1.12 %, 12.82 ± 1.27 %, 14.21 ± 1.62 %
and moisture content of 7.04 ± 1.21 %, 6.93 ± 1.17 %, 8.14 ± 1.15 %, respectively.
After pretreatment, the cellulose, hemicellulose, and lignin contents of RS, WS, and
SB were (80.00 ± 1.58 %; 67.00 ± 1.60 %; 70.00 ± 1.32 %); (3.00 ± 1.91 %;
6.54 ± 1.42 %; 4.36 ± 1.44 %); (2.01 ± 1.16 %; 4.98 ± 1.97 %; 5.13 ± 1.48 %),
respectively (Narra et al. 2015).
3.2 Dilute Acid Hydrolysis of Lignocellulosic Biomass
RS yielded maximum amount of reducing sugars (110 g l−1) followed by WS and

SB (90 and 95 g l−1) when treated at optimum conditions, i.e., 4 % H2SO4 at 121 °C
for 30 min. The substrate to acid ratio maintained was 1:5 (w/v). As the treatment
time increased from 30 min to 60 min, there was no substantial difference in the
reducing sugar yield (102, 85, and 91 g l−1). The acid hydrolysate obtained at
optimum conditions of hydrolysis contained major amount of xylose
(90.23 ± 2.34 g l−1), glucose (5.01 ± 1.29 g l−1), arabinose (7.48 ± 2.69 g l−1),
phenolics, furfural, and HMF. The major degradation products of pentose, hexose
sugars, and lignin are furfural, HMF, and phenolics, respectively (Kuhad et al.
2010). These toxins have the ability to decrease the activities of several yeast
enzymes, e.g., alcohol dehydrogenase, aldehyde dehydrogenase, and pyruvate
dehydrogenase during fermentation (Modig et al. 2002). In order to reduce the
inhibitor concentrations in acid hydrolysates, sequential addition of overliming and
activated charcoal was used. Overliming followed by activated charcoal treatment

resulted in reduction of furfural (96.31, 95.15, and 96.20 %), HMF (93.36, 92.18,
and 92.19 %), and phenolic (93.36, 92.18, and 92.19 %) in RS, WS, and SB,
respectively. As the acid hydrolysate was rich in xylose it was used to produce
ethanol using pentose utilizing yeast strain Pichia stipitis NCIM 3499.
3.3 Delignification of Lignocellulosic Biomass
Delignification of acid pretreated solid biomass was carried out with 0.5 % NaOH at
121 °C for 30 min to maximize reducing sugar yield. The cellulose content in the
pretreated biomass increased substantially with simultaneous reduction in lignin
content after alkaline pretreatment (Narra et al. 2015). The cellulose content was
found to be increased by 95.02, 74.02, and 79.48 %, while the lignin removals were
78.16, 61.15, and 63.90 % in RS, WS, and SB, respectively. It was also observed
that during the treatment most of the pentosan was solubilized. The increase in
cellulose content during pretreatment might be due to removal of lignin which
might have increased the enzyme effectiveness by eliminating nonproductive
adsorption sites and increasing access to cellulose and hemicelluloses (Lu et al.
2002). Kumar et al. (2009) reported that solubilization of other components in the
aqueous alkali solution is also responsible for increase in the cellulose content.
These findings were consistent with earlier published data on effects of delignifi-
cation of different lignocellulosic biomass RS, WS, and rapeseed straw by alkaline
pretreatment (Narra et al. 2012; Nopparat et al. 2013).
3.4 Enzymatic Saccharification of Delignified Cellulosic

Material
When delignified cellulosic substrates were subjected to enzymatic saccharification,

an increase in reducing sugar concentration was observed till 40 h time period and
thereafter it remained almost constant. Maximum yield of saccharification from RS,
WS, and SB was 266, 242, and 254 mg g−1 substrate, respectively (Fig. 1). Tween
80 1 % (v/v) addition also found to increase the saccharification yield by 12.1, 11.4,
and 10.6 %, respectively (Fig. 2). According to Kaar and Holtzapple (1998), all
through the enzymatic saccharification process, thermal deactivation of enzymes
was prevented by the addition of Tween. This outcome may be due to the surface
activity of Tween which resulted in shorter enzyme contact with air-liquid interface.
The decrease in surface tension of the solution not only permits the saccharifying
exoglucanase more active sites to cellulose, but it also prevents the nonproductive
part of the exoglucanase to the lignin surface which yielded in increased sugar level
(Hematinejad et al. 2002).
Fig. 1 Enzymatic hydrolysis 300

Rice straw
Reducing sugars (mg g -1

of RS, WS, and SB at
250 Wheat straw
different time intervals.
Temperature 42 °C, substrate 200
substrate)
load 10 % (w/v), enzyme load
9 FPU g−1 substrate, pH 4.8, 150
rpm 16 100
50
0
6 12 24 36 40 48
Time (h)
Fig. 2 Influence of Tween 80 350

Control Tween 80
Reducing sugars (mg g-1
on enzymatic hydrolysis of 300

RS, WS, and SB. Substrate
load 10 % (w/v), enzyme load 250
substrate)
9 FPU g−1 substrate, pH 4.8, 200

time 48 h, rpm 16, Tween 80
150
1 % (w/v)
100
50
0
RS WS SB
Lignocellulosic biomass
3.5 Fermentation of Hemicellulosic and Enzymatic

Hydrolysate
Maximum amount of ethanol production from RS, WS, and SB was obtained up to
5.1, 4.9, 5.2 g l−l, respectively. Table 1 shows the profile for ethanol fermentation
by Pichia stipitis 3499 NCIM. It was observed that there was increase in ethanol
Table 1 Fermentation profile of hemicellulosic hydrolysate of lignocellulosic biomass using

Pichia stipitis
Time (h) Ethanol (g l−l) Sugar (g l−l) Ethanol yield (g g−1)
RS WS SB RS WS SB RS WS SB
0 0.04 0.05 0.05 20.00 19.80 20.00 0.00 0.00 0.00
6 1.62 2.25 1.97 19.12 18.45 19.26 0.08 0.11 0.99
12 3.11 3.78 3.95 12.58 13.54 12.95 0.16 0.19 0.19
24 5.10 4.90 5.20 6.21 6.92 6.09 0.26 0.25 0.26
36 3.27 4.17 4.19 5.98 5.21 5.04 0.16 0.21 0.21
48 2.61 3.09 3.35 5.26 4.97 4.70 0.13 0.16 0.17
60 2.21 2.85 3.01 4.18 4.26 4.13 0.11 0.14 0.15
Table 2 Fermentation profile of enzymatic hydrolysate of lignocellulosic biomass using

Saccharomyces cerevisiae
Time (h) Ethanol (g l−l) Sugar (g l−l) Ethanol yield (g g−1)
RS WS SB RS WS SB RS WS SB
0 0.08 0.07 0.08 40.00 40.00 40.00 0.00 0.00 0.00
6 10.18 6.95 6.29 11.89 13.28 14.09 0.25 0.17 0.16
12 12.84 9.01 7.58 10.37 12.04 12.54 0.32 0.22 0.19
24 13.53 11.26 9.26 8.43 9.30 9.01 0.34 0.28 0.23
36 14.00 13.90 12.90 7.27 6.95 8.15 0.35 0.35 0.32
48 13.84 11.78 11.27 5.31 5.29 6.26 0.34 0.29 0.28
60 12.63 11.05 10.98 4.98 4.42 5.01 0.32 0.32 0.27
and increase in the growth of fermenting yeast but the consumption of reducing
sugars was relatively poor. The incomplete utilization of reducing sugars by fer-
menting yeast may be due to some kind of inhibitors present in the acid hydrolysate
which might have not been removed during the detoxification process. Similar
observation was also made by Kuhad et al. (2010) that the yeast could not tolerate
the higher amount of sugar concentration. The ethanol yield obtained from cellu-
losic hydrolysate (RS, WS, and SB) using Saccharomyces cerevisiae was 0.35,
0.35, and 0.32 g g−1, respectively (Table 2).
3.6 SSF
Maximum ethanol yield achieved at 60 h with 10 % solid load from RS, WS, and
SB (23.23, 18.29, and 17.91 g l−1) which was equivalent to 51.29, 48.22, and
45.19 % of maximum theoretical yield (Fig. 3). The earlier reports have shown that
increase in substrate load beyond certain extent could have caused decrease in
hydrolysis rate due to the product inhibition. The extent of inhibition usually
Fig. 3 SSF of RS, WS, and 25 Rice straw

SB at different time intervals. Wheat straw
Temperature 42 °C, substrate 20 Sugarcane bagasse
load 10 % (w/v), enzyme load
Ethanol (g l-1)
9 FPU g−1 substrate, pH 4.8,

15
rpm 16
10
0
12 24 48 60 72
Time, h
depends on the ratios of substrate to enzyme load (Wang et al. 2011; Xin et al.
2010). The present results also revealed that higher ethanol yields at lower solid
loads may be due to lower glucose accumulation compared to higher solid loads.
The current results were in accordance with Naveen et al. (2011) that at 8 % solid
loading, higher ethanol yields were observed compared to 12 % solid loading when
pretreated switchgrass was used for SSF studies by Kluyveromyces marxianus
IMB3.
Enhanced ethanol yields were achieved by the addition of 1 % Tween 80 to the
reaction mixture in comparison to the control (Fig. 4). The yields were increased by
8.39, 9.26, and 8.14 % with 10 % solid loading at 60 h from RS, WS, and SB,
respectively. Maximum ethanol yield from RS, SB, and WS were found to be
25.18, 19.57, and 18.19 g l−1, respectively. Kaar and Holtzapple (1998), Zhu et al.
(2014) similarly have found that addition of surfactant enhanced the rate and extent
of hydrolysis as the surfactants could have changed the nature of the substrate either
by increasing the available cellulose surface area or by eliminating inhibitory lignin.
The present results have shown that 1 kg untreated RS, WS, and SB contained
410, 370, 380 g, and 252, 205, 207 g cellulose and hemicelluloses, respectively.
This amount can be theoretically enough for production of 232, 217, 215 g and 129,
105, 106 g ethanol from cellulose and hemicellulose, respectively. Based on the
best yields attained in the current work, the ethanol production from RS, WS, and
SB through SSF was found to be 201, 162, 156 g kg−1 raw biomass, respectively.
Whereas, the ethanol production from RS, WS, and SB through SHF was found to
be 95, 75, 82 g kg−1 raw biomass, respectively (Table 3). The present results
revealed that SSF could prove enhanced ethanol production from delignified bio-
mass compared to SHF. The reason could be that the optimum condition for sep-
arate enzymatic hydrolysis might be higher than 42 °C, and secondly the product
inhibition exerted by saccharification might have occurred during the process.
Whereas in case of SSF, pre-hydrolysis was carried out at 42 °C for 6 h followed by
yeast addition to ease the problems caused by production inhibition.
Fig. 4 Influence of Tween 80 30

on SSF of RS, WS, and SB. Control
Substrate load 10 % (w/v), 25 Tween 80
enzyme load 9 FPU g−1
Ethanol (g l-1)
substrate, pH 4.8, time 60 h, 20

rpm 16
15
10
0
Rice straw Wheat straw Sugarcane
bagasse
Tween 80 1% (v/v)
Table 3 Comparison of ethanol yields from SSF and SHF of different lignocellulosic biomass
Process SSF SHF
Lignocellulosic substrates RS WS SB RS WS SB
Optimum conditions SSF: Temperature: 42 °C, SH: Temperature: 42 °C,
substrate load: 10 % substrate load: 10 %
(w/v), enzyme load: 9 (w/v), enzyme load: 9
FPU g−1 substrate, FPU g−1 substrate,
duration: 48 h, Tween 80: duration: 48 h, Tween 80:
1 % (v/v) 1 % (v/v)
Fermentation:
Temperature: 30 °C,
duration: 24 h, sugar
concentration: 2 %
Theoretical ethanol yield, g kg−1 substrate
Cellulosic substrate 232 217 215 232 217 215
Hemicellulosic substrate 129 105 106 129 105 106
Ethanol yield, g kg−1 substrate
Cellulosic substrate 129 108 95 23 21 21
Hemicellulosic substrate 72 54 61 72 54 61
4 Conclusion
Cellulosic ethanol is considered to be a globally accepted alternative fuel. The

current results demonstrated that SSF could ascertain improved ethanol production
from delignified lignocellulosic biomass compared to SHF in terms of total ethanol
production time and ethanol yield. RS yielded higher amount of ethanol followed
by WS and SB using in-house cellulases produced by Aspergillus terreus and
in-house thermotolerant yeast strain Kluyveromyces sp.
Acknowledgements The authors are thankful to the Director, Sardar Patel Renewable Energy
Research Institute (SPRERI), Vallabh Vidyanagar, Gujarat for allowing us to carry out research at
SPRERI. The financial support from Department of Biotechnology (DBT), Government of India is
highly acknowledged.
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Liquid Hot Water Pretreatment of Paddy
Straw for Enhanced Biomethanation
Abhinav Trivedi, Virendra Kumar Vijay and Ram Chandra
Abstract Indian agriculture produces nearly 150–160 million tons of paddy straw
per annum. 70–80 % of total produced rice straw is burned in the open field for
cultivation of the next crop. Proper disposal of paddy straw in an economical and
environment friendly manner is a serious concern in India, where rice is the major
agricultural crop in some states. The work presented in the paper is focused on
pretreatment of paddy straw using liquid hot water treatment followed by
biomethanation. Results observed during batch process are encouraging and work is
being carried out for bigger models. Under batch condition with *5–7 kg of paddy
straw can yield 1.0 m3 of biogas with average methane content of 60.0 and
25–30 % carbon dioxide content during retention period of 40 days.

Keywords Agricultural residue Lignocellulosic biomass Biomass pretreat-

ment Liquid hot water pretreatment Biogas from paddy straw Biomethane
production
1 Introduction
Paddy straw is among ubiquitous lignocellulosic agricultural waste biomass which

has been rarely utilized as a source for biofuel production. For the year 2013,
annual production of rice was reported as 159.200 million metric tons in India
(FAOSTAT 2013). With such colossal production of rice, around 238.50 million
metric tons of paddy straw is generated per annum with an average of 1.0–1.5 kg of
paddy straw per kilogram of the rice grain harvested (Maiorella 1985) of which
two–third is burnt in the field itself (Phutela 2011). Some amount of unburnt residue
is used as a fuel for modern biomass-based power plant, while some amount is used
Abhinav Trivedi (&) V.K. Vijay Ram Chandra

Biogas Production and Enrichment Laboratory, Centre for Rural
Development and Technology, Indian Institute of Technology Delhi,
Hauz Khas, New Delhi 110016, India
e-mail: abhinavtrivedi22@gmail.com

DOI 10.1007/978-81-322-2773-1_2
16 Abhinav Trivedi et al.
to fuel biomass cookstoves in rural area for cooking and heating applications
(Gadde et al. 2009).
The portion of the paddy straw residue that remains in the field as uncollected is
subsequently plowed back into the soil (residue incorporation into soil), which
serve as biofertilizer for upcoming crops. This is widely practised across the
country and it provides a nutrient source for upcoming crops but also conductive to
crop diseases (Hrynchuk 1998). This activity stimulates methane emissions due to
decomposing paddy straw in the field (Yuan 2013).
The research paper published in recent years has been perceived that open field
burning of paddy straw residues contributes toward emissions of polycyclic aro-
matic hydrocarbons, i.e., PAHs (Korenaga et al. 2001), as well as polychlorinated
dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs), referred
to as dioxins (Gullett and Touati 2003). It has also been reported that open burning
of one ton of paddy straw emits 3 kg particulate matter, 60 kg CO, 1460 kg CO2,
199 kg ash, and 2 kg SO2 (Jenkins and Bhatnagar, 2003a, b). These compounds are
toxic and carcinogenic for humans and have a long term impact on human health
(Meesubkwang 2007).
Anaerobic digestion of rice straw for methane production is considered as the best
option among all thermochemical and biochemical processes for providing energy in
form of methane, thus reducing air pollution up to considerable limits and at the same
time producing digestate solids, which is a potential biofertilizer for land application
(Nand 1999). The anaerobic digestion technology is a most efficient way in term of
energy output/input ratio for handling of biomass resources to produce energy and
biofertilizer. Furthermore, raw biogas after enrichment is equivalent to natural gas and
can be alternatively used in mobile and static internal combustion engines.
The present research focused on evaluation of liquid hot water (LHW) pretreatment
method for delignification of paddy straw and subsequently anaerobic digestion of the
substrates. In this method of pretreatment the destruction of lignocellulosic structure
occurs by super-heated water at temperature 200 and 240 °C. The work was carried out
using nonstirred reactor having volume of 2.0 liter, which can be operated to maxi-
mum working temperature of 300 °C. The degraded paddy straw samples were
evaluated for its biomethane potential under laboratory conditions.
2.1 Characterisation of Paddy Straw
The characterization of paddy straw was carried out using proximate and ultimate
analyses. Initially paddy straw was dried at 105 °C using hot air oven and ground to
nearly 1.0 mm size using grinder. Percentage of moisture content, total solids,
volatile solids, and ash contents (proximate analysis) in paddy straw was estimated
using standard methods as described by APHA while basic elements such as car-
bon, hydrogen, and nitrogen (ultimate analysis) were determined using ‘Vario EL’
Liquid Hot Water Pretreatment of Paddy Straw … 17
elemental analyzer (Perkin Elmer, USA Made). 20 mg of homogenized samples

were packed in tin foil and placed into the carousel of the automatic sample feeder
of the analyzer and analyzed as per procedure described in the instruction manual of
the instrument supplied by the manufacturer of CHN elemental analyzer.
2.2 Pretreatment of Paddy Straw
Fresh paddy straw samples were pretreated in a hydrothermal reactor at reactor

operating temperatures of 200 and 240 °C for pretreatment time duration of
15–20 min. During pretreatment the water-to-paddy straw ratio was maintained as
8:1, this ratio is maintained in order to get nearly 10 % of total solids concentration
in the digesting substrate. Appropriate amount of NaOH was added in the pretreated
paddy straw substrate to maintain pH around 7.5–7.8. To maintain the homogeneity
of the substrates in reactor bottles, the bottles were shook daily manually. The
prepared substrates were kept at 37 °C temperature in an incubator for stabilization
of pH before being fed to anaerobic digester (Chandra et al. 2012).
2.3 Experimental Setup
The biogas production potential of pretreated paddy straw samples was estimated
using 1.0 L glass bottles (Schott Duran). Each reactor consisted 30 g of
hydrothermally pretreated paddy straw and 240 mL of process water along with
480 mL of fresh slurry taken from cow dung-based biogas plant. The C/N ratio of
substrate was adjusted in a range of 20–25 by addition of 0.75 g of urea in the
substrate. Biogas production was monitored for 40 days after which biogas pro-
duction had been found ceased. The reactor bottles contained the desired substrates
along with inoculum were placed in an incubator and maintained constantly at
37 °C operating temperature. The daily biogas volume was measured by using
inverted water displacement system. The biogas produced from various anaerobic
reactors was analyzed for methane as well as carbon dioxide contents using a gas
chromatograph (Agilent 7890) equipped with Porapak Q column and thermal
conductivity detector (TCD).
3.1 Properties of Paddy Straw
The results of proximate and ultimate analysis of untreated paddy straw are shown
in Table 1. The proximate analysis revealed that untreated paddy straw contains up
Table 1 Proximate and Sl. No. Properties parameter Observed value (%)
ultimate analysis of
nontreated paddy straw Proximate properties
1 Moisture content (wet basis) 08.00
2 Total solids (wet basis) 92.00
3 Volatile solids (dry basis) 81.50
4 Ash content (dry basis) 18.50
Ultimate properties
5 Carbon 35.20
6 Hydrogen 5.596
7 Nitrogen 0.745
8 C/N ratio 47.248
to 8.0 % moisture and 92.0 % total solids on wet weight basis, while 81.50 and
18.50 % volatile solid matter and ash matter, respectively, on dry weight basis. The
ultimate analysis resulted into 35.2 % carbon, 5.5 % hydrogen, and 0.745 %
nitrogen contents on dry weight basis. Upon elemental analysis, it has been found
that the amount of nitrogen content present in rice straw biomass is very low
(C/N ratio = 47.2) to maintain an appropriate range of C/N ratio (20–30) for
effective methane fermentation.
3.2 Effect of Liquid Hot Water Pretreatment on Structure

of Paddy Straw
Figs. 1, 2, 3, and 4 show the scanning electron microscopic images of nontreated

paddy straw and various hydrothermal pretreated paddy straw such as 200 and
Fig. 1 SEM image of

nontreated paddy straw
Fig. 2 SEM image of

pretreated paddy straw (200 °
C)
Fig. 3 SEM image of

pretreated paddy straw (240 °
C)
240 °C, respectively. It is clearly evident from Fig. 1 that the microfibrils are very
tightly packed in case of nontreated paddy straw. However, in case of hydrothermal
pretreated paddy straw the original structure of paddy straw has been visibly dis-
rupted depending upon the level of pretreatment temperature. The microfibrils are
loosed and open for microbial degradation. The brightness in the various images is
due to charging effect during SEM imaging of volatile fatty acids released during
the pretreatment process.
Fig. 4 Cumulative biogas production for various substrates of paddy straw
Table 2 Cumulative biogas production and average methane content in the biogas
Sl. No. Treatment Cumulative biogas Biogas Methane
production production production
(mL) (m3/kg TS) (m3/kg TS)
1 Nontreated paddy straw 3215 0.107 0.058
2 Hydrothermal pretreated paddy 6540 0.218 0.144
straw (200 °C)
3 Hydrothermal pretreated 740 0.024 Not
paddy straw (240 °C) measured
3.3 Biomethane Potential Analysis
The cumulative biogas and methane production yields observed during the course
of investigation are shown in Table 2. The input feed material in the substrates
remained constant as 30.0 g which were fed to each anaerobic reactor. Nontreated
paddy straw substrate resulted to produce 3215 mL of biogas over a retention
period of 39 days. After that biogas production was completely ceased. However, in
case of hydrothermal pretreated paddy straw (200 °C) substrate the biogas pro-
duction was observed to be 6540 mL over a retention period of 37 days and
afterward there was no occurrence of biogas production. In case of hydrothermal
pretreated paddy straw (240 °C) substrate the biogas production was observed to be
740 mL over a retention period of 21 days. After that biogas production was
completely ceased.
The pattern of biogas production with respect to retention time is shown in

Fig. 4. It is clearly evident that in case of non-pretreated substrate of paddy straw
the biogas production rate was slow and lasted for longer duration of retention time
in comparison to the hydrothermal pretreated paddy straw (200 °C) substrate. The
biogas production from hydrothermal pretreated paddy straw (240 °C) substrate
was found very low in comparison to hydrothermal pretreated paddy straw (200 °C)
substrate as well as non-pretreated substrate of paddy straw. This might be due to
lower pH level of the digesting substrate during the anaerobic digestion process.
The average concentration of methane in the produced biogas was found as
54.0 % in case of non-pretreated substrate of paddy straw. However, the average
methane content in the biogas produced from hydrothermal pretreated paddy straw
(200 °C) substrate was observed to be 66.0 %. The specific biogas and methane
production from nontreated paddy straw substrate was found to be 0.107 and
0.058 m3/kg TS, respectively. However, the specific biogas and methane production
from hydrothermal pretreated paddy straw (200 °C) substrate was found to be 0.218
and 0.144 m3/kg TS, respectively.
4 Conclusions
The results of experiments so far conducted on batch scale biogas production assays
revealed that liquid hot water pretreated paddy straw substrate results in the increase
in amount of biogas and methane production yield. The biogas and methane
production yields after liquid hot water pretreated paddy straw at 200 °C was found
more than 2.0 times of untreated paddy straw substrate. There are many other
processes for pretreatment of lignocellulosic waste but most of these processes are
in the laboratory–bench or pilot–plant stage and at the present time and the
economic feasibility of these available processes are not certain in Indian context.
Further, the work has been extended in the same laboratory to explore the potential
of liquid hot water pretreatment at lower temperatures and toward designing farm
scale biogas digestion for paddy straw.
Acknowledgments Authors gratefully acknowledge the financial support of the Indian Institute
of Technology Delhi for conducting this research. Authors also thanks to biogas research group for
their support during the course of investigation.
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Accessed 07 Oct 2015
Improving Yeast Strains for Pentose
Hexose Co-fermentation: Successes
and Hurdles
Shalley Sharma, Sonia Sharma, Surender Singh,

Lata and Anju Arora
Abstract Bioethanol, a second generation biofuel, is considered to be one of the

best alternatives to conventional petroleum-based liquid fuels. In the present sce-
nario, it is being majorly produced by fermentation of hexoses coming from the
cellulosic fraction of the lignocellulosic biomass. Biomass also comprises of up to
33 % hemicellulose, therefore, its fermentation would lead to enhanced bioethanol
fermentation productivities. Bioethanol can be produced through biochemical as
well as thermochemical processes. A biochemical process, which is environmen-
tally favorable involves the use of microbes, e.g., yeast and bacteria. Bioethanol
production from yeast, i.e., Saccharomyces cerevisiae has already been commer-
cialized. However, demerit of this commercially viable strain is that it utilizes only
hexoses, while pentoses are left unused. This paper discusses different strategies for
improving the potential of yeast strains for mixed sugar fermentation to ethanol.
There are ways to genetically improve yeast strains to enable them to ferment
mixture of hexoses and pentoses. However, there are several physiological hurdles
which can limit the success of conventional genetic approaches like cofactor
imbalance, excessive by product formation, glucose repression, etc., which need to
be tackled, in order to obtain enhanced yield. Metabolic engineering of the yeast
strains is a way for enhancing bioethanol fermentation efficiency.

Keywords Bioethanol Hexose Pentose Fermentation Metabolic engineering
1 Introduction
In recent years, research over the bioethanol production has increased, particularly
focusing on the bioconversion of forestry, agricultural, and municipal residues into
ethanol. With increasing demand and limited resources of the fuels, bioethanol
Shalley Sharma Sonia Sharma Surender Singh Lata Anju Arora (&)
Division of Microbiology, ICAR-Indian Agricultural Research Institute,
New Delhi 110012, India
e-mail: anjudev@yahoo.com

DOI 10.1007/978-81-322-2773-1_3
24 Shalley Sharma et al.
production has become a necessity, but most of the production is based on the
agricultural crops (such as corn, wheat, sugarcane, rice, etc.) which may raise
debate over food versus. fuel. Solution to the problem is utilization of lignocellu-
losic biomass (it consists of agricultural wastes, i.e., the leftover biomass after crop
processing) as it is rich in cellulose and hemicellulose and hence is a good source of
sugars to be fermented for the production of bioethanol. Also, lignocellulosic
biomass is more abundant than crops, which is more economical and also does not
hamper the stability of environment (Stephanopoulos 2007). Hydrolysis of ligno-
cellulosic biomass yields a mixture of sugars, i.e., hexoses (glucose, mannose, and
galactose) and pentoses (xylose and arabinose), of which glucose is the most
abundant sugar followed by xylose.
Although lignocellulosic biomass is rich in hexose as well as pentose sugars, its
efficient conversion into ethanol is not an easy task as pentose fermentation is not an
efficient process because pentoses are not readily utilized by yeasts and bacteria (Chu
et al. 2007) as in the case of hexoses. Even S. cerevisiae, well known fermentative
yeast is unable to utilize xylose, when grown on media containing xylose as the sole
carbon source (Kuhn et al. 1995). However, there are reports suggesting the presence
of xylose transport and xylose metabolizing genes such as xylose reductase (XR),
xylulokinase, etc., in this yeast. Earlier it was thought to be the reason that xylose
could not be fermented by yeasts and the strategies employed for its fermentation
involved: conversion of xylose to xylulose in vitro using glucose isomerase and then
fermentation of this xylulose to ethanol (Chandel et al. 2011). Nevertheless, this
scenario changed with the discovery of pentose fermenting yeasts during 1980s
(Hahn-Hägerdal et al. 2007; Chandel et al. 2008a). As mentioned above, commercial
strain of S. cerevisiae is unable to utilize and ferment pentose sugars. This has led the
researchers to study the metabolic pathway of this yeast. Studies suggest that S.
cerevisiae can uptake xylose via glucose transporters and hence the rate of transport
becomes slow (van Vleet et al. 2009). Metabolic studies of S. cerevisiae found out that
this organism has the transport system for xylose and all the enzymes required for the
xylose metabolic pathway (Gárdonyi et al. 2003a) but is still unable to utilize it for
biomass conversion. This is due to the fact that xylose fermentation requires oxygenic
conditions which result in the oxidation of ethanol by the end of the process, thus
lowering the fermentation efficiency of the strain.
Several research groups have been trying to genetically modify the strain so that
it can utilize xylose for the production of ethanol. Efforts going on in this area of
research have been discussed in this chapter.
2 Xylose Transport and Metabolism
In order to understand the co-fermentation of glucose and xylose, strong under-

standing of the xylose transport and its metabolic pathway is required. Pentose
fermenting yeast species include Pachysolen tannophilus, Scheffersomyces
(Candida) shehatae, and Scheffersomyces (Pichia) stipitis. Although, these strains
Improving Yeast Strains for Pentose Hexose … 25
are xylose fermenting, but the rate of fermentation is much lower than that for
glucose fermentation (Jeffries 1985). This is due to the fact that these are intolerant
to high ethanol concentration (Maleszka and Schneider 1982) as well as by-product
formation, such as xylitol.
Xylose transport plays an important role in its metabolism which can occur only
after the uptake of the sugar. There are different transport systems in different
microorganisms for the uptake of xylose. These include—facilitated diffusion and
active mechanism. In active transport system, metabolic energy is required and
uptake occurs against a concentration gradient. Active transport systems can be
categorized depending upon the type of metabolic energy used, such as
chemo-osmotic, direct energization, or group translocation. While facilitated diffu-
sion does not require any metabolic energy for the uptake of sugars. As both the
systems need carrier proteins for sugar uptake making these systems specific to
the substrate type and the maximum rate of uptake and transport is dependent upon
the expression and activity level of the carrier protein and other enzymes involved in
the transport system (e.g., transmembrane diffusion gradient in the case of facilitated
diffusion). Studies also suggest that these systems can be subjected to competitive
inhibition by other sugars or their analogs, due to substrate specificity. Since there
are many chemo-osmotic proton symport-based transport systems, so they are
strictly pH-dependent (McMillan 1993). Previous studies on xylose-utilizing yeast
strains clearly indicate the presence of two import systems in xylose transport: a high
capacity, low-affinity facilitated diffusion system for both, xylose and glucose; and a
high-affinity proton symport system specific for xylose (Does and Bisson 1989,
1990; Kilian and Vanuden 1988; Lucas and Vanuden 1986).
After being transported into the cells, xylose metabolism starts through a
two-step oxidoreductive isomerization and is converted to xylulose (Webb and Lee
1990). Xylulose is being converted to xylulose-5-phosphate through the action of
enzymes, NADH- or NADPH-dependent xylose reductase (EC 1.1.1.21; XR), and
NAD-dependent xylitol dehydrogenase (EC 1.1.1.14; XDH). Whole pathway has
been presented in the Fig. 1. There is an alternate pathway for the conversion of
Embden-Meherhof-Parnas
(or Entner-Doudoroff pathway)
Xylose Xylitol Fructose,6-P

Reductase
Pentose Fructose,1,6-P
Xylose Xylitol Xylulose-5-P phosphate
dehydrogenase pathway
Xylose
Xylulokinase G,3 P
Isomerase
Xylulose Fermentation
Ethanol
Fig. 1 Xylose metabolism to produce bioethanol

xylose to xylulose which is catalyzed by xylose isomerase, but this pathway is

generally adopted by bacteria (Mishra and Singh 1993) and some anaerobic fungi
(Mishra and Singh 1993) but not by yeast strains. Xylose metabolism has been
extensively studied in Pichia stipitis and the xylose is metabolized in the strain as
illustrated above.
2.1 Xylose Metabolism in S. Cerevisiae
Xylose uptake in yeasts can occur by both facilitated diffusion and active transport
processes. In S. cerevisiae, a yeast generally recognized not to metabolize xylose,
xylose is transported by facilitated diffusion (Jeffries 1983; Lucas and van Uden
1986). Although S. cerevisiae possess facilitated diffusion system for sugar uptake,
but inefficiency to metabolize xylose is due to the fact that this system exhibits more
affinity toward glucose than xylose. However, it has also been studied that
S. cerevisiae possesses homologs for genes involved in xylose metabolism, but
lacks the functional metabolic pathway. These homologs are GRE3, YPR1,
YJR096 W (xylose reductase), YLR070C, SOR1, YDL246C (xylitol dehydroge-
nase), and YKS1 (xylulokinase).
As stated earlier, Saccharomyces cannot utilize xylose but it can grow on
xylulose, suggesting that this yeast is unable to convert xylose to xylulose, therefore
is unable to metabolize xylose for ethanol fermentation (Toivari et al. 2004). While
in most of the yeast strains other than Saccharomyces, xylose uptake occurs after
the depletion of glucose, its metabolism does not start immediately. Metabolism
starts after a lag period of productive processes, especially for the formation of
xylose metabolizing enzymes (Gong et al. 1981).
In a study by Toivari et al. (2004), it was found that when these endogenous
genes of Saccharomyces for xylose metabolism were overexpressed, xylitol for-
mation was increased, suggesting the existence of efficient xylose metabolic
pathway. As for example, overexpression of GRE3 and ScXYL2 enabled the yeast to
grow on xylose, even in the presence of glucose under aerobic conditions. While
the accumulation of xylitol could be due to low XDH activity and the specificity of
Gre3p toward NADPH because XRs cannot supply NAD+ required for the activity
of XDH. So, this reaction cannot occur under anaerobic conditions due to inability
of NAD+ regeneration, resulting in non-utilization of xylose. Due to the NADPH
specificity of Gre3p, redox imbalance is created which results in the accumulation
of xylitol and hence xylose metabolism cannot occur anaerobically in yeast.
2.2 Native Pentose Fermenting Yeasts
There have been several reports of pentose fermenting yeast strains which can not
only utilize xylose but can efficiently, ferment it for ethanol production. These
include Pichia, Pachysolen, Brettanomyces, Candida, Clavispora, Kluyveromyces,

Schizosaccharophora, etc. According to the literature, Candida shehatae,
Pachysolen tannophilus, and Pichia stipitis have been reported to be the best native
yeast species capable of efficient xylose fermentation with high yield (Mcmillan
1993). However, it has always been debated as to which of these three strains
should be used for commercial processes, but the research has been focused over
xylose fermentation through these three yeast species.
Native strains of S. cerevisiae exhibit similar characteristic as that exhibited by
the commercial strain by not utilizing xylose as the carbon source. While “torula
yeast” aka Candida utilis was grown over xylose but it was unable to produce
ethanol as this yeast is strictly aerobic. With the discovery of pentose fermenting
yeast species, mentioned above, intensive screening efforts have been made and it
has been observed that some yeast species can convert xylose to ethanol directly
under oxygen-limited conditions (Skoog and Hahn-Hägerdal 1990; Fonseca et al.
2007).
Considering the presence of pentose fermenting yeast species in nature, many
strategies can be adopted for improving the co-fermentation efficiencies of the yeast
species. These may include genetic modification through recombinant DNA tech-
nology or through evolutionary approaches, so that simultaneous utilization and
fermentation of glucose and xylose can be achieved in the improved strain.
3 Scope for Improvement
Since many pentose fermenting yeasts are known, their metabolic pathways need to be
studied for improving the fermentation efficiencies of the native as well as commercial
strains. Various strategies (Fig. 2) have been employed for the improvement of the
Fig. 2 Possible strategies for metabolic engineering of yeast

Fig. 3 The rate limiting steps for xylose metabolism and fermentation and possible remedial
measures
strains. Possible strategies include metabolic engineering (Fig. 3) and evolutionary

engineering. These have been discussed in detail below.
3.1 Metabolic Engineering Aspects
Study of xylose metabolism in yeast suggests that the strains can be engineered for
the efficient utilization and fermentation of xylose sugar. Strains capable of pentose
fermentation can be engineered and screened for their co-fermentation potential.
This can be done through metabolic engineering to incorporate the genes encoding
the enzymes required for xylose metabolism. Metabolic engineering is defined as
the strategy to improve the product formation through the modification of a specific
biochemical reaction or the introduction of a new one through recombinant DNA
technology (Bailey 1991; Stephanopoulos et al. 1998). S. cerevisiae has been
engineered to express these genes and it shows a theoretical yield of 0.51 g g−1
from xylose fermentation, but with low maximal productivities (Ostergaard et al.
2000).
Characteristics of an ideal microorganism for the efficient production of

bioethanol/biofuel include: specific productivity, broad substrate range, and high
ethanol tolerance as well as tolerance to the inhibitors present in lignocellulosic
hydrolysates (Aristidou and Penttila 2000).
3.1.1 Genetic Engineering Approach
Since most of the xylose metabolism has been studied in P. stipitis, efforts have
been made to genetically engineer the commercial strain of the yeast, i.e., S.
cerevisiae by expressing the genes from P. stipitis, involved in xylose metabolism.
In one such effort, a research group has expressed XYL1 and XYL2 genes (en-
coding XR and XDH from P. stipitis) in S. cerevisiae, which led to enhanced
bioethanol production utilizing xylose. When an endogenous gene, XKS1 which
encodes for XK was overexpressed in the recombinant strain of S. cerevisiae (al-
ready harboring XYL1 and XYL2 gene from P. stipitis), ethanol yield from xylose
was enhanced but it was observed that the rate of fermentation is still low and a
huge amount of xylitol was accumulated during the process, making this process
unsuitable for commercial applications. So, it was concluded that various metabolic
strategies need to be configured out for the construction of a genetically improved
strain for bioethanol production. These strategies are for optimization of the ratio of
the genes XR/XDH/XK (Eliasson et al. 2001; Matsushika and Sawayama 2008),
heterologous expression of transporters involved in xylose uptake and the use of
enzymes with decreased/altered coenzyme affinity (Matsushika et al. 2008a, b),
these enzymes include XDH and XR which show affinity toward NAD+ and
NADH, respectively. All these strategies can be applied over S. cerevisiae strain,
expressing XR, XDH, and XK.
First report of successful construction of a recombinant strain of S. cerevisiae
came from the works of Ho and coworkers (Ho et al. 1998). This strain could
efficiently ferment xylose alone as well as exhibit co-fermentation of xylose and
glucose (Ho et al. 1998). In this study XR and XDH genes from P. stipitis and XKS
gene from S. cerevisiae were used for the transformation of S. cerevisiae and
co-fermentation of glucose and xylose was observed in the resultant recombinant
strain. While in another report (Rodriguez et al. 1998) wherein overexpression of
the endogenous gene XKS from S. cerevisiae was carried out, strain could not grow
over xylulose. Toivari and coworkers overexpressed the endogenous genes XKS 1,
PsXR, and XDH and the recombinant strain successfully fermented xylose to
ethanol (Toivari et al. 2001). Many such reports have come ever since Ho et al.
made an effort to genetically improve the yeast strains (Matsushika et al. 2009,
Matsushika et al. 2008; Karhumaa et al. 2005; van Vleet et al. 2009). As we analyze
these reports, it can be inferred that the controlled expression of XKS gene in
S. cerevisiae can improve the xylose utilization ability of the strain as well as xylose
fermentation properties. Jeffries and coworkers have been very active in this field to
genetically modify the S. cerevisiae strain through recombinant DNA technology
and they have generated some good reports. One of these reports suggests that the
recombinant strain of S. cerevisiae which was subjected to spontaneous or chemical

mutations, could overcome growth inhibition caused by the overexpression of
ScXKS and PsXKS gene (Ni et al. 2007).
Since, aerobic conditions are required for xylose fermentation, it was found in
one of the study carried out by (Shi et al. 2002, 1999) that respiratory deficient
strains can accumulate more ethanol than the normal ones. They created a strain,
whose cytochrome pathway was inhibited by way of heme protein disruption in
cytochrome c. Results were quite interesting as this mutant produced more ethanol
(0.036 g g−1) than the wild type (0.027 g g−1).
Classic strain improvement approaches result in random changes in the genome,
which may be undesirable and may have adverse effect on the strains and the
overall productivity of the yeast strains for ethanol production. However, genetic
engineering approaches target the specific genes in the entire genome, involved in
the metabolic pathway. Only limitation of this approach is the little knowledge of
the metabolic pathways and the target points needed to generate a desired strain
with higher mixed sugar fermentation efficiencies.
Cofactor Imbalance
Although, there have been several reports of genetically engineered strains har-
boring genes from other pentose fermenting yeasts, most of these genetically
modified strains have some flaws. These flaws are due to the fact that cofactor
imbalance occurs as XR and XDH are specific to NADPH and NAD, respectively,
and regeneration of NADH requires aerobic conditions. To address this issue of
cofactor imbalance, researchers have been trying to construct strains with altered
affinities toward these cofactors. These modifications can be of three types:
increasing the affinity of XRs toward NADH than NADPH, increasing the affinity
of XDHs toward NADPH than NAD, and modification of both XRs and XDHs.
It is worthwhile to mention that there are two yeast species of whom XRs have
more affinity toward NADH than NADPH, these are Candida parapsilosis and
Spathospora passalidarum. Among the two yeast species, S. passalidarum exhibits
xylose fermentation efficiently under anaerobic conditions (Hou et al. 2009).
Specificity of XRs and XDHs of various strains has been altered by different
groups. Some of these efforts have been listed in Table 1:
Basis of these mutations is the fact that XRs possess an IPKS (Ile-Pro-Lys-Ser)-
conserved motif and that Lys residue interacts with the 2’-phosphate group of
NADPH (Lee 2008).
Another important factor to be kept in mind while developing a potential strain
for xylose utilization and henceforth its efficient fermentation is the choice of the
host. Generally, S. cerevisiae is used as the host, but its strains also show varied
efficiencies during fermentation. Therefore, it should possess the ability to ferment
xylulose and be tolerant to inhibitors present in the lignocellulosic biomass
(Eliasson et al. 2000; Garay-Arroyo et al. 2004; Nilsson et al. 2005; Sonderegger
et al. 2004; Yu et al. 1995).
Table 1 Mutations caused in the variants to alter the affinities of XRs and XDHs
Yeast Mutation Purpose Reference
XR mutants K274M NADPH to NADH Petschacher et al. (2005)
C. tenuis preference
K274R NADPH to NADH
preference
S275A NADPH to NADH
preference
N276D NADPH to NADH
preference
R280H NADPH to NADH
preference
S. stipitis K341R/N343D NADPH to NADH Dmytruk et al. (2008)
preference
E223A Strict NADPH Khattab et al. (2011)
dependence
E223D Strict NADPH
dependence
S271A Strict NADPH
dependence
K270 M NADPH to NADH Kostrzynska et al.
preference (1998)
XDH mutants 1208R NAD to NADP Watanabe et al. (2005)
S. stipitis preference
F209S NAD to NADP
preference
N211R NAD to NADP
preference
D270G NADP preference Metzger and Hollenberg
(1995)
D270G/D210G NAD to NADP
preference
Evolutionary Genetic Approach
Apart from the genetic engineering approach, evolutionary genetic approaches may
be employed as well for the construction of an improved strain. Here a genetically
modified strain is further subjected to improvement strategy through evolutionary
engineering. This approach includes exposing a strain to varying stress conditions
and then screening the best isolate among others.
Mutagenesis is the most common approach used under this strategy.
In this approach, firstly, a recombinant strain is chosen with desired traits, which
is further mutagenized and then subjected to stress conditions to select the best
strain of industrial relevance. An example of this approach is the construction of a
vector carrying the expression cassettes for xylose utilization (Demeke et al. 2013).
In this strategy, a diploid recombinant industrial strain is mutagenized and those
mutants which are able to grow on xylose are selected. Their genome is shuffled and
the best D-xylose utilizers are selected in D-xylose pretreated spruce hydrolysate.
The cultures are then subjected to evolutionary adaptations and the best clones are
selected at different stages and the best strain from the last stage is assumed to be
the best performer for xylose utilization and fermentation.
An advantage of this study is that no preliminary knowledge of the metabolic
networks or metabolic pathways is required as in the case of genetic engineering
approach, where a gene is being targeted. Multigenic traits can as well be addressed
with this approach. Only limitation associated with this issue is the identification of
genetic modifications of the improved strain as the genetic changes are random due
to the random mutagenesis of the genetically modified strain.
3.1.2 Other Strategies
Efforts have been made to improve pentose fermentation efficiency of yeast strains
by employing strategies other than metabolic engineering. These may include tar-
geting the native PPP. As an explanation to this, enzymes xylulokinase, transal-
dolase, and transketolase have been supplemented by heterologous native genes,
which have been imported from P. stipitis; for example, PsTAL1 may be expressed
to supplement ScTAL1 (Jin et al. 2003). It was evident from the enzyme kinetic
studies that the transaldolase from the Pichia was more efficient than from the
Saccharomyces. This lead to the conclusion that there might be the existence of
more efficient PPP enzymes involved in xylose consumption and hence bio-
prospecting might lead to the identification of such enzymes. Apart from targeting
PPP, other strategies for strain improvement include mutagenesis, adaptation,
genome shuffling through protoplast fusion, etc. (Fig. 2).
Mutagenesis
Mutations can improve the strains and these are of two types, either random
mutations or site-directed mutagenesis. After a mutation has been produced, effi-
cient screening process is required to select the desired mutant. Recombinant strains
have been mutagenized showing enhanced ethanol production than the wild type
strain. In one such report, a recombinant strain of S. cerevisiae, TJ1 was mutated
with the chemical mutagen, ethyl methane sulfonate (EMS). It exhibited lower XR
and higher XD and XKS activities than the parental strain leading to 1.6-fold
increase in ethanol production (Tantirungkij et al. 1994). Another example is a
mutant of S. cerevisiae TMB 3001 which can utilize xylose even under anaerobic
conditions. It was developed by sequential EMS mutagenesis and then adaptation
under microaerophilic and anaerobic conditions (Sonderegger et al. 2003).
A similar strategy was employed over the mutagenized strains of S. cerevisiae
(3399 and 3400) which showed improved growth on glucose and xylose (Wahlbom
et al. 2003). As is evident from the different experiments carried out by the
researchers that most of the xylose utilizing and fermenting yeast species do not
work best under anaerobic conditions, which is required for the co-fermentation of
glucose and xylose, natural selection and random mutagenesis can be employed in
such cases (Matsushika et al. 2009). Apart from EMS, other mutagens used for
mutagenesis include 2-deoxyglucose, UV, etc. 2-deoxyglucose (2-DOG) has been
used to produce a mutated strain, which displayed substantial improvement in
xylose utilization (Sreenath et al. 2000). According to another report, a UV
mutagenized P. stipitis NRRL Y-7124 exhibited higher ethanol production than the
wild type strain (Bajwa et al. 2009).
Another strategy is site-directed mutagenesis to obtain mutants with improved
xylose utilization and fermentation efficiencies. Site-directed mutagenesis was used
to target NAD+ dependent XDH from P. stipitis, so that a structural zinc atom could
be introduced to completely reverse the specificity of the enzyme (Watanabe et al.
2005). After the screening process, the selected mutants exhibited thermo stability
and increased catalytic activity with NADP+. Same experiment was carried out to
reverse the specificity of PsXDH toward NADP+. To add to this date for muta-
genesis, one of the S. cerevisiae mutant (MA-R5), showed high ethanol yield and
low xylitol yield from xylose, under the influence of a strong promoter. This strain
also exhibited fermentation with a mixture of sugars, with sugars being, glucose and
xylose (Watanabe et al. 2007; Matushika et al. 2008; Matsushika et al. 2009).
Genome Shuffling Through Protoplast Fusion
Genomes of the modified and improved strains can be mixed or shuffled to produce a
new improved strain of fermentative importance. Protoplast fusion is a means of
genome shuffling between different yeast species. It has advantages such as promotion
of high frequency of genetic information. Protoplast fusion provides characteristic
advantage such as promotion of high frequencies of genetic information between
organisms for which poor or no genetic exchange has been demonstrated or which are
genetically uncharacterized (Heluane et al. 1993). For efficient protoplast fusion, a
fusogenic reagent is required, such as PEG (polyethylene glycol). In the presence of
PEG, protoplasts fuse and form diploids or hybrids. This technique has been used for
fusing the pentose and hexose fermenting yeasts and the resultant hybrids displayed
higher ethanol formation and yielded more biomass. An example of this work is the
transfer of xylose-utilizing genes from P. tannophilus to S. cerevisiae (Heluane et al.
1993). Hybrids produced were morphologically similar to S. cerevisiae but were able
to utilize pentose like P. tannophilus did. Similar work was done by Lin and
coworkers who fused Schizosaccharomyces pombe and Lantinula edodes which
resulted in a fusant capable of utilizing xylan as the sole carbon source (Lin et al.
2005). A thermo tolerant S. cerevisiae and mesophilic xylose-utilizing C. shehatae
were electro fused leading to the formation of a fusant exhibiting 90 % fermentation
efficiency and higher temperature tolerance up to 40°C (Pasha et al. 1997). A fusant F6
was obtained by fusing C. shehatae and S. cerevisiae, displaying increased ethanol
production of 28 % than its parent strain (Li et al. 2008). Li and coworkers used the
combinatorial strategy of firstly adapting the C. shehatae strain for ethanol tolerance
and then mutagenizing it by UV irradiation and then screened a mutant RD-5 which
was deficient in respiration. Then, the protoplasts of this mutant strain were fused with
that of S. cerevisiae and the fusant was obtained which showed 28 % higher ethanol
production than the parent C. shehatae strain. Pure genome shuffling experiments
include the several rounds of shuffling and then finally screening strains for better
ethanol yield, lesser xylitol production and enhanced tolerance to inhibitors. In one
such experiment, genomes of six UV mutagenized P. stipitis strains were used for
shuffling and 3rd and 4th round of genome shuffling, improved mutant colonies were
pooled, regrown, and spread on hardwood spent sulphite liquor (HWSSL) gradient
plate again (Bajwa et al. 2010). Two mutants from the 4th round were able to grow in
80 % (v/v) HWSSL while the other two mutants from the 3rd round could grow in
85 %/85 % (v/v) HWSSL. The conclusion of this study was that the mutants after
protoplast fusion exhibited more inhibitor tolerance toward HWSSL than the parent
strains.
Adaptation
Adaptation approach is an alternative approach for the improvement of yeast strains

for xylose utilization and fermentation as it is time consuming and may not produce
the desired results. Yet, there have been reports of successful enhancement of
ethanol production form yeast strains using this strategy. For example, modified
strains of P. stipitis and C. shehatae have been obtained for the fermentation of
detoxified and partially detoxified hydrolysates (Zhu et al. 2009). Another example
includes ethanologenic yeast adapted to inhibitors due to repeated subculturing over
the medium with furfural and HMF up to 10–20 mM concentration. This yeast
strain could grow more efficiently in the presence of inhibitors than its parent strain
(Liu et al. 2004). Using combinatorial strategy, a nonrecombinant strain of S.
cerevisiae was developed which could grow upon xylose, efficiently than its parent
strain. This combinatorial strategy includes natural selection and breeding. This
strategy was employed by Attfield and Bell to produce a nonrecombinant strain of
S. cerevisiae (MBG-2303), which could grow aerobically on xylose and exhibited
considerable increase in biomass yield as compared to the parent strain (Attfield
et al. 2006).
Evolutionary adaptation approaches have been adopted for recombinant strains
to enhance their fermentation efficiencies. Earlier, these efforts were applied on the
genetically engineered strains but a new strategy involves genetic engineering,
followed by mutagenesis with EMS and then two-step evolutionary adaptation
(under sequential aerobic and oxygen limited conditions) (Wisselink et al. 2009).
Improved strain demonstrates a fourfold increase in the specific growth compared to
the parent strain. It was also found during the study, that crucial enzymes of xylose
metabolism (XR, XDH, and XKS) remain unchanged, indicating that this strategy
created new genetic traits letting the mutants to metabolize xylose (Liu and Hu
2010). These traits still need to be studied.
Although no prior information is required for the modification of the strains,

using the above-mentioned strategies, there are some limitations associated with
these strategies. Major drawback of the above-discussed strategies is the screening
as it becomes tedious to screen a single strain with the desired traits amongst others.
Therefore, a high-throughput technology is required for the screening processes.
Also, there are a very less no. of selection criteria for the improved strains through
evolutionary engineering.
Since researchers have developed a number of strategies for improvement of
yeast strains for enhancing lignocellulosic ethanol production, various efforts have
been made to improve strains using these strategies. A very few of them have been
listed in Table 2.
Of all the methods described here, genetic engineering is the most robust
technique in which all the underlying processes are known. But this strategy has not
generated any potent strain of industrial relevance as there is a no. of constraints
associated with this method. Therefore, other strategies are used in conjunction with
genetic engineering approach.
4 Constraints
Although pentose fermenting yeasts have been discovered, but the problem of
co-fermentation of glucose and xylose still remains unsolved. This is due to the fact
that glucose fermentation precedes that of xylose but by the time glucose is com-
pletely fermented, yeast strains being ethanol intolerant are unable to utilize xylose
efficiently. Along with ethanol intolerance, cofactor imbalance is another problem.
This problem arises as a result of affinity of XR and XDH for NADPH and NAD,
respectively, which leads to the accumulation of NADP and NADH causing
cofactor imbalance. This issue has already been discussed in the previous sections
of this review. To regenerate these cofactors, oxygen is required; hence, aerobic
conditions become mandatory for xylose metabolism. Another disadvantage of
aerobic fermentation is the concurrent utilization of ethanol even when xylose is not
completely utilized. All these problems can be addressed by genetic manipulations
or by evolutionary engineering techniques allowing the organism to grow in the
presence of mixed sugars and hence fermentation to occur. One more problem of
concern is the presence of inhibitors after pretreatment which adversely affect
microbial growth and fermentation. This issue can be resolved by letting the strains
grow in the presence of inhibitors and then screening out for the best strain capable
of efficient fermentation. Although all these issues have been addressed by various
research teams and several improved strains have been developed, but a strain of
commercial importance has not been produced till date.
36
Table 2 Strain improvement strategies and ethanol yield obtained

Yeast strain Yeast Method of cultivation Sugar concentration Ethanol Ethanol Reference
improvement yield (gg−1) productivity
strategy (gL−1h−1)
S. cerevisiae ScF2 Genome Fermentative 50 gL−1 glucose + 50 0.40 NA Zhang et al.
shuffling product gL−1 xylose (2012)
S. cerevisiae A Industrial strain Anaerobic batch 50 gL−1 glucose + 50 0.42 NA Martinez et al.
fermenstation gL−1 xylose (2001)
S. cerevisiae and Co-culture Fermentative 30 gL−1 glucose + 30 0.41 NA Rouhollah et al.
P. stipitis gL−1 xylose (2007)
P. stipitis and Co-culture Fermentative 30 gL−1 glucose + 30 0.36 NA Rouhollah et al.
K. marxianus gL−1 xylose (2007)
S. cerevisiae ITV01 and Co-culture Aerobic batch 75 gL−1 glucose + 30 0.40 NA Gutierrez-Rivera
P. stipitis Y-7124 fermentation gL−1xylose et al. (2012)
S. cerevisiae Fusant 1 Protoplast fusant Fermentative 30 gL−1 glucose + 20 0.19 NA Yan et al. (2009)
gL−1xylose
S. cerevisiae YD43-4 Protoplast fusant Fermentative 75 gL gL−1 0.348 NA Chmielewska
glucose + 30 et al. (2003)
gL−1xylose
S. stipitis BCRC21777 Adaptation Adapted to increasing Rice straw hydrolysate NA 0.44 Huang et al.
concentration (2009)
S. cerevisiae TMB 3008 Recombinant NA 50 gL−1 xylose 76 0.27 Jeppsson et al.
(2002)
S. cerevisiae BH42 Recombinant NA 50 gL−1 glucose + 50 56 NA Sonderegger et al.
gL−1 xylose (2004)
P. stipitis FPL-UC7 Recombinant Fermentative Xylose 80 g/L in 0.38 0.41 Shi et al. (1999)
complex medium
P. stipitis FPL-Shi21 Recombinant Fermentative Xylose 80 g/L in 0.46 0.43 Shi et al. (1999)
complex medium
Shalley Sharma et al.
S. cerevisiae (δX-27) Recombinant Fermentative NA 0.35 NA Kato et al. (2013)

5 Future Prospects
As utilization of pentoses, particularly, xylose has become a prerequisite for cost

effective bioethanol production from lignocellulosic biomass, most of the research
has centered on developing strategies for metabolic engineering of the yeast and
bacterial strains to develop a novel strain with the desired characters. Although,
various groups have been doing research and have developed new strains through
genetic engineering approaches, but no successful lead has been achieved as fer-
mentation efficiency of xylose fermentation has been observed to be insignificant
for commercial purposes. Hence, strategies should be emphasized over developing
strains with higher substrate tolerance, tolerance toward hydrolysate inhibitors and
ethanol tolerance. The approach should be focused on efficient utilization of pen-
toses along with hexose for enhancement in overall fermentation efficiency of the
lignocellulosic substrate. Efforts are already under process to develop a modified
strain of commercial importance using high-throughput screening techniques and
better expression systems for efficient production of membrane proteins. Earlier
efforts included integration of genes into the plasmids and then transformation of
the yeasts strains with these recombinant plasmids which lead to the development
of inefficient strains, but, recently chromosomal integration of these genes has been
successfully reported in yeast strains for commercial production of bioethanol from
xylose (Matsushika et al. 2009). So, deep understanding of the metabolic pathways
of sugar utilization in yeast is required to combine biochemical engineering with
metabolic engineering strategies for the betterment of the microbial (yeast and
bacterial) strains with respect to fermentation efficiency improvement.
6 Conclusion
A lot of work has been done for the improvement of yeast strains with better xylose
utilization and fermentation abilities. This work has generated a lot of data for
reference. Of the various strategies employed for the betterment of the yeast strains,
genetic engineering is the most precise one, as it targets the specific genes involved
in the pentose metabolism. Though studies suggest that none of the strategies can
alone be used for the improvement of the strains. So, a combinatorial strategy is
required. These combinatorial studies include strategies as listed in the previous
sections, such as genetic engineering, evolutionary engineering like mutagenesis,
adaptation, mutations, and genome shuffling through protoplast fusion. Some
studies also suggest that there are some hidden traits of the xylose metabolism
which are still uncovered and a complete understanding of these traits can help in
constructing a strain of commercial reputation.
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Pretreatment of Paddy Straw to Improve
Biogas Yield
Rupali Mahajan, Harmanjot Kaur, Raman Rao and Sachin Kumar
Abstract Biofuels have been considered as an alternative energy source to cope up

the future energy demand. There are various biomass-based energy sources
including biogas, bioethanol, biobutanol, etc. Biogas is one of the biofuels which
can be produced from the lignocellulosic biomass. Cellulose, hemicellulose, lignin,
and several inorganic materials constitute lignocelluloses. Lignocelluloses are often
the sole component of different agricultural waste such as paddy straw. Paddy straw
is one of the lignocellulosic biomass that can be used as feedstock for biogas
production because of its great profusion, sustainable supply, and low price. This
type of biomass is not fully biodegraded in anaerobic digestion process at industrial
scale due to their intricate physical and chemical structure that consequently leads
to lower energy resurgence in terms of biogas yield. The biodegradability of these
lignocelluloses waste can be increased by a pretreatment. Therefore, breakdown of
complex materials to simpler ones or to make cellulose accessible to hydrolytic
enzymes is the key step for better digestion to biogas. The aim of this paper is to
review promising pretreatment technologies which have greatly improved the
production of biogas. Effective parameters in paddy straw pretreatment involve
increased fortification by lignin and hemicelluloses. Various pretreatment methods
such as mechanical, thermal, chemical, biological, and their advantages and dis-
advantages as well as effects on the improvement of biogas production are
discussed.
Keywords Biogas Pretreatment Lignocelluloses Paddy straw
Rupali Mahajan Harmanjot Kaur Raman Rao S. Kumar (&)

Biochemical Conversion Division, Sardar Swaran Singh National
Institute of Bio-Energy (Formerly Sardar Swaran Singh National Institute
of Renewable Energy), Kapurthala 144601, Punjab, India
e-mail: sachin.biotech@gmail.com

DOI 10.1007/978-81-322-2773-1_4
44 Rupali Mahajan et al.
1 Introduction
With advancement in the technology, the energy requirement for the urbanized
industries has been tremendously increased all around the world. Moreover, the
easily accessible fossil fuels have also been decreased during almost two centuries
and have resulted in the increased prices. Therefore, supply of energy becomes one
of the major causes of concern for the globe (Cheremisinoff 2002). Conversely the
incineration of fossil fuel carriers such as petrol, natural gas, etc., liberates CO2,
SOx, and NOX which causes gigantic environmental problems and adversely affects
the ecosystem along with their negative impact on health. Currently, there is a real
apprehension about the environment due to its significance to a sustainable future.
The stipulation for a better way of living, as well as alleviation of environment
obliges us to search about new kinds of technologies, habits, and also to develop
way of thinking with least negative impacts (Mussgnug et al. 2010). As Richard
Wright says “In the brilliance of universal climate change, poignant in the path of
renewable energy sources seems indispensible”.
The agricultural sector has long been acknowledged as one of the most fasci-
nating activities for effectual execution of large scale use of renewable energy
especially conversion of feedstock from the plant or animal origin. Therefore
agricultural waste such as wheat straw and paddy straw can play an integral role in
meeting the increasing societal demands of energy in sustainable manner.
Specifically lignocellulosic waste has enormous potential for energy generation
(Chandra et al. 2012a). Rice is ranked as third largest agricultural crop grown in the
world with total cultivated land of 161, 42 million hectare with a gross yield
production of 678.69 million tons. It is estimated that about 905 million tons per
annum of lignocellulosic biomass is produced from cultivated rice. In order to
obtain better yield from these lignocellulosic materials pretreatment plays an
imperative role. Further for the better production of biogas the biodigestibility of
the waste can be enhanced by pretreatment. Moreover, it has been observed that in
anaerobic digestion most of the contents of biomass such as carbohydrates, pro-
teins, and fats are converted into simple derivatives and at last transformed into
biogas with the assistance of different types of anaerobic microorganisms.
Anaerobic digestion (AD) of biodegradable waste and other agricultural residues is
extensively used as it yields biogas rich in methane and carbon dioxide and is
suitable for production of energy. A range of pretreatment technologies have been
developed in the topical years in order to amplify the accessibility of AD of sugars
and other small molecules in biogas substrates predominantly in lignocellulosic
waste materials such as paddy straw (Fredriksson et al. 2006). These pretreatment
technologies basically aim to
• Increase the yield of biogas
• To make anaerobic digestion faster
• To avert processing tribulations such as high electricity requirement for amal-
gamation and development of floating layers
• To make use of new substrates.
Pretreatment of Paddy Straw to Improve Biogas Yield 45
Fig. 1 Anaerobic digestion process
Anaerobic digestion generally involves four steps (as shown in Fig. 1), i.e.,
I. Hydrolysis: This step occurs at the beginning of the process as lignocelluloses
have a recalcitrant structure which is naturally designed to prevent the enzy-
matic degradation; hydrolysis occurs as extracellular enzymes, produced by
hydrolytic microbes that decompose complex organic polymers into simple
monomers.
II. Acidogenesis: These small molecules are converted to the mixture of volatile
fatty acids (VFAs) and alcohol by microorganisms of anaerobic and faculta-
tive group called acidogenic bacteria.
III. Acetogenesis: The VFAs are further converted to acetate, carbon dioxide, and
hydrogen by Acetogenic bacteria and further act as substrate for methanogens.
IV. Methanogenesis: In this step, the anaerobic bacteria called as methane formers
converts organic acid into biogas. The main constituents of biogas are methane
and carbon dioxide with traces of H2S, H2, N2, etc. These methanogens are
quite sensitive to the pH changes (Zheng et al. 2014).
The degradation of lignocelluloses is a complicated process as lignocellulosic
waste material such as paddy straw has a recalcitrant composition and often contain
high amount of lignin which is difficult to degrade. Therefore, consecutively a
pretreatment process is quite indispensable to disrupt the naturally intractable lignin

shield that impairs the accessibility of enzymes and microbes to cellulose and
hemicelluloses to improve the yield of biogas from paddy straw (Teghammar et al.
2014). The present paper paves the way for integrating the waste obtained from
rice, by reviewing different methods for their pretreatment and production of biogas
with its positive and negative aspects.
2 Material Composition of Paddy Straw
2.1 Cellulose
It is the main component of lignocellulosic biomass. It is a polysaccharide that

consists of a linear chain of D-glucose linked by β−1, 4 linkages. The cellulose
strains are allied together to make cellulose fibrils (Li et al. 2010). Cellulose fibers
are connected by a number of intra and intermolecular hydrogen bonds. Therefore
cellulose has high solubility in water and the majority of organic solvents
(Swatloski et al. 2002).
2.2 Hemicellulose
Hemicelluloses are assorted branched polymers containing pentoses and hexoses.

They are located in the secondary cell walls of the lignocellulosic biomass (Girio
et al. 2010). They are easy to hydrolyze because of its amorphous nature and
branched structure as well as due to its low molecular weight. As hemicelluloses
covers the cellulose fibrils and affects its digestibility and availability for enzymatic
hydrolysis (Agbor et al. 2011). The accessibility to celluloses can be increased by
the removal of hemicelluloses.
2.3 Lignin
Lignin is an aromatic polymer synthesized from propanoid precursors. The cellu-

lose and hemicelluloses are creased by lignin. It is accountable for structural
stringency and integrity. The major chemical phenylpropane units of lignin make a
complicated matrix consisting primarily of syringyl, guaiacyl, and p-hydroxy
phenol. Therefore, the pretreatment of lignocellulosic biomass results in improved
rate of hydrolysis. By the removal of lignin, the pretreatment provides better surface
accessibility for enzymes by increasing the pore volume (Demirbas 2008). Figure 2
shows the percentage material composition of lignocellulosic biomass.
Fig. 2 Composition of paddy

straw
cellulose
Lignin 45-50%
20-25%
Hemicellulose
25-30%
3 Pretreatment Methods
The basic idea behind pretreating a particular substrate for biogas process is to
increase the yield of methane and also to minimize the required time in order to get
the maximum yield. If the degradation process is fast, it will result in the higher
total degradation of the substrate in the biogas process and therefore would result in
the better yield of methane.
3.1 Physical Pretreatment
Physical pretreatment can result in increased pore size and accessible surface area.
It further helps in reducing the crystallinity and degree of polymerization of cel-
lulose. Among physical pretreatments diverse types of techniques such as
mechanical, thermal, as well as other techniques such as freeze pretreatment and
hydrothermal pretreatment can be used to improve the biodegradability or enzy-
matic hydrolysis of lignocellulosic wastes.
3.1.1 Mechanical Pretreatment
The main intent of mechanical pretreatment is to bring reduction in particle size as

well as crystallinity of lignocellulosic materials. The specific surface area increases
with the reduction in particle size and also reduction in the degree of polymer-
ization. This effect can be obtained by grinding or milling, finally depending upon
the particle size of the material. Further depending up on the nature of biomass, type
of milling, and also the extent of milling; hydrolysis yield can be increased by 5–
25 % (Delgenes et al. 2002). One of the disadvantages of using mechanical pre-
treatment is that the power constraint of this method is comparatively high
depending upon the characteristics as well as the final particle size of the biomass.
3.1.2 Thermal Pretreatment
Thermal pretreatment is basically responsible for structural alteration of the insol-

uble fraction to make it more amenable to biodegradability. In this the substrate is
held at high temperature of 125–190 °C under pressure for up to 1 h. The high
temperature is accountable for disrupting the hydrogen bonds that hold together
crystalline cellulose and lignocelluloses complexes. Thermal pretreatment is
effective up to certain temperatures. The maximum range of temperature depends
upon the nature of substrate and also on pretreatment retention time (Garrote et al.
1999). Although various studies have revealed that thermal pretreatment is used to
increase the yield of biogas up to certain range of temperature, above which biogas
yield starts decreasing. Moreover at high temperatures, certain dark colored lignin
and xylose breakdown products are produced. These compounds comprise of
phenolic and heterocyclic compounds such as furfural. These compounds act as
inhibitory compounds and inhibit the activity of AD microorganisms and hence
affect the yield of biogas.
Liquid Hot Water (LHW) Pretreatment
Liquid hot water pretreatment is used to make cellulose accessible to AD

microorganisms by solubilizing the hemicelluloses. Further the formation of inhi-
bitors can also be avoided by maintaining the pH range between 4 and 7. The pH
range between 4 and 7 minimizes the formation of monosaccharides and other
degradation products that can further catalyze hydrolysis of cellulosic material
during pretreatment. Therefore, by maintaining the pH range of 4 and 7 the auto-
catalytic formation of fermentation inhibitors is avoided during pretreatment
(Kohlmann et al. 1995; Mosier et al. 2005; Weil et al. 1997). The major advantage
of using liquid hot water is that the solubilized hemicelluloses and lignin products
are present in lower concentrations due to higher water input. Due to the lower
concentrations of hemicelluloses and lignin the risk on degradation products like
furfural is reduced. Further it also helps reducing condensation and precipitation of
lignin compounds.
3.1.3 Hydrothermal Pretreatment
In hydrothermal pretreatment water is used under high temperature and pressure so

that it can penetrate into the biomass, hydrate cellulose, and remove hemicelluloses
and part of lignin. The major advantages of this pretreatment are
• No addition of chemicals
• No requirement of corrosion resistant materials for hydrolysis reactors
• No need for size reduction of biomass (Taherzadeh and Karimi 2008).
• Enhances accessible and susceptible surface area of the cellulose and makes it
more accessible to hydrolytic enzymes (Zeng et al. 2007).
Pretreatments of paddy straw biomass were investigated by Chandra et al.
(2012b) hydrothermal pretreatment was done which was further followed by
addition of 5 % NaOH at 200 °C for 10 min, in order to maintain the pH suitable for
the fermentation of methane and to circumvent the formation of inhibitor. Various
experimental studies have also revealed that addition of NaOH to paddy straw
results in enhanced biodegradability of paddy straw biomass which is apparent from
the augmented production of biogas. The result showed that biogas and methane
production yield as 315.9 and 132.7 L/kg VS, respectively, increase of 225.6 % in
biogas production and 222.0 % in methane production relative to untreated paddy
straw. Hydrothermal pretreatment resulted into enhanced biogas and methane
production by accelerated prehydrolysis of biomass contents during the treatment
process. Moreover, it has taken longer hydraulic retention time as compared to
untreated substrates. The reason for taking longer HRT to convert the available
volatile degradable matter into biogas is that inadequate bacterial population were
present in the substrate during the process of degradation. Further an increase in
HRT may provide ample time for methanogens to convert organic matter to
methane and carbon dioxide (Kaparaju et al. 2010). Therefore, it is quite apparent
that the hydrothermal pretreatment is potentially effective for pretreatment to
enhance biogas yield from paddy straw biomass followed by proper amount of
NaOH addition.
3.1.4 Freeze Pretreatment
This pretreatment is requisite to amend the structural and chemical composition of

feedstock sequentially to provide efficient hydrolysis. For efficient hydrolysis the
cell structures need to be broken down and the pore size must be increased to
convert carbohydrates to fermentable sugars. Freeze pretreatment is one of the
unique approaches that boost the enzyme digestibility of paddy straw (Keshwani
and Cheng 2009). According to the study of Chang et al. (2011), with the help of
freeze pretreatment the enzymatic hydrolysis yield was increased by 84 %. In this
pretreatment the paddy straw was pretreated by freezing at −20 °C for 2 h and the
frozen straw was thawed at room temperature for 1 h. The result showed that freeze
pretreatment can reduce sugar yields from 226–77 to 417–27 g/kg and 93–84 to
138–77 g/kg substrate for 150 U cellulose and 100 U xylanase. Further it was
reported that acetate buffer was used to preincubate paddy straw for 1 h and the
solute of acetate buffer penetrated in the pores of paddy straw. The pore size and the
accessible surface area expanded as the buffer solute freeze. This further results in
breakdown of some structures and enhanced the rate of enzymatic hydrolysis
(Tuankriangkrai and Benjakul 2010).
3.2 Chemical Pretreatment
Chemical pretreatment has been investigated under diverse conditions using the
series of different chemicals, mainly acids, and bases of diverse strengths
3.2.1 Alkali Pretreatment
As discussed earlier, lignocellulosic materials due to their structure and composition

are defiant to hydrolysis. Lignocellulosic materials swell by the addition of alkali
and further results in partial solubilization of lignin. Additionally, alkali pretreat-
ment due to its low price, high recoverability, safe handling, and minor environ-
mental effects have been extensively used in improving the biodegradability. There
have been numerous reports which show that alkali pretreatment is quite effective
for anaerobic digestion. According to He et al. (2008) noteworthy increment was
seen in the biogas yield in batch tests using paddy straw. The paddy straw used was
pretreated with 6 % NaOH for 3 weeks at ambient temperature. The biogas yield
was increased by 27.3–64.5 %. The enhancement in yield was accredited to NaOH
pretreatment that increased biodegradability of paddy straw.
Ca(OH)2 is one of the alkalis that has been comprehensively used in improving
the biodegradability due to its high recoverability, low cost, safe handling, and
inconsequential environmental effects. Song et al. (2012) reported that the lignin,
cellulose, and hemicelluloses content of paddy straw were significantly degraded
when pretreated with 9–81 % Ca(OH)2 (w/w TS) for 5–89 day pretreatment time
and 45–12 % inoculums content. Further the methane yield was also increased up to
225–300 ml/g VS. These studies verified that alkali pretreatment can act as a
potential pretreatment option to increase the gas yield from lignocellulosic sub-
strates. Further it is important to note that pretreatment time considerably affects the
degradation of core components of paddy straw and the extensive treatments
increased the diminution in cellulose and hemicelluloses contents under certain
conditions. In general, this pretreatment technology is economically not so attrac-
tive due to high costs of alkalis but it may be useful for acidic substrates or
substrates rich in lignin content that could otherwise not be anaerobically digested
(Chang et al. 1997).
3.2.2 Acidic Pretreatment
Acid pretreatment disparately from alkali pretreatment does not result in lignin
disruption but is considered to break down hemicelluloses and interrupt ether bonds
between lignin and hemicellulose. Further for lignocellulosic biomass dilute acid is
particularly favored over concentrated acid for acid pretreatment (Kalnappert et al.
1981) Dilute acid primarily hydrolyzes up to 100 % of hemicelluloses into its
constituent sugars for example galactose, arabinose, and xylose depending on the
pretreatment conditions (Zheng et al. 2014). Zhao et al. (2010) reported that dilute
acid pretreatment with mixture of acetic-propionic acid significantly enhanced 35–
84 % production of methane. Further it was also revealed from the study that acid
pretreatment at concentration of 0–75 mol/L for 2 h at solid to liquid ratio of 1:20
(w/v), was able to remove about 34–19 % lignin and 21–15 % hydrolysis rate was
obtained. 0–75 mol/L of acid concentration can enhance the biodegradability of
paddy straw and can remove the hemicelluloses and lignin to some extent. This was
represented by the increased rate of hydrolysis because it could contribute more
hydrogen ions which help solubilizing hemicelluloses and lignin. As a result, the
cellulose can be more easily converted into fermentable sugars due to increased
accessibility caused by disruption of barriers by lignin and hemicelluloses. The
removal of lignin is not associated with the longer pretreatment time. It is more
likely that alteration in the structure of lignin results in removal of hemicelluloses,
in so doing enhancing the cellulose digestibility in the residue solids. The lignin
removal depends up on the certain fixed value of acid concentration, i.e., the
quantity of lignin removed depends on certain amount of hydrogen ion in the
reaction liquid. It can be concluded that hemicelluloses can be effectively removed
with the help of dilute acid pretreatment but it proves to be ineffective for lignin
removal. Another study by Hsu et al. (2010) reported that when paddy straw was
pretreated with 1 % (w/w) sulfuric acid for 1–5 min at 160 °C, followed by
enzymatic hydrolysis, the maximal sugar yield of 83 % was achieved. The pore size
of pretreated solid residues increased as a result of complete release of sugar such as
glucose and xylose. This further resulted in efficient enzymatic hydrolysis of 70 %.
3.2.3 Oxidative Pretreatment
Oxidative pretreatment is one of the effective pretreatments as it helps in breakdown

of lignin with hydrogen peroxide. Hydrogen peroxide (H2O2) is a strong oxidant
and has been used for biomass pretreatment for biogas production (Hendriks and
Zeeman 2009). It degrades biomass into oxygen and water without leaving any
residues. Further it also prevents the formation of any secondary products (Teixeira
et al. 1999). H2O2 treatment is a nonselective oxidation process and partially breaks
down lignin and hemicelluloses and releases a cellulose fraction with high
degradability to the anaerobic microorganisms. Further the inhibitors might be
generated as lignin is oxidized to form soluble aromatic compounds. The H2O2
concentration should also be considered because high concentration of hydrogen
peroxide was found to inhibit the AD process due to toxicity of an excess of

hydroxyl ions to methanogens (Song et al. 2013a).
Song et al. (2013b) reported that methane yield of 288 mL/g VS was obtained
when paddy straw was pretreated with 2–68 % H2O2 concentration and 1–08
substrate/inoculum for 6–18 days at ambient temperature of 25 ± 2 °C. The cellulose
and hemicelluloses content in the pretreated paddy straw decreased remarkably as
compared to untreated paddy straw. There was considerable decrease in the cellu-
lose, hemicellulose, and lignin concentrations pretreated with H2O2 ranging from 0.9
to 22.0 %, 3.7 to 60.3 %, and 0.4 to 12.5 %, respectively. The results showed that
H2O2 was particularly effective in the hemicelluloses decomposition of paddy straw.
There is great effect of pretreatment time (PT) on the degradation of the main
constituents of agricultural wastes. The extensive treatments could certainly con-
tribute to increased reductions in the contents of cellulose and hemicellulose under
firm conditions. At the higher H2O2 concentration (HC) it has been observed that
there is higher reduction in cellulose, hemicellulose, and lignin contents. The higher
H2O2 augmented the accessibility of contents to the anaerobic microorganisms.
This further facilitates the usage of low molecular weight soluble compounds by the
microorganisms and thus resulting in increased biodegradability. One possible
disadvantage of this pretreatment is that the proportion of CO2 increases in the
produced biogas by the increased volume of oxygen introduced in the system.
Moreover this pretreatment is not usually carried out, apparently due to its high
cost.
3.2.4 Sodium Carbonate–Sodium Sulfite Pretreatment
Sodium carbonate (Na2Co3) and sodium sulfide are the main constituents of green
liquor which shows promising enzymatic hydrolysis (Yang et al. 2012a). Even
though sodium sulfide is precisely good for delignification but the elemental sulfur
leads to the difficulty of handling process of spent liquor as well as environmental
contagion (Yang et al. 2012b). The considerable amounts of hemicelluloses can be
degraded by sulfite. Moreover the sulfite can reduce the degree of polymerization of
cellulose. It can further also increase the lignin hydrophilicity by sulfonation (Zhu
et al. 2009). Na2SO3 and Na2CO3 have low environmental risks and barriers and are
considered potentially advantageous.
Yang et al. (2012b) used different ratios of Na2SO3 and Na2CO3 at 140°C,
chemical charge of 12 % for 60 min to pretreat paddy straw. It was observed that
when pretreated with only Na2CO3 at ratio of 1:0, the increasing Na2CO3 charge
may strengthen the alkaline degradation of polysaccharides. Further the lignin
macromolecules are degraded by breaking down ester bonds between lignin,
hemicelluloses, and cellulose (Gaspar et al. 2007). The paddy straw at the ratio of
0:1 is pretreated with only Na2SO3, the sulfite (SO32−), or bisulfite (HSO31−) leads
to the lignin sulfonation to increase its hydrophilicity and may subsequently pro-
mote the process of enzymatic hydrolysis. Better delignification was observed with
the higher ratio of Na2SO3 as compared to Na2CO3 at the same chemical charge.
The delignification capacity of Na2SO3 was remarkable even at low chemical
charge. While in case of Na2CO3 chemical charge resulted in momentous impact on
its improved delignification. The pretreated paddy straw showed the highest sugar
recovery of 74.5, 82.7, and 59.5 % for total sugar, glucan, and xylan, respectively.
The Na2CO3 pretreatment at the ratio of 1:0 showed sugar recovery of 67.1, 74.4,
and 53.7 % for total sugar, glucan, and xylan, respectively. Its analogous deligni-
fication ratio was 40.3 % and polysaccharides conservation ratio was 88.9 %.
3.2.5 NMMO Pretreatment
N-methylmorpholine-N-oxide is an environmental friendly organic solvent used for

pretreatment. More than 98 % of this solvent can be recovered without the pro-
duction of any noxious waste pollutants and chemical derivatization (Adorjan et al.
2004). Compared with other volatile organic cellulose solvents, NMMO possess the
advantages of low toxicity, low hydrophobicity, low viscosity, low volatility with
potentially negligible environmental impact, nonflammable properties, thermal
stability, enhanced electrochemical stability and wide assortment of anion and
cation combinations, and high rate of reaction (Zheng et al. 2009).
Teghammar et al. (2012) reported that 328 Nml CH4/gCH when paddy straw
was pretreated with NMMO. During this pretreatment the paddy straw was digested
with 50 % w/w NMMO solution at 130 °C. As a result the produced methane was
seven times more than the methane production of untreated paddy straw samples.
This further corresponds to theoretical yield of 79 %. Additionally, it was also
reported that 1 to 3 h of pretreatment is considered quite efficient for paddy straw.
Extensive pretreatments resulted in less production of biogas. Furthermore, even
though the experiments went on for six weeks, the paddy straw can be efficiently
digested in about one week of anaerobic digestion. This means that just a couple of
days would have been enough to convert the pretreated paddy straw to biogas
provided if the digesting bacteria need no lag phase. This improvement is pre-
sumably due to the breakdown of the crystalline structure of the lignocelluloses. In
addition, the NMMO solvent converts the crystallinity of the dissolved cellulose,
which further increases the biodegradability (Jeihanipour et al. 2009). The best
results were obtained when paddy straw was digested for 1 h. Further the extensive
pretreatments resulted in lower preliminary digestion rate and also less production
of methane. The chemical pretreatment unlike physical and biological pretreatment,
may leave chemical residues which may perhaps influence the downstream
anaerobic digestion process. For NMMO pretreatment, it is possible to recover
nearly 100 % of the used NMMO liquid, so chemical residues might not be a
problem (Dadi et al. 2006).
3.3 Biological Pretreatment
It is one of the alternative methods that is cost effective and does not cause any
environmental pollution. One of the advantages of biological pretreatment is that it
can take place at lower temperature. Further as compared to other pretreatments it
does not require any chemicals. One of the disadvantages of biological pretreatment
is that it could be slower than other nonbiological methods.
3.3.1 Fungal Pretreatment
In context of anaerobic digestion, quite a lot of fungi are known for their ability to
remove lignin from lignocellulosic biomass. This predominantly includes white rot
fungi. The lignins are essential to remove for efficient production of biogas as
lignins could either cause problems during digestate use or can impede anaerobic
digestion (Barr and Aust 1994). Fungal pretreatment is one of the methods which
has been investigated to confiscate phenolic toxins before the process of anaerobic
digestion (Dhouib et al. 2006). Sahni and Phutela (2013) reported pretreatment of
paddy straw with standard culture namely C. versicolor MTCC 138 and isolated
lignocellulolytic culture, that is, Fusarium sp. In the case of control, there was very
little or insignificant change in almost all components, that is, cellulose, hemicel-
lulose, lignin, and silica. In case, when paddy straw is pretreated with C. versicolor
MTCC 138 and Fusarium species, the lignin content decreased with the increase of
incubation period with maximum reduction of 17.5 and 27.1 % in 20 days,
respectively. These observations clearly indicate that C. vesicolor MTCC 138 and
Fusarium sp. are lignocellulolytic fungus.
3.3.2 Enzyme Addition
Enzymes required to breakdown lignocellulosic biomass are produced by

microorganisms of anaerobic digestion and therefore, they are already present in the
digester. Further to enhance the process of breakdown, an addition of enzyme
mixture can be done. This enzyme mixture may include cellulose, hemicellulose,
pectin, and starch degrading enzymes. Practically the enzyme additives can be
applied in three different ways:
• By direct addition to a single stage anaerobic digester,
• By addition to hydrolysis and acidification vessel of a two-stage system or
• By addition to a dedicated enzymatic pretreatment vessel.
Various studies have been conducted to analyze the addition of enzymes to
anaerobic digester (Rintala and Ahring 1994). It was suggested in some of the
studies that there was no significant effect when the enzymes were directly added to
the biogas reactor. Moreover, in such case enzymes are quickly degraded after
addition (Binner et al. 2011). A number of studies have indicated that higher
substrate solubilisation is obtained by the addition of enzymes to first stage of a
two-stage anaerobic digestion process and consequently leads to higher yield of
biogas for example addition of cellulosic enzyme cocktails on paddy straw leads to
better yield of biogas (Quemeneur et al. 2012). Some studies showed that higher
substrate solubilisation or biogas yields in batch AD tests can be achieved by
enzymatic pretreatment in a dedicated vessel for example with various agriculture
residues with cellulolytic enzyme cocktail. Continuous anaerobic digestion of dif-
ferent agricultural residues pretreated in a dedicated vessel leads to small increases
in biogas yield (Quinones et al. 2011).
3.3.3 Anaerobic Microbial Pretreatment
This pretreatment is also acknowledged as two-stage digestion or dark fermenta-

tion, pre-acidification. The pretreatment technology is quite simple kind of in which
first step of anaerobic digestion is alienated from methane production. Although the
pH during methane production must be between 6.5 and 8, the pH value of the first
digester must lie between 4 and 6, which inhibits production of methane and causes
accumulation of volatile fatty acids (Deublein and Steinhauser 2010). This pre-
treatment method is having a positive effect on the methane concentration in biogas.
During the pre-acidification step, CO2 is formed in addition to H2 and volatile fatty
acids. CO2 formed can be present in three forms: in the form of carbonate ion CO2– 3
at higher pH values, in the form of bicarbonate ion HCO3– at neutral pH and in
acidic environment; it is present in the form of carbon dioxide. Due to low pH most
of the carbonate is present in the form of CO2. The CO2 thus produced is released
into hydrolysis gas produced from the pre-acidification step due to its volatility.
Therefore, the concentration of CH4 obtained is much higher which shows that
there is less CO2 in the gas phase of methanogenesis. The advantage of using
two-stage digestion is that the microorganisms are less responsive to many chem-
icals in the first stage as compared to second stage. Thus in the first step itself large
number of inhibiting chemicals can be broken down. On the whole, the two-stage
digestion is quite useful for the array of substrates and added permanence of feed
with a steady pH.
3.3.4 Aerobic Microbial Pretreatment
This type of pretreatment can be conceded in naturally occurring mixed cultures.

Aerobic pretreatment is basically carried out to solubilise the substrate as some
aerobic microorganisms produce large amounts of cellulose, hemicelluloses, and
lignin degrading enzymes. While in case of anaerobic microbial pretreatment, the
chemicals that might otherwise hinder the step of methanogenesis in the anaerobic
digester are wrecked by microorganisms. Moreover, the pH and temperature are
also quite suitable for hydrolysis enzymes. There have been no studies showing the
comparison of aerobic microbial pretreatment to anaerobic microbial pretreatment.

Broadly, the advantage of an aerobic process is that it is significantly faster. While
one of the disadvantages is that a lot of the organic matter is despoiled to CO2
which could otherwise be degraded to CH4 if the duration of pretreatment is too
long (Jagadabhi et al. 2010).
3.4 Combined Pretreatment
These are complex but are typically most effective than the other processes using
one mechanism.
3.4.1 Chemical + Microwave Irradiation
Microwave irradiation leads to considerable energy savings due to high heating

efficiency and also it reduces the reaction time and eventually results in increase in
reaction rate. However the processing by microwave irradiation may lead to certain
side effects such as production of heat-induced inhibitors such as phenolic com-
pounds and furfural (Hsu et al. 2010). Therefore, it becomes essential to control
these pretreatment conditions to evade the formation of these inhibitors. Thus the
microwave treatment has not been used individually but usually it has been used to
provide heat for assisting acid or alkaline pretreatment at relatively low tempera-
tures without compromising pretreatment effects (Liu and Cheng 2009; Cheng and
Liu 2010). Zhu et al. (2005) used microwave and 1 % alkali (NaOH) at 700 W for
30 min. The results indicated that higher rate of hydrolysis was obtained when
paddy straw was pretreated with microwave and NaOH. Additionally, the higher
glucose content was observed in the hydrolysate as compared to paddy straw
pretreated with alkali alone. Gong et al. (2010) also reported that sugar yield of
paddy straw can be effectively improved by the combination of microwave and
chemical pretreatment with organic acid. It was obtained from the results that sugar
yields of 71.41 and 80.08 % with 46.1 and 51.54 % removal of lignin was achieved
at optimal conditions of 230 W microwave intensity, 1:15 solid liquid ratio, 25 %
acid concentration and 5 min irradiating time.
3.4.2 Steam Explosion
The combination of heating and sudden pressure referred to as steam explosion,

increases the accessibility of the substrate. During steam explosion the substrate is
heated up to a temperature ranging between 160 °C and 220 °C, in a closed vessel
to further cause a rise in pressure. The pressure is abruptly released after a certain
time period of around 5 to 60 min. The sudden pressure drop results in rapid
evaporation of intracellular water and thus causing a phenomenon known as steam
explosion. In one of the studies carried out by Bauer et al. (2009) the methane yield
was found to be increased by 20 % in the paddy straw pretreated with steam
explosion than the untreated paddy straw.
4 Advantages and Disadvantages
There is no distinct pretreatment technology that is suitable for all substrates and
systems of anaerobic digestion. The diverse pretreatment technologies discussed
above may be suitable for one or the other economic situation of particular region.
Table 1 Advantages and disadvantages of different pretreatment technologies (Taherzadeh and

Karimi 2008; Hendriks and Zeeman 2009)
Pretreatment Advantages Disadvantages
process
Mechanical • Increased surface area • Augmented demand of energy
• Makes substrate easier to • Elevated maintenance cost.
handle
• Improves fluidity in digester
Thermal • Possibility to ensure • Possible formation of compounds which are
anaerobic process difficult to degrade with overall reduction of
stabilization methane yield
• High efficiency in improving • High energy consumption
organic matter solubilisation
Liquid hot • Increases the accessibility of • Elevated heat demand
water enzyme • Only effectual up to certain temperature
Oxidative • Strong oxidizing power • High maintenance cost
ensuring short reaction time • Possible formation of less biodegradable
• High solubilisation byproducts
improvement
• No addition of chemical to
the substrate
Acid • Solubilises hemicellulose • High cost of acids
• oxidization problems
• Formation of inhibitors
Alkali • Breaks down lignin • High concentration of alkali in digester
• High cost of chemical
Microbial • Low energy expenditure • No lignin breakdown
• No chemical addition • Slow in process
Enzymatic • Low energy utilization • High cost of enzymes
• No addition of chemicals • Continuous addition required
• High reaction time
Steam • Breaks down lignin and • Effective up to certain temperature
explosion solubilises hemicelluloses • High heat and electricity demand
Table 2 The influence of different pretreatment methods on the breakdown of lignocelluloses

Pretreatment Cellulose Hemicellulose Lignin Increase in specific
method decrystallisation degradation degradation surface area
Mechanical + +
Liquid hot ND + – +
water (LHW)
Concentrated + + +
acid
Diluted acid + +
Alkaline – + +
Oxidative ND +/- +
Biological +
Steam + + +
explosion
Here, + = major effect
− = minor effect
ND = Not determined
No symbol means that it is unclear if there is effect or not
Further the selection of pretreatment method depends on the composition of the

substrate. The greatest challenge for the pretreatment technologies is to increase the
bioavailability of substrate. This could only be achieved if the right pretreatment
technology is combined with right substrate. Energy balance and cost effectiveness
are the most important parameters for selecting the right choice. The erroneous
choice can make the process extravagant. Further, to obtain better gas yield, it is
quite essential to make the process economically feasible. The Table 1 given below
summarizes all the advantages and disadvantages of different pretreatment methods.
In the recent years, the influence of pretreatment technologies on anaerobic
digestion process has been investigated but still these technologies need to be
optimized for the better yield of biogas. The Table 2 given below summarizes the
influence of different pretreatment methods on the breakdown of lignocelluloses.
5 Conclusion
Pretreatment in paddy straw is basically employed to remove the recalcitrance of

lignocellulosic biomass for increased biogas yield. Pretreatment can further
potentially reduce the lignin content and thus results in increased accessible surface
area. A successful pretreatment method should be able to
(1) Improve the feedstock’s digestibility for microbes in anaerobic digestion
(2) Avoid formation of inhibitors
(3) Require low energy input
(4) Avoid loss of carbohydrates
(5) Avoid the need of waste disposal

(6) Be cost effective and environmentally friendly.
Pretreatment is predominantly apt for substrates where there is nonavailability of
sterically degradable biomass for enzyme attack. Finally, it should be noted that it is
not realistic to treat all the substrates with one single pretreatment technology.
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Bioethanol Production from Fermented
Cashew Apple Juice by Solar
Concentrator
Y.P. Khandetod, A.G. Mohod and H.Y. Shrirame
Abstract In the present study, it is used for bioethanol production from locally
available cashew apple fruit juice (variety Vengurla-4) grown in the Konkan region
of Maharashtra, India. The objective of this study is to show the ways of ethanol
production. Fermentation followed by distillation are the processes for ethanol
production. For fermentation, Saccharomyces cerevisiae was the cheapest strain
available for the conversion of substrate. Raw fermented cashew apple juice after
fermentation is used for ethanol production. Distillation process using concentrating
solar cooker having the per day rate of distillation of the system was 1170 ml with
distillation efficiency 33.41 %. The concentration of ethanol increased from initial
12 to 18.6 % in first distillation, which was further increased to 35.5 % after second
distillation. As the boiling point of ethanol is 78.5 °C, the temperature obtained was
higher, i.e., 109.2 °C. Same amount of water also got evaporated, which diluted
distillated product. Therefore, the higher concentration of ethanol was not obtained.
The specific gravity and acid value of double distilled ethanol at 35.5 % concen-
tration were 0.947 and 1.044 mg KOH/g, respectively.
Keywords Cashew apple juice Saccharomyces cerevisiae Fermentation

Distillation Ethanol
List of Abbreviations and Symbols

% Percent
°
C Degree centigrade
Aa Aperture area of concentrator
Ac Collector area required
Acont Total surface area of the container
Ar Area of receiver
AS Surface area of paraboloid
Cc Cubic centimeter
Y.P. Khandetod A.G. Mohod H.Y. Shrirame (&)

Department of Electrical and Other Energy Sources, College of Agricultural
Engineering and Technology, DBSKKV, Dapoli, Maharashtra, India
e-mail: hemantshrirame@gmail.com

DOI 10.1007/978-81-322-2773-1_5
64 Y.P. Khandetod et al.
Cc Specific heat of fermented cashew apple juice

Ccont Specific heat of the material of container
Ce Specific heat ethanol
Cw Specific heat of water
Cpr Specific heat of the material of receiver
CR Concentration ratio
D Aperture diameter of paraboloid
E East
dc Diameter of cylinder
et al. And others
etc. Et cetra
FAO Food and Agriculture Organization
Fig. Figure
F′UL Overall heat loss coefficient
F′ηo Optical efficiency factor
F Focal length of paraboloid
G.I. Galvanized iron
Hs Total incident radiation on reflecting surface
Hc Convective heat coefficient
hc Height of the container
h Hour
hrs/day Hours per day
i.e. That is
Ib Solar insolation
°
K Degree Kelvin
K Thermal conductivity
H Convective heat coefficient
J/kg K Jules per kilogram per Kelvin
kcal/m2 h Kilo calorie per square meter per hour
kg/m s Kilogram per meter per second
kg/m3 Kilogram per cubic meter
W/m K Watt per meter per Kelvin
W/m2 K Watt per square meter per Kelvin
kW/m2 Kilo watt per square meter
L Length of the circumference of paraboloid
m Meter
Ml Milli-lit
m/s Meter per second
m3 Cubic meter
(MC)′w Total heat capacity
Mc Mass of cashew apple juice
Mcont Mass of the empty container with lid
Mr Mass of receiver
Mw Mass of water
Bioethanol Production from Fermented … 65
N North
NGr Grashof number
NNu Nusselt number
NPr Pradalt number
nref Reference density of water
nt Thermal efficiency
Pevp Evaporation rate of product
Qa Heat available kcal
Qf Average energy falling at Dapoli
Qd Energy delivered by paraboloid
Qloss Total heat loss Kcal
Qr Energy required for vaporization
qconv Heat transfer rate by convention
qrad Heat transfer rate by radiation
R Aperture radius of parabolic
S Arc length of paraboloid
Sr. No. Serial number
TSS Total soluble solid
Ta Ambient temperature
Tr Surface temperature of receiver
Td Distillation point of ethanol
Tf Final temperature of fermented cashew apple juice
Ti Initial temperature of fermented cashew apple juice
TV Vaporization temperature of cashew apple juice
Tw Water temperature in the container
Vc Volume of container
W Watt
Wu Weight of ethanol sample
W/m2 Watts per square meter
λV Latent heat of vaporization of cashew apple juice
Ρ Density of air
ρc Density of cashew apple juice
Z Depth of paraboloid
Τ Specular reflectance of reflector
Α Absorptance of absorber
Β Volumetric coefficient of expansion of the fluid
Φ Intercept factor
Μ Viscosity
Ε Emissivity
ΔT Positive temperature difference between the surface of container and
fluid
Π Pie
1 Introduction
Energy plays a crucial role in modern life. The main problem of the world is the
increasing demand for fossil fuels but the storage strata are limited. Nowadays,
alternative fuel like ethanol has more demand in developed and developing coun-
tries for the fulfillment of requirement for fuel. However due to increasing petro-
leum shortage, renewable resources can be used for production (Ames 2001).
Glucose rich substrates can be fermented to produce ethanol which is a clean energy
source (Yu and Zhang 2004). Demand for ethanol as an alternative renewable fuel
sources has steadily increased (Sheoran et al. 1998) which helps the world to secure
its future supply of energy by reducing its dependency on fossil fuels. Biomass with
higher sugar content can produce ethanol by fermentation and distillation. Ethanol
is a clear, colorless liquid with sweet flavor, but at higher concentration it has
burning taste. In India, research and development efforts for using ethanol blended
transport fuels started in 1980 (Linoj Kumar and Prakash 2006). Nowadays, fer-
mentation technology produces nearly 80 % ethanol as clean fuel and for which
Saccharomyces cerevisiae is considered to be the most important yeast strain
because of its biotechnological application in the field of fermentation technology
(Sreenath and Jeffries 1996). Ethanol produced by fermentation has been found to
serve considerably as transportation fuel for cars, trucks, and trains. In India and
Brazil, ethanol is blended with petrol at 5–10 and 24 % by volume, respectively.
India is the second largest fruit producer in the world after China (Costa et al.
2009). In the Konkan region, cashew is a traditional crop mainly grown on hill
slope as rained permissible horticulture crop. Cashew (Anacardium occidentale L.)
belongs to family Anacardiaceae. North Brazil is the origin of cashew. In the world,
a total of 32 countries produced the cashew fruit, and the major cashew apple
producing countries, based on the FAO report, their production are approximately,
Vietnam 8.4, Nigeria 5, India 4, Brazil 1.6, and Indonesia 10 lakh tons, respec-
tively. In India, Maharashtra, Goa, Karnataka, and Kerala are leading states along
the west coast and Tamil Nadu, Andhra Pradesh, Orissa, and West Bengal along the
east coast for cashew growing (Hubballi 2006).
The total cashew plantation area in Konkan region of Maharashtra is about
1,73,601 hectares with annual production of about 1.5 lakh metric tones (Haldankar
et al. 2007). The region is endeavored with the average solar energy availability of
450–600 W/m2 for 7–8 h in a day and 250 days in a year. Solar energy is the
cheapest, inexhaustible, and ample source of energy, which is a direct form of
energy. Utilization of solar energy for thermal applications like distillation, cook-
ing, heating, and drying is well recognized in tropical and semitropical regions.
Ethanol obtained using distillation process using concentrating solar cooker pro-
vides a solution for energy conservation in ethanol production.
2.1 Cashew Apple Fruit
Cashew apple fruits of variety Vengurla-4 were used which is grown in the Konkan
region of Maharashtra, India. The cashews are collected/harvested from cashew nut
orchard of the Central Experiment Station, Wakawali (in the year 2009) of Konkan
region, Maharashtra, India. Cashew apple seems as an alternative raw material for
ethanol production, due to its vast availability and high concentration of reducing
sugars (Pinheiro et al. 2008).
2.2 Processing
Cashew apple (Anacardium occidentale L.) fruits were picked at mature and ripe
stages Plate 1. Basket press and mechanical juice extractor were used for the
extraction of cashew apple juice. Basket press juice extractor is a manually operated
press as given in Plate 2. Cashew apple juice obtained from basket press had good
quality as compared to mechanical juice extractor, therefore juice obtained from
basket press was used and the overlook of the extracted juice is shown in Plate 2.
2.3 Physical Properties of Cashew Apple
The Vengurla-4 variety of cashew apple was used for ethanol production. An
average weight, length, diameter, and volume of Vengurla-4 variety is reported in
Table 1. The cashew apple had an average length of 4.9 cm and diameter of 2.7 cm.
The cashew apple was red in color and it had an average volume 56.66 ml. The
average juice recovery was 67.30 % w/w.
Plate 1 Cashew apple fruits

of variety Vengurla-4
Plate 2 Overlook of the

extracted cashew apple juice
Table 1 Physical properties Sr. No. Physical property Particulars

of cashew apple
1 Weight (g) 54.24
2 Length (cm) 4.9
3 Diameter (cm) 2.7
4 Volume (ml) 56.66
5 Color Red
6 Juice recovery (%) 67.30 w/w
2.4 Chemical Properties of Cashew Apple Juice
The data on chemical properties of cashew apple are reported in Table 2.
2.4.1 Determination of TSS
In cashew apple, TSS was 11.8 °Brix, it indicates the percentage of sucrose by
weight in grams per 100 ml of water, which is determined with the help of hand
refractometer of Erma (Japan) manufacturing and values were corrected at 20 °C
Table 2 Chemical properties Sr. No. Chemical property Particulars

of cashew apple
1 Total soluble solid (°Brix) 11.8
2 pH 4.4
3 Sugar (%) 11.3
4 Acidity (%) 0.3
using temperature correction chart with the range 0–90 %, scale (1) 0–42 %, (2) 42–
90 % (A.O.A.C 1975).
2.4.2 Determination of PH
The pH of cashew apple juice was measured using digital pH meter and the pH of
apple was 4.4.
2.4.3 Determination of Sugar
The monosaccharides sugars include glucose, fructose, and galactose, which are
derived from different sources. First 50 ml sugar solution will be taken in 250 ml
conical flask, then 5 g of citric acid and 50 ml of water will be added in it. For the
complete inversion of sucrose, solution will be boiled for 10 min and after that it
will be kept for cooling, then this solution will be transferred to 250 ml volumetric
flask to neutralize it with 1 N NaOH using phenolphthalein indicator. For inversion
at room temperature, 50 ml of aliquot of clarified and deleaded solution will be
transferred to 250 ml flask, then 10 ml of HCl will be added in it and solution will
be allowed to stand at room temperature for 24 h. Finally solution will be neu-
tralized with concentrated NaOH solution. For the determination of total sugar
aliquot will be taken for testing. Results will be expressed on percent basis
(Ranganna 2009). In cashew apple juice sugar was found to be 11.3 %.
2.4.4 Determination of Acidity
A known quantity of sample (cashew apple juice) was titrated against 0.1 N sodium
hydroxide (NaOH) solution using phenolphthalein as an indicator. A known
quantity of sample was blended in pestle and mortar with 20–25 ml distilled water
and then it was transferred to 100 ml volumetric flask and filtered. A known volume
of aliquot was titrated. The results were expressed as a percentage of anhydrous
citric acid (Ranganna 2009). The acidity was found to be 0.3 %.
The chemical analysis of cashew apple revealed that the cashew apple is rich in
sugar content. The acid sugar blend in the cashew apple was good and indicated
that the apples could be successfully utilized for the production of alcohol.
2.5 Microorganism
The yeast Saccharomyces cerevisiae was used for the fermentation process
(Pinheiro et al. 2008). For the fermentation, the juice was supplemented with
diammonium hydrogen phosphate (DAHP) as 1 g/l and potassium metabisulfite
(KMS) as 0.03 g/l. The juice was inoculated with pure culture of Saccharomyces
cerevisiae var. ellipsoideus (NCIM-3315) as 0.30 g/l.
2.6 Solar Concentrating Cooker
Different types of concentrating cookers like Flat absorber with flat reflectors,
parabolic concentrator, compound parabolic concentrator, Fresnel lens, and cylin-
drical parabolic concentrator are used for various applications with lower temper-
atures to higher temperatures (Garge and Prakash 2005). For this study, the
parabolic solar cooker (SK-14), which concentrated the incoming solar radiation at
the focal point, was used as heat source for the distillation of fermented cashew
apple juice. The concentrating system followed the sun, so that maximum sun rays
were always focused on reflecting surface. A solar concentrating collector SK-14
having aperture diameter of 1.4 m and depth 0.35 m with focal length of 0.30 m was
used for distillation. The distillation of cashew apple juice was carried out on solar
concentrating collector by placing the container at focal point to get maximum
concentration of ethanol.
3.1 Fermentation Process for Cashew Apple Juice
Known volume of cashew apple juice was taken and TSS and pH content of cashew
apple juice was adjusted to 24 °Brix and 3.5, respectively, by addition of sugar. The
acidity was adjusted by addition of citric acid. The juice was supplemented with
diammonium hydrogen phosphate (DAHP) as 1 gm/l and potassium metabisulfite
(KMS) as 0.03 g/l. After adjusting TSS, pH, and acidity, the measured volume of
juice was taken in fermentation flask. The juice was inoculated with pure culture of
Saccharomyces cerevisiae as 0.30 gm/l. The fermentation flask was incubated at
28 ± 2 °C. The start of fermentation was indicated by evolution bubbles. The end of
fermentation was indicated by cessation of foaming and bubbling after fermentation
assembly was dismantled.
First observation was taken after 24 h and then observations were continuously
taken at alternate days till total soluble solid of juice was found to be constant. The
fermentation was allowed to continue for 10 days at the end of which the total
Table 3 Changes in total Sr. No. Time (h) TSS (°Brix) pH Acidity (%)
soluble solids TSS, pH and
acidity in cashew apple juice 1 0 24 3.50 0.6144
during fermentation process 2 24 18.4 3.47 0.7936
3 72 12.2 3.53 0.9472
4 120 8.8 3.63 0.6656
5 168 8.6 3.65 0.704
6 216 8.0 3.70 0.768
7 264 8.0 3.68 0.793
7 312 8.0 3.79 0.768
9 360 8.0 3.66 0.704
soluble solid was found to be 8.0 °Brix. There was no further decrease in total
soluble solid after 8 days of fermentation. Table 3 shows changes in TSS, pH, and
acidity in cashew apple juice during fermentation process.
3.2 Performance of Solar Concentrating Collector

for Distillation of Fermented Cashew Apple Juice
The parabolic solar cooker SK-14, which concentrated the incoming solar radiation
at the focal point, was used as heat source for distillation of fermented cashew apple
juice. The angle of focus to attend the required temperature for distillation was fixed
based on preliminary testing. The present study on distillation of cashew apple juice
was carried out to get maximum concentration of ethanol which is shown in Plate 3.
The performance of solar parabolic concentrator was firstly evaluated without
load (i.e., without juice in the container). The solar pyranometer is used for the
measurement of solar radiation. Measurement of solar radiation was made when
Plate 3 Solar parabolic

concentrator for distillation of
ethanol
pyranometer faces the sky. It was observed that the maximum temperature of the
container was 225.7 °C reached at 13.00 h when solar radiation was 594 W/m2 with
36.4 °C ambient temperature. Then there was a gradual decrease in the temperature
along with time and solar intensity which is presented in Fig. 1.
Solar parabolic concentrator with load was used for the distillation of ethanol.
When container was fully loaded with cashew apple juice with its designed
capacity, the maximum temperature of inside container was 109.2 °C. The maxi-
mum solar insolation was 611 W/m2 at 13.00 h. during the test Fig. 2 shows the
temperature profile at full load test.
inside temp focal point temp

ambient temp solar radiatian
240 650
Solar radiation (W/m 2)

220
200 550
Temperature oC
180
160 450
140
120 350
100
80 250
60
40 150
20
0 50
Time (h)
Fig. 1 Performance of parabolic solar concentrator for without juice in the container
Inside temp. focal point temp

ambient temp solar radiatian
240 650
220
Solar radiation (W/m 2 )
200 550
Temperature o C
180
160 450
140
120 350
100
80 250
60
40 150
20
0 50
Time (h)
Fig. 2 Performance of parabolic solar concentrator for with juice in the container
3.2.1 Distillation Efficiency of Solar Parabolic Concentrator
The solar parabolic concentrator was manually adjusted towards the sun as the sun
inclination changed. The condensation was provided to collect ethanol. The
receiver was mounted on parabolic solar concentrator with its full load capacity,
i.e., 3.00 kg of fermented cashew apple juice for production of ethanol. During the
test, actual distillation was obtained between 9.00 and 16.00 h. The maximum
distillation rate was obtained between 11.00 and 13.00 h. when a maximum solar
intensity ranged from 450 to 600 W/m2 was available and the distillation rate
decreased after 13.00 h, as solar intensity decreased. The distillation efficiency of
parabolic solar concentrator with time for different solar insolations is given in
Table 4. Higher efficiency of the system was found to be 33.4 % at 13.00 h when
solar intensity was 500 W/m2 and ambient temperature 31.5 °C.
The relationship between cumulative distillation rate and solar radiations versus
time is shown in Fig. 3. The maximum distillation observed was 580 ml when solar
radiation was 620 W/m2 at 13.00 h and minimum distillation rate was measured at
16.00 h. The maximum range of total distillation rate calculated during the whole
day, i.e., from 8.00 to 17.00 h was 2230 ml from 3.00 kg of fermented cashew apple
juice. Same trend was observed which is shown in Figs. 4 and 5 with minimum
difference in solar radiation and ethanol distillation rate.
Water heating and cooling tests are recommended and both test performed with a
load of water in aluminum made container. The first test covered the water heating
test using parabolic concentrator till the steady state temperature of water was
reached under clear sky condition. Then the test of water cooling was done by
shading the concentrator. The water heating and cooling curves were plotted
between difference in temperature of water and ambient temperature versus time,
i.e., (Tw–Ta) is given in Fig. 6 which show the time required for water boiling. For
this test, water was required 55 min to reach the steady state temperature at 9.30 h
when average solar insolation was 563 W/m2. The analysis was done for both the
Table 4 Performance of parabolic concentrator from water heating test water heating and cooling
test
Time (h) Water τo (min) F′UL (W/m2 K) F′UL/C F′no Avg. F′no Thermal
temperature efficiency (ηt)
°C initial
final
09.30 27.2 36.5 25 12.83 0.32 0.0946 0.1202 33.41
09.40
09.40 36.5 47.7 25 12.83 0.32 0.1242
09.50
09.50 47.7 61.2 25 12.83 0.32 0.1267
10.00
10.00 61.2 80.6 25 12.83 0.32 0.1274
10.10
Cumulative Distillation (ml) Solar radiation(W/m2)
2400 700
2200
Cumulative Distillation (ml) 2000 600
Solar radiation (W/m 2 )

1800
1600 500
1400
1200 400
1000
800 300
600
400 200
200
0 100
Time (h)
Fig. 3 Cumulative distillation rate and solar radiations versus time

2400 700
Cumulative Distillation (ml)
2200
Solar radiation (W/m 2)

2000 600
1800
1600 500
1400
1200 400
1000
800 300
600
400 200
200
0 100
Time (h)
curves of water heating and cooling. The water cooling curves give overall heat loss
coefficient (F′UL) and optical efficiency factor (F′no) was computed by water heating
test.
3.2.2 Overall Heat Loss Coefficient (F′UL)
Cooling Curve Analysis
The calculated value of [ln (Tw − Ta)] for each set of the data points that plotted
with this value on Y-axis and time on X-axis was drawn (Fig. 7). Different points of

2400 700
Cumulative distillation (ml)

2200
2000 600
1800
1600 500
1400
1200 400
1000
800 300
600
400 200
200
0 100
Time (h)
Tw-Ta
70
60
50
Cooling curve
(Tw-Ta)
40
30
20
Heating
(Tw -Ta )
10 curve
Time (min)
Fig. 6 Water heating and cooling curve test
the plot were fitted to a least square linear regression equation. The slope of the line
equals to (−1/τ0), where τ0 is defined as the time constant for cooling.
Substituting known values of time constant for cooling (τ0) in Eq. 1, surface area
of the container (Acont), and total thermal capacity of the container in the Eq. 1 and
the value of the heat loss factor (F′UL) of the cooker was evaluated.
0 ðMCÞ0W
FUL ¼ ð1Þ
Acont s0
Fig. 7 Graph for cooling ln(Tw-Ta) Linear (ln(Tw-Ta))

curve 5
y = -0.0398x + 4.0812
4
R² = 0.9859
ln (Tw-Ta)
3
Time (h)
where,
Acont = Surface area of the container, m2
(MC)′w = Heat capacity, J/K
Ccont = Specific heat of container, J/kg K
Cw = Specific heat of water, J/kg K
Mcont = Mass of the container with cover, kg
Mw = Mass of water, kg
τo = Sensible cooling constant
By regression analysis of the test, the value of the time constant (τo) is 25 min at
the average ambient temperature of 31.5 °C. With the cylinder surface area
0.0791 m2 and values of cooling time constant (τo), overall heat loss factor (F′UL)
was found to be 12.83 W/m2. The overall heat loss factor (F′UL) was found higher,
because of increase in thermal resistance between the receiver cylinder and the
surrounding.
3.2.3 Optical Efficiency
The optical efficiency factor indicates the theoretical upper limit of the overall
efficiency of solar paraboloid concentrator which is related to the performance of
reflecting surface and its reflectance. The determination of optical efficiency factor
(F′no) is by using water heating test. This is found after analyzing the sensible
heating curve as shown in Fig. 6.
h i
ðFUL
0
ÞAcont
Aaperture
Twf Ta
Ib TwiIT a
es=so
0
¼ ð2Þ
b
Fgo
1 es=so
where
Twi = Temperature of water at the beginning of the interval, C
Twf = Temperature of water at the end of the interval, C.
τ = Duration of the interval (e.g. 10 min or 600 s), s.
Ib = Average intensity of beam radiation incident on the aperture of concentrator
during the interval, W/m2.
Ta = Average ambient air temperature during the interval, C.
Acont = Total surface area, m2.
Aaperture = Aperture area of the cooker, m2.
Equation 2 was used for the determination of optical efficiency factor (F′no). The
result of the above test is given in Table 4. The optical efficiency factor (F′no) was
found 0.1202 for average solar insolation of 500 W/m2. The major factor, which
affects this value, i.e., reflectance of the reflector and absorptance of receiver
cylinder.
3.3 Characteristic Components of Ethanol
After distillation, the value of ethanol concentration was calculated by using

standard ethanol curve shown in Fig. 8. Percentage of ethanol available in raw
cashew apple juice was 12 %. After first distillation process, it was increased up to
18.6 %. The value of ethanol concentration solution obtained from second distil-
lation was 35.5 %. Ethanol production of 35.5 % by volume is possible by using
solar concentrating collector. Table 5 shows ethanol concentration during first
distillation and second distillation and Plate 4 shows ethanol samples of first dis-
tillation and second distillation.
After second distillation, the analysis of ethanol–water mixture was done. The
characteristic components of ethanol, i.e., the specific gravity and acid values were
obtained 0.947 and 1.044 mg KOH/g, respectively. The efficiency of distillation
unit for the production of 35.5 % ethanol is about 33.4 %. The temperature of the
Fig. 8 Standard curve of Spectrophotometer reading Linear (Spectrophotometer reading)

Spectrophotometer reading
ethanol 0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0 12 24 36 48 60
Series concentration
Table 5 First distillation, second distillation and ethanol concentration

Sr. No. Time (h) 1st distillation (ml) 2nd distillation (ml)
1 8.00 0 0
2 9.00 0 0
3 10.00 130 50
4 11.00 410 240
5 12.00 500 310
6 13.00 580 380
7 14.00 430 170
8 15.00 140 20
9 16.00 40 0
10 17.00 0 0
Total 2230 1170
Ethanol concentration 18.6 % 35.5 %
Plate 4 Ethanol samples of

first distillation and second
distillation
fermented juice was much higher than boiling point of ethanol, i.e., 78.5 °C. Due to
higher temperature, same amount of water also got evaporated, which diluted
distillated product. Therefore, the higher concentration of ethanol was not obtained
by using solar parabolic cooker. It was possible to get 95 % ethanol by further
distillation in laboratory. For obtaining 100 % ethanol concentration, the benzene
would be used as a dehydrating agent.
4 Conclusions
This study could establish that cashew apple juice commercially for any industrial
application. Ethanol production from cashew apple juice was carried out by using
solar concentrating collector/cooker. The two processes, i.e., fermentation and
distillation are involved in ethanol production. Saccharomyces cerevisiae was used
as inoculation for better fermentation (Pinheiro et al. 2008). The total distillation
rate per day of the system was obtained 2230 ml. The average distillation efficiency
of the system was found to be 33.4 with 35.5 % ethanol concentration.
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Potential Role of Xylose Transporters
in Industrial Yeast for Bioethanol
Production: A Perspective Review
Nilesh Kumar Sharma, Shuvashish Behera, Richa Arora

and Sachin Kumar
Abstract Sustainable development in lignocellulosic bioethanol production has

major challenge due to high cost of production. There are several issues such as
efficient utilization of pentose sugars present in lignocelluloses, economical pro-
duction of lignocellulolytic enzymes with high specificity and economical product
recovery, etc. In line, genetically modified yeast strains have been approached to
utilize pentose and hexose sugars for bioethanol production. However, these strains
showed limited xylose consumption. For efficient utilization of xylose, it is nec-
essary to provide efficient molecular transportation of xylose to the yeast cells. The
yeast strains which have been found prominent are Saccharomyces cerevisiae,
Candida intermedia, C. tropicalis, Kluyveromyces marxianus and Scheffersomyces
stipitis which have been engineered and examined for xylose transporter genes for
xylose utilization. Several transporter genes of interest have been targeted; how-
ever, there are major bottleneck in this approach such as xylose transporters which
are significantly inhibited by glucose and other hexose sugars. Hence, there are
several molecular approaches that have been applied to engineer the yeasts which
could improve the xylose transportation. This review has been focused to discuss
the molecular advancements in xylose transporter genes and its complexity.
Keywords Genetic engineering Xylose transporter Industrial yeasts Xylose
1 Introduction
For the last three decades, biofuels produced from renewable feedstocks have
received much publicity because of their potential to replace conventional fossil
fuels. However, the use of readily fermentable agricultural feedstocks like sugar-
N.K. Sharma Shuvashish Behera Richa Arora S. Kumar (&)

Biochemical Conversion Division, Sardar Swaran Singh National Institute
of Bio-Energy (Formerly Sardar Swaran Singh National Institute
of Renewable Energy), Kapurthala 144601, Punjab, India
e-mail: sachin.biotech@gmail.com

DOI 10.1007/978-81-322-2773-1_6
82 N.K. Sharma et al.
cane juice and corn for the production of bioethanol is less desirable because of its
negative connotation in the food versus fuel debate. Instead, lignocellulosic bio-
mass from hardwood, softwood, and agricultural residues is generally considered as
a more sustainable source of feedstocks for biofuels (Kumar et al. 2008; Solomon
2012; Behera et al. 2014a; Sharma et al. 2016). Pentose sugars derived from the
hydrolysis of lignocelluloses make up nearly one-third of the reducing sugars with
xylose as the major component (Rydholm 1965; Lee et al. 1979; Sharma et al.
2014). Hemicellulose, upon hydrolysis, yields D-xylose as the major component
and a number of other sugars, somewhat depending on the source of the hemi-
cellulose, including glucose, D-mannose, D-galactose, and L-arabinose (Wenzl
1970; Jeffries 1983) (Fig. 1). Yeasts capable of fermentation of xylose, thus pro-
ducing ethanol, have been identified by some researchers (Maleszka and Schneider
1982; Behera et al. 2014b). Pachysolen tannophilus (Maleszka 1982), Candida
shehatae, and Scheffersomyces stipitis (Preez and Bosch 1986) appear to generate
the highest yields of ethanol from xylose, and these yields compare favorably with
that of ethanol from glucose in Saccharomyces cerevisiae (Skoog and
Hahn-Hägerdal 1988). Efficient conversion of xylose to ethanol occurs only under
aerobic conditions in some yeast and under anaerobic conditions in others (Does
and Bisson 1989). The explanation for this finding appears to lie in the specificity of
the cofactor required for xylose reductase activity. In C. utilis, the cofactor for
xylose reductase is NADPH, whereas NAD+ is required for xylitol dehydrogenase
activity, resulting in an imbalance of cofactor production and consumption in the
absence of an endogenous electron acceptor such as molecular oxygen
(Bruinenberg et al. 1983). Xylose reductase utilizes either NADPH or NADH as
cofactor in the more efficient xylose-fermenting yeasts such as C. shehatae,
P. tannophilus, and S. stipitis, thus permitting fermentation of xylose under
anaerobic conditions (Does and Bisson 1989). Uptake of xylose into the cell may
also be the cause of rate limiting for catabolism. The kinetics of xylose uptake has
been investigated in C. shehatae, S. stipitis, and Rhodotorula gracilis. In all cases,
both high- and low-affinity xylose transport systems were observed (Does and
2%
3%
5% Glucose Xylose Arabinose
Mannose Galactose
33%
57%
Fig. 1 Percentage sugar content in lignocellulosic biomass

Potential Role of Xylose Transporters … 83
Bisson 1989). Xylose fermentation and subsequent ethanol production have been
studied in several other yeast species, including S. stipitis, C. shehatae, and
P. tannophilus; however, their ethanol productivities are very poor as compared to
S. cerevisiae when glucose-based substrates are used (Prior et al. 1989;
Hahn-Hägerdal et al. 1994; Nobre et al. 1999). Efforts to establish a xylose-utilizing
pathway in S. cerevisiae by insertion of the genes encoding xylose reductase and
xylitol dehydrogenase from S. stipitis or other organisms have resulted in only poor
ethanol production from xylose (Hamacher et al. 2002; Sharma et al. 2014).
Various steps, including the uptake of xylose, have been suggested to limit the
metabolism of xylose in metabolically engineered S. cerevisiae (Eliasson et al.
2000). Although a number of research articles have been published that deals with
monosaccharide transport in baker’s yeast, little study has been done in the field of
specificity of the sugar-transporting system (Cirillo 1961, 1963).
Therefore, this review has been focused to discuss the molecular advancements
in xylose transporter genes and its complexity in yeast cells.
2 Transport of Sugars
There are number of machineries that are available in industrial yeast for assisting
sugar transport. It covers a complete range from energy-independent to
energy-dependent transport system. However, adaptation of machinery depends on
a large number of factors including substrate availability, type of strain, growth
environment, physical conditions, and availability of cofactors (Weusthuis et al.
1994) (Fig. 2).
2.1 Glucose
Glucose transport research studies on industrial yeasts including S. cerevisiae and

Kluyveromyces, Scheffersomyces, Candida, and Schizosaccharomyces species show
the existence of different machineries present in all the yeast to facilitate glucose
transportation inside the cell. S. cerevisiae has high-affinity glucose transport sys-
tem including energy-independent carrier-mediated facilitated diffusion; however,
energy-dependent proton symport system is not available in S. cerevisiae (Jansen
et al. 2002). Glucose transport is facilitated by carrier-mediated facilitated diffusion
together with phosphorylation by glycolytic kinases such as Hxk1p and Hxk2p and
Glk1p in S. cerevisiae. S. cerevisiae has the highest glucose influx rate as compared
to other yeasts (Teusink et al. 1998). In higher concentration of glucose, the glucose
permeates freely with the help of low-affinity system (Gamo et al. 1995; Naftalin
2008). In other yeasts, glucose accumulation, arisen from a proton electrochemical
Fig. 2 Sugar transportation mechanism in yeast
gradient across the cell membrane against a concentration gradient, is driven by

energy. In fact, the energy cost is quite high for active proton symport of glucose
(Stambuk et al. 2003).
With the help of molecular experiments, transportation of glucose and other
sugars in the yeasts has identified several genes encoding high- and low-affinity
transporters. The huge number of putative glucose transporters has been identified
including Hxt1p, Hxtl4p, and Snf3, belonging to a sugar permease family (Boles
et al. 1997). The HXT genes affect glucose (HXT2), galactose (GAL2), glucose and
mannose (HXTI), or glucose, fructose and galactose (HXT4) uptake. However,
none of the genes has been identified in S. cerevisiae specifically fructose uptake
(Reifenberger et al. 1997; Boles and Hollenberg 1997) (Table 1).
Table 1 Glucose transport S/N Yeast Mechanism Control

systems in some
biotechnologically industrial 1 C. utilis Proton symport Repressible
yeast (Walker 1998) 2 C. shehatae Proton symport Repressible
Facilitated Non-repressible
diffusion
3 K. marxianus Proton symport Repressible
Proton symport Repressible
4 S. stipits Proton symport Non-repressible
5 S. cerevisiae Facilitated Repressible
diffusion
Facilitated Constitutive
diffusion
Notes
• Control refers to whether or not the transport system is
repressible by glucose or is constitutively expressed
• Facilitated diffusion is an energy-independent system mediated
by protein carriers, whereas proton symport refers to active
energy-dependent ATPase-mediated proton motive force
2.2 Xylose
Studies concerning to pentose sugar transport by yeasts again reveal that different
transport systems exist in different yeasts. Pentose transportation in yeasts generally
occurs by membrane potential due to proton symport, while sometimes facilitates
diffusion through low-affinity transport systems (Singh and Mishra 1995). Different
machineries for sugar uptake exist in different yeast species such as K. marxianus,
S. stipitis, C. shehatae, and Rhodotorula glutinis. These yeasts have been used as a
model organism in the study of pentose transport (Young et al. 2014). Under
aerobic environment, a bioelectrogenic proton symporter is perceptible, during
high-affinity D-xylose uptake in R. glutinis. A low-affinity system involving a
proton symport mechanism may also exist in this yeast (Singh and Mishra 1995). In
S. stipitis, xylose transport is a rate-limiting step under aerobic growth conditions
and uptake of this sugar is mediated by both low- and high-affinity carriers (Du
et al. 2010). In C. shehatae, both machineries exist including facilitated diffusion
and proton symport mechanisms for D-xylose transport. The former mechanism is
operative under conditions of repression but it is absent in starving cultures of this
yeast (Walker 1998). S. cerevisiae is not able to utilize xylose but sometimes in
starvation xylose uptake occurs under aerobic conditions by a low-affinity system
(Toivari et al. 2004). In general, xylose uptake is inhibited in the presence of
glucose in most of the xylose-fermenting yeasts (Wang et al. 2014), which reveals
the preferential utilization of glucose in cells grown on complex sugar mixtures of
hemicellulosic hydrolysates. The sequential uptake pattern represents a major
Table 2 Xylose transport systems in some biotechnologically industrial yeast (Walker 1998)
S/N Yeast Mechanism Comments
1 C. shehatae Proton symport Xylose both respired and fermented
Facilitated –
diffusion
2 C. utilis Proton symport Only high-affinity system operates in glucose
de-repressed cells
3 M. reukaufii Proton symport Xylose only respired
4 P. stipitis Proton symport Xylose both respired and fermented
5 R. glutinis Proton symport Xylose only respired
Facilitated –
diffusion
6 S. cerevisiae Facilitated Xylose non-metabolizable
diffusion
7 K. marxianus Facilitated Xylose metabolizable
diffusion
8 S. pombe Facilitated Xylose non-metabolizable
diffusion
factor, which limits the biotechnological exploitation of pentose-fermenting yeasts

in the bioconversion of wood hydrolysates (Spencer-Martins 1994; Nijland et al.
2014) (Table 2).
3 Factor Affecting Xylose Transporters
Xylose transporters have its own importance in the xylose uptake in the yeast cells.
However, a lot of efforts including the cloning of the transporter genes of other
species have been made to sort out the transportation issues. However, several other
factors as discussed below play the major role in inhibition of xylose transporter
(Nevoigt 2008).
3.1 Co-expression of GXF1 and GXS1
When glucose/xylose transporter genes from C. intermedia were cloned in S.

cerevisiae, i.e., GXF1 (glucose/xylose facilitator) and GXS1 (glucose/xylose proton
symporter), Gxf1p was observed fully functional in S. cerevisiae. However, the
symporter Gxs1p exhibits very low-glucose/xylose transport activity, which could
not be able to produce the sufficient protein. Therefore, it is evident that
co-expressions of GXS1 and GXF1 inhibit the transportation of pentose sugars in S.
cerevisiae (Leandro et al. 2006, 2008; Young et al. 2011).
3.2 Extracellular Glucose
Similar transport system is accompanied for xylose and glucose in S. stipitis as S.

cerevisiae (Farwick et al. 2014). However, xylose at 100 mM concentration did not
inhibit glucose uptake, while glucose inhibited the xylose uptake. Thus, xylose did
not compete with glucose for uptake of glucose in P. heedii which also shows the
involvement of different carrier systems (Does and Bisson 1989; Barnett et al.
2008).
3.3 Acetic Acid
Acetic acid is fungistatic, act by dissipating plasma membrane proton gradients and
depressing cell pH when they dissociate into ions in the yeast cytoplasm. Acetic
acid disrupts proton gradients by diffusing from the acidified external medium into
the cells and dissociating in the more neutral environment of the cytoplasm (Walker
and White 2011). Therefore, proton gradient looses to drive nutrient uptake. Higher
concentration of acetic acid also changes growth and thermal death profile. C.
shehatae could tolerate up to 0.4 ml/l acetic acid at pH 4.5, and shifted temperature
range from 5–34 °C to 21–27 °C and resulted in decreasing cell mass yield by 64 %
on D-xylose (Rodrigues et al. 1992; Singla et al. 2012).
4 Role of Transporter Genes
Several transporter genes have been identified for both glucose and xylose trans-
portations in different yeast strains. Each transporter gene has a different role in the
system. Hence, many genetic engineering approaches have been applied to increase
the xylose and glucose uptake in industrial yeasts (Young et al. 2011). Some of the
functions of major transporter genes have been summarized in Table 3.
4.1 AT5G17010 and AT5G59250
The directed movement of carbohydrate into, out of or within a cell, or between

cells occurs by means of some agents such as a transporter or pore. The genes
AT5G17010 and AT5G59250 are D-xylose-proton symporter-like protein 2 and
protein 3 of A. thaliana, respectively (Büttner 2007), which facilitates the
translocation of a specific substance or associated substances from out to in and vice
versa. These genes have been cloned in S. cerevisiae to improve xylose transporter
Table 3 List of transporter genes and their role

S/N Transporter Function Source References
genes organism
1 AT5G17010 D-xylose-proton Arabidopsis Hector et al. (2008)
symporter-like 2 thaliana
2 AT5G59250 D-xylose-proton A. thaliana Hector et al. (2008)
symporter-like 3
3 HXT1 Hxt1p (MFS transporter, SP S. cerevisiae Sedlak et al. (2004)
family)
family)
family)
family)
7 GXF1 Glucose/xylose facilitator 1 C. intermedia Leandro et al.
(2006)
8 GXS1 Glucose/xylose symporter 1 C. intermedia Leandro et al.
(2008)
9 XUT4 High-affinity xylose S. stipitis Young et al. (2011)
transporter (Putative)
(HGT3)
10 XUT5 Putative xylose transporter S. stipitis Young et al. (2011)
11 XUT6 Sugar transporter, putative S. stipitis Young et al. (2011)
(STL12)
12 XUT7 Xylose transporter, high S. stipitis Young et al. (2011)
affinity, putative similarity to
STL13
13 RGT2 Glucose transporter/sensor S. stipitis Hamacher et al.
(2002)
14 SUT1 Hexose transporter S. stipitis Weierstall et al.
(1999)
15 STP2 sugar transport protein 2 A. thaliana Hamacher et al.
(2002)
16 STP3 Sugar transport protein 3 A. thaliana Hamacher et al.
(2002)
17 TRXLT1 – Trichoderma Saloheimo et al.
reesei (2007)
18 HUP1 H(+)/hexose cotransporter 1 Chlorella Wolf et al. (1991),
kessleri Hamacher et al.
(2002)
19 CNBC3990 Uncharacterized protein Cryptococcus Young et al. (2011)
neoformans
(continued)
Table 3 (continued)
S/N Transporter Function Source References
genes organism
20 DEHA0D02167 – Debaryomyces Young et al. (2011)
hansenii
21 ALI0C06424 – Yarrowia Young et al. (2011)
lipolytica
22 YALI0B06391 – Y. lipolytica Young et al. (2011)
efficiency and significant improvement, i.e., xylose uptake rate increased by 45 % in

comparison of isogenic control strain lacking the A. thaliana transporters (Hector
et al. 2008).
4.2 HXTs Family
Several studies have been carried out to resolve the problems related to
co-expression of glucose and xylose (Boles and Andre 2014). An approach has
been applied in which xylose uptake properties recombinant xylose-utilizing S.
cerevisiae has been modified by expression of heterologous and homologous
permease-encoding genes. In this mutant strain main hexose transporter genes have
been deleted and engineered for xylose utilization (Young et al. 2011). After
screening in xylose plates, the enhanced growth was shown, whereas, when the
clone was retransformed into the host, it did not support significant growth on
xylose. After long-term adaptive mutation it was revealed that Hxt1p, Hxt2p,
Hxt4p, and Hxt7p had the capability to uptake of the xylose (Saloheimo et al.
2007).
In S. cerevisiae, HXT2 gene was identified by its ability to complement the
defect in the glucose transport of a snf3 mutant, when it was present on the mul-
ticopy plasmid pSC2 (Kruckeberg and Bisson 1990). It has 12 highly hydrophobic
regions that can form transmembrane domain. It has been observed that an hxt2 null
mutant strain lacks a high-affinity glucose transport when under derepressing
(low-glucose) conditions. However, the hxt2 null mutation did not incur a major
growth defect on glucose-containing media. Biochemical studies of this protein
suggest that wild-type levels of high-affinity glucose transport require the products
of both the HXT2 and SNF3 genes; these genes are not linked (Kruckeberg et al.
1990, Jansen et al. 2002).
To reveal glucose transport properties of HXT family transporters, HXT1,
HXT2, HXT3, HXT4, HXT6, and HXT7 have separately expressed in a HXT1–7
null strain. The Km values from high to low affinity determined from
counter-transport and initial-uptake experiments are Hxt6p 0.9 and 1.4 mM, Hxt7p
1.3 and 1.9 mM, Hxt2p 2.9 and 4.6 mM, Hxt4p 6.2 and 6.2 mM, Hxt3p 28.6 and
34.2 mM, and Hxt1p 107 and 129 mM. Same Km values were obtained for each
transporter from both the methods counter-transport and initial uptake, which show
the essential facilitated diffusion transporters for transporting glucose in S. cere-
visiae (Maier et al. 2002).
4.3 STP2
The AtSTP2 gene (sugar transport protein 2) of Arabidopsis thaliana encodes a

high-affinity, low-specificity monosaccharide carrier that can transport a number of
hexoses and pentoses at similar rates. AtSTP2 has 12 putative transmembrane
helices and a molecular mass of 55.0 kDa (Truernit et al. 1999).
5 Conclusion
There are different biotechnological applications like synthetic biology, systems

biology, and metabolic engineering which have been followed to tackle the prob-
lems ensuring the stable supply of transportation fuels for the future. All genetic
engineering methodologies must go beyond the different levels of genetic infor-
mation. The transport physiology of the recipient organism is a very important part
which depends on the expression of that genetic information, although the
recombinant yeasts can be utilized to convert xylose into ethanol. However, the
xylose consumption rate is very slow, which is not sufficient for efficient production
of ethanol from lignocellulosic materials. There are some other problems such as
inefficient co-fermentation of glucose and xylose using recombinant strains.
Further, it is also necessary to consider the kinetics and energetics of sugar transport
systems in relation to the overall metabolism of engineered organisms. The efforts
are also required to study the expression pattern of different transporter genes
affecting xylose uptake.
Acknowledgments Authors are very much thankful to Ministry of New and Renewable Energy,
New Delhi, Govt. of India for the financial support and providing all research facilities to carry out
research work. One of the authors (Nilesh Kumar Sharma) also acknowledges Sardar Swaran
Singh National Institute of Bio-Energy, Kapurthala for providing Junior Research Fellowship and
I. K. Gujral Punjab Technical University, Jalandhar for his Ph.D. registration (Pro. reg. 1422002).
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Part II
Chemical Conversion
Transesterification of Neem Oil Using Na
Ion-Doped Waste Fishbone
Himadri Sahu and Kaustubha Mohanty
Abstract High cost of biodiesel produced from various known sources till date has
focused the attention of researchers on low cost and renewable raw materials such
as nonedible oils and waste cooking oils. Catalyst plays a significant role on the
yield and quality of biodiesel. Heterogeneous catalysts are better over conventional
homogeneous catalysts as they can be easily recovered from the reaction mixture by
filtration and can be reused after activation, thereby making the process economi-
cally viable. Heterogeneous catalysts are mostly solid and can be prepared from
various materials such as industrial wastes (e.g., red mud, aluminum dross, fly slag,
blast furnace, and steel slag) and natural wastes (e.g., chicken egg shells, rice husks,
fish skeletal, shells of crab, oyster, snail, shrimp, etc.). The use of such wastes to
develop catalysts can be a win-win situation as this takes care of the waste disposal
issues. Current focus is to develop catalysts from natural sources. In the present
work, heterogeneous catalyst was developed from one natural hydroxyapatite, i.e.,
cooked fish bone and was doped with Na ion. Doping of metalloid ions and
calcining not only results in higher surface area but also increases the ion exchange
capability. The catalyst has been prepared employing wet impregnation method.
This developed catalyst was used for transesterified neem oil to produce biodiesel
with a acid value conversion closer to 96.7 %. The final transesterified product was
analyzed and the parametric effects on the acid value conversion were studied.
Keywords Transesterification Neem Hydroxyapatite Fishbone Catalyst
Himadri Sahu Kaustubha Mohanty (&)

Department of Chemical Engineering, Indian Institute of Technology Guwahati,
Guwahati 781039, India
e-mail: kmohanty@iitg.ernet.in

DOI 10.1007/978-81-322-2773-1_7
98 Himadri Sahu and Kaustubha Mohanty
1 Introduction
Globally in excess ninety one million tons of fish are caught and around 55 % of
this are used for human consumption, while rest were discarded (Rustad 2003). The
discard materials generate undesirable impact on environment. To reduce any
adverse environmental effects, it is advisable to use these discard materials for high
value-added products. Few fish species are exported after extracting the bones.
These discarded bones are the cheap source of calcium phosphate. So this is also
termed as hydroxyapatite, with stoichiometric formula Ca10(PO4)6(OH2).
However, there are few differences between synthetic and raw hydroxyapatite.
Natural hydroxyapatite has and better dynamic response to the environment, high
catalytic activity, and good thermochemical stability than the synthetic hydroxya-
patite (Obadiah et al. 2012; Best et al. 2008). As far as solubility and activity is
concerned, crystallographic structure plays an important role. In line to synthetic
hydroxyapatite, the natural hydroxyapatite has perturbed nanostructures and non-
stoichiometric composition with low hydroxyl content. As physical and chemical
properties depend on structure, natural hydroxyapatite will lead to good results
(Leventouri 2006). Because of these properties, hydroxyapatite can be used as a
heterogeneous catalyst. Although natural hydroxyapatite has favorable structure but
the surface area is bit low, which can be increased by doping metal ions on to it.
In the present work, raw hydroxyapatite collected from waste fish bone was used
as a base material for metal doping to conduct transesterification of neem
(Azadirachta indica) oil. After the literature review, it was found that calcined
Sodium silicate alone has given very good result in transesterification reaction (Fan
et al. 2013; Long et al. 2011; Yin et al. 2010). So, sodium silicate metal ion was
doped on to the raw hydroxyapatite by wet impregnation method to increase the
surface area of hydroxyapatite. In this catalyst both the hydroxyapatite and Na/Si
will act as an active source for the adsorption of reactants.
2 Materials and Detailed Methodology
2.1 Materials
From IIT Guwahati hostel mess-cooked waste fish bone was collected. Few
chemicals namely, 25 % aqueous ammonia solution, methanol and sodium silicate
(Na2O3Si·9H2O) were procured from Merk, India and were used as it is without any
further treatment.
Transesterification of Neem Oil Using Na Ion-Doped Waste Fishbone 99
2.2 Na/Si Doping on Hydroxyapatite Methodology
Fish bone was washed properly to remove proteinaceous part and sun dried for
2 days. After that oily matters were removed completely by dipping in acetone for
24 h and dried at 60 °C. The fishbone was crushed and raw hydroxyapatite was
obtained. Sodium Silicate was used as precursor for metal ions during wet
impregnation (Wang et al. 2008). Following the method, 1:1 wt. proportions of raw
hydroxyapatite and sodium silicate, at first 30 g of sodium silicate was added to
100 mL of water to get a solution of sodium silicate. To this solution, 30 g of fine
powder of raw hydroxyapatite was added gradually and blended rigorously using a
mechanical agitator under aggregate reflux time of 30 min. 25 % aqueous ammonia
solution was used to maintain pH at 11. The entire solution was mechanically
stirred for 2 h at 700 rpm at 60 °C. The final solution was kept for 24 h at room
temperature so as to allow the deposition of sodium and silica ions on raw
hydroxyapatite. After that, mother liquor was vacuum filtered and obtained sticky
precipitate was dried in hot air oven. The dried mass was then calcined at 650 °C
for 3 h and final Na/Si-doped hydroxyapatite was sent to lab for characterization.
2.3 Procedure for Transesterification of Neem Oil Using

Raw, Calcined, and Na/Si-Doped Hydroxyapatite
Transesterification of purified neem oil was conducted in a batch reactor having

conventional heating framework. For a standard analysis, 30 g of neem oil,
methanol, and catalyst (2 wt%) were taken in a 3-necked glass reactor of 1 L
volume and was heated up to 70 °C and then agitated for a particular time period.
After that, the entire reaction mixture was transferred to a rotavapor for removing
excess methanol. The reaction mixture was then centrifuged to separate the catalyst
and the mixture was kept in a separating funnel overnight to separate the glycerol
produced during the reaction. Then the acid value conversion was calculated by gas
chromatography. Following this standard method, effect of oil to methanol ratio and
catalyst loading on acid value conversion (%) with respect to reaction time were
also studied.
2.4 Catalyst and Oil Characterization
2.4.1 Catalyst Characterization
Bucker X-ray D8 advance diffractometer was used to gather powder X-ray

diffraction information for structural analysis. For X-ray radiation source, a Cu Kα
(λ = 0.154 nm, 45 kV, 40 mA) anode was used, ranging 2θ between 2° and 90° with
0.5 s/step scan rate and 0.05 increment. Surface analyzer device (Beckman-Coulter;
Model: SA3100) was used to measure BET surface area, average pore measure-
ment, and pore volume (t-plot strategy) by physical adsorption–desorption of N2 at
the boiling point (77 K). Preceding the examination, raw hydroxyapatite and
Na/Si-doped hydroxyapatite were subjected to a preheating for 90 min at 423 K
under vacuum. A thermal analyzer (NETZSCH: STA449F3 Jupiter) was used for
the thermo gravimetric analysis of the powder sample. Argon was passed through
the instrument with a heating rate of 10 K min−1 using a ceramic crucible up to
1000 °C. A scanning electron microscope (JEOL-JSM-6390LV) furnished with an
EDAX PV 9760 detector for energy dispersive X-ray spectroscopy (EDX) was used
to investigate nearby chemical arrangement. The samples were dispersed in
methanol and deposited on an Al foil before mounting on sample holder, then
coated with gold–palladium to make the specimen fit for microstructures
investigations.
2.4.2 Purified Neem Oil and Transesterified Neem Oil

Characterization
The acid Number of purified neem oil (Azadirachta indica) and transesterified
product was determined by colorimetric titration as per ASTM D974 (ASTM
standard 2009) and saponification number of raw oil was calculated as per ASTM
D5558 (ASTM standard 2006). The acid number and saponification value of
purified neem oil was found to be 23.8 mg KOH/g oil and 190 mg KOH/g oil. The
physical properties such as density, viscosity, flash point, and pour point were
calculated by ASTM standards. Fourier transform infrared spectroscopy (FTIR)
provides information with respect to various functional groups present in the
sample. DRS connected with Excalibur Bio-Rad spectrophotometer (Model FTS
3500 GX) FTIR analyzer was utilized for the same. The IR range was between 400
and 4000 cm−1 at a scan rate of 40 and at a step size of 4 cm−1.
3.1 Characterization of Na/Si-Doped Hydroxyapatite

Catalyst
XRD patterns of raw hydroxyapatite and Na/Si-doped hydroxyapatite are delin-

eated in Fig. 1. Well-resolved characteristic peak of most elevated intensity for
hydroxyapatite was acquired at 2θ value of 31.77° corresponding to 211 planes.
The phase formed was clear and matches well with standard pattern reported in
Fig. 1 X-ray diffraction patterns of raw hydroxyapatite and Na/Si-doped hydroxyapatite showing
characteristic peaks
literature (Rocha et al. 2005). The standard corresponding plane for hydroxyapatite
(namely, 100, 101, 200, 002, 211, 202, 301, 130, 131, 113, 203, 222, 132, 321,
004, 240, 241, 502, 323, and 511) was well observed in case of the synthesized
catalyst. The diffraction peak at 2θ = 21.8° with plane 2, 0, 0 corresponds to the Si
support (Musić et al. 2011) as registered on the XRD patterns (Fig. 1).
The principal evidence for framing of raw hydroxyapatite was in the form of a
strong complex broad FTIR band (Fig. 2) focused at about 1000–1120 cm−1
because of asymmetric stretching mode of vibration for PO4 group (Varma and
Babu 2005). The band at 570 cm−1 corresponds to P–O stretching vibration of the
PO4 group. As a major peak of phosphate group, the vibration peak could be
distinguished in the region between 1120–960 cm−1 for both raw hydroxyapatite
and Na/Si-doped hydroxyapatite, which were due to P–O asymmetric stretching of
PO43−. Many of the compounds can be acknowledged to be analogs of ethers,
especially when an alkoxy group was available, featuring the P–O–C linkage at
1240 cm−1. In case of Na/Si-doped hydroxyapatite the bands at 780–980 cm−1 and
peak in the range of 900–1200 cm−1 are resulting to the peroxide formation (M–O–
O–M) and the M–O–M bonding, respectively (Maensiri et al. 2006).
Figure 3 shows a typical nitrogen adsorption–desorption isotherm of raw, cal-
cined and Na/Si-doped hydroxyapatite. The isotherms display a type IV curve with
a hysteresis loop which corresponds to mesoporous materials. Raw, calcined, and
Na/Si-doped hydroxyapatite have BET surface areas of 10.865, 167.86, and
57.124 m2 g−1, respectively. The total pore volumes were found to be 0.0894,
0.4926, and 0.4943 cc g−1 for raw, calcined, and Na/Si-doped hydroxyapatite,
Fig. 2 FTIR spectrum of raw hydroxyapatite and Na/Si-doped hydroxyapatite
Fig. 3 Adsorption and desorption isotherms of N2 for raw hydroxyapatite and Na/Si-doped
hydroxyapatite
respectively. It can be seen from Table 1 that, the BJH pore volumes were close to
the BET pore volumes for the samples.
Figure 4 demonstrates the TGA analysis of raw, calcined, and Na/Si-doped
hydroxyapatite. In case of raw hydroxyapatite, initial weight loss of 6.84 %
occurred in between the temperature regime of 90 and 150 °C because of the
evaporation of water molecules. After that, the weight loss was found to be about
35 % which was due to the decomposition of carbonate as mentioned below.
CaCO3 ! CaO þ CO2 ð1Þ
Table 1 Different surface area (m2 g−1) measurements and pore volume (cc g−1)
Parameter Hydroxyapatite
Raw Calcined Na/Si
Langmuir surface area (m2 g−1) at PS/P0 = 0.9814 5.7 164.7 55.9
One point BET surface area (m2 g−1) at PS/P0 = 0.3 8.8 165.1 56.1
Adsorbed BET Surface area (m2 g−1) 10.8 167.8 57.1
Adsorbed t-plot Surface area (m2 g−1) 27.2 161.2 57.3
Total pore volume (cc g−1) at PS/P0 = 0.9814 0.08 0.49 0.49
Total BJH pore volume (cc g−1) 0.1 0.49 0.54
Fig. 4 Thermal gravimetric analysis of raw, calcined, and Na/Si-doped hydroxyapatite

Fig. 5 SEM micrograph of raw hydroxyapatite and Na/Si-doped hydroxyapatite
For calcined hydroxyapatite the total weight loss at 1000 °C was 12.3 % and
after 450 °C the degradation was thought to be the aftereffect of continuous
dehydroxylation. However, in case of Na/Si-doped hydroxyapatite, no crest was
found within a temperature region of 30–1000 °C and the reported weight loss was
only 4.18 %. It confirms the stability of Na/Si-doped hydroxyapatite.
The morphologies of raw and Na/Si-doped hydroxyapatite ares presented in
Fig. 5. These SEM micrographs gave understanding into the structure with respect
to pore sizes and distribution. As can be seen from the micrographs, surface
morphologies of the Na/Si-doped hydroxyapatite appear as a polycrystalline
material. For Na/Si-doped hydroxyapatite, the pores are uniformly distributed on
the surface. The calcination at 650 °C resulted in decomposition of carbonate which
helped in forming uniform pores. Hence, the surface area as well as the pore volume
was higher in case of Na/Si-doped hydroxyapatite than raw hydroxyapatite that was
evident from the BET results.
The EDX analysis (Fig. 6) of raw hydroxyapatite and Na/Si-doped hydroxya-
patite showed the presence of C, O, Mg, P, Ca, Na, and Si and the corresponding
weight percentages are tabulated in sight. The wt% analysis confirmed the oxide
formation of Si.
Fig. 6 EDX analysis of raw

hydroxyapatite and
Na/Si-doped hydroxyapatite)
3.2 Parametric Effects on Acid Value Conversion
Figure 7a shows the effect of neem oil to methanol molar ratio on acid value
conversion (%) with respect to reaction time. It can be clearly observed that the time
required to reach highest conversion (%) at lower concentration of Na/Si-doped
hydroxyapatite 1:2 and lower oil to methanol ratio (1:10), was approximately
195 min. But with increase in oil to methanol ratio the time required to reach
highest conversion gradually decreased to 135 min.
Further, the reduction in acid value (%) versus reaction time data was obtained at
various Na/Si-doped hydroxyapatite, constant oil to methanol ratio (1:20), and
reaction temperature (70 °C) are presented in Fig. 7b. It was evident from the
analysis that the equilibrium conversion was achieved after three and half hour.
For Na/Si-doped hydroxyapatite 2:1 the stability over certain reaction time
(160 min.) was studied at constant oil to methanol ratio (1:20) and reaction tem-
perature (70 °C) and 2 wt% catalyst. The catalyst was regenerated after complete
separation from the reacting mixture. It was first washed with methanol several
times and then dried for 2 h at 110 °C. Then the dried sample was again calcined at
650 °C for 1 h and finally the regenerated catalyst was further used. Figure 8
depicting the acid value conversion of purified neem oil with respect to time (min.).
Fig. 7 Effect on reduction of

acid value (%) with respect to
reaction time by varying a oil:
methanol and b different
catalyst
It is clear that the conversion after regeneration does not vary significantly up to 4th
regeneration. But after 5th regeneration, the conversion was decreased to 90.3 from
96.7 % after 160 min.
The physical properties of the transesterified product obtained at constant oil to
methanol ratio (1:20) and reaction temperature (70 °C) and 2 wt% Na/Si-doped
hydroxyapatite 2:1 catalyst was analyzed by following ASTM standards. The
physiochemical properties of both neem oil and transesterified product are pre-
sented in Table 2.
Fig. 8 Acid value conversion of neem oil with respect to time for Na/Si-doped hydroxyapatite 2:1
and the regenerated Na/Si-doped hydroxyapatite 2:1
Table 2 Physicochemical properties of neem oil and transesterified product

Properties Unit ASTM test method Neem oil Transesterified neem oil
Acid value mg KOH/g oil D 974 23.8 0.78
Density g cm−3 D 4052 910 872
Viscosity mm2 s−1 D 445 52.1 4.82
Flash point °C D 93 – 165
Pour point °C D4419–90 3 −4
3.3 FTIR Results of Purified and Transesterified Neem Oil
Figure 9 presents the graph in between wave number (cm−1) and transmittance (%)
for transesterified neem oil produced at constant reaction temperature (70 °C), oil to
methanol ratio (1:30) and Na/Si-doped hydroxyapatite 2:1. At first it is clear from
figure that alkane is present without phosphorous and sulfur, which confirms that
leaching of catalyst did not occur and it was separated clearly. Also, a C=O group is
identified at 1742 cm−1 which refers to ester. Along with other wavenumbers,
functional groups are also presented in Table 3.
Fig. 9 FTIR of purified and transesterified neem oil
Table 3 FTIR frequencies of purified and transesterified neem oil

Frequency (cm−1) Functional group Frequency (cm−1) Functional group
722 –CH2 1742 –C=O
1163 C–O–C 2766 –CH2
1375 O–CH2 2912 –CH2
1379 O–CH2 3018 =C–H
1436 (CO)–O–CH3 3422 –OH
4 Conclusion
An active, reusable solid catalyst was prepared from cooked waste fish bone. The
method of reusing fishbone waste to prepare catalyst could help waste recycling,
contaminants minimization, cost reduction, and making the catalyst environmen-
tally viable. This high efficient and low-cost fishbone catalyst could make the
process of transesterification economic and fully ecologically friendly. So, it can be
anticipated that the low-cost catalyst could be utilized in a large-scale industrial
process of biofuel, which will lead to cheap and ecologically viable process.
Acknowledgments Authors acknowledge the analytical facilities, i.e., FESEM, EDX, BET, and
TGA analysis provided by Central Instruments Facility (CIF), IIT Guwahati. Use of XRD facility
(procured through FIST Grant No. SR/FST/ETII–028/2010 from Department of Science and
Technology, Government of India) at Department of Chemical Engineering, IIT Guwahati is also
acknowledged.
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Assessment of the Seed Oils
of Persea americana and Melia dubua
for Their Potentialities in the Production
of Biodiesel and Possible Industrial Use
Kariyappa S. Katagi, Ravindra S. Munnolli, Sangeeta D. Benni

and Sneha S. Kulkarni
Abstract The seed oils of Persea americana and Melia dubua species are selected
for this analytical study. These seed species yield 63.6 and 28 % of nonedible oil,
respectively. The details of component fatty acids (CFAs) are collected from the
literature. The necessary analytical data, viz., iodine value (IV) and saponification
value (SV) of seed oils and major fuel properties of biodiesel, viz., cetane number
(CN), higher heating value (HHV), and lower heating value (LHV) of the fatty acid
methyl esters (FAMEs) are empirically computed. These parameters of biodiesel of
seed oils under investigation are compared and evaluated in reference to existing
biodiesels. This work reports the suitability of especially Persea americana for the
biodiesel production.
Keywords Seed oil Persea americana Melia dubua Biodiesel Fatty acid
methyl esters
1 Introduction
Sustainable development has been the issue of immense importance. Renewable

raw materials add significantly to the sustainable progress. Plant oils cover the
larger section of the existing utilization of renewable raw materials in the chemical
industry (Liu 2013). Seed oils find various applications in production of surfactants,
soaps, detergents, solvents, lubricants, paints, cosmetics, etc. The unusual fatty
acids used in formulations of protective coatings, plastics, urethane derivatives,
K.S. Katagi (&)

Department of Chemistry, Karnatak University’s Karnatak Science College,
Dharwad 580001, India
e-mail: kskatagi@gmail.com
R.S. Munnolli S.D. Benni S.S. Kulkarni
Department of Chemistry, Research Centre, KLS’s VDR Institute of Technology,
Haliyal 581329, India

DOI 10.1007/978-81-322-2773-1_8
112 K.S. Katagi et al.
dispersants, cosmetics, lubricant additives, biolubricants, pharmaceuticals, textiles,

variety of synthetic intermediates, and stabilizers in plastic formulations, etc. (FAO
2008; Kucuk 1994).
The fast depletion of natural resources of energy such as petroleum and coal has
triggered the emergence of biodiesel as a potential substitute for energy. It is derived
from animal fat or from plant seed oils. The advantages of biodiesel include its domestic
origin, renewability, biodegradability, higher flash point, inbuilt lubricity, higher cetane
number, low viscosity, improved heating value, etc. also with lowered emissions of
particulates, COX and SOX (Leduc et al. 2009; Mustafa and Havva 2010).
Presently, due to apprehension about energy security, fuel cost and safe envi-
ronment, biodiesel is considered for part or full replacement of petro-diesel. The seed
oil has 2.25 times higher energy density than starch or proteins (Mehta and Anand
2009; Banas et al. 2007). Currently, seed oils such as Palm seed oil, Soybean seed
oil, Sunflower seed oil, Coconut oil, etc., which are edible are also used for biodiesel
production this has resulted in a competing situation for plant seed oils being used
for food and for possible industrial applications. Thus, due to increased obligation,
biodiesel practice would continue to grow at a fast rate to meet the energy needs. As
a result, the cost of edible seed oils led to escalating prices constraining to secure
sufficient supply in various parts of the world. Consequently, there is a need to
identify nonedible/alternative sources of plant oils to be used for energy-related
applications. Thus, those plant crops which require only a minimum land for their
crop production yielding significantly good amount of nonedible seed oils are to be
endorsed (Dennis et al. 2010; Manjula et al. 2009).
Researchers in the recent times, are focusing on nonedible seed oils for the
biodiesel production (Altun 2011; Balusamy and Marappan 2010; Banapurmath
et al. 2008) to mitigate the consequences of usage of edible oils for the production
of biodiesel which otherwise, would lead to adverse effect on food security.
1.1 Persea americana
The Persea americana (Avocado) belongs to Lauraceae plant family. It is a tree,

native to Mexico and Central America, its fruit is shown in Fig. 1. It has a large
berry with single seed. Fruits have a green skinned, fleshy body that is pear-shaped,
or egg-shaped, or spherical. The tropical and subtropical regions are suitable cli-
matic conditions for cultivation of this species.
It is one of the 150 varieties of avocado pear. Its parts such as, seeds, stems,
leaves, roots, bark, etc., are used for the curing of various diseases affecting
humans. The drugs used in medicine today are directly or indirectly derived from
this plant due to its bioactive constituents such as; alkaloids, steroids, tannins, etc.,
(Glossary of Indian medicinal plants 1976).
The seed of Persea americana (avocado seed) has varied applications in ethno–
medicine, like in the treatment of diarrhea, dysentery, toothache, intestinal parasites,
dermatological treatments, and in beautification. The seed oil is used in the
Assessment of the Seed Oils of Persea americana and Melia dubua … 113
Fig. 1 Persea americana

fruit
treatment of obesity. The fruit possesses fatty, subtly flavored, smooth, and creamy
texture. Fruits are consumed as milk shakes and intermittently added to ice cream in
countries such as Mexico, Brazil, South Africa, and India (Ramos et al. 2004).
1.2 Melia dubua
Melia dubua belongs to Meliaceae family. It is an indigenous tree to India,

Southeast Asia, and Australia. It has been cultivated as a source of firewood, also
called as Maha Neem or Forest Neem (Glossary of Indian medicinal plants 1956a,
b). The seeds are shown in Fig. 2.
1.3 Component Fatty Acids in Seed Oils
Listed below in Table 1 are the fatty acid components with their chemical formulae
and molecular weights that make up the two seed oils under investigation. There are
Fig. 2 Melia dubua seeds

Table 1 Fatty acids that constitute the two seed oils under investigation
Fatty acid Molecular formula Molecular weight
Lauric acid CH3(CH2)10COOH 200.32
Myristic acid CH3(CH2)12COOH 228.38
Palmitic acid CH3(CH2)14COOH 256.43
Stearic acid CH3(CH2)16COOH 284.48
Oleic acid CH3(CH2)7CH=CH(CH2)7COOH 282.47
Linoleic acid CH3(CH2)4CH=CHCH2CH=(CH2)7COOH 280.46
Arachidic acid CH3(CH2)18COOH 312.54
seven fatty acids out of which oleic acid and linoleic acid are the two unsaturated
acids. Their content in higher proportion makes the oil as much more unfavorable
for biodiesel synthesis.
2.1 General Procedures
2.1.1 Oil Extraction
The seeds are ground, powdered and the oil extracted using petroleum ether
(boiling range: 40–60 °C) in Soxhlet extractor for 24 h. The organic extract is
filtered and dried over anhydrous sodium sulphate. The petroleum ether is removed
under vacuum.
2.1.2 Transesterification
Triglycerides in the seed oils on reaction with alcohol usually methanol or ethanol
in presence of base or acid as a catalyst, yield FAMEs or fatty acid ethyl esters
(FAEEs) along with glycerol as byproduct.
Triglyceride þ Alcohol
FAMEs or FAEEs þ Glycerol
ðSeed oilÞ ðMethanol=EthanolÞ ðBiodieselÞ ðByproductÞ
The seed oil is treated with methanol or ethanol in 1:6 molar ratio in the presence
of base or acid catalyst, then organic and aqueous phases are separated using a
separating funnel. The lower layer containing glycerol, alcohol, and most of the
catalyst is drained out. The upper layer containing alkyl esters, some alcohol, traces
of the catalyst is washed thoroughly with deionized water. Then, the residual
methanol is removed by rotary evaporation at around 70 °C. The product obtained
is a mixture of FAMEs or FAEEs and is used as biodiesel (Katagi et al. 2011).
3 Computational Analysis of Biodiesel Properties of Seed

Oils
The selected seed oils and the computed biodiesel properties are compared for fuel
properties with those of ASTM, DIN D6751, and EN 14214 standards.
3.1 Saponification Value (SV) and Iodine Value (IV)
These parameters of the seed oils are calculated to establish their suitability for
biodiesel synthesis. The SV and IV of seed oils are evaluated using Eqs. (1) and (2),
respectively, based on composition of fatty acids (Kalayasiri et al. 1996). The
results obtained are comparable with experimental values.
X 560 Ai
SV ¼ ð1Þ
Mwi
X 254 Ndb Ai
IV ¼ ð2Þ
Mwi
where Ai is the percentage of component fatty acids, Ndb is the number of carbon–
carbon double bonds, and Mwi is the molecular mass of each component.
3.2 Cetane Number (CN)
Generally, biodiesel exhibits higher CN than conventional petro-diesel and results

in higher combustion efficiency. CN is significant in knowing the suitability of
biodiesel. The CN of FAMEs is computed from the following Eq. (3), which suits
closely with the experimental values (Krisnangkura 1986).
5458
CN ¼ 46:3 þ 0:225 IV ð3Þ
SV
3.3 Higher Heating Value (HHV)
It is known that the processed vegetable oils used in diesel engines are the complex
chemical mixture of FAMEs. The HHV of biodiesel is calculated using Eq. (4) in
accordance with regression model (Demirbas 1998).
HHV ¼ 49:43 ð0:015 IVÞ ð0:041 SVÞ ð4Þ

3.4 Lower Heating Value (LHV)
LHV of biodiesel is computed from Eqs. (5) and (6) based on bond energy values
of different FAMEs. The methods deployed for the calculation of lower heating
value are quite general and their predictability is reasonable (Mehta and Anand
2009).
For FAMEs,
3 2
C C C
LHV ¼ 0:0109 0:3516 þ 4:2000 þ 21:066 0:100 Ndb ð5Þ
O O O
3 2
H H H
LHV ¼ 0:0011 0:0785 þ 2:0409 þ 20:992 0:100 Ndb ð6Þ
O O O
where C, H, and O are the number of carbons, hydrogen, and oxygen, respectively.
Ndb has the same meaning as stated above.
Biodiesel property of selected seed oils of Persea americana and Melia dubua are
computed based on the data from the literature (Shanbhag and Badami 1974;
Thakkar 1983). Details of component fatty acids (CFAs) and other analytical values
of seed oils and properties of biodiesel are depicted in Tables 2 and 3 respectively.
The computed parameters of biodiesel from Persea americana are complimentary
to quality biodiesel and make it a befitting candidate for the synthesis of biodiesel.
A comparison of the properties of different seed oils and corresponding bio-
diesels is made in Table 4. One nonedible seed oil and three edible seed oils are
included for purpose of comparison.
Iodine value of seed oils should not exceed 120 mg I2 g−1 which best fits
especially for Persea americana as per the limitation laid by European standard
organization EN 14214 for biodiesel. However, the IV is on the higher side for
Melia dubua because of the higher proportion of unsaturated acids, oleic acid and
linoleic acid. This is clear as is shown in Fig. 3a. There is consistency in the SV.
This signifies that Persea americana is ideal for the biodiesel as per the conclusion
derived by Knothe (Knothe 2008).
Literature reveals that, biodiesel standards of USA (ASTM D6751), Germany
(DIN 51606), and European Organization (EN 14214) have set CN value as 47, 49,
and 51 respectively (Krisnangkura 1986). The computed values of CN for the
FAMEs of seed oils of Persea americana and Melia dubua are 59.8 and 41.5,
respectively, whereas, the CN of petro-diesel is 42.0. The empirically calculated CN
value of FAMEs of Persea americana meet the ASTM standards, however; Melia
dubua exhibits relatively lesser CN than the expected value. Comparisons shown in
Fig. 3b confirm the facts mentioned above.
Table 2 Analytical values of Persea americanaa and Melia dubuab seed oils
Source/seed species Persea americana Melia dubua
Seed oil percentage 63.6 28.0
Molecular weight of oil (g mol−1) 762.2 880.2
Total saturated fatty acids (TSFAs) percentage 61.7 13.9
Total unsaturated fatty acids (TUSFAs) percentage 38.3 86.1
Saponification value (mg KOH g−1) 240.3 208.5
Iodine value (mg I2 g−1) 40.2 140.0
Percentage of component fatty acids: 12:0 (Lauric) 36.3 NA
14:0 (Myristic) 14.3 NA
16:0 Palmitic) 7.9 9.4
18:0 (Stearic) 1.1 1.5
18:1 (Oleic) 29.6 20.5
18:2 (Linoleic) 8.7 65.6
20:0 (Arachidic) 1.1 NA
a
Shanbhag and Badami (1974)
b
Thakkar (1983)
Table 3 Computed biodiesel properties of Persea americana and Melia dubua plant species
Source/seed species Persea americana Melia dubua
Cetane number 59.8 41.5
Higher heating value (HHV; MJ kg−1) 39.1 39.0
Lower heating value C/O (LHV; MJ kg−1) 37.1 38.1
Lower heating value H/O (LHV; MJ kg−1) 37.3 38.0
Table 4 Comparison of properties of biodiesels from Persea americana and Melia dubua with
existing biodiesels and petro-diesel
Fuel Persea Melia Pongamia Soybeana Rapeseeda Sunflowera Petro
property americana dubua pinnataa (SB) (RS) (SF) diesela
(PA) (MD) (PP) (PD)
SV (mg 236.3 201.4 182.3 194.6 197.1 193.0 NA
KOH g−1)
IV (mg I2 42.9 141.6 58.4 120.5 108.1 132.3 NA
g−1)
CN 59.8 41.5 63.1 45.0 59.0 49.0 42.0
HHV 39.1 39.0 40.5 39.8 37.0 40.6 46.0
(MJ kg−1)
LHV 37.1 38.1 34.3 33.5 32.8 33.5 43.1
(MJ kg−1)
NA Not applicable
a
Indicates the data obtained from Ref. (Ramadas et al. 2005)
(a) (b)
(c) (d)
Fig. 3 Graphical comparison of biodiesel properties with standard fuel and petro-diesel. a IVs of
biodiesels compared with that of EN standard. b CNs of biodiesels compared with that of
petro-diesel. c HHVs of biodiesels compared with that of petro-diesel. d LHVs of biodiesels
compared with that of petro-diesel
The carbon, hydrogen, and oxygen content of a fuel is directly proportional to its
HHV and it is evaluated using the Eq. (4). The HHV is seen to be almost same at
about 39.1 MJ kg−1 for the biodiesel derived from the seed oils under investigation
are slightly lower than that of petro-diesel (43 MJ kg−1) or petroleum (42 MJ kg−1),
but is higher than that of coal (32–37 MJ kg−1). The LHVs are estimated using two
Eqs. (5) and (6). The computed LHVs of respective biodiesels are in the range of
37–38 MJ kg−1. The European Biofuels Technology Platform 2011 reported the
LHV for biodiesel as 37.1 MJ kg−1 (Biofuels Technology Platform 2011). This is
slightly lower than the LHV of petro-diesel (43 MJ kg−1). It may be noted that
heating values are a function of mainly carbon content in the fuel. Obviously, it is
expected to be higher for petro-diesel because of the presence of mostly carbon and a
little of hydrogen in petro-diesel. However, biodiesel is a mixture of fatty acid esters
(oxygenates) reducing the carbon content per unit mass. It is also interesting to
observe that the oxygen in biodiesel molecules promotes relatively better combus-
tion efficiency and yields higher cetane number as is evident for most of the bio-
diesels including the two under investigation in this work. Figure 3b–d support the
statements made above.
It may be noted that higher SV infers higher proportion of fatty acids per unit
mass of oil and subsequent higher proportion of FAMEs in the biodiesel. Also,
because, the FAMEs are oxygenates, improved combustion efficiency and CN is

expected which is indicated by corresponding values for Persea americana. It is
comparable with that of biodiesel from rape seed oil or Pongamia pinnata. Also,
the computed value of LHV is more favorable in comparison to any other biodiesel
listed above.
The analytical data for seed oils other than those investigated in this paper are
obtained from the literature (Ramadas et al. 2005). It signifies, Persea americana to
be an ideal candidate for biodiesel production with mostly favorable parameters
especially with regard to IV, CN and heating values when compared to other seed
oils/biodiesels.
5 Conclusion
The FAMEs of seed oil of Persea americana meet the major specifications of US
biodiesel standard and European standards and can be utilized for the production of
biodiesel and also has the possible industrial relevance. However, further research is
required to evaluate these FAMEs for other properties like, tribological studies and
long-term engine testing, etc.
Acknowledgments The authors acknowledge the support of authorities of Sri. S.S. Basavanal
Library, Karnatak University, Dharwad for facilities provided during data collection. The authors
also acknowledge Karnatak Science College, Dharwad and KLS’s VDR Institute of Technology,
Haliyal for extending encouragement in completing this work.
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www.biofuelstp.eu (2011) Fact sheet european biofuels technology platform
Investigation of CI Engine Fueled
with Ethanol Nano Additives Blended
Diesel
V. Karthickeyan, P. Balamurugan and R. Senthil
Abstract Experimental investigation on the performance and emission character-

istics of ethanol–cerium oxide blended diesel in a single cylinder four-stroke diesel
engine is presented. Solubility of cerium oxide in diesel and ethanol is investigated
with diethyl ether. Four blended diesel test fuels have been prepared using ethanol,
diethyl ether, and cerium oxide in diesel. A computerized CI engine with eddy
current dynamometer test rig with gas analyzer has been used. Result shows that the
prepared blended fuel is capable of replacing neat diesel as fuel for diesel engine
with lower CO2 emission. The blended fuel has higher thermal efficiency with
nominal variations in fuel consumption, due to nano additives. NOx emission is also
observed lower when compared to neat diesel.
Keywords Diesel Ethanol Cerium oxide Computerized diesel engine

Performance Emission
1 Introduction
In the present scenario of energy and environment, the drive for research in the field
of IC Engines is always targeted toward reducing the dependence on fossil fuels and
the emission from the engines. These could be achieved with the exploration of
different alternate fuels from renewable sources of energy. Investigation on
preparation of different blended diesel fuels and their performance on engines are
being carried out by researchers across the world.
V. Karthickeyan P. Balamurugan (&)

Department of Mechanical Sciences, Sri Krishna College of Engineering and Technology,
Coimbatore 641008, Tamil Nadu, India
e-mail: balamuruganp@skcet.ac.in
R. Senthil
Department of Mechanical Engineering, University College of Engineering Villupuram,
Villupuram 605103, Tamil Nadu, India

DOI 10.1007/978-81-322-2773-1_9
122 V. Karthickeyan et al.
Aksoy et al. (1990) carried out investigation on the properties of triglyceride oils
obtained from seeds of fermented raisins of Turkish origin. The technical properties
of the derivatives of pot still distillation have been determined, to be recommended
as an alternative to conventional fuels. Gowen Marcia (1989) explored sectoral
differences between biofuel and fossil fuels with regard to production costs,
economies of scale, subsidies, and other economic incentives in developing
countries. Malaya Naik et al. (2008) investigated the production of biodiesel from
high FFA Karanja oil. The production of biodiesel was discussed using the
mechanism of dual process. It was observed that high FFA Karanja oil yield 96.6–
97 % biodiesel by dual step process.
Experimental investigation on the performance and emissions of a diesel engine
fuelled with ethanol–diesel blends was conducted by Huang et al. (2009). The fuel
consumptions of the engine fuelled by the blends were found to be higher that of
pure diesel. However, it was observed that the blended fuel gave comparable
thermal efficiency like pure diesel, with marginal deviations at different loads and
speed. Also, the CO emissions of blends were found higher than those of diesel.
However, it was found that the engine fuelled with blends emitted less NOx at low
engine speed. Also, smoke emissions from the engine with blended fuel were lower
than that of diesel, the reductions ranging from 16.7 to 87.5 %.
The effect of thermal insulation of CI engine with ethanol was investigated by
Karthickeyan and Srithar (2011). Yttria-stabilized zirconia was used as insulation to
control the ignition delay of the low heat rejection engine. Benefits of insulation
were investigated with ethanol and diesel operated in glow plug assisted engine. It
was observed that highest efficiency was obtained with ethanol with insulated
combustion chamber.
The effect of metal-based additives (FeCl3 and FBC) in diesel engine with waste
cooking palm oil is investigated by Kannan et al. (2011). BSFC decreased by 8.6 %
and BTE increased by 6.3 %. CO, HC and smoke emission of FBC added biodiesel
decreased by 52.6, 26.6 and 6.9 %, respectively, when compared without FBC.
Performance of high alcohols and diesel fuel in diesel engine was compared by
Campos-Fernandez et al. (2012). The existence of oxygen in the molecular structure
of 1-butanol and 1-pentanol offsets its reduced low heating value, showing better
combustion and brake thermal efficiency, while power and torque were found
similar to that of diesel fuel. With regard to BSFC, butanol blends exhibited slightly
better behavior than pentanol blends and neat diesel fuel. Ramesh Kumar and
Nagarajan (2012) experimentally investigated the effect of thermal barrier coating
using alumina (Al2O3) in SI engine. It was found that the combustion chamber
temperature increased resulting in limiting the preignition and knocking. It was also
observed that the partially insulated SI engine and when fuelled with alternate fuel
ethanol 20 % improved performance with a reduction of HC and CO emissions up
to a maximum of 48 and 50 %, respectively.
Experimental investigation on tobacco seed oil methyl ester (TSOME) in diesel
engine found that effective power and torque which were increased was analyzed by
Parlak et al. (2013). However, on the contrary, as TSOME contains only 10 % of
oxygen in its content, blended fuel was found to cause an increase of about 6 % in
Investigation of CI Engine Fueled … 123
NOx emission. Golimowski et al. (2013) evaluated the performance of common rail
diesel tractor engine using pure plant oil. The engine generated maximum power of
102 kW when fuelled with diesel, and 90 kW with raps oil. The engine operating on
vegetable oil displayed better efficiency over diesel. Experiments with Pongamia oil
methyl (PME) ester and diesel blends (B20 and B100) were investigated by
Prabhahar and Rajan (2013) in a 4S direct injection diesel engine with and without
coated piston. Ceramic coating material (TiO2) was applied on the piston crown
using plasma spray method. From the experiment it was observed that it is 100 %
biodiesel. The efficiency of the engine was improved with a decrease in BSFC by
about 10 % at full load. Also, the coated engine showed lower carbon monoxide and
hydrocarbon emissions, however, with an increase of 15 % of nitrogen oxide
emission. Kim and Choi (2013) carried out experiments using biodiesel fuel
(BDF) and ethylene glycol mono-n-butyl ether (EGBE) to investigate the reduction
of smoke and NOx emission in IDI engines. Better smoke reduction was observed
with the BDF and the mixture of BDF and EGBE when compared to neat diesel.
Also, the simultaneous application of cooled EGR was found to have augmented
reduction effects of emissions. The prepared alternate fuel was validated by com-
paring with the results available in the literature. From the literature survey, it has
been found that even though different researches have been carried out on the
alternate fuels, very few works have been carried out with respect to nano additives
in fuel. In this context, this research work shall be highly significant and unique one.
In the present work, performance and emission characteristics are investigated
experimentally in direct injection diesel engine using ethanol–cerium oxide blended
diesel fuel and the results are presented.
2 Preparation of Biofuel Blends
The present experimentation is mainly intended to examine the characteristics of

new blended diesel combinations at different loading conditions. The new fuel is
prepared by blending ethanol and cerium oxide with diesel in different proportions.
Ethanol addition to diesel fuel results in different physiochemical changes in diesel
fuel properties, particularly reductions in cetane number, viscosity, heating value,
etc. Since ethanol is having polar molecule and its solubility in diesel is prone to be
affected by temperature and water content, high percentage addition of ethanol to
diesel is difficult. In order to mix ethanol and diesel, an emulsifier or cosolvent like
diethyl ether should be added. Cerium oxide nano particles as an additive (Sajith
et al. 2010) could possibly exhibit high catalytic activity because of their large
surface area per unit volume, leading to improvement in the fuel efficiency and
reduction in the emissions. Hence by adding the cerium oxide nano particle the fuel
reduces the NOx emissions and prolongs combustion, which leads to a reduction in
unburnt hydrocarbons (by up to 15 %) and a decrease in fuel consumption up to 5–
9 %. In this, an ultrasonic bath has been used to mix diesel, ethanol, and diethyl
Table 1 Composition of four trial samples of test fuels

NEAT DIESEL 100 % diesel
D89E10DEE1 89 % diesel + 10 % ethanol + 1 % DEE
D89E10DEE1CEO10 89 % diesel + 10 % ethanol + 1 % DEE + 10 gm cerium oxide
Table 2 Properties of diesel and other trial sample prepared

Properties Neat D89E10DEE1 D89E10DEE1CEO10 D89E10DEE1CEO15 D89E10DEE1CEO20
Diesel
Diesel content (%vol) 100 89 89 89 89
Ethanol content (%vol) 0 10 10 10 10
Density at 150 °C (kg/m3) 843 831 833 836 838
Viscosity at 40 °C (Ns/m2) 2.48 1.86 1.88 1.92 1.92
Flash point (°C) 52 89 86 86 85
ether with cerium oxide. The sonicator resulted in better mixing of components
resulting ultimately with an essential solution of cerium oxide.
In order to carry out the investigation on performance and emission character-
istics, four combinations of blended diesel fuels have been prepared using the
cerium oxide solution, ethanol, and diesel. The compositions of individual com-
ponents of the four trial samples are given in Table 1. The physiochemical prop-
erties of the prepared fuels namely, density, viscosity, calorific values, flash point,
etc., have been determined and the values are presented in Table 2.
3 Experimental Setup
The schematic diagram of computerized single cylinder four-stroke diesel which is

used for experiment is shown in Fig. 1.
The setup includes air chamber with U-tube manometer, digital temperature
indicator, digital speed indicator, fuel measuring unit, and fuel tank. The system
also includes engine jacket cooling water circuit with calorimeter temperature
indicator and glass tube rotameter for measuring cooling water, valves, and
instrumentation. Various measuring devices such as pressure sensors, flow meters,
and temperature sensors are fitted at suitable locations. The specification of the
engine used and uncertainties of various instruments are listed in Tables 3 and 4,
respectively.
Fig. 1 Schematic diagram of computerized IC engine test rig
Table 3 Specifications of Make Kirloskar

test engine
Cylinder number 1
Type Four-stroke, stationary, constant speed,
direct injection, air cooled, and diesel
engine
Max. power/speed 4.4 kW/1500 rpm
Bore 87.5 mm
Stroke 110 mm
Compression ratio 17.5:1
Injection timing 23.4° bTDC
Table 4 List of measurement Measurement Uncertainty (%)

uncertainty
Load ±0.2
Speed ±0.1
Time ±0.2
Manometer ±1
CO2 ±0.2
NOx ±0.2
The experiment is carried out by varying the brake load using the eddy current
dynamometer. During which the data of fuel consumption, engine temperature,
engine speed, and exhaust gas temperature are recorded using NI USB-6210, 16 bit,
250 kS/d data acquisition system. U-tube manometer is used to measure the air
intake. Exhaust gas has been investigated using AVL make 444 five gas analyser.
4 Experimental Procedure
Figure 2 shows the experimental setup of computerized engine test rig with eddy
current dynamometer.
Initially, the diesel is taken in fuel tank. Before the engine is started at no load
condition, the cooling water line is regulated to ensure continuous flow of water.
Simultaneously the data acquisition system and the AVL gas analyzer, which are
connected to engine, are switched ON to keep track of the performance and
emission. After starting, the engine is given sufficient time to attain a constant
steady state speed of 1500 rpm. Initially, the engine is investigated at no load
condition to measure the necessary data. Consequently the load is increased to full
load condition gradually with an increment of 25 %. At every loading, after
reaching steady state condition, all required data are measured using data acqui-
sition system and the gas analyzer. Then the entire steps are repeated with four
blended biodiesel trial samples.
Fig. 2 Computerized engine

coupled with eddy current
dynamometer
Experimentation has been carried out on the computerized single cylinder

four-stroke diesel engine connected to data acquisition system, with one fuel after
another. There are totally five fuels (including, neat diesel) tested. This section
compares the performance and emission characteristics of four combinations of
blended diesel fuels with those of neat diesel. By the end of the present work, a
suitable replacement to diesel engine shall be recommended.
Figure 3 shows the variation of brake thermal efficiency with brake power. As
expected, it has been observed that the efficiency of the engine increases with the
increase in load and brake power. As the blended diesel contains cerium oxide as
one of the constituents, it facilitates the production of oxygen in the combustion
chamber of engine.
Larger the quantity of cerium oxide, larger will be the oxygenation of fuel. This
results in the higher efficiency of blended diesels with higher cerium oxide content.
Due to this, it is found that D89E10DEE1CEO15 and D89E10DEE1CEO20 have
higher brake thermal efficiency at every loading conditions. Also, these two fuels
are found to have higher values of efficiency than that of the neat diesel at all
operating conditions.
For the same reason, it has been observed that fuels with higher cerium oxide
composition have lower brake specific fuel consumption than their counterpart. This
has been substantiated in Fig. 4, showing the variation of brake specific fuel con-
sumption with brake power. Figure 5 shows the variation of percentage of carbon
dioxide emitted from the engine at different operating conditions investigated with
the different test fuels. Being a green house gas, any recommended fuel shall have
the lowest value of carbon dioxide emission. It has been found that the carbon
dioxide percentage increases with increase in brake power. Also in all the operating
conditions, the blended diesels are found to have lower emission when compared to
that of neat diesel. Among the prepared test fuels, D89E10DEE1CEO20 is observed
NEAT DIESEL
30 D89E10DEE1
Brake Thermal Efficiency, %
D89E10DEE1CEO10
25 D89E10DEE1CEO15
D89E10DEE1CEO20
20
15
10
0
0 1 2 3 4
Brake Power, kW
Fig. 3 Effect of brake power on brake thermal efficiency

Brake Specific Fuel Consumption, kg/kWhr

NEAT DIESEL
D89E10DEE1
0.8
D89E10DEE1CEO10
D89E10DEE1CEO15
D89E10DEE1CEO20
0.6
0.4
0.2
0 1 2 3 4
Brake Power, kW
Fig. 4 Effect of brake power on brake specific fuel consumption
NEAT DIESEL
7
D89E10DEE1
D89E10DEE1CEO10
D89E10DEE1CEO15
6
D89E10DEE1CEO20
CO2, %
2
0 1 2 3 4
Brake Power, kW
Fig. 5 Effect of brake power on carbon dioxide
to have the lowest carbon dioxide percentage at every load. However, the test fuel
D89E10DEE1CEO15 is having only marginally higher emission than test fuel
D89E10DEE1CEO20. The amount of cerium oxide present in the blended diesels
prepared, contributes for the complete combustion due to the presence of more
oxygen produced. The complete combustion ensures emission of less carbon dioxide
from the engine. Hence the test fuel D89E10DEECEO20 with more cerium oxide
has the lowest emission of carbon dioxide.
Figure 6 shows the effect of engine loading on NOx emission from the engine.
From the figure, it has been inferred that the NOx emission increases with the
increase in engine loading, due to the higher temperature of the cylinder attained
(Campos-Fernandez et al. 2012).
In the entire range of operating conditions, the NOx emission of blended diesels
is found to be always less than that of neat diesel. Also, among the blended diesel
1400
NEAT DIESEL
D89E10DEE1
1200
D89E10DEE1CEO10
D89E10DEE1CEO15
NOx, PPM 1000 D89E10DEE1CEO20
800
600
400
200
0
0 1 2 3 4
Brake Power, kW
Fig. 6 Effect of brake power on oxides of nitrogen
fuels, D89E10DEE1CEO20 is found to have the lowest NOx emission next to test
fuel D89E10DEE1CEO15. Even though the high temperature of engine at higher
engine loading condition causes the increase in NOx emission, the presence of
cerium oxide in the blended diesel acts favorable to facilitate complete combustion
inside the cylinder. This results in less NOx emission for test fuel
D89E10DEE1CEO20 next to D89E10DEE1CEO15 at every engine loading
condition.
With the foregoing discussions it has been observed that in the entire range of
operating conditions, the test fuel D89E10DEE1CEO20 has a 3.2 % increase in
efficiency and 0.6 % decrease in emission when compared to the neat diesel. The
test fuel D89E10DEE1CEO15 is found to exhibit better characteristics with respect
to performance and emission than the neat diesel. Hence, the blended diesel fuel
D89E10DEE1CEO15 may be considered as a promising alternative to the neat
diesel, for the single cylinder four-stroke diesel engine.
6 Conclusion
A computerized single cylinder four-stroke diesel engine test rig has been used to
investigate for a suitable alternative to the neat diesel. For this, four trial blended
diesel fuel samples have been prepared using ethanol, cerium oxide, and diesel.
Experiments have been repeated with different loading conditions and fuels. Based
on the experimental data, the performance and emission characteristics of test fuels
have been compared with diesel and investigated. It has been found that the test fuel
D89E10DEE1CEO15 has a 0.05 % increase in volumetric efficiency, 3.2 %
increase in brake thermal efficiency, 0.6 % decrease in CO2, and 252 PPM decrease
in NOx. Economic feasibility study also shows that the average one gallon of diesel
and ethanol costs around 3.97$ and 3.41$ (Rashedul et al. 2014), respectively. The
nano additives costs may be less than 1$ per kg. If the government takes forward
and increase the production of ethanol it may reduce the price with decreasing the
sample preparation rate. Based on the above investigation, the test fuel may be
considered as a potential alternative for the conventional diesel in the test engine.
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Designer Biodiesel: An Optimization
of Fuel Quality by Blending Multiple Oils
Anuchaya Devi, Renuka Barman and Dhanapati Deka
Abstract Liquid biofuels such as biodiesel, derived from vegetable oils are gaining
worldwide importance as a substitute for diesel fuel owing to their environmental
advantages and renewable sources in nature. Since biodiesel has the similar fatty
acid composition as the parent oil, its fuel properties are directly affected by the
composition of the precursor vegetable oil. Various blends of some locally available
vegetable oils such as Jatropha, Nahor, Yellow Oleander, Karanja, and Coconut
were prepared by mixing at different volumetric ratios in the range of 1:1–1:10
(Jatropha: Coconut; Nahor: Coconut; Yellow Oleander: Coconut; Pongamia:
Coconut, Jatropha: Yellow Oleander; Nahor: Yellow Oleander, Pongamia: Yellow
Oleander, Pongamia: Jatropha, and Pongamia: Nahor). Biodiesel was prepared in
laboratory scale from the pure vegetable oils and selected blends through a single
step conc. H2SO4 catalyzed transesterification process and their fuel properties were
analyzed in accordance with the standard test method. Fatty acid composition of the
vegetable oils and the blends were elucidated by 1H NMR. The effect of fatty acid
profile/composition on fuel properties such as cetane number, viscosity, calorific
value, and iodine number has been measured.
Keywords Biodiesel
1
Blending Fatty acid composition Oxidation stability
H NMR
1 Introduction
The substitution of fossil fuels to safe future energy demand goes on with the first
and foremost disquiet over the last decade. With this correlation, biodiesel (Knothe
et al. 2005; Mittelbach and Remschmidt 2004) is an option of selection for all sorts
of petroleum-based diesel fuel. The outstanding plausible alternate of the usual
Anuchaya Devi Renuka Barman Dhanapati Deka (&)

Biomass Conversion Laboratory, Department of Energy, Tezpur University,
Napaam, India
e-mail: dhanapati@tezu.ernet.in

DOI 10.1007/978-81-322-2773-1_10
132 Anuchaya Devi et al.
diesel fuel known to be biodiesel has generally been constituted with fatty acid
methyl esters which are often produced from triglycerides found in vegetable oils
via the process of transesterification using methanol/ethanol. Transesterification is
an effective way to overcome all these problems associated with biodiesel (Shay
1993). In recent years, biodiesel has recently gained worldwide popularity as a
solution of energy crisis and also as domestic, renewable and biodegradable
alternative diesel fuel (Singh and Singh 2010). The bright feed stocks for biodiesel
production are demonstrated as the vegetable oils which are highly replenished and
can be manufactured in industrial level. Biodiesel which is a fatty acid methyl esters
(FAME) of several vegetable oils and animal fats have been found appropriately fit
for utilizing in diesel engine in the form of a fuel. FAME as biodiesel is envi-
ronmentally harmless, non-toxic and rapidly deteriorated by atmosphere (Azam
et al. 2005). The overall fuel properties including oxidation stability, Cetane
number, Calorific value etc. have been robustly affected by the characteristics of
each of the fatty acid esters (Knothe and Kenar 2004). The main practical trou-
blesome associated with the biodiesel utilizations in the current hours can be put
forward as oxidative stability, low-temperature properties, and a minor enhance-
ment in NOx exhaust production, even though the last difficulty may become paler
over time with the invention of novel exhaust emission control technology. To
solve the aforementioned troubles at the same time have demonstrated intricacies on
account of the clarification to one of the difficulty always creates disturbances to
another ones. The above circumstances can be mainly traced to the belief of fuel
features on the structure of fatty acid (Knothe 2005). Hence, it has been revealed
that the cetane number which is directly connected to the ignition quality of a diesel
fuel lessens with shortening the chain length, extension in branching, and rises in
unsaturation in the chain of fatty acid (Harrington 1986). Normally, the greater the
cetane number, the superior the ignition quality. Conversely, saturated esters with
greater value of cetane numbers have lower cold-flow traits. Fatty acids with
unsaturated or polyunsaturated functionalities possessing poorer melting points are
enviable for better low-temperature traits and have small value of cetane numbers as
well as reduced oxidative stability which are unwanted for the biodiesel. Remedy to
one of these complexities always brings about the growing problematic behavior in
the other characteristics and have incorporated the application of additives or
changing the composition of fatty acid either via physical procedures, for instance
winterization, or through genetic adjustments. This has led to the concept of
designer biodiesel, with optimized fatty acid compositions for superior fuel prop-
erties. To achieve the biodiesel fuel with encouraging features, it is beneficial for
the fuel to constitute with merely one major part probably in a large concentration.
The characteristics of the different fatty ester components encompassing biodiesel
decide the on the whole fuel features of the biodiesel fuel. Thus, the qualities of the
several fatty esters are detected by the structural characteristics of the fatty acids and
the alcohol functionalities consisting of them (Knothe 2005, 2008). As per our
objective is concerned, in this manuscript, different oil seeds from locally available
oil bearing trees has been collected, extracted and prepared model feedstock oils by
blending of composed vegetable oils at different ratio in order to modify their fatty
Designer Biodiesel: An Optimization of Fuel … 133
acid compositions. Additionally, the fuel properties of biodiesel/methyl esters of

model oils based on fatty acid compositions has also been predicted and verified the
outcomes experimentally. The optimum blending ratio of various feedstock oils for
production of biodiesel with superior fuel quality has also been accomplished.
2 Experimental
Methanol (extra pure), Petroleum Ether (40–60 °C), H2SO4 (98 % pure), Na2SO4
(99 % pure), and NaHCO3 (99 %) were procured from Merck India Ltd, Mumbai.
Each of the reagents and chemicals had been made use with no additional purifi-
cation. Various oil bearing seeds such as Yellow oleander (Thevetia peruviana),
Nahor (Mesua ferrea Lin.), Karanja (Pongamia pinnata), and Jatropha (Jatropha
curcas) were collected from different localities of Assam, India. Jatropha curcas
and Pongamia pinnata seeds were supplied by Kaliabor Nursery, Nagaon (Assam),
India. Coconut oil (Cocos nucifera; 100 % pure, according to manufacturer)
involved in the experiment was obtained from a local supplier in the Tezpur uni-
versity campus and used without any further purification.
The components of the fatty acids of the vegetable oils and the blends were
confirmed by using 1H Nuclear Magnetic Resonance Spectroscopy (1H NMR). 1H
NMR spectra were processed using “ACD NMR Processor Academic Edition”
(Elyashberg et al. 2008). All 1H NMR analyses were carried out at 25.5 °C with
CDCl3 and TMS as solvent and internal standard, respectively. The 1H NMR
spectra were carried out on a Jeol JNM-ECS400 spectrometer.
2.1 Mathematical Treatment for Determining Biodiesel Fuel

Properties
Based on the fatty acid compositions, important fuel parameters such as cetane
number, viscosity, calorific value, iodine value of the vegetable oils, and model oils
were determined using the empirical relations as follows (Ramírez-Verduzco et al.
2012).
X
fb ¼ zi fi ð1Þ
where, zi is the wt% individual fatty acid/ester determined from 1H NMR and fi is a
function that represents a fuel parameter such as cetane number, viscosity, calorific
value, and iodine value (the subscripts b and i refer to the biodiesel and the pure ith
FAME, respectively).
The individual fuel parameters of each FAME component were calculated as
follows:
Cetane number; CN ¼ 7:8 þ 0:302 Mi 0:20 N ð2Þ
Calorific value ðMJ=kgÞ; CVi ¼ 46:19 1794 Mi 0:21 N ð3Þ
Viscosity ðcStÞ; lnðvÞ ¼ 12:503 þ 2:496 lnðMi Þ 0:178 N ð4Þ

X
Iodine value ðmg I2 =gÞ; IV ¼ f254 ðno: of double bonds wt%Þg Mi
ð5Þ
where, Mi is molecular weight of individual FAME, N is the number of double

bonds and ƞi is the kinematic viscosity at 40 °C of the ith FAME. For calculation,
the entire saturated portion in all vegetable oils and their blends were approximated
to be C16 fatty esters except for coconut oil where C12 fatty esters was considered
to be the saturated fraction and Nahor oil where C18 fatty esters was considered to
be the saturated fraction because 1H NMR method do not allow quantification of
individual saturated fatty acids/esters. Oxidation stability as induction period
(IP) was predicted based on the oxidation stability (IP) of individual FAME
reported in literature and then putting the values in the general expression (1).
2.1.1 Acid Value
The determination of acid value was done by titration of the material in ethanol with
aqueous KOH. The calculations of the acid values of the various vegetable oils
were done as per ASTM D 664. For this purpose, 0.5 g of sample was dissolved in
ethyl alcohol by heating gently on a water bath and then the titration was performed
against 0.1 M KOH solution utilizing phenolphthalein as an indicator. The acid
value was calculated as follows:
56:1 V N
Acid value ðAVÞ ¼
W
where,
V volume of standard KOH used (mL).
N normality of standard KOH.
W weight of oil (grams).
2.1.2 Iodine Value
Iodine value was determined according to AOAC Official Method 993.20. 0.4 g of
the oil was weighed into a conical flask and made miscible in CCl4 (25 mL). Then
Wij’s solution (25 mL) was transferred to it and mixed appropriately. Having done
this, concentrated HCl (5 mL) and KI (10 %, 15 mL) were poured into the flask.
The flask was then placed in the dark for 30 min and then titration was performed
against standard sodium thiosulphate solution by making use of starch as an indi-
cator. The similar method was repeated for the blank test.
The calculation of Iodine Value (IV) is given by:
Iodine value ðIVÞ ¼ 1:269ðV2 V1 Þ=W
Here,
V1 volume of thiosulphate utilized for the test (in mL).
V2 volume of thiosulphate utilized for the blank test (in mL).
W weight of oil under investigation (in g).
2.1.3 Oxidation Stability
The oxidation stability was measured via the induction period (IP) of the prepared
sample. The IP value was calculated using Rancimat method EN 14112 on a
Metrohm 873 Rancimat instrument. The moisture free biodiesel samples (3 g) were
examined underneath a steady flow of air at the rate of 10 L/h into the vessel having
double distilled water. At 110 °C, the biodiesel samples were heated and a temper-
ature correction factor ΔT as recommended by the test method had been set to 1.5 °C.
To the measuring and recording device the electrode was fixed. The finish point of the
induction period was presented as soon as the conductivity begins to rise quickly. The
instant, at which the conductivity of these measuring solutions is obtained continu-
ally along with achieving an oxidation curve at that point of inflection, is called the
induction period which supplies the characteristic numerical value of the oxidation
stability for that particular biodiesel sample (Jain and Sharma 2011).
2.1.4 Calorific Value
The calorific value of the vegetable oils and the methyl esters and their blends were
measured using an oxygen bomb calorimeter according to ASTM D 2015 standard
method.
2.1.5 Kinematic Viscosity
Kinematic viscosities of the prepared samples were determined by falling-ball

viscometer (HAAKE, Type C) following the standard method ASTM D1298. The
time of falling-ball is directly proportional to its viscosity. The outcomes obtained

during the experiment are the falling time for a definite distance which is evaluated
by electronic means and changed into the units of viscosity.
The dynamic viscosity ‘η’ (in mPa s) has been calculated as given below:
g ¼ K ðq1 q2 Þt
Here,
K ball constant (in mPa s cm3/g s).
ρ1 density of the ball (in g/cm3).
ρ2 density of the liquid to be measured at the measuring temperature (in g/cm3).
t falling time of the ball (in s).
The dynamic viscosity ‘η’ and the kinematic viscosity ‘ν’ are related with as
shown below:
g
v¼
q
ν kinematic viscosity (in mm2/s) (1 mm2/s = 1 cSt);

η dynamic viscosity (in mPa s);
ρ density of the liquid sample (in g/cm3).
2.1.6 Density
Densities of the prepared samples of biodiesel were recorded in accordance with

ASTM D 287 standard.
2.1.7 Cloud Point and Pour Point
Cloud point and Pour point were measured by following ASTM D 2700-91 and
ASTM D 97-96, respectively. The cloud point (CP) is the temperature at which wax
gives a cloudy outward show. To determine the Cloud point, samples were cooled
and examined visually until first cloud appeared. Pour point (PP) is the minimum
temperature at which the fuel transforms into semi solid and drops its flow prop-
erties (Giakoumis 2013).
2.2 Oil Extraction
The seeds were dried in sunlight and in an oven at 110 °C to remove any traces of
moisture. The moisture free seeds were dehulled, kernels removed, and grinded
using a standard mixture grinder. Oil was extracted by the Soxhlet extraction
method using petroleum ether (40–60 °C) as solvent (Dobush et al. 1985) [AOAC
963.15., 1976]. The extracted oil has been dried over Na2SO4 (anhydrous) and
filtered for removing solid particles and impurities and stored in volumetric flasks.
2.3 Preparation of Model Oils (Mixed Oil Feedstocks)
Various blends were prepared by mixing Jatropha, Nahor, Yellow oleander,

Karanja (Pongamia pinnata), and Coconut oils in different volumetric proportions,
such as by mixing (Jatropha with Coconut), (Nahor with Coconut), (Yellow ole-
ander with Coconut), (Pongamia with Coconut), (Jatropha with Yellow oleander),
(Nahor with Yellow oleander), (Pongamia with Yellow oleander) (Pongamia with
Jatropha) and (Pongamia with Nahor) oil in volumetric ratios 1:1–1:10. The fatty
acid compositions of these model feedstocks were evaluated and fuel properties of
their corresponding methyl esters were predicted based on empirical relations.
A number of fuel features for instance oxidation stability, iodine number, acid
value, calorific value and kinematic viscosity of biodiesel from the mixed feed-
stocks were determined and compared to predicted values.
2.4 Experimental Methods for Determination of Selected

Biodiesel Fuel Properties
For determining Oxidation stability, Acid value, Iodine value, Kinematic viscosity,
Calorific value, Density, Cloud point, and Pour point and in order to validate our
predicted results (biodiesel fuel parameters of the oils and blends), biodiesel was
prepared in laboratory scale from the pure vegetable oils and selected blends
through a single step conc. H2SO4 catalyzed transesterification process, and their
fuel properties were analyzed according to standard test methods. In a typical
reaction for this purpose 10 g of preheated oil was added to a 100 mL round bottom
flask containing 25 mL MeOH (alcohol) and conc. H2SO4 (2 wt% of oil) and stirred
vigorously at 600 rpm with a magnetic stirrer. The contents in the flask (reaction
mixture) were heated at 100 ± 5 °C for 24 h. As soon as the reaction completed, the
entire contents in the flask was moved to the separating funnel and allowed to settle
for getting two distinct phases. The layer of the upper phase contained biodiesel and
lower phase was having glycerin. The excessive volume of methanol present in the
upper phase of the collected layer was evaporated using rotary vacuum evaporator.
The methyl ester (biodiesel) present in upper phase was washed with distilled water
till neutralizing unutilized H2SO4 and the glycerine was removed. Finally, biodiesel
is dried over anhydrous Na2SO4. Fuel properties like Acid value, Iodine value,
Oxidation stability, Calorific value, Density, Kinematic viscosity, Cloud point, and
Pour point were recorded according to standard methods discussed above and
compared with the predicated values. Calorific values were determined using an
oxygen bomb calorimeter.
3.1 Fatty Acid Profiles/Compositions of Feedstocks
Table 1 shows the fatty acid compositions of single and model oil feedstocks
(created by blending) evaluated using 1H NMR spectroscopy.
Samples prepared were characterized using 1H NMR technique. The 1H NMR
spectra of Coconut oil, Jatropha oil, Nahor oil, Pongamia oil and Coconut and
Jatropha blend (1:1 blending ratio) are shown in Figs. 1, 2, 3, 4, and 5. From Fig. 5,
the effect of blending on the patterns of 1H NMR of the oils was seen. The 1H NMR
of highly saturated coconut oil shows less number of peaks because the unsaturated
fatty acids were not present. The characteristic linolenic acid peak at 0.98–0.96 ppm
was absent and peak intensities of characteristic linoleic and oleic acids appearing
near *2.77 and *2.0 ppm were very low due to the presence of only trace
amounts linoleic acid (0.72 %) and oleic acid (4.8 %). However, when blended with
unsaturated oil like Jatropha, the pattern of 1H NMR spectrum changed drastically,
the peak intensities of unsaturated peaks at 2.77 and 2.0 ppm increased and reached
to a maximum. This means that 1H NMR could be used to detect the changes in
fatty acid compositions of model oils due to blending. In our study we found that as
the percentage of unsaturation was increased, the intensities of peaks at 0.98–0.96,
2.77 and 2.0 ppm were also increased.
From the 1H NMR spectrums of Nahor and Pongamia, we have seen that both
are showing oleic, linoleic and linolenic acids characteristic peaks. However, the
peak area for linoleic acid is little smaller in Nahor than Pongamia oil. This is
because of the presence of more amounts of linoleic acid (22.22 wt%) in Pongamia
than Nahor (13.21 wt%). Also, it is seen that Pongamia is more saturated compared
to Nahor.
Table 1 Fatty acid composition of single and model oil feedstocks

Sl. No. Sample’s name Fatty acid profile/composition (wt%)
Linolenic Linoleic Oleic Total OSI
(C18:3) (C18:2) (C18:1) saturated (h)
1 C (Coconut) 0 0.72 4.8 94.48 22.81
2 J (Jatropha) 0.5 36.99 53.51 9 4
3 K (Karabi) 1.78 23.81 56.66 17.75 6.07
4 N (Nahor) 3.42 13.21 65.47 17.9 6.25
5 P (Pongamia pinnata) 4.51 22.22 51.8 21.47 5.77
6 C+J (1:1) 0 17.23 20.36 62.41 15.71
7 C+J (2:3) 0 20.08 28.42 51.5 13.34
8 C+J (1:2) 0 18.99 26.46 54.55 14
9 C+J (1:3) 0 23.15 33.41 43.44 11.57
10 C+J (1:4) 0 24.29 33.38 42.34 11.32
11 C+J (1:5) 0 24.51 39.99 35.5 9.86
12 C+J (1:8) 0 24.72 39.39 35.9 9.94
13 C+J (1:10) 0 25.88 43.80 30.32 8.73
14 C+N (1:1) 1.81 3.39 26.51 68.29 16.51
15 C+N (1:2) 2.65 4.03 33.29 60.03 15.37
16 C+N (1:3) 3.7 3.22 39.54 53.54 13.98
17 C+N (1:5) 3.94 4.76 44.19 47.13 12.58
18 C+N (1:8) 4.09 5.07 49.08 41.76 11.43
19 C+N (1:10) 4.22 6.32 56.27 33.19 9.59
20 C+K (1:5) 0 17.18 27.54 55.28 14.19
21 C+K (1:10) 0 22.09 40.53 37.38 10.3
22 K+N (1:1) 3.5 15.52 62.67 18.31 13.4
23 K+N (2:3) 3.89 14.07 57.72 24.32 7.57
24 K+N (1:2) 3.68 14.65 62.68 19.01 6.4
25 K+N (1:3) 3.56 14.81 57.61 24.02 7.51
26 K+N (1:8) 5.21 9.53 66.67 18.59 6.41
27 K+J (1:1) 1.29 18.69 64.1 15.92 2.65
28 K+J (2:3) 1.33 30.53 50.66 17.48 5.89
29 K+J (1:2) 0.8 32.32 52.19 14.69 5.28
30 K+J (1:3) 4.9 21.73 56.05 17.32 5.92
31 K+J (2:4) 1.05 29.42 52.19 17.34 5.89
32 C+P (1:4) 2.52 11.3 37.06 49.25 12.96
33 C+P (1:5) 2.41 14.92 33.49 49.18 12.87
34 C+P (1:10) 3.19 19.42 41.8 35.59 9.88
35 P+K (2:1) 3.49 18.51 53.11 24.89 7.62
36 P+J (1:1) 2.51 26.56 51.01 19.92 6.45
37 P+N (6:1) 4.54 20.35 45.84 23.27 7.05
38 P+N+C (2:2:1) 1.32 14.28 38.57 45.83 12.2
The used symbols represent:
J Jatropha (Jatropha curcas); N Nahor (Mesua ferrea Lin); C Coconut (Cocos nucifera); K Karabi
(Thevetia peruviana); and P Karanja (Pongamia pinnata)
1.0 COCONUT OIL C(5)-1.ESP
0.9
0.8
0.7
Normalized Intensity
0.6
0.5
0.4
0.3
0.2
0.1
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
Chemical Shift (ppm)
Fig. 1 1
H NMR spectrum of coconut (Cocos nucifera) oil
1.0 JATROPHA OIL J(4)-3.ESP
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
Fig. 2 1
H NMR spectra of Jatropha (Jatropha curcas) oil
1.0 NAHOR OIL N(3)-1.ESP
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
Fig. 3 1
H NMR of Mesua ferrea (Nahor) oil
1.0 PONGAMIA PINN(KARANJA)-1.ESP
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
Fig. 4 1
H NMR spectra of Karanja (Pongamia pinnata) oil
3.2 Comparison of Experimental and Predicted Properties

of Biodiesel
All the predicted and experimental results of cetane number, calorific value,
kinematic viscosity, iodine number and oxidation stability that we obtained, are
shown in Table 2.
1.26
JATROPHA OIL J(4)-3.ESP
1.0
0.9
0.8
1.30
1.25
COCONUT+JATROPHA ,1,1.ESP
0.7
1.0
0.6
0.9
0.5
0.8
0.4
0.88
0.7
1.26
COCONUT OIL C(5)-1.ESP
0.89
0.3 CH 3COOCH 2-
Jatropha oil -CH-CH-
2.31
0.6
1.0 -CH2CH2CH=CH-
5.33 5.34
5.34
0.86
2.02
2.04
0.2 -CH=CHCH2CH=CH-
2.00
2.06
2.29
5.36
2.33
-CH2 COOR
1.30 1.61
0.91
2.30
0.5
2.77
0.9
4.29
4.28
4.17
4.15
5.37
4.31
4.14
2.76
4.32
7.27
4.12
2.79
0.00
5.32
5.39
5.27
0.00
0.1
0.4
0.8
0.88
0.3
0.7 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
2.31
0.89
0.2
0.86
0.6 Coconut oil +Jatropha oil (1:1) mix
2.33
1.61
2.29
4.29
2.02
5.34
5.34
4.28
4.17
2.04
5.35
5.36
4.32
4.15
2.00
4.31
7.27
4.14
2.77
5.37
0.1
0.5
0.4
0.88
1.28
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
0.3 Chemical Shift (ppm)
Coconut oil
2.31
0.87
0.90
0.2
2.29
1.61
2.33
0.00
4.29
4.28
4.17
4.16
7.27
4.32
4.31
4.14
4.13
7.27
5.27
0.1
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
Fig. 5 Effect of fatty acid composition on 1H NMR Spectrum of oils
And it is found that the predicted values that we calculated are nearly equivalent
to those obtained experimentally. Thus, it is clear that the method we used is the
correct one which can be successfully used for the determination of fuel properties
of biodiesel. It is seen from Table 2 that cetane number, kinematic viscosity and
oxidation stability increase with the increase in saturation. But unlike these prop-
erties, iodine value and density decrease with the increasing degree of saturation.
Calorific value increases with increasing chain length. Here, Coconut FAME is the
most saturated biodiesel which is illustrated by less viscosity and greater cetane
number and also considered as a feedstock that differentiates by a lot from the
others.
Table 3 lists the experimental values of some important fuel properties such as
density, IV, viscosity, calorific value, OSI, cloud point, and pour point of 15
selected samples. It is seen from Table 3 that, highly saturated coconut biodiesel is
having highest oxidation stability >24 h and lowest iodine value and viscosity.
When Coconut is blended with highly unsaturated oil such as Jatropha or Nahor or
Karabi or Pongamia oil, we are getting significant changes in the fuel properties.
Viscosity, iodine value and calorific value were found to be increased with the
addition of unsaturated nonedible oils. Overall, the trends for variation of fuel
properties were found to be similar to the empirical results presented in Table 2.
Additionally, all the characteristics of the biodiesel obtained from the feedstocks
were in accordance to the specifications of ASTM D6751 and EN 14214.
3.3 Effect of Coconut Oil Blending Ratio on Iodine Value

and Viscosity
The effect of blending ratio (percentage of Coconut oil in the blends) on iodine
value and viscosity are described in Fig. 6a–c. From the results we could see that as
the percentage of unsaturated oils in the blends increased (percentage of Coconut
oil in blends decreased) the iodine values also increased and reached its maximum
value when the blends contained no added Coconut oil. In all cases the presence of
10 % (1:10 = C:J/P/N) coconut oil in blends was sufficient to reduce the iodine
values to <100 mg I2/g the minimum limit as per the specifications of EN14111.
Figure 6a–c illustrated the interdependence between iodine number (and vis-
cosity) and degree of unsaturation. Degree of unsaturation is found to show a
relationship with the iodine number and a statistical connection is established. It is
found from the above graph that the iodine value increases with the increasing
unsaturation in the blends, as the compositions in the blends becomes richer in
unsaturated fatty acids (mainly linoleic and/or linolenic acids). An increase in the
concentration of jatropha (or nahor or pongamia) in the blends, which is highly
unsaturated, leads to an increase in degree of unsaturation in the blends. Therefore,
an extremely strong correlation exists between IV and unsaturation degree sub-
stantiating the outcomes of many earlier literature (Sayyed et al. 2013).
Biodiesel is a combination of more than a few fatty esters having each con-
stituents added to the whole value of kinematic viscosity. Hence, the constituents
having smaller viscosity can lessen the total kinematic viscosity of biodiesel to a
satisfactory range albeit larger value of viscosity may present. Double bonds reduce
the kinematic viscosity (Knothe and Steidley 2005). Viscosity of biodiesel enhances
with rising degree of saturation and increment in the number of carbon atoms chain.
It has been observed that long chain saturated esters (C18:0) have higher viscosity
than short chain saturated or long chain unsaturated fatty esters. Because of this
combined effect, we can see that coconut methyl ester is the only biodiesel which
significantly distinguishes, as observed, in the number of carbon atoms in the chain,
obviously demonstrates the least value of viscosity (Giakoumis 2013). In the pre-
sent study, the viscosities of biodiesel obtained from the blended samples were
Table 2 Comparison of predicted and experimental biodiesel fuel properties

Cetane Calorific Kinematic Iodine value OSI (h)
number CN value (KJ/g) viscosity (IV) (mg I2 g−1)
CV (cSt) ν
Sample X X Y X Y X Y X Y
code
J 52.26 39.66 40.49 4.27 5.05 110.95 109.00 4.00 2.0
K 57.03 39.78 39.79 4.33 5.11 94.25 90.00 6.07 5.82
N 60.95 39.91 39.75 4.61 6.17 88.16 72.54 6.25 3.87
C 61.00 37.65 38.44 2.56 1.99 5.354 8.20 22.81 >24
P 57.89 39.76 40.20 4.30 3.12 94.43 84.33 5.77 3.79
C+J (1:1) 56.42 38.08 38.53 3.51 4.35 47.16 43.61 15.71 13.47
C+J (2:3) 60.35 39.32 38.60 2.70 4.64 58.97 46.83 13.34 11.78
C+J (1:2) 61.22 39.35 – 3.97 – 55.43 – 14.00 –
C+J (1:3) 60.39 39.51 – 4.10 – 77.54 – 11.57 –
C+J (1:4) 60.43 39.56 38.73 4.14 4.85 70.48 54.385 11.32 10.6
C+J (1:5) 60.00 39.62 38.88 4.20 4.97 76.52 49.66 9.86 7.98
C+J (1:8) 60.71 39.65 – 4.24 – 76.38 – 9.94 –
C+J (1:10) 59.60 39.69 38.98 4.26 5 82.15 69.52 8.73 6.73
C+N (1:1) 58.64 38.46 – 3.13 – 33.27 – 16.51 –
C+N (1:2) 67.09 39.57 38.42 4.49 4.24 56.33 51.65 15.37 13.77
C+N (1:3) 67.17 39.71 38.53 4.62 4.41 49.07 43.47 13.98 13.01
C+N (1:5) 66.84 39.82 38.58 4.72 4.59 56.33 55.14 12.58 12.5
C+N (1:8) 63.25 39.88 – 4.76 61.45 11.43 –
C+N(1:10) 64.76 39.89 38.79 4.71 6 70.05 60.12 9.59 10.11
K+C (5:1) 57.33 39.51 – 2.7 – 53.21 – 14.19 –
K+C(10:1) 61.13 39.66 – 4.26 – 72.82 – 10.3 –
K+N (1:1) 59.32 39.85 – 4.61 – 89.58 80.34 13.4 –
K+N (2:3) 61.41 39.86 – 4.5 – 83.83 – 7.57 –
K+N (1:2) 60.58 34.79 – 4.5 – 88.54 – 6.4 –
K+N (1:3) 61.75 39.88 – 4.57 – 84.18 – 7.51 –
K+N (1:8) 61.25 39.9 – 4.6 – 87.13 – 6.41 –
K+J (1:1) 57.61 38.73 – 4.26 – 90.52 – 2.65 –
K+J (2:3) 57.01 39.65 – 4.29 – 99.5 – 5.89 –
K+J (1:2) 56.51 39.77 – 4.29 – 102.54 – 5.28 –
K+J (1:3) 58.8 39.77 – 4.31 – 98.23 86.21 5.92 –
K+J (2:4) 57.33 39.77 – 4.3 – 98.19 – 5.89 –
C+P (1:4) 62.66 39.50 38.62 4.17 4.19 57.81 55.506 12.96 10.5
C+P (1:5) 62.24 39.54 38.98 2.63 3.96 60.71 59.36 12.87 9.1
C+P(1:10) 60.18 39.66 – 4.25 – 77.62 – 9.88 6.2
P+K (2:1) 59.49 39.76 – 4.34 – 86.52 – 7.62 –
P+J (1:1) 57.63 39.78 – 4.3 – 96.06 – 6.45 –
P+N (6:1) 55.05 39.55 – 4.28 – 86.21 – 7.05 –
(continued)
Table 2 (continued)
Cetane Calorific Kinematic Iodine value OSI (h)
number CN value (KJ/g) viscosity (IV) (mg I2 g−1)
CV (cSt) ν
Sample X X Y X Y X Y X Y
code
P+N+C 63.98 39.66 38.47 4.4 5.41 61.12 53.01 12.2 8.3
(2:2:1)
The used symbols represent:
X Predicted and Y Experimental
J Jatropha (Jatropha curcas); N Nahor (Mesua ferrea Lin.); C Coconut (Cocos nucifera); K Karabi
(Thevetia peruviana); and P Karanja (Pongamia pinnata)
Table 3 Experimental determination of fuel properties of 15 selected feedstocks

Samples Density Viscosity Calorific Iodine value OSI (h) Cloud point Pour point
name @ 15 °C @ 40 °C value (mgI2g−1) (°C) (°C)
(g/c c) (cSt) (kJ/g)
J 0.878 5.05 40.49 109 2.00 9 −3
K 0.882 5.11 39.79 90 5.82 6 −8
N 0.891 6.17 39.75 72.54 3.87 7 3
C 0.852 1.99 38.44 8.20 >24 4 −6
P 0.884 3.12 40.2 84.33 3.79 15 6
C+J 0.867 4.35 38.53 43.61 13.47 8 5
(1:1)
C+J 0.872 4.34 38.60 46.83 11.78 0 −5
(2:3)
C+J 0.887 4.85 38.73 54.385 10.6 10 −2
(1:4)
C+J 0.891 4.97 38.88 49.66 7.98 9 −3
(1:5)
C+J 0.895 5.0 38.98 69.52 6.73 – –
(1:10)
C+N 0.877 4.24 38.42 51.65 13.77 4 2
(1:2)
C+N 0.889 4.41 38.53 43.466 13.01 9 5
(1:3)
C+N 0.890 4.59 38.58 55.14 12.48 12 8
(1:5)
C+N 0.893 6.0 38.79 60.12 10.11 16 10
(1:10)
C+P 0.810 4.19 38.62 55.506 10.51 12 7
(1:4)
C+P 0.887 3.96 38.98 59.36 9.06 19 9
(1:5)
C+P+N 0.897 5.41 38.47 53.01 8.27 5 1
(1:2:2)
The used symbols represent
J Jatropha (Jatropha curcas); N Nahor (Mesua ferrea Lin.); C Coconut (Cocos nucifera); K Karabi (Thevetia
peruviana); and P Karanja (Pongamia pinnata)
(a) (b)
80
90 Iodine value(mg I2 g−1)
Iodine value (mg I2 g−1) 70
80 Viscosity (cSt)
Viscosity (cSt) 60
70
60 50
50 40
40
30
30
20 20
10 10
0 0
C+J C+J C+J C+J C+J C+J C+J C+J
(1:0) (1:1) (2:3) (1:2) (1:4) (1:5) (1:8) (1:10)
Blending ratio, coconut: jatropha (v/v)

Blending ratio, coconut: nahor (v/v)
(c) 90
Iodine value(mg I2 g−)
80
Viscosity (cSt)
70
60
50
40
30
20
10
0
C+P (1:0) C+P (1:4) C+P (1:5) C+P (1:10)
Blending ratio, coconut: pongamia (v/v)
Fig. 6 a Effect of blending ratio (C:J) on iodine value and viscosity. b Effect of blending ratio (C:
N) on iodine value and viscosity. c Effect of blending ratio (C:P) on iodine value and viscosity
always found to be lower than the parent oil (such as Jatropha curcas, or Mesua
ferrea Lin., Pongamia pinnata or Thevetia peruviana). The viscosities of blends
containing 50 % or more coconut oil were found to significantly lower.
3.4 Effect of Blending Ratio on Calorific Value
Figure 7a–c show a correlation between calorific value and different blending ratio
of Coconut and Jatropha (C+J), Coconut and Nahor (C+N) and Coconut and
Pongamia(C+P). Coconut biodiesel exhibited the lowest calorific value due to the
existence of short chain fatty acid methyl esters (C8:C12) in large amounts. It is
found that, calorific value increases gradually as the proportion of Coconut oil in
the blends were declined. This is due to the fact that, with addition of unsatuarted
oil (such as Jatropha, Nahor and Pongamia) amount of oleic acid (C18:1) in the
blends also increases. Thus, the amount of long chain methyl esters (C18:1 and
C18) in the biodesel obtained from these blends also increases which is responsible
for the increased calorific values.
(a) (b)
40 40.5
39.5 40
Calorific value (KJ/g)

39 39.5
39
38.5
38.5
38
38
37.5
37.5
37 37
36.5 36.5
Blending ratio, Coconut: Jatropha (v/v) Blending ratio , Coconut: Nahor (v/v)
(c) 40
39.5
39
38.5
38
37.5
37
36.5
C+P (1:0) C+P (1:4) C+P (1:5) C+P (1:10)
Blending ratio, coconut: pongamia (v/v)
Fig. 7 a Effect of blending ratio (C:J) on calorific value. b Effect of blending ratio (C:N) on
calorific value. c Effect of blending ratio (C:P) on calorific value
4 Conclusions
In conclusion, we devised an improved fuel quality of biodiesel from four locally

available nonedible vegetable oils namely, Jatropha curcas (Jatropha), Mesua
ferrea Lin. (Nahor), Thevetia peruviana (Karabi/Yellow oleander), Pongamia
pinnata (Karanja) by modifying their fatty acid/ester composition via blending with
Coconut oil, a highly saturated short chain oil. Fatty acid/ester composition were
modified by blending the different nonedible vegetable oils in different volumetric
ratios (1:1–1:10) with Coconut oil. Hence, the mixtures of Jatropha, Nahor, Karabi,
Karanja, and Coconut oil can be used as feedstocks for biodiesel production. It has
been endowed with that by blending different oil feedstocks prepared using this
method can influence magnificent increase in the quality enhancement of the bio-
diesel which is going to be a breakthrough in domain of industry and human
civilization.
References
Azam MM et al (2005) Prospects and potential of fatty acid methyl esters of some non-traditional
seed oils for use as biodiesel in India. Biomass Bioenergy 29:293–302
Dobush et al (1985) The effect of apparatus, extraction time, and solvent type on lipid extractions
of snow geese Can. J Zool 63:1917–1920
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Biodiesel Production
from Moringa oleifera Oil and Its
Characteristics as Fuel in a Diesel Engine
Mohd. Zeeshan, Mohit Vasudeva and A.K. Sarma
Abstract Biodiesel is emerging as a promising source for alternative energy,

owing to the increasing demand for fuel derived from fossil fuel resources. For
developing countries like India, biodiesel can help reduce the dependency on the
fossil fuel being imported. Present study is focused on the production of biodiesel
from a nonedible oil source (Moringa oleifera) along with the engine testing of its
blends as fuel. A single-step transesterification process is carried out for the
extraction of biodiesel from M. oleifera oil with NaOH as catalyst. Properties of
biodiesel extracted are tested and met the ASTM D6751 and EN 14214 standards.
Engine performance and exhaust emission characteristics of 10, 15, 20 (%vol.)
biodiesel blends and diesel are examined on a four-stroke diesel engine at com-
pression ratio of 16. An average increase in brake power of 2.2 % and decrease in
brake thermal efficiency of 3.7 % is observed for biodiesel blends compared to
diesel. Overall increase in brake specific fuel consumption of 4.1 % for biodiesel
blends is observed compared to diesel. On an average carbon monoxide and
hydrocarbon emissions of biodiesel blends decreased by 4.9 and 74.4 %, respec-
tively, compared to diesel. Carbon dioxide and NOx emissions of biodiesel blends
increased by 19.5 and 13.9 %, respectively, on an average compared to diesel.
M. oleifera can be considered as a potential feedstock for biodiesel production and
used as diesel engine fuel without making any engine modifications.

Keywords Moringa oleifera Transesterification Engine performance Exhaust
emissions
Mohd. Zeeshan (&)

Mechanical Engineering Department, Lovely Professional University, Phagwara, India
e-mail: er.mdzeshan@gmail.com
Mohit Vasudeva
Mechanical Engineering Department, Baddi University of Emerging
Sciences and Technology, Baddi, India
A.K. Sarma
R&D-I, Sardar Swaran Singh National Institute of Renewable Energy,
Kapurthala, India

DOI 10.1007/978-81-322-2773-1_11
150 Mohd. Zeeshan et al.
1 Introduction
It is the utmost requirement of the time to develop an alternative to the fossil-based

petroleum fuels as the dependency on fossil fuels is increasing exponentially with
the increase in fossil fuel-based vehicles. The increase in fossil fuel-based vehicles
is also contributing to the increase in atmospheric pollutants. Researchers all over
the world have been working for decades for an alternative source of fuel that can
reduce the overall dependency on petroleum fuels and also help in the reduction of
the harmful engine emissions. Use of biodiesel and ethanol as alternative auto-
motive fuel is quite advantageous (Kalam and Masjuki 2008; Ali et al. 2013;
Giakoumis et al. 2012), as their blends do not pose any engine problem and reduces
the harmful emissions. Use of 5 % ethanol blends with motor spirit is already
deployed in different countries (Knothe et al. 2005). Moreover, biofuel develop-
ment for an alternative and renewable energy source for automobiles has become
important in the national effort for maximum self-reliance.
Moringa oleifera is an indigenous oilseed tree of Sub-Himalayan regions of
northwest India, Africa, Arabia, and Southeast Asia, the Pacific and Caribbean
islands, and South America (Rashid et al. 2008). It is now also distributed in the
Philippines, Cambodia, and Central and North America (Rashid et al. 2008). In
India, cultivation of Moringa is mainly in the southern states of Tamil Nadu,
Karnataka, Kerala, and Andhra Pradesh with Andhra Pradesh leading in production
(Rajangam et al. 2001). M. oleifera has an oil content of nearly 35 % by weight
(Azam et al. 2005). Oil from M. oleifera is obtained by cold pressing of seeds in an
oil expeller machine. The oil of M. oleifera is referred to as “ben oil” due to its
behenic acid contents that gives the oil a specific order of behenic acid, which also
provides resistance to oxidation for oil. Various studies have been conducted so far
to assess the properties of biodiesel derived from various resources such as palm,
pongamia pinnata, jatropha curcas, rice bran, rapeseed/canola, sunflower, safflower,
etc. and their effects on diesel engines when used fully or partially as fuel with
petro-diesel (Mofijur et al. 2014; Kaya et al. 2009; Kalbande et al. 2008; Qiu et al.
2011; Vasudeva et al. 2013; Yuan et al. 2008).
The present study work is focused on extraction of biodiesel from M. oleifera oil
through transesterification process and evaluating the performance and exhaust
emission characteristics of its biodiesel blends with diesel on a stationary
four-stroke diesel engine at a compression ratio of 16.
Crude M. oleifera oil, procured from AOS Products Private Ltd., Ghaziabad is used
as a feedstock for biodiesel/methyl ester extraction. The physicochemical properties
of the oil had been shown in Table 1. All the necessary instruments, chemicals, and
Biodiesel Production from Moringa oleifera … 151
Table 1 Physical and Properties

chemical properties of
Moringa oleifera oil C16:0 Palmitic acid (saturated) (%) 6.5
C18:0 Stearic acid (saturated) (%) 6.0
C18:1 Oleic acid (monosaturated) (%) 72.2
C18:2 Linolic acid (polysaturated) (%) 1.0
C20:0 Arachidic acid (saturated) (%) 4.0
C20:1 Eicosenoic acid (monounsaturated) (%) 2.0
C22:0 Behenic acid (saturated) (%) 7.1
Other fatty acids (%) 1
FFA content (%) 1.66
Density at 15 °C (g/cc) 0.92615
Kinematic viscosity at 40 °C (cSt) 35.2567
Flash point (°C) 153
experimental test setup are generously provided by Sardar Swaran Singh National
Institute of Renewable Energy, Kapurthala, Punjab.
Methyl esters of M. oleifera oil is extracted by a single-step transesterification
process using a Reactor Ready setup RS27 standard. A molar ratio of oil to
methanol (1:6) is used for the carrying out the transesterification reaction in the
presence of NaOH (1 %wt.) as alkali catalyst. Transesterification reaction is carried
out at a temperature of 60° for a time of 2 h with constant stirring at a rate of
500 rpm. After transesterification reaction the mixture is allowed to settle in a
separating funnel. After a period of 20 h glycerol is settled at the bottom of the
separating funnel separating the biodiesel/methyl ester on top of it. It is then washed
with warm distilled water for 3–4 times, then dried in rotary evaporator and filtered
using a filter paper. Properties of the M. oleifera methyl esters (MOME) after
washing and drying are determined and are shown in the Table 2.
Blends of M. oleifera methyl esters extracted are further designated as B10 for
10 % by volume of MOME, B15 for 15 % by volume of MOME and B20 for 20 %
by volume of MOME in the blend. B0 is designated for neat diesel.
The M. oleifera Methyl Ester (MOME obtained after the process was tested for
the percentage conversion of oil to biodiesel in a Gas Chromatography System
7890A, manufactured by Agilent technologies, U.S.A. and the system conforms
Table 2 Properties of M. oleifera methyl esters (MOME)

Property parameters MOME ASTM D6751 EN 14214
Calorific value (MJ/kg) 40.05 – –
Cetane number 56.3 >47 >51
Density (g/cc) 0.87327 – 0.86–0.9
Flash point (°C) 150.5 >130 >120
Fire point (°C) 155.5 – –
Kinematic viscosity (in cSt) 4.941 1.9–6 3.5–5
Table 3 Engine specifications

Engine Make Kirloskar, single cylinder, four-stroke diesel engine
Power rating 3.5 kW
Engine speed 1500 rpm
Cylinder bore 87.50 mm
Stroke length 110.00 mm
Swept volume 661.45 cc
Compression ratio 16
Dynamometer Type eddy current (water cooled) with loading unit
Load indicator Digital supply 230 VAC
Rotameter Engine cooling 40–400 LPH
Software “EnginesoftLV” engine performance analysis software
with ASTM D6584 for testing. Conversion efficiency of biodiesel was measured in
terms of C17:0 and was found to be 68.146 %.
The experimental test setup consists of a computerized Kirloskar-make single
cylinder four-stroke diesel engine connected to an eddy current (water cooled) type
dynamometer for loading. It is equipped with instruments and sensors for mea-
surement of cylinder pressure, crank angle, fuel flow, airflow, temperature, and load
applied. EnginesoftLV software is used for online study of engine performance
parameters such as brake power, brake mean effective pressure, brake thermal
efficiency, specific fuel consumption. Table 3 shows the engine specifications. An
exhaust gas analyzer AVL DiGas 444 manufactured by AVL India Private Ltd., is
used to record the exhaust emission characteristics of the engine during testing
process.
Engine performance and exhaust emission characteristics of a four-stroke diesel

engine are evaluated at different load conditions with B0, B10, B15, and B20 as
fuel.
3.1 Engine Performances Characteristics
Engine performance parameters in terms of brake power, brake thermal efficiency,

and brake specific fuel consumption are evaluated with increase in engine load
conditions.
Figure 1 shows the brake power output of the engine for different blends with
increasing load conditions. Brake power gives the measure of the net power
Fig. 1 Variation of brake power with load
delivered by the engine at the crank shaft measured with the help of a
dynamometer. Results show an overall 3.2 % increase in brake power for B10,
1.9 % increase for B15 and 1.4 % increase for B20 compared to that of diesel.
Figure 2 shows the variation of brake thermal efficiency with load for biodiesel
blends and diesel. Brake thermal efficiency is defined as brake power of an engine
as a function of the thermal input from the fuel and is used to evaluate the con-
version of heat from a fuel to mechanical energy in an engine.
An average decrease of 5.8 % in brake thermal efficiency is observed for B10
compared to diesel. Whereas B15 and B20 have an average decrease of 4.2 and
1.3 % in brake thermal efficiency compared to diesel. Higher brake power along
with lower brake thermal efficiency results in higher specific fuel consumption.
Results showing the variation in brake specific fuel consumption (BSFC) with load
for biodiesel blends and diesel is shown in Fig. 3. Brake specific fuel consumption
is the ratio between mass fuel consumption and brake power. It is a type of com-
parison ratio which looks at an engine’s fuel efficiency in terms of amount of fuel
the engine consumes versus the power it produces. A nominal 0.25 % increase in
BSFC for B10 is observed compared to diesel. Whereas an average increase of 9.7
Brake Thermal Efficiency %
35 B0 B10 B15 B20

30
25
20
15
10
0
15 30 45 60 75
Load %
Fig. 2 Variation of brake thermal efficiency with load

0.9
B0 B10 B15 B20
0.8
0.7
BSFC [kg/kWh]
0.6
0.5
0.4
0.3
0.2
0.1
0
15 30 45 60 75
Load %
Fig. 3 Variation of brake specific fuel consumption with load
and 2.2 % for B15 and B20, respectively, is observed in comparison to diesel.
Higher BSFC and lower brake thermal efficiency are due to the lower energy
content of M. oleifera biodiesel and its blends. The decrease in brake thermal
efficiency can also be attributed to the significantly lower efficiencies of biodiesel
especially at lower compression ratios due to a reduction in the calorific value and
an increase in fuel consumption as compared to B0.
3.2 Exhaust Emissions Characteristics
Exhaust emission characteristics as carbon monoxide (CO), carbon dioxide (CO2),

hydrocarbons (HC), and nitrogen oxide as NO for biodiesel blends and diesel are
evaluated at different load conditions.
0.16
B0 B10 B15 B20
0.14
0.12
CO (% vol.)
0.1
0.08
0.06
0.04
0.02
0
15 30 45 60 75
Load %
Fig. 4 Variation of carbon monoxide emission with load

Figure 4 shows the variation in carbon monoxide emissions of the engine with
load for biodiesel blends and diesel. Results show an overall decrease of 12.1 % in
CO emissions for B10, a nominal 0.4 % increase for B15, and 3.3 % decrease for
B20 compared to diesel. Reduction in CO emissions may be attributed to the higher
oxygen content in biodiesel resulting in better combustion of fuel particles. Also
due to the higher cetane number of M. oleifera biodiesel, the probability of fuel-rich
zones formation decreases, which results in lower CO emissions of biodiesel
blends.
Figure 5 shows the variation of the hydrocarbon emissions of engine with load
for different biodiesel blends and diesel. Unburnt hydrocarbons in emissions are due
to incomplete or improper combustion of fuel in the engine. Results show an overall
decrease in HC emissions of 69.7, 76.8, and 76.7 % for B10, B15, and B20,
respectively, compared to that of diesel. Improved combustion of biodiesel blends
due to the presence of excess oxygen content can be attributed to the reduction in its
hydrocarbon emission. Variation in carbon dioxide emissions per unit brake power
with load for biodiesel blends and diesel is shown in Fig. 6. An increase in CO2
emissions for biodiesel blends is observed. CO2 emissions pose a threat to the
environment as it a major constituent for the cause of global warming. But as
products of combustion it is more acceptable compared to CO which is a result of
incomplete combustion. Results show an overall increase of 16.5 and 39 % in CO2
emissions per unit brake power for B15 and B20, respectively, compared to diesel.
A higher CO2 emission for biodiesel blends is a result of complete combustion of
fuel. Figure 7 shows the variation of emissions of nitrogen oxide as from the engine
with load for different biodiesel blends and diesel. Oxide of Nitrogen is highly
dependent on the temperature and pressures inside the combustion chamber.
Exhaust NO for biodiesel blends increased as compared to that of diesel for all
range of loading conditions. There was 5.6 % increase in NO emission for B10,
12.5 % increase for B15, and 23.8 % increase for B20 compared to that of diesel.
140
B0 B10 B15 B20
120
100
HC [ppm]
80
60
40
20
0
15 30 45 60 75
Load %
Fig. 5 Variation of hydrocarbon emission with load

CO2 / Brake Power [%/kW] B0 B10 B15 B20

3
2.5
1.5
0.5
0
15 30 45 60 75
Load %
Fig. 6 Variation in carbon dioxide emissions per unit brake power with load
250
B0 B10 B15 B20
200
NO [ppm]
150
100
50
0
15 30 45 60 75
Load %
Fig. 7 Variation in emissions as nitrogen oxides as NO with load
4 Conclusions
Following conclusions are drawn from the study:

• In the present study, M. oleifera oil shows the potential to be a feedstock for
biodiesel production and the performance of blends up to B20 are closely
comparable with that of diesel.
• An average increase of 2.2 % in brake power of engine is observed compared to
diesel. In addition, average decrease in brake thermal efficiency of 3.7 % is
observed for biodiesel blends with an overall increase in brake specific fuel
consumption of 4.1 % compared to diesel.
• The use of the biodiesel blends shows effective reduction of 4.9 % in CO
emissions and 74.4 % reduction in HC emissions on an average for different
loading conditions.
• As a result of improved combustion of biodiesel blends and higher temperature

inside the combustion chamber, an increase of 19.5 and 13.9 % is observed for
CO2 and NO emission.
Blends of biodiesel/methyl esters extracted from M. oleifera oil can be used as
fuel in diesel engine without making any engine modification.
Acknowledgments The authors would like to acknowledge the support of Sardar Swaran Singh
National Institute for Renewable Energy for providing resources to conduct the research work. The
authors would also like to thank Mr. Balwant Singh and Ms. Neetu Singh for their technical
assistance in carrying out the research work.
References
Ali MH, Mashud M, Rubel MR, Ahmad RH (2013) Biodiesel from Neem oil as an alternative fuel
for Diesel engine. Procedia Eng 56:625–630
Azam MM, Waris A, Nahar NM (2005) Prospects and potential of fatty acid ethyl esters of some
non-traditional seed oils for use as biodiesel in India. Biomass Bioenergy 29:293–302
Giakoumis EG, Rakopoulos CD, Dimaratos AM, Rakopoulos DC (2012) Exhaust emissions of
diesel engines operating under transient conditions with biodiesel fuel blends. Prog Energy
Combust Sci 38:691–715
Kalam MA, Masjuki HH (2008) Testing palm biodiesel and NPAA additives to control NOx and
CO while improving efficiency in diesel engines. Biomass Bioenergy 32:1116–1122
Kalbande SR, More GR, Nadre RG (2008) Biodiesel production from non-edible oils of Jatropha
and Karaja for utilization in Electric Generator. Bioenerg Res 1:170–178
Kaya C, Hamamci C, Baysal A, Akba O, Erdogan S, Saydut A (2009) Methyl ester of peanut
(Arachis hypogea L.) seed oil as a potential feedstock for biodiesel production. Renew Energy
34:1257–1260
Knothe G, Gerpen JV, Krahl J (2005) The biodiesel handbook. AOCS Press Champaign, Illinois,
USA
Mofijur M, Masjuki HH, Kalam MA, Atabani AE, Fattah IMR, Mobarak HM (2014) Comparative
evaluation of performance and emission characteristics of M. oleifera and Palm oil based
biodiesel in a diesel engine. Ind Crops Prod 53:78–84
Qiu F, Li Y, Yang D, Li X, Sun P (2011) Biodiesel production from mixed soybean oil and
rapeseed oil. Appl Energy 88:2050–2055
Rajangam J, Azahakia Manavalan RS, Thangaraj T, Vijayakumar A, Muthukrishan N (2001)
Status of production and utilisation of moringa in southern India. Development potential for
Moringa products, Dar es Salaam, Tanzania
Rashid U, Anwar F, Moser BR, Knothe G (2008) Moringa oleifera oil: a possible source of
biodiesel. Bioresour Technol 99:8175–8179
Vasudeva M, Sharma S, Mohapatra SK, Kundu K (2013) Optimizing the compression ratio of
compression ignition engine fuelled with esters of crude rice bran oil. Int J Theor Appl Res
Mech Eng 2(3):131–134
Yuan X, Liu J, Zeng G, Shi J, Tong J, Huang G (2008) Optimization of conversion of waste
rapeseed oil with high FFA to biodiesel using response surface methodology. Renew Energy
33:1678–1684
Growth and Lipid Production
from Scenedesmus sp. Under Mixotrophic
Condition for Bioenergy Application
Monika Prakash Rai and Shivani Gupta
Abstract Microalgae are seen as an alternative and renewable feedstock for

biodiesel production. They are effective in producing large amount of oil which can
be converted into biodiesel by the process of transesterification. The present work
aims to cultivate microalgae under optimized culture condition and lipid was
extracted for the production of fatty acid methyl esters (FAMEs). The biomass and
lipid content of the species was tried to enhance under photoheterotrophic (mixo-
trophic) condition by adding glycerol as the organic carbon source. Glycerol is the
by-product in transesterification reaction for biodiesel production, its utilization in
algae cultivation will support the biorefinery system. The green microalgae,
Scenedesmus sp. was cultured in Fogg’s medium and culture conditions were
optimized for best growth. The algae were allowed to grow mixotrophically with
glycerol at concentrations ranging from 0–5 % (v/v) and significant improvement
was noticed in growth and lipid accumulation. The growth of the Scenedesmus
sp. was increased up to 1 % glycerol (v/v) and recorded 0.414 g l−1 that is
approximately 2 times in comparison with photoautotrophic cultivation
(0.223 g l−1). The lipid accumulation of 36.47 % was estimated in cultures with 1 %
glycerol which is, nearly, 3 times that of autotrophic culture (12.55 %). Maximum
lipid accumulation (52.32 %) was achieved under mixotrophic cultivation using
5 % glycerol, but growth is inhibited at very high concentration. Oil from
Scenedesmus sp. grown under 1 % glycerol was subjected to acid-based transes-
terification for its proposed application in biofuel. The lipid enhancement in
Scenedesmus sp. in the presence of glycerol provides a potential route for economic
biodiesel production.
Keywords Scenedesmus sp. Mixotrophic condition Glycerol FAME
M.P. Rai (&) Shivani Gupta

Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125,
Gautam Budh Nagar, Noida, India
e-mail: mprai@amity.edu; monika1778@gmail.com

DOI 10.1007/978-81-322-2773-1_12
160 M.P. Rai and Shivani Gupta
1 Introduction
Energy is the prerequisite for survival, sustainability, and growth of any country, its
economy and population. Increasing industrialization creates a burden on the fossil
fuels as they are limited owing to their nonrenewable nature. This has created
various issues of global concern like increase in crude oil prices and global
warming due to burning of fossil fuels in different forms, liquid (oil) and gas
(Malcata 2011). This motivates researchers across the globe to quest for alternative
sources of energy which are renewable and grown on industrial scale to meet
energy demands (Chisti 2007a). Biodiesel, as word signifies, is a diesel from a
biological entity that replaces conventional diesel and has gained enormous pop-
ularity in recent years (Miao and Wu 2006). Among all candidates, viz., plant
sources, oil from animal tissues, and algae, microalgae have been proved to be the
most efficient one (Chisti 2007a). They have many advantages as compared to other
feedstock like less land requirement and per land usage productivity is more, can
make use of arable land, ease of culture and harvest, can feed on waste water,
majority of industrial effluent, flue gas, and mitigate extra atmospheric CO2 to keep
up the healthy balance (Li et al. 2014; Smith et al. 2009; Liu et al. 2008; Chisti
2007b). The main challenge in algae biofuel production is to increase cellular lipid
accumulation to make this feedstock more economically viable and cost-effective
(Bhatnagar et al. 2011). Many researchers have tried different approaches to
enhance lipid accumulation like applying variety of culture conditions, CO2 bub-
bling in the culture growing photoautotrophically to enhance biomass (Mata et al.
2010), photoperiods (Perez and Izquierdo 2011), and light and salt stress in various
strains (Mohan and Devi 2014). According to Marquez et al. (1993), photo-
heterotrophic growth rate is a combination of autotrophic and heterotrophic growth
rates. There have been reports in which some microalgae yield 3–10 times higher
biomass when grown mixotrophically (Li et al. 2014; Bhatnagar et al. 2011).
Scenedesmus sp. is known for its high growth rate and lipid content; it can
achieve higher lipid accumulation under mixotrophic condition using glycerol,
glucose, acetate, and others (Andruleviciute et al. 2014; Dittamart et al. 2014;
Makereviciene et al. 2012). The lipid content of Scenedesmus quadricauda showed
excellent enhancement when grown mixotrophically (33.1 % dry cell weight)
compared to grown photoautotrophically and heterotrophically (14–28 %) (Zhao
et al. 2012). The use of glycerol increases the usage of resources in the algal oil
process more as it is the by-product of transesterification process; so recycling
makes process more economically feasible. The present study aims to analyze the
most effective concentration of glycerol to obtain high biomass and increased lipid
accumulation in Scenedesmus sp. for biodiesel application.
Growth and Lipid Production from Scenedesmus sp. … 161
2.1 Culture Maintenance
The axenic culture of Scenedesmus sp. was maintained in a batch mode in a 1 l

Erlenmeyer flask by taking 300 ml of Fogg’s media including 0.2 g MgSO4 · 7H2O,
0.1 g CaCl2 · 2H2O, 0.2 g K2HPO4, and 0.0745 g Na2EDTA and trace elements
2.86 mg H3BO3, 1.81 mg MnCl2 · 4H2O, 0.222 mg ZnSO4 · 7H2O, 0.39 mg Na2MoO4
· 2H2O, 0.0494 mg Co(NO3)2 · 6H2O, 0.0557 mg FeSO4 · 7H2O, and 0.079 mg
CuSO4 · 5H2O in 1000 ml distilled water (Nigam et al. 2011). The microalgae were
cultured in a temperature controlled incubator at 25 °C by providing 24 h continuous
illumination (40 W, white tube light) of 3000 lx light intensity with intermittent
shaking at 120 rpm. Exponentially growing cells were inoculated by keeping the
optical density 0.1 O.D. This was referred to as autotrophic control culture.
2.2 Mixotrophic Culture
The Scenedesmus sp. was grown in Fogg’s media supplemented with different
concentrations of technical glycerol, viz., 0, 0.5, 1, 3, and 5 % (v/v). Other media
components were kept the same as in autotrophic cultures.
2.3 Growth Curve Analysis
2.3.1 Determination of Dry Weight
The optical density of the Scenedesmus cultures was measured spectrophotomet-

rically (UV/Vis Shimadzu) at 660 nm after every 24 h for both autotrophic and
photoheterotrophic cultures. After stationary phase, photoheterotrophic cultures
with different concentrations of glycerol were harvested by centrifugation
(Eppendorf 5810R) at 4000 rpm for 15 min. The pellet was then washed twice with
double distilled water and dried in oven at 105 °C to obtain dry cell mass.
2.3.2 Determination of Chlorophyll Content
Chlorophyll content of the microalgae was measured at alternate days to estimate

cell growth. 10 ml of homogenized algal suspension was taken and centrifuged at
4000 rpm for 10 min. Chlorophyll was extracted by using the same volume of
methanol (95 %) in a water bath at 60 °C for 30 min. Centrifugation was performed
to discard the pellets and the absorbance of supernatant was taken at 650 and
665 nm against 95 % methanol in blank (Mackinney 1941).
Chlorophyll contentðlgnmlÞ ¼ f2:55 102 OD ð650 nmÞ

þ 0:4 102 OD ð665 nmÞg 103
2.4 Lipid Estimation
The total lipid was extracted by mixing chloroform: methanol (2:1 v/v) with dried
algal samples using Bligh and Dyer method (Bligh and Dyer 1959).
Lipid accumulationð% DCWÞ ¼ lipid production ðgl1 Þ=

ðbiomass production ðgl1 Þ 100
2.5 Production of FAME by Transesterification
The extracted lipid was transesterified by using methanol and a mixture of acidic
catalyst (Rai et al. 2013). The method was used for transesterification of dried lipid
extract to obtain the fatty acid methyl esters (FAME). The FAME thus produced
was preserved for further analysis.
2.6 Statistical Analysis
All the experiments were performed in triplicates for better validation. Also they
were repeated in lieu to ensure reproducibility of the results and the average value
has been recorded and represented in the results.
3 Result and Discussion
The present work was aimed to explore the efficacy of microalgae Scenedesmus
sp. for biodiesel production. The work was focused to assess the maximum lipid as
well as biomass production of alga under different culture conditions and its effi-
cient conversion to FAME. Apart from lipid, the amount of producible biomass is
one of the major factors to determine the potentiality of algae for bioenergy
feedstock, and it is necessary to provide growth conditions which favor high bio-
mass content (Mandotra et al. 2014). Cellular growth and lipid productivity of
Scenedesmus sp. were investigated under various conditions. Biomass and oil
production were evaluated under photoautotrophic and photoheterotrophic condi-
tions using Fogg’s medium and organic carbon glycerol.
Fig. 1 Growth curve of 8 0.5

Scenedesmus sp. in Fogg’s Chlorophyll Content
Chlorophyll Content (µg/ml)

0.45
Biomass Production (g /l)

7
medium (by estimating dry Biomass Production
0.4
cell weight and chlorophyll 6 (g/l)
0.35
content)
5 0.3
4 0.25
3 0.2
0.15
2
0.1
1 0.05
0 0
0 10 0 200 300 400
Time (h)
3.1 Growth Curve Analysis
The microalgae were grown under photoautotrophic condition using normal Fogg’s
medium including major macro- and microelements (Nigam et al. 2011). The
Scenedesmus sp. growth pattern in Fogg’s medium is shown in Fig. 1. During lag
phase (up to 72 h) the growth of microalgae is very little and microorganisms
adapted to growth conditions. Cellular growth between fourth and sixth days (i.e.,
by 144 h) increases sharply that strongly express the exponential phase. The
maximum concentration was achieved after 264 h (beginning of stationary phase)
and reached 0.23 g l−1. The growth curve plotted by taking chlorophyll content of
algae is showing parity with the curve plotted by taking dry cell weight (Fig. 1).
3.2 Effect of Glycerol on Algae Growth
To determine the effect of organic carbon source on the growth of Scenedesmus sp.,
glycerol was added at different concentrations 0, 0.5, 1.0, 3.0, and 5.0 % (v/v). The
growth curve was plotted in mixotrophic condition at various concentrations of
glycerol as given in Fig. 2. During the first six days (up to 144 h), the growth of
algae in various concentration of glycerol was very similar (Fig. 2). The fastest
growth was obtained during the exponential phase in the culture containing 1 %
(v/v) glycerol. The highest concentration obtained after stationary phase (after
288 h) of the media containing 1 % volume fraction of glycerol was 0.42 g l−1. This
concentration is almost 2 times higher than the cells obtained in photoautotrophic
control, which was 0.23 g l−1. Results show that, under photoheterotrophic con-
ditions (using glycerol), growth of Scenedesmus sp. is much better compared to
autotrophic control. In mixotrophic cultivation, the microalgae could obtain energy
from both photosynthesis and oxidation of organic carbon compounds. Thus, they
could utilize some part of the energy for cell division, but the excess energy was
Fig. 2 Effect of 0.5

concentration of glycerol (0, 0% Glycerol
0.45
Biomass Production (g/l)

0.5, 1.0, 3.0, and 5.0 %) on 0.4
0.5% Glycerol
growth of Scenedesmus sp. 1% Glycerol
0.35
0.3 3% Glycerol
0.25 5% Glycerol
0.2
0.15
0.1
0.05
0
0 100 200 300 400
Time (h)
stored in the form of lipid granules. Similar kind of results was also obtained by
researchers using glycerol to increase the growth of Scenedesmus
sp. Makarevičienė et al. (2012) have achieved 1.3 times higher growth of alga under
mixotrophic condition using 5 g l−1 technical glycerol compared to autotrophic
growth.
Microalgae growth was not obtained well in all concentrations of glycerol. The
algal growth was lower in the growth medium containing more than 1 % volume
fraction of the glycerol. High concentration of glycerol, i.e., 3 and 5 % showed an
inhibitory effect on biomass production. In our previous research, Chlorella
pyrenoidosa showed best growth at 0.5 % (v/v) glycerol and was inhibited above
this concentration (Rai et al. 2013). Various microalgae have different levels of
inhibitory concentration of carbon. After a certain concentration, growth decreases
with increasing carbon concentration. Impurities such as free fatty acids, residue of
catalyst, soap, etc., could be the possible reason for growth inhibition (Liang et al.
2010).
3.3 Effect of Glycerol Concentration on Lipid Content

of Scenedesmus sp.
The lipid content obtained under photoautotrophic and with glycerol under pho-
toheterotrophic conditions was estimated. Accumulation of lipid in microalgae cells
is improved by applying photoheterotrophic conditions (in the presence of glyc-
erol). The cellular lipid content increases by increasing the amount of glycerol as
given in Fig. 3. Under photoheterotrophic cultivation with the use of glycerol,
microalgae have increased lipid content approximately 3 times, i.e., from 12.55 to
36.47 % along with double increase in biomass concentration (g l−1) as compared to
photoautotrophic condition. The lipid content of 36.47 % is achieved in the growth
medium containing 1 % (v/v) glycerol, the lowest lipid concentration (13.02 %) is
Fig. 3 Effect of glycerol 600

concentration (0, 0.5, 1.0, 3.0, Biomass Production (mg/l)
and 5.0 %) on biomass 500 Lipid production (mg/l)
production (mg l−1), lipid Lipid accumulation (% biomass)
production (mg l−1), and lipid
accumulation (% biomass) of 400
Scenedesmus sp.
300
200
100
0
Glycerol Glycerol Glycerol Glycerol Glycerol
0% (v/v) 0.5% (v/v) 1% (v/v) 3% (v/v) 5% (v/v)
estimated in the growth medium containing 0.5 % (v/v) glycerol under photo-
heterotrophic cultivation.
The lipid content (% biomass) of microalgae in 3 and 5 % (v/v) glycerol is
higher than the content in 1.0 % glycerol, as the lipid accumulation is higher
through carbon dependent metabolic pathways; but showed very poor effect on
biomass concentration (Fig. 3). Hence, the overall productivity will be the highest at
1 % volume fraction of glycerol. Some early results also showed compatibility of
mixotrophic culture condition using glycerol for lipid enhancement in microalgae
like Chlorella sp., Scenedesmus sp., Chodatella sp., etc., (Rai et al. 2013;
Makarevičienė et al. 2012; Li et al. 2014). Crude glycerol is proved to be an
effective carbon source to enhance growth and cellular lipid accumulation in algae
and also it provides an effective way for disposal of surplus glycerol in the pro-
duction of biodiesel.
3.4 Transesterification of Algal Oil
The algae were cultivated in 5 l Erlenmeyer flask and 30.127 g wet biomass was
collected and dried to produce 4.845 g dry cells. Lipid was extracted by means of
solvent extraction using chloroform and methanol (2:1) and 1.406 g of lipid pro-
duced from Scenedesmus sp. was converted to fatty acid methyl esters (FAME) by
the method of transesterification. The total yield of fatty acid from 1.406 g cells was
1.052 g that shows more than 75 % recovery (Table 1).
Table 1 Production of FAME from Scenedesmus sp. by transesterification

Wet weight (g) Biomass production (g) Lipid production (g) FAME (g)
30.127 4.845 1.406 1.052
4 Conclusion
Growth and lipid production of freshwater green microalgae Scenedesmus sp. was
investigated using Fogg’s medium for photoautotrophic and glycerol for photo-
heterotrophic cultivation. Utilization of glycerol in the cultivation of microalgae,
that is a by-product of biodiesel industry will help not only in the removal of waste
of the environment, but also makes the biofuel production sustainable. Our results
show that the maximum biomass production is obtained under photoheterotrophic
conditions by adding 1 % by volume fraction of glycerol into the Fogg’s medium.
The presence of organic carbon source (glycerol) in the growth medium increases
the growth of microalgae but the growth is inhibited at very high concentration of
glycerol. Cultivation of Scenedesmus sp. under photoheterotrophic condition not
only shows better growth but also higher lipid accumulation. The highest lipid
accumulation was achieved in the medium containing 5 % glycerol but growth
decreases. Hence, the cells grown under 1 % volume fraction (v/v) of glycerol
showed highest overall productivity in terms of both biomass and lipid and the
process could be utilized for the formation of fatty acid methyl esters. The FAME
produced in the present research will be characterized thoroughly for its bioenergy
applications.
Acknowledgments The present work is financially supported by a project grant (Ref.No.

DST/TSG/AF/2009/101) from Department of Science and Technology, Govt. of India, New Delhi.
Authors are grateful for the financial support.
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Experimental Evaluation of Combustion
Parameters of a DI Diesel Engine
Operating with Biodiesel Blend at Varying
Injection Timings
Abhishek Sharma and S. Murugan
Abstract There is a limited reserve of the petroleum fuels and the world is already
facing acute energy crisis. Besides this, the petroleum fuels are the dominant global
sources of carbon dioxide emission. This research work provides an overview on
the usage of a blend of jatropha methyl ester (JME) with oil obtained by the
pyrolysis of waste tyre in diesel engine while varying the injection timing (IT) in
order to improve the combustion parameters of the engine. Experiments were
conducted in a single cylinder diesel engine at the advanced (24.5 °CA bTDC),
standard (23 °CA bTDC), and retarded IT (21.5 °CA bTDC) for the blend and
results were compared with diesel fuel and also with JME operation. The com-
bustion parameters of the diesel engine were calculated in terms of cylinder pres-
sure history, cumulative heat release, maximum heat release rate, maximum
cylinder pressure, and mass fraction burned. For the blend with advancement of IT,
the maximum cylinder pressure and cumulative heat release rate are enhanced.
Keywords Biodiesel Combustion Diesel engine Injection timing Tyre

pyrolysis oil
1 Introduction
Conventional petroleum fuels for example diesel and gasoline are the principal
source of energy for the development of any country and their demand is expanding
with accelerating rate due to increasing industrialization, overwhelming rise in the
automobiles, and growing population. Speedy boom of fuel costs, lack of fossil fuels,
and reducing fossil fuel reserves have forced to search for their substitutes which
Abhishek Sharma (&) S. Murugan

Internal Combustion Engines Laboratory, Mechanical Engineering Department,
National Institute of Technology Rourkela, Rourkela 769008, India
e-mail: drasharma58@gmail.com

DOI 10.1007/978-81-322-2773-1_13
170 Abhishek Sharma and S. Murugan
might meet the constantly growing claim of the energy. Also, the petroleum fuels are
the topmost sponsors to pollute air and main contributors of greenhouse gases, and
are reflected to be the leading reason behind the climate change. Automobiles sig-
nificantly contaminate the environment by emissions of pollutants like carbon
monoxide (CO), carbon dioxide (CO2), nitric oxides (NOx), sulfur dioxide (SO2),
unburnt or partially burnt hydrocarbons, and particulates (Sher 1998). These cir-
cumstances have forced researchers and expertise to look for alternatives to fossil
fuels that would preferably be economical and environmental friendly.
Biodiesel has gained a promising extender for diesel and is known as the methyl
or ethyl esters of fatty acid prepared from renewable sources such as virgin and used
vegetable oils (edible and non-edible) and animal fats (Knothe 2005). It is con-
sidered as clean burning and renewable fuel, and various advantages of biodiesel
are nontoxic, biodegradable, which can mitigate exhaust emissions of a diesel
engine. Engine fueled with biodiesel results in considerable decrease of hydro-
carbons (HC), CO, and particulate matters. It is considered to have almost no sulfur,
no aromatics and improves the combustion quality of the fuel due to their high
cetane number. As a renewable source of supply, biodiesel imparts a promising
influence on the surroundings, and it can replace the diesel fuel in compression
ignition (CI) engines. Extensive consumption of biodiesel will cut back the relay on
fossil fuels, which is presently the key source of energy for the world. For countries
without or restricted petroleum reserves, the usage of biodiesel will cut back
imports of fossil fuels, thus resulting in substantial foreign exchange savings. In
India, the biodiesel production from renewable sources such as edible and
nonedible vegetable oil has been established in the last three decades. Use of edible
oils like soybean, sunflower oil, and coconut oil to produce biodiesel is not practical
as these oils are being consumed as food in India and there is an enormous gap in
their demand and supply in the country. Several researchers in India have tried
different non-edible feed stocks such as Jatropha curcas (Sharma and Murugan
2014, 2015b), Pongamia pinnata (Srivastava and Verma 2008), Madhuca indica
(Ghadge and Raheman 2005), Linseed caster, jojoba, etc., for the production of
biodiesel. Thus in Indian conditions, solely non-edible oil seed plants can be
considered for the biodiesel production.
Several studies have been carried out to investigate the potential application of
tyre pyrolysis oil (TPO) as a replacement of base fuel in both petrol engines and
diesel engines. The TPO shows longer ignition delay compared to diesel fuel owing
to higher viscosity of the test fuel (Murugan et al. 2008). A Kirloskar diesel engine
was used to examine the influence of an ignition improver (Di ethyl ether) when it
was admitted at three different flow rates (65,130 and 170 g/h) (Hariharan et al.
2012). Beside this, ignition delay of TPO can be upgraded by blending it with high
cetane fuels.
In an earlier investigation, reported by the authors, experiments were conducted
in a diesel engine with different blends of jatropha methyl ester (JME) and TPO.
Experimental Evaluation of Combustion Parameters … 171
The TPO was blended with JME in the blend from 10 to 50 % in interval of 10 %
on a volume basis. Experimental investigation revealed that, the combustion
parameters of the engine fueled with JMETPO20 (80 % JME + 20 % TPO) blend
were better than other blends considered in that study but poorer than that of diesel
operation (Sharma and Murugan 2013). The inferior combustion behavior of the
blend was achieved due to the relatively higher density and poor volatility of the
blend. Injection timing (IT) plays a vibrant role on the behavior of a CI engine, as
the pressure and temperature change significantly when piston approaches near top
dead center. In the recent past, research works have been documented study of the
influence of IT on the behavior of a CI engine running with different alternative
fuels (Sayin and Gumus 2011; Xu et al. 2014; Sayin and Canakci 2009). This test is
carried out to estimate the influence of IT on the combustion characteristics of a CI
engine running on the optimum blend, i.e., JMETPO20.
For this experiment, the JME was collected from a commercial biodiesel plant in
Raipur, India. The TPO was collected from a pilot pyrolysis plant located at
Rourkela, India. The TPO was blended with the JME on a 20/80 % volume basis
and the blend was kept under observation for 30 days, to ensure its stability. The
details of preparation of JME and TPO have been described by the authors else-
where (Sharma and Dhakal 2013). It was noticed that the TPO was not separated
from the JME in the blend. Table 1 gives properties of diesel, JME, TPO, and the
JMETPO20 blend.
Table 1 Properties of diesel, JME, TPO, and the JMETPO20 blend

Properties ASTM test method Diesel JME TPO JMETPO20
Specific gravity D 4052 0.830 0.881 0.913 0.887
Viscosity (cSt) D 445 2.6 5.6 3.35 5.2
Heating value (MJ/kg) D 4809 43.8 39.4 38.1 38.82
Flash point (°C) D 93 50 156 49 132
Fire point (°C) D 93 56 171 58 145
Cetane number D 613 50 55 28 52
Carbon (%) D 3178 86.2 77.1 86.92 79.26
Hydrogen (%) D 3178 13.2 11.81 10.46 11.31
Nitrogen (%) D 3179 Nil 0.119 0.65 0.23
Sulfur (%) D 3177 0.3 0.001 0.95 0.18
Oxygen by difference (%) E 385 Nil 10.97 1.02 9.02
3 Experimental Section
The combustion tests were carried out in a Kirloskar make single cylinder, four
stroke, direct injection, diesel engine, with a rated power of 4.4 kW. The layout of
the test setup is shown in Fig. 1. The specifications of the test are given Table 2.
It was observed during the stability test that, the TPO is miscible with the JME
and also found that the blend was stable during the test. First experiment was started
with base fuel diesel and after that it was moved over to JME, and then to the blend
at the standard IT of 23 °CA bTDC for obtaining the base data. Further, the tests
were done at varied IT for the blend. The standard IT was advanced and retarded by
adjusting the number of shims fitted under the plunger in the pump. Addition or
removal of a single shim of thickness 0.25 mm changes the IT by about 1.5 °CA.
The experiments were carried out using the blend at three different injection tim-
ings, of 24.5, 23, and 21.5 °CA bTDC, respectively. Three shims were used in the
fuel pump while engine runs at standard IT of 23 °CA bTDC.
The study was carried out with 1.5 °CA advanced and retarded IT for the blend,
and the findings were compared with reference fuel diesel and JME. The Kistler
type piezoelectric pressure sensor was attached on the cylinder head which mea-
sures the cylinder pressure of the engine. A top dead center encoder was used to
detect the crank angle (CA). The engine setup was attached with a control panel,
which is connected with the pressure sensor, and to convert the signal from the
pressure sensor to the analog voltage signal, which was fed to the computerized
data acquisition system (DAS).
Valve
Exhaust temperature
Fuel tank indicator
U-Tube Smoke meter

manometer
Air box
Pressure transducer
Exhaust gas analyser
Computer
Crank position
Data acquisition system sensor
Load
bank
Dynamometer
Fig. 1 Schematic diagram of the test setup

Table 2 Test engine Model Kirloskar TAF 1

specifications
Engine type Single cylinder, four stroke, constant
speed, direct injection diesel engine
Rated power 4.4 kW
Speed 1500 rpm (constant)
Bore 87.5 mm
Stroke 110 mm
Displacement 661 cm3
volume
Compression ratio 17.5
Nozzle opening 200 bar
pressure (bar)
Start of fuel 23 °CA bTDC
injection
Dynamometer Eddy current
Injection type 3-Hole pump-line-nozzle injection
system
4.1 Cylinder Pressure History
Figure 2 compares the cylinder pressure variation at full load with crank angle for
diesel, JME, and the JMETPO20 blend and at different injection timings. The
highest cylinder pressure for diesel, JME, and blend, are approximately 80 bar at
371.6 °CA, 81 bar at 370.3 °CA, and 80 bar at 371 °CA, respectively, with the
standard IT.
At full load and standard IT, the start of combustion process occurs by about
348.51, 347.15, and 348.36 °CA in the case of base fuel diesel, JME, and blend,
respectively. For the blend with advancement and retardation of IT by 1.5 °CA,
Fig. 2 Variation of the 100

cylinder pressure with crank 90
angle at different injection 80
timings 70
Pressure (bar)
60
50
40
Diesel
30
JME
20 24.5 CA bTDC (JMETPO20)
23 CA bTDC (JMETPO20)
0
330 340 350 360 370 380 390 400
Crank Angle (degree)
the start of combustion occurs at about 346.21 and 356.14 °CA, respectively. It can
be observed for blend that at the advanced IT and full load, combustion starts earlier
and causes increased cylinder pressure in comparison with standard IT. This is a
result of faster burn at premixed combustion because of longer ignition delay
(Ganapathy et al. 2011).
4.2 Cumulative Heat Release (CHR)
The CHR rate pattern at full load, with respect to the crank angle for diesel, JME,
and JMETPO20 blend at different injection timings is shown in Fig. 3. The rate of
heat release and CHR is determined to evaluate the rate of pressure rise in the
cylinder and combustion characteristics for different operating conditions. The CHR
value is higher for diesel compared to the JME and blend owing to higher calorific
value of diesel.
The CHR values of 2210, 2124, and 2018 kJ are obtained for the blend at full load
and injection timings of 24.5, 23, and 21.5 °CA bTDC, respectively. The maximum
value of the CHR for blend is attained at CA of 400° and an IT of 1.5 °CA
advancement, because mixing process of fuel with the air is superior due to the
availability of more time with advanced IT.
4.3 Maximum Heat Release Rate (MHRR)
Figure 4 demonstrates the variation of the MHRR for base fuel diesel, JME, and
JMETPO20 blend with brake power at different injection timings. It can be
observed that with the growth in the brake power, the MHRR rises throughout the
combustion period for all the test fuels in this investigation. The MHRR values for
diesel, JME, and blend are 56.4, 52.43, and 50.36 J/°CA, respectively, at full load
and original IT.
Fig. 3 Variation of the CHR 2500

rate with crank angle at Diesel
Cumulative Heat Release (J)
different injection timings 2000

JME
24.5 CA bTDC (JMETPO20)
1500
1000
500
0
325 340 355 370 385 400
-500
Crank Angle (deg)
Fig. 4 Variation of the 60
Maximum Heat Release Rate

MHRR with brake power at 55
different injection timings 50
45
(J/ CA)
40
35
Diesel
30 JME
25 23 CA bTDC (JMETPO20)
20
0 1.1 2.2 3.3 4.4
Brake Power (kW)
The MHRR for blend is found to be higher in comparison with standard IT. This
is owing to increase in the volume of fuel delivered throughout the delay period
because of the longer ignition delay (Sharma and Murugan 2015a).
4.4 Maximum Cylinder Pressure (Pmax)
The effects of injection timings on Pmax are compared between diesel, JME, and the
JMETPO20 blend and are shown in Fig. 5. The Pmax values for diesel, JME, and
blend at the standard IT and full load are found to be about 80.96, 80.6, and
79.95 bar, respectively.
It is apparent from the figure that advancing the IT, rises the Pmax for the blend.
It is due to early starting of fuel injection that results in a more amount of mixture
accumulated that undertakes combustion process, which results in an increase in
Pmax (Ganesan 2010). While retarding the IT, a lower Pmax is obtained as a result of
less fuel being accumulated in the delay period for blend. For the blend, at full load
and an advanced and retarded IT of 24.5 and 21.5 °CA bTDC, respectively, the
Pmax values are found to be 83.66 and 78.32 bar.
Fig. 5 Variation of the Pmax 100

Maxim Cylinder pressure (bar)
with brake power at different 95 Diesel

90 JME
injection timings 24.5 CA bTDC (JMETPO20)
85 23 CA bTDC (JMETPO20)
75
70
65
60
55
50
0 1.1 2.2 3.3 4.4
Brake Power (kW)
Fig. 6 Variation of the MFB 100

Diesel
with crank angle at different 90
Mass Fraction Burnt (%)

JME
injection timings 80
70
60
50
40
30
20
10
0
325 340 355 370 385 400 415
Crank Angle (degree)
4.5 Mass Fraction Burnt (MFB)
Figure 6 shows the MFB of the fuels tested in full load condition with respect to the
crank angle for diesel, JME, and the JMETPO20 blend at different injection tim-
ings. The MFB provides an information about the percentage of fuel that has burned
at any given engine crank angle during the combustion process. Numerous key
MFB parameters are explored to record the rate and nature of combustion phe-
nomenon. The study of the MFB duration provides a better outlook on the rate of
burning during the combustion process.
The 10–50 % MFB duration indicates the rate of the early part of combustion
while the 10–90 % MFB duration provides a sense of the overall combustion
duration (Jacobs 2011). It can be observed from the figure that at original IT, the
MFB duration for the JME is shorter in comparison with diesel fuel due to the
oxygen present in the JME, which supports for a faster combustion. The lowest
value of MFB duration for the blend is recorded at advanced IT, because of rapid
burning of more fuel accumulated owing to longer delay period.
5 Conclusions
The aim of this investigation was to study the combustion process of a CI engine
fueled with biodiesel blend, when the experiments were carried out at three different
injection timings: 24.5, 23, and 21.5 °CA bTDC. Based on the test results obtained
for the JMETPO20 blend, the following specific conclusions are given:
• The rise in the Pmax and CHR was found at advanced IT, while the reverse trend
was noticed at retarding IT.
• Advancing the IT by 1.5 °CA compared to standard IT, the maximum cylinder
pressure was found to be higher by 2.7 bar at full load.
• The MHRR and CHR were found to be higher with advancing the IT by 1.5 °CA
in comparison with standard IT. The MHRR and CHR at advanced IT of
24.5 °CA bTDC were noticed 51.5 J/°CA and 2210 J, which were higher by about
1.2 J/°CA and 86 J than standard IT.
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combustion and emission characteristics of jatropha biodiesel engine. Appl Energy 88:4376–
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high free fatty acids. Biomass Bioenerg 28:601–605
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Performance and Emission Characteristics
of CI Engine Fueled with Alternative Fuel
with Special Reference to Modification
for Combustion: A Literature Review
Himansh Kumar, Y.K. Yadav and A.K. Sarma
Abstract The future of alternative fuels for compression-ignition engine has

become imperative due to exhaustion of petroleum products and its major contri-
bution in pollution. As Dr. Rudolf Christian Karl Diesel had invented the
compression-ignition engines and the first exhibition of compression-ignition
engines was carried out at World’s fair in Paris fueled with peanut oil (Alternative
Fuel) in 1911. However, due to the ample availability of petrodiesel and the
unmatched properties of alternative fuel, researchers ignored it. But, after 1970 due
to the hike in price and increased demand-supply gap, researchers had investigated
the new alternative fuel source to overcome the dependency on crude oil and foreign
exchange. This review paper is about the various types of alternative fuels and the
modification needed in compression-ignition engines to run effectively fueled with
biofuels. All the modifications are considered on single cylinder four-stroke CI
engine with rated power of 5.2 kW at 2000 rpm. Six types of modifications in
compression-ignition engines are detailed and almost every performance and
emission test results were described, i.e., BSFC, BTE, hydrocarbon emission, oxide
of nitrogen emission, and carbon monoxide emission in this review paper. The
outcomes of this survey shows that each and every modification have their own
advantages and disadvantages but for modification of guide vanes at the inlet pas-
sage increased the breathing capacity of the CI engine and due to this brake thermal
efficiency was increased and emissions were decreased.
Keywords Compression-ignition engine Modification Alternative fuel

Emissions Performance Biodiesel
List of symbols
BSFC Brake specific fuel consumption
CI Compression ignition
BTDC Before top dead center
Himansh Kumar (&) Y.K. Yadav A.K. Sarma

Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala 144601, India
e-mail: himansh.rmu@gmail.com

DOI 10.1007/978-81-322-2773-1_14
180 Himansh Kumar et al.
BTE Brake thermal efficiency

NO2 Nitrogen dioxide
NOx Nitrogen oxides
CFPP Cold filter plugging point
BP Brake power
CO Carbon monoxide
ppm Parts per million
CO2 Carbon dioxide
rpm Revolutions per minute
HC Hydrocarbon
wt Weight
CNG Compressed natural gas
D-base Engine without modification
Md1 First type of modification
Md2 Second type of modification
Md3 Third type of modification
Md4 Fourth type of modification
Md5 Fifth type of modification
Md6 Sixth type of modification
1 Introduction
The past scenario shows that the production of petroleum oil was no longer than the
supply and demand. Therefore, different techniques were used to prepare the alter-
native fuels which can take petroleum fuel place. As alternative fuels are defined as
any material or substance, other than petroleum used to provide energy to power for
power producing device such as engine. So the term biodiesel was referred to any
petrodiesel equivalent processed fuel, prepared from biological sources (Jaichandar
and Annamalai 2011; Subramaniam et al. 2013; Kumar et al. 2010). It is a processed
fuel, which was ready to be used in common compression-ignition engines vehicles
and it was distinguished as SVO or WVO (Xue et al. 2011; Pandey et al. 2012). As
biofuel contained no petroleum source, it can be blended up to any percentage with
petrodiesel to create a biodiesel–diesel blend. Some of the commonly used alter-
native fuels are shown in Fig. 1.
The prime objective of this review paper is to discuss about CI engine modifi-
cations required for fruitful run of bioliquid fuels in conventional compression
ignition. Almost every modern CI engines would run on biodiesel quite happily
provided that the biodiesel has enough high quality, moreover biodiesel required
much less engine modification but for low class engines there were some basic
problems such as failure of rubber seal, cold starting, fuel injector failure due to high
viscosity, poor atomization, less lubrication, etc. (Heywood 1998; Bari et al. 2002).
Performance and Emission Characteristics of CI Engine Fueled … 181
Alternative fuels
Bio Solid fuel from Bio Gaseous fuel

nature
(For direct burning) Bio Liquid fuel
(Produced specially for C.I engine)
• Syngas from bio mass

• Bio CNG
• Briquette
• Bio Fuel (including biodiesel, Fischer-Tropsch and diesel blends)

• Bio methanol and other alcohols
• Green diesel
• Coal-derived liquid fuels
• Fuels (other than alcohol) derived from biological materials
• Tyre derived oil
• Lubricant derived oil
• Plastic oil
• Oil from algal is also used in compression ignition engine
Fig. 1 Classification of alternative fuels focused on bio liquid fuel
Engine modification is used to overcome all of these difficulties. In CI engine

modification, like preheating of alternative fuel and intake of air through exhaust
gasses, was also a good method for using alternative fuel efficiently. Some little
changes like use of separator or additional fuel filter to protect the CI engine from
water and foreign waste that had missed during prefiltering of biodiesel was also a
modification in CI engine (Prasad et al. 2011). Use of nonrubber products like
synthetic rubber (Viton) for rubber seal and special grade lubricant oil for biodiesel
is also come in the category of modification (Pulkrabek 2003; Agarwal 2006).
The design methodology for modification in compression-ignition engines,
performance and emission analysis were discussed in this review paper. The out-
come of this study was indicated that the following modifications allowed the CI
engine to efficiently utilize the biodiesel as a petrodiesel substitute (Papagiannakis
2013; Pandiyarajan et al. 2011).
2 Types and Designs Available for CI Engine
CI engine is a heat engine in which combustion takes place when the air–fuel
mixture self-ignites due to increased suction air temperature in the combustion
chamber caused by high compression. Various designs are available for CI engine
on the basis of injection of fuel in the combustion chambers like direct, indirect, and
common rail injection and on the basis of geometry of combustion chamber like
open, divided combustion chamber (Ganeshan 2012).
CI engine researches aimed to improve the brake thermal efficiency, BSFC and
to reduce emissions, noise pollution, and vehicle vibration. With different types of
designs, BTE increases up to 7 % to values as high as total 40 % (Pulkrabek 2003).
In present scenario CI engine emission control requirement is one of the major
considerations in design and operation.
3 Modification in CI Engine with Respect to Alternative

Fuel
The physicochemical properties of alternative fuels are closed to petrodiesel but due
to their inherently high kinematic viscosity and less volatility were created poor
atomization, evaporation, and air–fuel mixing formation. This is also creates
improper combustion, lower BTE, and higher pollutant emission. The increased
kinematic viscosity of alternative fuel created operational problems like poor engine
starting, missing in ignition, and decrement in brake thermal efficiency. In
long-term operation, failure problems like coking in nozzle, carbon deposition on
fuel injector and valve seat of CI engine, and lubricating oil dilution was reported
(Çetinkaya et al. 2005). So these problems are created due to direct use of biofuels
in CI engines without any modifications. Almost every CI engine runs on biofuel
and it blends quite happily, but still it needs some modification for proper com-
bustion and increased its efficiency. In this review paper, six types of modifications
are discussed and these are given below (Sects. 3.1–3.6).
3.1 Using Guide Vanes at Air Inlet Passage (Md1)
The guide vanes at inlet passage were installed to increase the breathing capacity of CI
engine. Guide vanes were used to increase the turbulent flow, kinematic velocity, and
scrolling strength of suction air (Brasz 1996). Simulation technique was used to
evaluate the accurate inlet guide vane angle by analyzing the turbulent flow, kinematic
velocity, vorticity, and scrolled air strength created by the inlet guide vanes (Bari and
Saad 2015). It was reported that due to inlet guide vanes CI engine performance was
increased and exhaust emissions decreased compared to any other engine that runs
with biodiesel without using inlet guide vanes (Payri et al. 2004). Five inlet guide vane
models ranging from of 25° to 45° were used and tested on a generator CI engine
fueled with biodiesel. Brake specific fuel consumption was increased in between 0.69
and 1.77 % with use of inlet guide vanes compare to without modification and 35° inlet
guide vane angle indicated the maximum performance (Saad et al. 2013; Naser and
Gosman 1995). In terms of brake thermal efficiency the guide vanes were improved in
the range of 0.7–1.82 % compare to without modification, also 35° inlet guide vane
angle showed the highest performance (Bari and Saad 2015). There was an increased
of NOx emission in the ranges of 0.48–3.79 % with use of guide vanes compared to
without guide vanes. Carbon monoxide was also reduced due to inlet guide vanes at air
inlet passage in the ranges of 1–8.85 % to the highest reduction by 40° inlet guide vane
angle redirected by 35° inlet guide vane angle (Bari and Saad 2015; Naser and
Gosman 1995). With different inlet guide vanes, the alternative fuel emission was
reduced in the variation of 1.19–7.49 % and 35° inlet guide vane angle showed the
highest reduction (Bari and Saad 2015).
3.2 Using Preheated Alternative Fuel (Md2)
Higher kinematic viscosity of alternative fuel was one of the major problems of carbon
deposition on fuel injectors and engine valve seat also poor fuel atomization results
incomplete combustion (Kalam and Masjuki 2004; Masjuki et al. 2001). Preheating
and blending with petrodiesel fuel was the possible solution for decreasing the
kinematic viscosity of biodiesel (Utlu and Koc 2008; Pugazhvadivu and
Jeyachandran 2005). It was reported that at 135 °C, the kinematic viscosity of bio-
diesel was same as petrodiesel at 30 °C. The BTE was improved and the CO and
smoke emissions were reduced with preheated biodiesel (Pugazhvadivu and
Jeyachandran 2005). This waste heat recovery technique was used while the exhaust
gas temperature would ultimately reduce the overall energy requirement and create
healthy environment (Bari et al. 2002; Yilmaz and Morton 2011).
3.3 Using Thermal Barrier Coating on CI Engine Parts

(Md3)
The BTE of every range of CI engine was in the range of 28–36 % generally. It was
meant that the 70 and 64 % of the fuel energy content was lost in the form of losses
and radiation of heat. Experimentally 30 % of energy was wasted in the exhaust
gasses and the remaining was removed by the cooling water in the environment
(Aydın and Sayın 2014). Coating of ceramic materials was used to reduce the heat
loss from CI engine combustion chamber wall that results in energy saving which
was useful to increase the BTE of CI engine. The use of ceramic materials created
low thermal conductivity and high thermal expansion coefficient and this was useful
for insulation for CI engine radiated components (Shrirao and Pawar 2011). For the
daily application of biofuels, it was necessary to use CI engine with little modifi-
cation because its properties are not as same as that of petrodiesel (Hüseyin 2013).
Al2O3, 3Al2O3 · 2SiO2 (mullite), ZrO2, and AE2O4 (spinel), where A is divalent
and E are trivalent, are the common ceramics for coating on internal surface and
parts of combustion chamber (Hazar and Oztürk 2010). It was reported that the
BTE increased with decrease of BSFC and increased in NOx with different loads
Table 1 Coating materials with their properties

Materials/properties ZrO2 Al2O3 3Al2O3 · 2SiO2 (Mullite) AE2O4
(Spinel)
Melting point High High High High
Thermal conductivity Low High Low High
Coefficient of thermal expansion High Low Low Low
Phase Stable Unstable Unstable Stable
Oxidation resistance Low High Low High
Corrosion resistance Low Moderate to high High High
after using thermal barrier coating (Hazar and Oztürk 2010). Table 1 shows some
coating materials and their properties.
3.4 Using Dual Fuel Mode in the Compression-Ignition

Engine (Md4)
Conservation of fossil fuels is necessitated due to limited resources of stocks in

nature. Dual fuel CI engine was fueled with both liquid and gaseous fuels simul-
taneously. Minor modification was needed to convert a CI engines for dual fuel
operation (Tarabet et al. 2014). Gaseous fuel was used as a primary fuel to reduce
the consumption of petrodiesel for power generation in CI engine. Premixed
combustion was also used to increase the BTE and BSFC and decrease the emis-
sions as compared to CI engine without modification (Mohsin et al. 2014; Yoon and
Lee 2011). It was reported that the use of compressed natural gas in a dual fuel CI
engine reduced the engine noise, BSFC consumption, and NOx emissions
(Karabektas 2009; Makareviciene et al. 2013). Also the researchers were reported
that with the use of biogas–petrodiesel and biogas–biodiesel in a dual fuel mode,
BSFC increased and BTE decreased, respectively, compared to without modifica-
tion in CI engine (Duc and Wattanavichien 2007; Tippayawong et al. 2007).
However, NOx and CO2 emission of biogas-biodiesel was higher than biogas–
petroldiesel dual fuel CI engine; whereas CO and HC emission was lower (Bedoya
et al. 2009; Banapurmatha et al. 2008). Dual fuel operation results improved BTE,
BSFC, and lower emissions also less in noise pollution. Also it was a cheap
technique for power generation (Luijten and Kerkhof 2011; Mustafi et al. 2013).
3.5 Using Varying Combustion Chamber Geometry

in Compression-Ignition Engine (Md5)
The impact of combustion chamber geometry variation and use of different shapes of
piston crowns were increased the BTE and BSFC of compression-ignition engines
fueled with biodiesel also improved emissions and combustion. Hemispherical

combustion chamber, toroidal combustion chamber, and shallow depth combustion
chamber were reported to increase the efficiencies of CI engine. The test results
showed that the BTE for toroidal combustion chamber was highest compared to
other reported combustion chambers (Jaichandar and Annamalai 2012). Also the
reduction in particulate matters, CO, and UHC was reported for toroidal combustion
chamber with others (Subhash et al. 2013). An increment in NOx was reported for
the toroidal combustion chamber (Subhash et al. 2013; Hribernik and Kegl 2007).
The combustion characteristics showed that the results were improved for toroidal
combustion chamber with respect to without modification with CI engine
(Venkateswaran and Nagarajan 2010). Also due to proper air–fuel mixing in toroidal
combustion chamber results maximum cylinder pressure compared to two other
engine combustion chambers fueled with biodiesel (Venkateswaran and Nagarajan
2010; Prasad et al. 2011).
3.6 Using Intake Air Preheating in Compression-Ignition

Engine (Md6)
In slow speed engines, preheating of intake air to the heat exchangers by the use of hot
water of engine jacket and again heated by engine exhaust gasses with another heat
exchanger is useful for increasing the volumetric efficiency of compression-ignition
engines (Kumar and Balamurugan 2014). Thermocouples were used to calculate and
maintain the temperature at various locations in the slow speed compression-ignition
engines (Papagiannakis 2013). It is reported that the volumetric efficiency and other
performance parameters increases with increase in both preheated suction air and its
temperature (Pandiyarajan et al. 2011). It is very useful for complete combustion in
CI engine, in the case of biofuel (Papagiannakis 2013). There was a drawback for inlet
air preheating without using intercooler because it created incomplete combustion in
compression-ignition engines for high speed engine. Also it was very useful for full
load conditions because of increased cylinder pressure (Kumar and Balamurugan
2014).
4 Discussion
This review paper consisted of mainly six types of techniques for modification in CI
engine with respect to alternative fuel. Although all the techniques were useful for
modification in CI engine, but there are some advantages and disadvantages. The
following Table 2 is showing the techniques used and the changes happened due to
the modification.
Table 2 Modification in CI engine related to different researchers

S. No. Techniques used Related researchers Changes in CI engine due to
modification
1 Guide vane S. Bari, Idris Saad, Additional guide vane arrangement
J.J. Brasz attached in the air intake passage
2 Preheating the M. Pugazhvadivu, Nadir Introduce a heat exchanger to preheat
alternative fuel Yilmaz the intake fuel
3 Thermal barrier Selman Aydın, Selman Coating of ceramic material on the
coating on engine Aydın, P.N. Shrirao, combustion chamber inner wall, valves
parts Hüseyin Aydin seat, etc.
4 Dual fuel L. Tarabet, R. Mohsin, Additional injection system to inject the
technique for CI N.R. Banapurmatha two fuels simultaneously
engine
5 Varying piston S. Jaichandara, Different types of piston crown are used
bowl shapes G.V. Subhash
6 Intake air R.G. Papagiannakis, Use of turbocharger or heat exchanger
preheating K. Sureshkumar before the air inlet passage
4.1 Performance and Emissions Analysis of CI Engine

with Modification
In this review paper performance and emission analysis of CI engine such as BSFC,
BTE, UHC, CO, and NOx were compared for different modification and deter-
mined. For understanding the effects properly some coding was taken. For guide
vanes at intake air passage type modification symbol Md1 was taken likewise for
second modification, i.e., preheating of the alternative fuel Md2 was taken for third
modification, i.e., use of thermal barrier coating Md3 was taken, for fourth modi-
fication, i.e., dual fuel technique Md3 was taken, for fifth modification, i.e., varying
piston bowl shapes Md5 was taken, for sixth modification, i.e., intake air preheating
Md6 was taken, and finally standard engine D-base was taken. The following
graphs show the performance and emissions results of concerned research papers
and these results were compared to standard engine with respect to alternative fuel.
4.1.1 Brake Specific Fuel Consumption (BSFC)
BSFC is basically the ratio of mass fraction of fuel burned to brake power (Singh
et al. 2015). The following review paper shows the brake specific fuel consumption
with increased engine load with respect to different modification in CI engine. The
graph shows that the BSFC for Md1 type modification was almost similar with
D-base engine and fuel consumption was increased in all the remaining modifi-
cations because of low calorific value of alternative fuel, but in Md1 modification
complete combustion occurs due to the turbulent flow of intake air. Figure 2 shows
the BSFC of biodiesel with different modification with respect to load.
0.7
Md1
0.6
Md2
0.5
Md3
Kg/KWh
0.4
Bsfc
Md4
0.3 Md5
0.2 Md6
0.1 D-Base
0
20 40 60 80 100
% Load
Fig. 2 Variations in brake specific fuel consumption for different modifications with respect to
loads
4.1.2 Brake Thermal Efficiency (BTE)
Brake thermal efficiency is an important efficiency for CI engine, it means how

much thermal energy of fuel input was converted into shaft output (Singh et al.
2015). Figure 3 shows the comparison of BTE for CI engine without modification
to all modified engine fueled with alternative fuel. Generally 31.7 % efficiency is
reported on the papers on full load condition and after Md1 modifications become
32.3 %, hence, there is an increment of 0.6 % of the efficiency (Bari and Saad
2015). Although, all the remaining modifications have similar efficiency as that of
base fuel, except Md4 modification because bio gas is not sufficient to work like
biofuels comparatively. A better BTE in Md1 modification was possible due to the
proper mixture formation of alternative fuel with air. Better swirling of air in
combustion chamber leads to better combustion of alternative fuel and results
increased in brake thermal efficiency.
%ηth
(BTE)
D-Base
% Load
Fig. 3 Variation of BTE for different modifications in CI engine with respect to load
4.2 Emission Analysis
USA was the first country to start the control of air pollution by vehicle in 1964
(Karabektas 2009). After that, all countries had started a numbers of emission
regulations in form of emission standards for CI engine. Euro 6 was the latest
engine emission standards used in almost every country in the world. In this review
paper, the harmful emissions like carbon monooxide (CO), unburnt hydrocarbon
(UHC), and NOx were studied. It is reported in the books that the combustion of a
petroldiesel produces carbon dioxide and water after complete combustion but in
practice it can not be possible, so this study is to find out the best technique to
produce minimum pollutants by CI engine in the exhaust process (Heywood 1998).
4.2.1 Unburnt Hydrocarbon Emission (UHC)
Unburned hydrocarbon is produced due to the incomplete combustion in the com-

bustion phase because it is the unburned part of fuel which was exhausted in
atmosphere during exhaust stroke of CI engine (Singh et al. 2015). The unburnt
hydrocarbon emissions for Md1, Md2, Md3, Md4, Md5, and Md6 were compared to
petroldiesel and shown in Fig. 4. The UHC emissions were reduced from no load to
full load with modification of preheating the biodiesel (Md2) also for modification of
air inlet preheating (Md6). This was because of complete combustion of alternative
fuel with proper mixing of air and fuel, due to increase in intake of air temperature or
fuel. There was a reduction of 30 % UHC emissions in Md2 with respect to pet-
roldiesel and almost similar result for Md6 also for all entire range of load with diesel
fuel. For modification Md4 the UHC emission is high due to incomplete combustion.
4.2.2 Carbon Monoxide Emission (CO)
Carbon monoxide emission is mainly because of incomplete combustion, slow

burning of fuel during final phase of combustion, and due to lower air–fuel ratio. In
(PPM)
UHC
D-Base
% Load
Fig. 4 Variation of unburned hydrocarbons for different modifications with respect to load
volume
CO(%)
by
D-Base
% Load
Fig. 5 Variation of carbon monooxide emissions for different modification with respect to load
case of complete combustion CO was converted into CO2 and if the combustion
was incomplete because of less inducted air and temperature, CO2 turned in CO
(Singh et al. 2015). High emission of CO was due to rich air–fuel mixture as well as
when combustion occurs with lean mixture CO turned into CO2. Figure 5 shows the
comparison of CO emissions for different modification in CI engine with respect to
CI engine without modification. In every engine load conditions CO formation for
modifications Md1 and Md5 were lower to the standard CI engine but for remaining
modifications, CO was more than that of standard diesel engine. However, on full
load condition, CO formation was decreased significantly when compared to
standard CI engine. In modification Md1 and Md5, CO formation was hugely
reduced due to higher air swirl results better combustion. For remaining modifi-
cations, like Md2, Md3, Md4, and Md6, it increased to the standard CI engine.
4.2.3 Oxides of Nitrogen Emission (NOx)
NOx is formed because of chain reactions in air with the presence of nitrogen and
oxygen. These reactions are depended on reaction temperature. NOx emission was
induced after 1200 °C reaction temperature (Singh et al. 2015). Figure 6 shows the
[PPM]
NOX
D-Base
% Load
Fig. 6 Variation of oxides of nitrogen emission for different modifications with respect to load
variations in NOx formation for standard CI engine with different modifications.

The NOx emissions have a little bit decrement for Md2 and Md4 to the standard
engine is due to use of alternative fuel in huge quantity. Increased in NOx emission
was due to high combustion product temperature results complete combustion in
case of CI engine without modification. For standard CI engine, increase in NOx
emission was due to the high part of the combustion that was completed before
BTDC due to lower ignition delay of fuel.
5 Conclusion
From the above indicated modifications it is concluded that the use of alternative
fuel or its blends with petroldiesel in modified CI engine can create a good impact
on the efficiencies and emissions of the CI engines. It is very useful in developing
countries like India because India has great potential for alternative fuels. The
compression-ignition engines easily adopt the characteristics of alternative fuels
like higher flash point, kinematic viscosity, API density, and lower calorific value
due to these modifications. Stationary slow speed compression-ignition enginess
were suitable for fueled with straight vegetable oil and their blends without much
environmental impact and these types of modifications were very good for sta-
tionary engines for increasing their brake thermal efficiency. The main conclusions
derived from present investigation are summarized below.
• Modification like air intake in a combustion chamber through the guide vanes
(Md1) is very helpful for proper air–fuel mixing and proper combustion due to
increased in volumetric efficiency and turbulent nature of intake air.
• Alternative fuel preheating through exhaust gasses was also an optional solution
to reduce the kinematic viscosity of liquid alternative fuel because it created the
major problems like failure of rubber seals, injector coking, cold shut in winter
time, etc.
• Thermal barrier coating is also a useful modification technique for increasing the
efficiency and decreasing the emission, but due to insulation, inside temperature
gets increased and NOx emission gets increased, but by adding additives in the
alternative fuel it can be reduced.
• Dual fuel CI engine is also a good technique for using gaseous fuel, but the
negative point is that the efficiency gets decreased and emissions gets increased,
but the positive point is that the fuel cost gets decreased.
• Varying piston bowls type modification is also useful for proper mixing of the
air–fuel mixture and complete combustion and toroidal combustion chamber
type piston bowl is reported for good results.
• Intake air preheating by exhaust gasses is also a good modification technique for
increasing the BTE, BSFC and less emissions, but for stationary engine it was
not good because the breathing capacity decreased because of addition of heat
exchanger in the intake passage, but for automobile industries, by the use of
turbocharger and supercharger, the breathing capacity was also increased and an
intercooler was added to control the temperature of intake air.
Although all the modifications have some advantages and disadvantages, but the
modification Md1 (guide vanes at air inlet passage) creates more breathing capacity
of the engine and due to this thermal efficiency gets increased and emissions
decrease. Also this arrangement does not need much attention for modification in
compression ignition engine. So this type of modification is best suited for CI
engine in case of alternative fuel. This review paper concludes that alternative fuel
and its blends can hold a good future as a fuel for compression-ignition engines
with little modifications; further research is needed for elaboration of modification
in CI engine.
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Part III
Thermochemical Conversion
Upgradation of Bio-oil Derived
from Lignocellulose Biomass—A
Numerical Approach
Anjani R.K. Gollakota, Malladi D. Subramanyam,

Nanda Kishore and Sai Gu
Abstract The envisaged upgrading of lignocellulose biomass derived feed stock to

partially replace crude oil is receiving much attention due to increasing demand for
renewable and CO2 neutral energy sources. One prospective method is to convert the
biomass into bio-oil through pyrolysis and upgrading the pyrolytic oil into biofuel.
The bio-oil has a high content of oxygen and therefore lowers the stability over the
time and lowers the heating value as well. Hence, upgrading is desirable to remove
the oxygen and enhance the properties of the bio-oil resembling as a second gen-
eration fuel which is an alternative to conventional fossil fuels. Hydrodeoxygenation
(HDO) process is found to be very promising route to upgrade pyrolysis bio-oil for
producing liquid transportation fuels. The present study deals with the CFD mod-
eling of hydrodynamics and reaction kinetics in a fluidized bed reactor using
ANSYS Fluent 14.5. By integrating a lumped kinetic model with Eulerian multi-
phase fluid flow, a comprehensive model for the hydrodynamics and reaction
kinetics for the upgradation of bio-oil is developed. CFD analyses are performed and
influence of operating parameters are evaluated for the weight hourly space velocity
(WHSV) of 2 h−1 and a constant pressure of 8720 kPa at temperature in the range
623–673 K.

Keywords Bio-oil Upgradation Fluidized bed reactor Hydrodeoxygeneation

Hydrodynamics Reaction kinetics
A.R.K. Gollakota M.D. Subramanyam Nanda Kishore (&)

Department of Chemical Engineering, Indian Institute of Technology Guwahati,
Guwahati 781039, Assam, India
e-mail: nkishore@iitg.ernet.in; mail2nkishore@gmail.com
Sai Gu
Centre for Biofuel, School of Energy, Environmental and Agrifood,
Cranfield University, Cranfield, UK

DOI 10.1007/978-81-322-2773-1_15
198 A.R.K. Gollakota et al.
List of symbols
! External forces (N)
Fq
sq Stress-strain tensor (Pa)
;ls Energy exchange (kJ)
ess Coefficient of restitution for particle collisions
*
g Gravitational force (m s−2)
chs Collision dissipation energy (kg s−3)
rk Turbulent Prandtl numbers
A Arrhenius constant (min−1)
Al2O3 Alumina
b Buoyancy
C Model constant
Cd Drag coefficient
CO Carbon-monoxide
CO2 Carbon-dioxide
Co-Mo Cobalt-molybdenum oxide
D Diffusion coefficient of species (m2 s−1)
D Diameter (m)
E Activation energy (kJ mol−1)
Fr Friction
G Generation of energy (kJ)
H Specific enthalpy (kJ)
HDO Hydrodeoxygenation
HNV Heavy non volatiles
i,j Components
K Interface exchange coefficient
k Mean velocity gradients
l Liquid phase
LNV Light non volatiles
Max Maximum value
Ni-Mo Nickel molybdenum oxide
Nu Nusselt number
P Pressure (Pa)
p Secondary phase
Pr Prandtl number
Pt Platinum
q Heat flux (J s−1)
r Rate of reaction (mol l−1 s−1)
Re Reynolds number
s Solid phase
S Source term
T Temperature (K)
WHSV Weight hourly space velocity (h−1)
Upgradation of Bio-oil Derived from Lignocellulose … 199
Y Local mass fraction of species

α Volume fraction
ε Rate of dissipation (m2 s−3)
ρ Density (kg m−3)
v Velocity (m s−1)
l Viscosity (Pa s)
1 Introduction
The modern day energy consumption of the world reached to threshold in com-
parison to the earlier times owing to the present lifestyle and the increased popu-
lation. The transportation sector consumption constitutes one fifth of the total
energy produced according to (van Ruijven and van Vuuren 2009). With the
population growth and the convenience in the transportation, there is a definite need
in search of alternate source of energy to meet the current demands. This poses
major challenge of the near future as the present fuel reserves are depleting dras-
tically. Materialistic research work is being carried out in the fields of energy to find
the replacement fuels of gasoline and diesel. The alternative fuel that is equivalent
to the conventional fuels should be compatible, sustainable, and reduce the CO2
emissions thereby decreasing the adverse environmental effects. Biomass derived
fuels seems to be the future perspective in accomplishing the aforementioned cri-
teria, and produced within relatively short cycles to reduce the environmental
hazards. Unlike first generation, bio-fuels (based on food grains) have been
implemented till now creating a problem of food scarcity particularly food grains
and the energy efficiency per unit land of the crop is relatively low. To overcome
these difficulties, advanced bio-fuels are produced from a wide range of lignocel-
lulosic biomass feed stocks, including waste, and their production does not nec-
essarily compete with food or feed production. The possible option available to
produce advanced or second generation bio-fuels is the coprocessing of upgraded
pyrolysis oil (also known as bio-oil). Bio-oils are complex natured with a mixture
of several compounds with an appreciable proportion of water from both the
original moisture and reaction product including char content. These second gen-
eration bio-fuels from pyrolysis process are incompatible with the conventional
fuels mainly due to the higher oxygen content, high solids content, high viscosity,
high moisture content, and chemically unstable, along with higher contents of char
formation. Hence, upgradation of the bio-oil from the pyrolysis process is highly
advisable. Among the various upgradation processes hydrodeoxygenation
(HDO) appears to be the promising technique carried out in the presence of a
conventional catalyst using H2 gas. This process is also known as catalytic
upgradation of pyrolytic oil. Thus in this work, numerical investigation of upgra-
dation of pine pyrolytic bio-oil in the presence Pt/Al2O3 catalyst using H2 gas has
been carried out within the range of operating conditions as: temperature, T = 623–
673 K, weight hourly space velocity, WHSV = 2 h−1, and a constant pressure of
8720 kPa.
2 Literature Review
Substantial research work on HDO process is mainly initiated by Weisser and

Landa (1973) followed by Furimsky (1978, 1979, 1983) based on various feed
stocks and different reaction kinetics mechanisms of furan and tetrahydrofuran.
Further, Ternan and Brown (1982) studies on HDO of coal distillate liquids with
high oxygen content observed higher conversion rates of reactants to products by
the reaction of smaller oxygenated compounds (phenols). Later, Elliott and Baker
(1984) performed HDO studies on the products of the liquefaction process in the
presence of CO at a pressure of 138 bar and at temperatures ranging between 350–
450 °C in a batch reactor. Johnson et al. (1988) investigated the conversion of lignin
into phenols and hydrocarbons by mild HDO process. Train and Klein (1986)
developed Monte-Carlo simulations of HDO of lignin using the kinetics obtained
from the experimental results. Elliott (1988) performed two-stage catalytic
hydro-treatment in the presence of Co/Mo supported alumina catalyst. The reaction
results are significant and further confirmed by Baker and Elliott (1988). The two
stages are combined to a single-stage non-isothermal process with a yield of 33 %
of lighter hydrocarbons and high gas formation. Sheu et al. (1988) performed
experimental studies on the upgradation of pine pyrolytic oil by HDO process in a
trickle bed reactor. The authors developed two models, one for the overall oxygen
removal and the other for the compositional changes in hydro-treated oil. Piskorz
et al. (1988) confirmed the findings of Elliott results particularly the necessity of
high hydrogen gas pressure. Baldauf et al. (1994) performed hydroprocessing of
flash pyrolysis oil using the conventional Co–Mo, Ni–Mo catalysts in which the
major yield of about 60 % is the moisture content with high deoxygenation rates.
However, bio-oil contains all other kinds of oxygenated chemical groups like
aldehydes, ketones, carboxylic acids, esters, aliphatic and aromatic alcohols (Horne
and Williams 1996). Oasmaa and Czernik (1999) performed catalytic
hydro-treatment of peat pyrolysate oils in a batch wise reactor at a higher pressure
of 21.5 MPa for two hours at 390 °C with 10 wt% catalyst, 4 % CoO, 15.5 %
MoO3, on alumina support. Oxygen content is reduced from 22 to 3 % in the
process. Elliott (2007) reviewed the insights and achievements of HDO process in
the past 25 years. The important aspect in HDO process is the hydrogen con-
sumption. Recent results of De Miguel Mercader et al. (2010), and Venderbosch
et al. (2010) indicate that stabilization of pyrolysis oil by low severity HDO is
sufficient for upgradation process and reduces the hydrogen consumption. Xu et al.
(2013) proposed two-step catalytic hydrodeoxygenation of fast pyrolysis oil to
hydrocarbon liquid fuels. In the first step, organic solvents were employed to reduce
the coke formation and promote higher rates of HDO process. In the second step,
conventional reaction mechanism of reactants to products in the presence of catalyst

along with hydrogen gas is performed to obtain hydrocarbon fuel. Recently,
Parapati et al. (2014) mentioned that the customary practice of using two-stage
hydro-processing units at moderate temperatures also yields approximately similar
to the single-stage reactor. Hence, in order to increase the throughput and decrease
the extra financial commitments, a single-stage hydro-processing unit is sufficient.
Finally, on the basis of aforementioned detailed literature review, it is clear that the
HDO process has been extensively used for the upgradation process of bio-oils;
however, optimized operating conditions are not available. Therefore, in this work,
a numerical investigation has been carried out to optimize the range of temperature
for the upgradation of pine pyrolytic bio-oil using hydrodeoxygenation in the
presence of Pt/Al2O3 catalyst with WHSV = 2 h−1 and P = 8720 kPa at T = 623–
673 K.
3 CFD Modeling
The numerical modeling and simulations of reactive multiphase flows in fluidized

bed reactors are extremely complex because of the turbulence of flow of gas and
catalyst particles. Thus, to make the model reliable turbulent particle flow phe-
nomena, particle turbulence and the turbulence interaction between the fluid and
particle phases are included in the present simulation studies. In the present study,
Eulerian multiphase fluid flow approach has been chosen to see the particle inter-
action alongside pursuing the chemical reaction kinetics using a lumped parameter
model adopted/marked as benchmark experimental studies of Sheu et al. (1988). To
perform these reaction kinetic studies numerically, the chemical species transport of
five lumped parameter model is chosen in a commercial computational fluid
dynamic-based solver ANSYS Fluent 14.5. By integrating the five lumped kinetic
model (Sheu et al. 1988), with the turbulent flow model, a multiphase flow and
reaction model is established. The governing equations are described as follows:
3.1 Mass Conservation Equation
The continuity equation for the gas, oil, and particle phases is written as
@
*
aq qq þ r aq qq vq ¼ 0 ð1Þ
@t
The species equation of the gas phase is expressed as follows which represents
the coupling of hydrodynamics and reaction kinetics

@ @ @ @Yi
ðqYi Þ þ quj Yi ¼ qDi þ R i þ Si ð2Þ
@t @xj @xj @xj
3.2 Fluid–Fluid Momentum Equation
@ *

* *

*
aq qq vq þ r : aq qq vq vq ¼ aq qq g þ r : sq aq rp
@t
X
n ! ð3Þ
þ Kpq !vp ! vq þ F q
p¼1
The first term in the left hand of the equation represents the unsteady state accel-
eration; second term represents convective acceleration of the respective fluids
inside the reactor. The right hand side terms denote the summation of different
forces acted upon the fluid–fluid interaction.
3.3 Fluid–Solid Momentum Equation
@ *

* *

*
as qs vs þ r as qs vs vs ¼ as qs g þ r ss as rp
@t
Xn
rps þ Kls !vl !
vs ð4Þ
l¼1
This equation is quite similar to the Eq. 3 with an additional term on the right side
rps represents the solids pressure due to solids collision which is modeled using
kinetic theory of granular flow. In this equation Kls denotes interphase/momentum
exchange coefficients between liquid and solid phases as given by Gidaspow et al.
(1992) model which is a combination of Wen and Yu (1966) and Ergun and Orning
(1952), and is given as:

3 al as ! ! 2:65 150as ð1 al Þll 1:75as ql !us !
ul
Kls ¼ Cd ql us ul al þ þ ð5Þ
4 ds al ds2 ds
and Kpq is between the continuous phases and is given by Schiller and Naumann
(1933)

ap qp 1 þ 0:15Re0:687
Kpq ¼ ð6Þ
sp
3.4 Enthalpy Conservation Equation
@ @pq
aq qq hq þ r aq qq hq !
uq ¼ aq þ sq : ru !r !
q qq
@t @t
X n
6cq aq ap Nu Tp Tq ð7Þ
þ Sq þ
p¼1
dp2
Here, hq is the specific enthalpy of qth (primary) phase, !qq is the heat flux, Sq is the
source term that includes the sources of enthalpy (e.g., due to chemical reaction or
radiation), Qpq is the intensity of heat exchange between pth (secondary phase) and
qth phases, and hpq is the interphase enthalpy. Ranz and Marshall (1952) correlation
is applied for fluid–fluid multiphase interaction as follows
1=3
Nup ¼ 2:0 þ 0:6Re1=2
p Pr ð8Þ
and for fluid–solid multiphase interaction, Gunn (1978) correlation is applied and
given as

1=3
Nus ¼ 7 10af þ 5a2f 1 þ 0:7Re0:2
s Pr þ 1:33 2:4af þ 1:2a2f Re0:7
s ð9Þ
3.5 k e Turbulence Model
The turbulent kinetic energy (k) equation for the multiphase is defined as

@ @ @ lt @k
ðqk Þ þ ðqkui Þ ¼ lþ þ Gk þ Gb q YM þ Sk ð10Þ
@t @xi @xj rk @xj
The dissipation rate equation (ε) of the turbulent kinetic energy for all the phases is
given by

@ @ @ lt @ 2
ðqÞ þ ðqui Þ ¼ lþ þ C1 ðGk þ C3 Gb Þ C2q þ S
@t @xi @xj r @xj k
ð11Þ
3.6 Kinetic Theory of Granular Flow
The particle fluctuation is modeled by kinetic theory of granular flow. The equation
for the turbulent fluctuating energy of the particle phase is expressed by Gidaspow
and Ding (1990)

3 @ !
ðqs as hs Þ þ r qs as hs vs ¼ ps I þ T
2 @t
! þ r ðK rh Þ ch þ ;
: rv ð12Þ
s hs s s ls
where the first and second terms in the right hand side of Eq. (12) represent the
generation of energy by the solid stress tensor, and the diffusion of energy, where
Khs is the diffusion coefficient given by Gidaspow et al. (1992)
pffiffiffiffiffiffi
2 rffiffiffiffi
150qs ds hp 6 hs
Khs ¼ 1 þ as g0;ss ð1 þ es Þ þ 2qs as ds ð1 þ ess Þg0;ss
2
ð13Þ
384ð1 þ ess Þg0;ss 5 p
and chs is the collision dissipation of energy, ;ls is the energy exchange between the
fluid and solid phase. Other model equations can be found in Fluent users guide
(Fluent Inc. 2006).
3.7 Lumped Kinetics Model
The reaction pathway (Fig. 1) and kinetic parameters for the present simulation
study is adopted from experimental study of Sheu et al. (1988).
Fig. 1 Reaction pathway of hydro-processing of pine pyrolytic oil (Sheu et al. 1988)
The kinetic parameters (Sheu et al. 1988) that are used in the present simulation
studies are listed below:

r1 ¼ k1 q1 E1 ¼ 74 kJmol1 ; A1 ¼ 3860 min1 ð14Þ

r2 ¼ k1 q1 k2 q2 k3 q2 E2 ¼ 91:8 kJmol1 ; A2 ¼ 75400 min1 ð15Þ

r3 ¼ k3 q2 k4 q3 E3 ¼ 80:6 kJmol1 ; A3 ¼ 8300 min1 ð16Þ

r4 ¼ k2 q2 þ k4 q3 k5 q4 E4 ¼ 62:3 kJmol1 ; A4 ¼ 950 min1 ð17Þ

r5 ¼ k5 q4 E5 ¼ 69:6 kJmol1 ; A5 ¼ 4000 min1 ð18Þ
4 Simulation System and Numerical Procedure
4.1 Simulation System
A schematic representation of the fluidized bed reactor used in the present simu-
lation study is shown in Fig. 2. The dimensions of the reactor are chosen exactly
same as in the experimental study due to Sheu et al. (1988). The height of the
reactor is chosen to be 0.813 m and the width of the reactor is chosen to be
0.01564 m. The reactor is packed with Pt/Al2O3 catalyst volume fraction of 0.0286
with no oil and gas volume fractions at the inlet at time t = 0 s. Pine pyrolytic oil
Fig. 2 Schematic
representation of fluidized bed
reactor model for upgradation
of bio-oil
Table 1 Properties of three phases (oil, gas and catalyst) in present simulations
Phase Compound Density Viscosity Specific heat Thermal
(kg m−3) (Pa s) (Jkg−1 K−1) conductivity
(Wm−1 K−1)
Pine HNV 841.15 0.00092 1833.817 0.127
pyrolytic oil LNV 679.5 0.00040 2223.19 0.14
phase
Phenol 1030 0.1842 1430 0.19
Aromatics 880 0.00081 1699.84 0.131
Alkane 0.669 0.000018 2222 0.033
Gas phase H2 gas 0.8189 0.000008 14283 0.167
Water 0.5542 0.000013 2014 0.0261
vapor
Catalyst Pt/Al2O3 21450 0.000017 130 71.6
phase Coke 375 1.206 850 0.2
consists of various lumping groups including heavy nonvolatile (HNV), light

non-volatile (LNV), phenols, alkanes aromatics mixture, and H2 gas is introduced
from the bottom of the reactor to pass through the catalyst bed. The properties of the
three phases used in the present simulation studies are listed in Table 1.
4.2 Numerical Procedure
The governing differential equations (as presented in the previous section) are
solved using commercial computational fluid dynamics-based software ANSYS
Fluent 14.5 and all simulations are carried out in two-dimensional computational
domain. The grids are generated using Gambit 2.2.30 and are exported to Fluent
14.5 software. The structured hexahedral grid is used with a total mesh number of
6000 cells, 12170 faces with 7000 node points. The bottom inlet of the reactor is
designated as “velocity inlet” boundary in Fluent software. At the inlet, the
velocities, mass fractions, volume fractions, temperatures of the three phases are
specified according to the desired operating conditions. The outlet at the top of the
reactor is set as “pressure outlet” boundary; and for the other two walls, the
boundary conditions are specified as “wall” boundaries which are defined as no-slip
boundaries for all the phases.
4.3 Model Validation
The numerical solver used in the present study is validated by comparing the
present values of the mass fractions of the individual species of the oil phase with
the existing experimental literature of Sheu et al. (1988). As the research related to
the upgradation of bio-oil using HDO process through a numerical approach is very
scarce, and hence to the best of the knowledge only Sheu et al. (1988) reported their
experimental results in terms of numeric for the HDO process. The present results
are in close proximity with the existing literature values which boosted us the
confidence to proceed further in order to check the effect of temperature at a
constant pressure and at a constant catalyst loading values.
5 Results and Discussions
5.1 Hydrodynamics
Figure 3 shows the volume fraction distribution of all phases at different operating
temperatures ranging between 623 K < T < 673 K at a constant WHSV = 2 h−1 with
a bio-oil flow rate of 0.033 × 10−6 m3 s−1 and H2 gas flow rate of 100 cc min−1g−1,
whereas the velocity of the gas feed is used in the calculation of total velocities of
feed to the reactor at a constant pressure P = 8720 kPa. Figures 4, 5, and 6 illustrate
the prototype contours of volume fraction representing the flow behavior of cata-
lyst, pine oil, and hydrogen gas in the reactor for different time steps ranging
between t = 0 and t = 150 s at T = 673 K. At t = 150 s the bed has reached pseudo
steady state with no further deviations with respect to bed characteristics and
temperature, the averaged results are plotted and analyzed. From Fig. 3, it is seen
that the volume fraction of the catalyst is slightly decreased with increase in the
temperature. This denotes the utilization of the catalyst in continuous formation of
different product fractions leads to catalyst deactivation. Further, the formation of
the aromatics enables the coke formation which is deposited on the surface of the
Fig. 3 Volume fraction of all

phases varying with respect to
temperature
Fig. 4 Catalyst volume fraction images of a fluidized bed reactor at T = 673 K and 8720 kPa
Fig. 5 Pine oil volume fraction images of a fluidized bed reactor at T = 673 K and 8720 kPa
catalyst leads to catalyst deactivation. Hence, proper measures are to be taken to

maintain the optimum physiochemical properties.
The volume fraction of oil is slightly decreasing till T = 648 K and further there
is no deviation observed even with the increase in the temperature as seen in Fig. 3.
This denotes that the increase in the temperature enhances the thermodynamic
equilibrium which increases the cracking rate to convert HNV, LNV, phenol (oil
Fig. 6 H2 gas volume fraction images of a fluidized bed reactor at T = 673 K and 8720 kPa
phase nonvolatiles) to desired product streams (gas phase), thus decreasing the
volume fraction of the oil phase. Finally, the volume fraction of the gas increases
gradually till T = 648 K and further shows a constant trend. As mentioned earlier,
the converted oil phase reactants are transformed to gas phase products, thereby
enhancing the volume fraction of the gasses. In summary, it is observed that the
change in the volume fraction is significant at an intermediate temperature of
T = 648 K in oil and gas phases and further no significant changes is observed.
5.2 Reaction Kinetics
The bio-oil mixture containing many organic compounds undergoes several

hydro-treating processes simultaneously, for removing the oxygen in the form of
water and hydrocracking step for reducing heavy components to middle compo-
nents. Figure 7 depicts the prototype of the mass fraction contours of three species
(oil, gas, catalyst) of upgraded bio-oil in a fluidized bed reactor at WHSV = 2 h−1,
pressure P = 8720 kPa, and temperature T = 673 K. The initial and upgraded mass
fractions of three phases (oil, gas, and catalyst) in fluidized bed resulted through
HDO are presented in Table 2 and the corresponding values are in close proximity
with the experimental (literature) values. Table 3 denotes the product mass fraction
species of the lumped bio-oil after HDO at different temperatures and at constant
pressure, WHSV. These values are obtained through a post-processing tool of
CFD-POST which is an integral part of ANSYS Fluent 14.5. The respective
reaction pathway shown in Fig. 1 favors the formation of alkanes and aromatics,
Fig. 7 Upgraded pyrolytic oil species mass fractions of (oil, gas and solid catalyst) for
temperature T = 673 K and pressure P = 8720 kPa
Table 2 Mass fraction of lumped product species in the fluidized bed reactor at T = 673 K,
P = 8720 kPa and WHSV ¼ 2h1
Lumped Fraction Pine pyrolytic oil (wt%) (Sheu Hydrotreated pyrolytic oil (wt%)
et al. 1988) Experimental (Sheu Present
et al. 1988)
HNV 0.4932 0.2457 0.2102
LNV 0.3690 0.2941 0.4083
Phenol 0.1232 0.1063 0.1552
Alkane + Aromatic 0.0146 0.1952 0.1962
Coke + H2O + gasses 0 0.1587 0.000169
Table 3 Mass fraction of lumped compounds (wt%) in the fluidized bed reactor at different
temperatures and constant pressure P = 8720 kPa
Temperature Heavy non volatiles Light non volatiles Phenol Alkane + Aromatic
T = 623 K 0.2651 0.4389 0.1486 0.1538
T = 648 K 0.2414 0.4363 0.1456 0.1765
T = 673 K 0.2102 0.4083 0.1552 0.1962
which are replaceable to conventional gasoline and middle distillates from non-
volatile, volatile, and phenol compounds of unprocessed bio-oil. It is observed that
the mass fraction of heavy nonvolatiles are decreased drastically from 0.4962 wt%
to the range of 0.21–0.25 wt% signify the conversion of heavy nonvolatiles to
alkanes and aromatics. The massive formation of undesirable LNV attributes to
many secondary reactions occurred during hydro-treating and hydrocracking steps.
Very small quantities of LNV are converted to phenol which is further reduced to a
combination of alkanes and aromatics. This clearly shows that the phenol reaction
rates of formation and utilization are equal.
In summary, there is no much deviation in the mass fractions of product species
formed from HDO of unprocessed bio-oil at different temperature ranges. The
formation of alkane and aromatics is highly desirable at higher temperatures as
shown in Table 3 along with higher quantities of deleterious species (lighter non-
volatiles) needs to be processed further.
6 Conclusions
A CFD prototype for fluidized bed reactor for upgradation of pine pyrolytic bio-oil
using hydrodeoxygenation using Pt/Al2O3 catalyst is successfully modeled in
ANSYS Fluent 14.5 incorporating all the continuity, momentum, energy equations,
drag behavior exhibited by the fluid and solid particles, turbulence motion, and
lumped species reactions. Simulations are conducted at different operating tem-
peratures to examine the variation in mass fractions of product compounds. As
observed, there is a significant change in mass fractions of alkane and aromatic
mixture species as temperature increases. It is also noted that the higher nonvolatile
formation is due to the secondary reactions during hydrocracking mechanism.
Significant conversion of heavy nonvolatiles to alkane and aromatics is observed
through HDO process.
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Sustainability Assessment of Biomass
Gasification Based Distributed Power
Generation in India
Amit Kumar Singh Parihar, Virendra Sethi and Rangan Banerjee
Abstract Access to energy is a precursor to sustainable development. In India, at

present, the number of people without access to electricity remains high, despite
several efforts by the government. Non-feasibility of grid extension is one of the
major reasons for this poor energy access. Even in the areas with grid connectivity,
reliable electricity supply still remains a challenge. Biomass gasification based
renewable distributed power generation sources may provide electricity access by
generating it at the point of consumption using locally available resources. Most of
the prior studies have evaluated biomass gasification system based on cost of elec-
tricity generation neglecting its impact on environment and society. Instead, sus-
tainability aspects including social, technical, environmental, and resource should be
an integral part of this practice along with economic aspect and compared in trans-
parent way. This not only necessitates identification of appropriate indicators to
reflect relevant sustainability dimensions, but also requires development of general
framework comparing different qualitative and quantitative indicators. Framework
should bring out various trade-offs and bounce associated with indicators. This paper
analyses different indicators and assessment methodologies including single-criterion
and multi-criteria decision methods discussed in the literature for sustainability
assessment of power generation systems followed with specific focus on sustain-
ability assessment methodologies suggested for biomass gasification systems.
Research gaps with scope for conceptual contributions in the prevailing sustainability
assessment process from selection of appropriate indicator to assessment method-
ologies are discussed. Based on the literature review, this article identifies appropriate
criteria and indicators to be considered for assessing the sustainability of biomass
gasification based power generation systems. A total of 18 indicators representing
economic, technical, environment, resource, and social aspect of sustainability along
with their relevance and importance for sustainability assessment are discussed. The
set of indicators identified may be used for comparing sustainability of possible
distributed power generation systems in Indian context.
A.K.S. Parihar (&) Virendra Sethi Rangan Banerjee

Department of Energy Science & Engineering, Indian Institute of Technology Bombay,
Bombay, India
e-mail: aksparihar@iitb.ac.in

DOI 10.1007/978-81-322-2773-1_16
214 A.K.S. Parihar et al.
Keywords Sustainability assessment framework Criteria and indicators Life

cycle assessment Multi-criteria decision-making
1 Introduction
Access to energy is a precursor to sustainable development (Demirtas 2013). In

India, around 70 % of the total population lives in remote areas where grid electricity
is either not available or suffers with poor supply if available (Castellanos et al.
2015). While percentage village electrification has improved due to various rural
electrification schemes including RGGVY, percentage of household electrification
still remains low (Harish et al. 2014). Further, electrification through conventional
fuel-based power generation leads to environmental pollution due to GHGs emis-
sion. Renewable energy based distributed power generation systems are being
looked upon as potential source to solve this problem of providing electricity without
causing damage to the environment. Due to local availability of fuel and carbon
neutral characteristics, biomass gasification based distributed power generation
systems may be one potential option among various renewable based distributed
power generation systems. However, field level success of this technology has not
been able to provide desired success due to reasons including combination of cost
and non-cost factors. Therefore, there is a need to examine impact of non-cost
parameters along with cost parameters on long-term sustainable operation of these
systems. Most of the prior relevant studies have focused on estimating the cost of
electricity generation (Banerjee 2006; Nouni et al. 2007; Palit et al. 2011) neglecting
other parameters related to technical, environment, resource, and social aspect.
This paper analyses different indicators and assessment methodologies suggested
for sustainability assessment of distributed power generation systems with focus on
small scale biomass gasification based power generation system in India. The
typical scale being targeted in the present study ranges from few kW to 150 kW, as
this range is sufficient to meet electrical needs of a typical village or clusters of
villages in off-grid mode. Gaps in present methodologies are identified. Finally,
present study proposes 18 indicators belonging to economic, technical, environ-
ment, resource, and social aspect of sustainability along with discussion about their
relevance and importance for developing a general and transparent sustainability
assessment framework.
2 Sustainability Assessment
The Brundtland Report initiated the concept of “sustainability” defined as “to satisfy
our needs without compromising the ability of future generations to meet their own
needs” (Brundtland 1987). The process of evaluating most suitable technological
option considering multiple parameters may be termed as sustainability assessment.
Sustainability Assessment of Biomass Gasification … 215
Fig. 1 Flowchart for general sustainability assessment methodology
While economic, environment, and social are the three major dimensions suggested
for sustainability assessment, technical and resource dimensions are also reported for
sustainability assessment of renewable energy systems (Frangopoulos and Keramioti
2010; Afgan et al. 2000; Wang et al. 2009).
Figure 1 depicts the flow chart of general sustainability assessment process as
reported in the literature. The process starts with identification of probable alterna-
tives followed by identification of relevant criteria and indicators using which the
various alternates are to be compared. Wang et al. (2009) have discussed guiding
principle as well as elementary methods for selection of suitable criteria and indi-
cators for sustainability evaluation. Selected indicators are scored for each alternate
option and normalized on a common scale to facilitate comparison. Thereafter,
technology alternates are ranked using either single- or multi-criteria assessment
methodology. In case of multi-criteria assessment, selected indicators and criteria are
assigned weights to reflect relative importance. Finally these weightage and indicator
scores are aggregated using multi-criteria decision-making methods to rank various
alternate options (Wang et al. 2009; Frangopoulos and Keramioti 2010).
3 Sustainability Criteria and Indicators
In the sustainability assessment process, evaluation of alternatives is carried out

considering different criteria comprising of relevant indicators (NEEDS 2009). As
technology alternatives are being ranked using sustainability criteria and indicators,
selection of relevant indicators becomes important for sustainability assessment.
Several studies provide indicator selection methodologies (Wang et al. 2009;

Musango et al. 2011; Dale et al. 2013). Selected indicators should be simple,
quantifiable, and understandable. For sustainability assessment of renewable power
generation systems technical, economic, environmental, material requirement, and
social are some of the main criteria used (Wang et al. 2009; Afgan et al. 2000).
Some of the selected criteria and indicators reported in the literature for sustain-
ability assessment of power generation systems are depicted in Fig. 2. There is no
clear demarcation for few indicators as they have been grouped under different
criteria. For example, efficiency has been considered under economic criteria,
whereas it may be more appropriate under technical criteria. Some of commonly
used indicators in sustainability assessment studies are capital cost (Rs/kW), cost of
electricity generation (Rs/kWh), GHG emissions (kg CO2/kWh), number of jobs
created (Man-hr/kWh), etc. (Varun et al. 2009; Streimikiene 2010; Wang et al.
2009; Dale et al. 2013; Evans et al. 2010). In case of sustainability assessment of
renewable energy technologies capacity factor, land and water requirement are also
used for evaluation (Chatzimouratidis and Pilavachi 2009; Evans et al. 2009;
Manish et al. 2006).
Fig. 2 Different categories of indicators and sub-indicators for sustainability assessment

(compiled from Evans et al. 2009; Varun et al. 2009; Onat and Bayar 2010; Afgan et al. 2000,
2002; Dale et al. 2013; Evans et al. 2010; Manish et al. 2006; Buytaert et al. 2011; Dimakis et al.
2011; Streimikiene 2010; Wang et al. 2009)
In addition to the conventional indicators, some specific indicators including net

energy ratio (Manish et al. 2006), energy payback time (Varun et al. 2010) have
been deployed to assess sustainability of renewable energy technologies. The
concept of net energy ratio defined as ratio of total energy output of the system to
the total non-renewable energy input is important for assessing sustainability of
renewable energy systems as it reflects total non-renewable energy consumed as
against its renewable energy generation over its complete life cycle. Its less than
one value (<1) will imply that plant consumes more non-renewable energy during
its construction, operation, and decommissioning than it generates during its life-
time and therefore may not be considered as renewable and vice versa. Similarly,
energy payback time reported by Varun et al. (2010) is defined as ratio of total
primary energy required throughout its life cycle in (GJ) to annual primary energy
generation by the system in (GJ/year). These indicators are more relevant and useful
in comparing sustainability of renewable power generation systems.
4 Sustainability Assessment Methodology
Single-criterion analysis and multi-criteria analysis are the two major assessment
methodologies reported in the literature for sustainability evaluation of power
generation systems (Afgan et al. 2000; Bhattacharyya 2012). In single-criterion
analysis, probable technology options are compared with respect to a single indicator
at a time (Evans et al. 2010; Streimikiene 2010). Mostly, researchers have used cost
of electricity generation to compare different technological options (Nouni et al.
2007; Banerjee 2006). Nouni et al. (2009) have made economic comparison con-
sidering cost of grid extension as baseline. Using single-criterion analysis may be
simple but requires high data accuracy to avoid any misinterpretation (Afgan et al.
2000). Further, single-criterion analysis neglects other relevant aspect including
impact to environment and society which becomes imperative to be considered in
sustainability evaluation studies (Bhattacharyya 2012). Multi-criteria analysis on
other hand, overcome such limitations by comparing probable alternatives using
different set of criteria and indicators. However, indicators need to be assigned
weights to quantify their relative importance towards assessing the sustainability of
alternatives. The selected indicators may be assigned the weights by either using
equal weighing methods or by rank order methods. Though, assigning weightage
using equal weighing to all indicators is a simple process, it has certain limitations of
ignoring relative importance among criteria. In rank order weighing methods,
indicators are given different weights using different subjective, objective, or mix
weighing methods. In subjective weighing methods, preference of decision makers
dominates whereas in objective weighing methods, weights are decided mathe-
matically using available data. Mix weighing method incorporates both to avoid
limitation of both the methods. A detailed description of these methods is provided
in Wang et al. (2009).
Once the weights are assigned, next step in the process is the evaluation of
different technical options using multi-criteria decision-making methods. The lit-
erature provides detailed description of several multi-criteria decision-making
methods along with their pros and cons (Pohekar and Ramachandran 2004; Wang
et al. 2009; Bhattacharyya 2012). In evaluation of different renewable energy
systems, weighted sum method (WSM) and analytical hierarchy process (AHP) are
the most widely used methods. In weighted sum methods, the product of score and
the weight of each indicator are added to obtain the overall score of individual
alternative. Among different multi-criteria decision-making methods, AHP is found
to be the most popular method as it facilitates better comparison of indicators
through pairwise comparison of evaluation criteria and alternative (Bhattacharyya
2012; Stein 2013; Wang et al. 2009). Although inclusion of weights to indicator
may be helpful in making multi-criteria analysis simple, there is always scope of
inherent subjectivity and biasness. This could be avoided to some extent by
adopting participatory approaches in deciding weights to individual indicators.
Assessing sustainability of technologies using life cycle methodology has also
been discussed in the literature (Finkbeiner et al. 2010). Pairwise comparison is the
other method of sustainability assessment in which two indicators are compared
simultaneously (Manish et al. 2006). Using this approach, Manish et al. (2006) have
assessed the sustainability of power generation systems comparing life cycle GHG
emissions and life cycle cost against net energy ratio.
5 Sustainability Assessment of Biomass Gasification

Systems
The need for sustainability assessment of bioenergy systems is very well contex-
tualized in prior studies. Most of these studies focused on biofuel generation from
biomass rather than power generation as evident from Table 1 summarizing
bioenergy sustainability assessment studies.
Evans et al. (2010) have assessed sustainability of biomass based power gener-
ation systems using data reported in the literature. However, technologies considered
in the study include biomass based power generation using combined cycle operation
mostly at MW scale. The indicators considered in the study included cost of elec-
tricity generation, efficiency, greenhouse gas emissions, availability, limitations, land
use, water use, and social impacts. The study found biomass based power generation
systems unfavorable on land, water requirement, and social impacts. This is due to
the fact that the study has assessed sustainability over complete cycle of biomass.
However, this may not be true if agriculture residues are available as byproduct.
Further, score of some of the indicator may be different for small scale biomass
gasification based power generation using reciprocating engine. For example, for
smaller scale power generation, biomass gasification has better efficiency than bio-
mass combustion based power generation technologies (Mukunda 2011).
Table 1 Summary of studies on sustainability assessment of bioenergy systems

Reference Focus of the study Remarks
Dale et al. Selection of Indicators for • More focus on indicator selection as
(2013) socio-economic sustainability of compared to assessment methodology
bioenergy systems development
McBride et al. Identification of 19 measurable • Over complete supply chain, more
(2011) indicators to assess focus is on fuel supply side than
environmental sustainability of conversion technology
bioenergy system
Buchholz et al. Identification and ranking of
(2009) sustainability criterion for
bioenergy systems based on
expert feedback
Myllyviita et al. Framework development for • Discussion on economic, ecological,
(2013) sustainability assessment of social, and cultural dimensions of
wood-based bioenergy systems sustainability
• No discussion on technical and
resource aspect
• Discussion mostly around biomass
combustion based technologies
Sadamichi et al. Development of sustainability • Sustainability assessment methodology
(2012) assessment methodology for development considering GHG
bioenergy utilization in eastern Savings, Total Value Added and
Asia Human Development Index
• Methodology used as pilot for biomass
to fuel generation case study
Bürgi (2003) To compare biomass gasification • Carry out sustainability assessment
with other CDM projects using Multi-Attributive Utility Theory
(MAUT) Methodology to make
comparison with other CDM projects
• Technology discussed is biomass
gasification in dual fuel operation
mode
• No indicators selected for net energy
analysis
• Grid supply and diesel generators are
considered as baseline
• Assessment methodology included
equal weighing of criteria
Evans et al. Sustainability assessment of • For MW scale biomass based power
(2010) biomass power generation generation technology using turbine as
systems prime-mover
• Found technology favorable on price,
efficiency, emissions, availability
• Unfavorable on high land, water usage
and social impacts
Myllyviita et al. (2013) developed framework for sustainability assessment of

wood-based bioenergy systems considering economic, ecological, social, and cul-
tural dimensions of sustainability. The framework suggested in the study
incorporates weighing of criteria to calculate aggregate sustainability index to

compare sustainability of local heat production plant, a combined heat and power
production plant, and a wood pellet processing plant. However, study does not
include technical and resource criteria to assess sustainability which may also be
critical in deciding long-term sustainability of biomass gasification based power
generation system.
Burgi (2003) have applied multi-attributive utility theory (MAUT) to assess
sustainability of biomass gasification system operating in dual fuel mode to com-
pare it with other CDM projects. In the proposed methodology, values of selected
indicators are converted to a single utility value between −1 and 1 depending upon
its performance against baseline value. Electricity generation from diesel generator
and grid electricity supply are considered as base case scenario. The proposed
methodology considered equal weighing of indicators.
6 Gaps in the Literature
Based on the literature review, key gaps identified in the prevailing sustainability
assessment methodologies for power generation systems are briefly summarized in
Fig. 3 and discussed in following section.
• As sustainability assessment includes evaluation of technology options against
multiple contrasting criteria, assessment framework should be able to bring out
various trade-offs in transparent manner. Most of the earlier studies on sustainability
assessment appear to be limiting on this front. Saunders and Pope (2013) in their
study about conceptualizing and managing trade-offs in sustainability assessment
have discussed the concept of negotiable and non-negotiable trade-offs.
• Most of the prior studies lack to provide general framework for sustainability
assessment without incorporating location effect. To further explain, several
Fig. 3 Key gaps identified in the prevailing sustainability assessment methodologies

studies comparing different renewable energy systems reports wind based power
generation as most sustainable solution which may only be true for location
having favorable wind speeds.
• Almost all the multi-criteria decision-making methods used for sustainability
assessment offers weightage to assign relative importance to different criteria.
This process of criteria weighing may be subjective and therefore may introduce
bias. Further, some of the indicators used for sustainability assessment fail to
justify weightage against their importance. For example, significant weightage
given to energy efficiency may only be justified in the case of scarce input
resources. Further, there is very limited clarity regarding consideration of time and
scale effect on sustainability assessment methodologies being reported in the
literature.
• Sustainability assessment methodologies should consider impact of one indi-
cator on other by establishing interrelationship between identified indicators.
Prevailing studies lack in establishing this interlinkage in the assessment
methodologies. For example, part load performance characteristics of technol-
ogy would affect overall efficiency thereby cost of electricity generation.
• Most of the studies carried out in bioenergy domain are more focused on sus-
tainability assessment of bioenergy to biofuel conversion with almost negligible
focus on biomass gasification based distributed power generation technologies.
The studies carried out on biomass gasification based power generation systems
are mostly focused on economic aspect without incorporating impact on envi-
ronment and society.
Considering the above mentioned identified gaps, this article proposes that a set
of suitable indicators should be identified and considered for sustainability evalu-
ation of biomass gasification systems in Indian context. Detailed description of
these indicators is provided in following section.
7 Selection of Indicators for Proposed Study
There is no common consensus related to selection of sustainability criteria and

indicators for assessing the sustainability of distributed power generation systems as
these are dependent on variety of factors including location, size, etc. (Buchholz et al.
2009). Based on the literature review and scope of present study, a total of 18 indi-
cators belonging to five dimension of sustainability are proposed as depicted in Fig. 4.
The selected indicators including mix of qualitative and quantitative parameters
represent economic, environmental, technical, resource, and social dimensions of
sustainability.
Under economic criteria, capital cost of technology (Rs/kW) and cost of elec-
tricity generation (Rs/kWh) are considered since upfront requirement of higher
capital cost for deployment of renewable energy technologies may limit developers
to invest in distributed power generation systems for rural electrification. Further,
* Qualitative indicator; # Semi-qualitative indicator
Fig. 4 Proposed criteria and indicators for sustainability assessment of biomass gasification based
distributed power generation systems
cost of electricity generation from probable distributed power generation systems

has to be at par with the paying capacity or cost of electricity generation from
reference system to make it sustainable on longer term.
The technical criteria in the proposed study encompasses life cycle net energy
analysis, hazard and safety associated with the technology, part load performance
characteristics, thermodynamic analysis, reliability, and maintenance. Prior studies
on sustainability assessment have been limited in addressing these parameters
which are found to be the reasons for limited success of many small scale dis-
tributed power generation systems installed in the field. In case of biomass gasi-
fication based power generation systems, there are maintenance related issues
necessitating requirement of trained man power for day-to-day operation. Further,
part load characteristic of technology is considered as indicator because part load
operation of technology affects overall efficiency and therefore higher cost of
electricity generation. Hazard and safety and reliability have also been considered,
as technologies facing these issues may not be sustainable on longer run.
On environmental front, life cycle GHG emission, water pollution, land pollu-
tion, air and noise pollution are considered as sustainability indicators because any
technology has to fulfill regulatory pollution norms to be acceptable. The fourth
criterion of sustainability proposed in the current study is local resource availability
without which no technology can be sustainable on longer run. Water consumption,
material consumption, land area, fuel requirement are the indicators proposed to be
used for the current study. In case of biomass gasification systems, if the biomass
fuel is not available locally, then transporting it from some other location would add
to fuel cost resulting into higher cost of electricity generation. This may adversely
affect sustainability on economic front.
Finally, for any technology to be sustainable and socially acceptable, it should
have positive impacts on society. In the present case, change in the income level,
number of jobs created, and social acceptance indicators are the indicators proposed
to be used for sustainability assessment of biomass gasification based distributed
power generation systems.
8 Summary
This paper reviews sustainability criteria and indicators along with various sus-
tainability assessment methodologies discussed in the literature for assessing sus-
tainability of power generation technologies. In case of sustainability assessment of
renewable energy systems, technical and resource criteria are also considered in
addition to economic, environment, and social criteria. Sustainability assessment
methodologies using single-criterion analysis are mostly focused on assessing the
cost of electricity generation without considering their impact on environment and
society. Single-criterion analysis is although simple but heavily depends on the
accuracy of data. Limitations of single-criterion analysis are overcome using
multi-criteria analysis which evaluates different technology alternatives considering
multiple parameters. Several methods of multi-criteria decision-making are sug-
gested in the literature. However, weighted sum method and analytical hierarchy
process (AHP) are most widely used for sustainability assessment of power gen-
eration technologies. Prevailing assessment methodologies mostly use weights to
rank importance of indicators and appear to be limited in providing general and
transparent framework for sustainability assessment of renewable energy systems.
Total of 18 indicators representing economic, technical, environment, resource, and
social aspect of sustainability are identified for assessing the sustainability of bio-
mass gasification based distributed power generation systems. The set of indicator
set identified may be used for comparing sustainability of different distributed
power generation systems in Indian context.
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Overview of Biogas Reforming
Technologies for Hydrogen Production:
Advantages and Challenges
Priyanshu Verma and Sujoy Kumar Samanta
Abstract In the past two decades, production of biogas from biomass degradation
has drawn the attention of several researchers. Biogas is produced during anaerobic
degradation of plant and animal wastes, basically consisting of higher concentra-
tions of methane (CH4), carbon dioxide (CO2), and trace amounts of hydrogen
sulfide (H2S). This biogas is an extremely potential and interesting source for the
production of hydrogen gas (H2). Hydrogen gas finds tremendous quantum of
applications as an essential raw material to meet the several H2 demands such as
high temperature fuel cell, combustion engine, petrochemical and fertilizer indus-
tries, mostly ammonia production. Traditionally, large-scale production of H2 gas
involves a thermal reforming process that uses light hydrocarbons, mainly natural
gas. Biogas which is regarded as a renewable source of methane, reduces the
excessive burden on natural gas. It can also help to reduce the greenhouse gas
emissions. However, the present methods used for biogas reforming have several
technological limitations, which may depend on the quality of biogas produced, the
conversion efficiency of the process, and specific requirements for the integration of
H2 production, purification, transportation, and application. This study reviews
several biogas reforming methods, the types of catalyst used, the advantages and
disadvantages offered by each route during the processing.
Keywords Biogas Methane Hydrogen Reforming processes Catalyst
1 Introduction
Energy crisis is one of the major problems for sustainable development. This is
magnified especially due to the increase in global population, and consequently the
increase in energy demands as well as the gradual exhaustion of natural resources.
P. Verma (&) S.K. Samanta

Department of Chemical and Biochemical Engineering,
Indian Institute of Technology, Patna 801103, India
e-mail: priyanshuvermamickey@gmail.com

DOI 10.1007/978-81-322-2773-1_17
228 P. Verma and S.K. Samanta
These challenging issues are enforcing us to go for several alternative energy

sources that are sustainable in nature. Since the major energy sources used at
present are derived from unsustainable fossil resources, it is a better alternative way
to utilize renewable energy sources for sustainable development (Deublein and
Steinhauser 2008).
Presently, biogas is one of the main categories of renewable energy sources.
Biogas is a mixture of different gases produced by the anaerobic digestion or
fermentation of organic matter in the presence of microorganisms. It is primarily
composed of methane (CH4) and carbon dioxide (CO2), combined with traces of
other gases such as ammonia (NH3), hydrogen (H2), nitrogen (N2), oxygen (O2),
hydrogen sulfide (H2S), and water vapors (H2O). Chemical composition of biogas
has been shown in Table 1. It is generally produced in biowaste digesters, landfills,
and sewage sludge that include animal wastes, agricultural wastes, animal dung,
energy crop, etc. H2S is the most undesirable component of biogas. Due to its
toxicity and corrosiveness, it used to damage the vessels, tubes, and pipe fittings
involved in the overall processes (Chattanathan et al. 2014). CO2 and water vapors
also act as impurities since it reduces the energy value of biogas (Horikawa et al.
2004). An ideal energy source must be cheap, clean, renewable, and sustainable in
nature and biogas is one of them. However, CH4 and CO2, the main components of
biogas, are the major greenhouse gases, which upon release into the atmosphere
results in unfavorable conditions causing global warming. To overcome this
problem, it must be transformed or converted into some efficient and clean fuel such
as H2 (Deublein and Steinhauser 2008; Gupta 2009).
Hydrogen has the highest energy content per unit mass (140 MJ/kg) in com-
parison to any other fuel. In addition, it is a clean fuel that does not leave any
residue like ash after combustion. The comparable properties of hydrogen and other
fuels have been shown in Table 2.
Traditionally, H2 has been widely used in chemical industries, hydrogenation
process, food processing, pharmaceuticals, and fertilizer industries (mainly for
ammonia production) (Armor 1999). Recently, hydrogen has been heavily used in
fuel cells that directly convert chemical energy into electrical energy without any
involvement of mechanical operations (Hotza and Da Costa 2008). In recent
studies, these fuel cells have achieved exceptional progress in the form of
Table 1 Chemical Compound Formula Percentage

composition of biogas
(Deublein and Steinhauser Methane CH4 55–70 vol.%
2008; Gupta 2009) Carbon dioxide CO2 30–45 vol.%
Hydrogen sulfide H2S 500–4000 ppm
Ammonia NH3 100–800 ppm
Hydrogen H2 ˂1 vol.%
Nitrogen N2 ˂1 vol.%
Oxygen O2 ˂1 vol.%
Water vapor H2O ˂1 vol.%
Table 2 Comparative properties of hydrogen and other fuels (Gupta 2009)
Properties Units Hydrogen Methane Propane Methanol Ethanol Gasolineb
Chemical formula H2 CH4 C3H8 CH3OH C2H5OH CxHy (x = 4−12)
Molecular weight 2.02 16.04 44.1 32.04 46.07 100–105
Density (NTP)a kg/m3 0.08375 0.6682 1.865 791 789 751
Viscosity (NTP)a Pa s 8.81 × 10−4 1.10 × 10−3 8.01 × 10−4 9.18 × 10−2 0.119 0.037–0.044
Normal boiling point °C −252.8 −161.5 −42.1 64.5 78.5 27–225
Overview of Biogas Reforming Technologies …
Flash point °C <−253 −188 −104 11 13 –43

Flammability range in air vol.% 4.0–75.0 5.0–15.0 2.1–10.1 6.7–36.0 4.3–19 1.4–7.6
Auto ignition temperature in air °C 585 540 490 385 423 230–480
Higher heating value (at 25 °C and 1 atm) MJ/kg 141.86 55.53 50.36 19.96 29.84 47.50
Lower heating value (at 25 °C and 1 atm) MJ/kg 119.93 50.02 45.60 18.05 26.95 44.50
a
NTP = 20 °C and 1 atm
b
Properties of a range of commercial grades
229
transportation, power generation, and installations (Alves et al. 2013). This system
will facilitate many applications such as massive power generation and distribution,
domestic employment of generators, better portability of electronic devices due to
small size, and an auxiliary power source for vehicles. The conversion efficiency of
fuel cells is found to be around 60 % (Faghri and Guo 2005; Chen et al. 2007),
which is about two to three times more than that of the device presently employed
in combustion vehicles (i.e., only about 20–30 %) (Alves et al. 2013). These special
features make hydrogen energy as a superior alternative over all the alternative fuel
categories available to compliment/substitute fossil fuels.
Presently, steam reforming of fossil fuels has been the most frequently used
method of H2 production which generally requires natural gas, naphtha, and other
lighter hydrocarbons as raw materials (Holladay et al. 2009). Biogas reforming for
H2 production is an appealing and promising technique to convert biogas into H2,
especially due to the reduction in emission of greenhouse gases and its reliability
(Herle et al. 2004; Hotza and Da Costa 2008; Levin and Chahine 2010; Papadias
et al. 2012). Another advantage of using biogas as a feedstock for H2 production is
its availability (mostly local) which reduces the transportation costs. Therefore,
utilization of biogas in reforming process has better environmental and economical
advantages (Rand and Dell 2008). Three major techniques used for conversion
of CH4 present in biogas to H2 are steam reforming, dry reforming, and par-
tial oxidation that can be used for the production of liquefied hydrogen (Galvagno
et al. 2013).
Basically, biogas reforming involves CO2 reforming as well as dry reforming of
CH4. Since this process requires the stripping of oxygen from carbon dioxide and
stripping of hydrogen from methane, there are significant chances of coke deposition
on the catalyst surface. That is why most of the reforming studies have been based on
the broad and intense analysis of different catalysts. It used to minimize the costs and
the total energy involved in the complete process (Muradov et al. 2008).
The development, modification, and utilization of several catalysts having a
higher catalytic activity and stability in reforming processes can control the higher
temperature needs and increase the reaction rate. In addition, it can also slower the
carbon deposition rate and the poisoning of the catalyst, which are the major
problems encountered during reforming processes (Alves et al. 2013).
The present study is focused on the limitations of several biogas reforming tech-
nologies used for the production of hydrogen gas. Furthermore, this review also high-
lights the advanced possibilities of H2 production using biogas as a renewable resource.
2 Several Hydrogen Production Processes from Methane

or Biogas
The most practiced methane/biogas reforming processes for hydrogen production

are steam reforming (SR), dry reforming (DR), partial oxidation reforming (POR),
auto-thermal reforming (ATR), and dry oxidation reforming (DOR). There are
Overview of Biogas Reforming Technologies … 231
some other unconventional processes for the production of H2 from methane such
as thermal plasma reforming, catalytic decomposition, and solar reforming. Several
industries uses SR, POR, and DR methods for the large-scale production of H2 that
uses natural gas (≈95 % of CH4) or naphtha as major sources of hydrocarbons
(Shiga 1998; Alves et al. 2013).
Most of the studies based on biogas reforming to produce H2 reported several
exhaustive analyses using mixtures of CH4 and CO2 to simulate laboratory biogas
compositions. Even some studies also used the high purity CH4 (>99 %) for lab
scale reforming processes. Only a few studies reported usage of actual biogas,
directly produced from the anaerobic digestion of residual biomass (real biogas), in
their experiments (Araki et al. 2009, 2010). Thus, to settle on the reforming process
using biogas, its composition must be measured and broadly classified into three
different situations: (i) in actual form, with 55–70 % of CH4, 30–45 % of CO2 and
500–4000 ppm of H2S (Table 1); (ii) partially treated for H2S removal; and
(iii) purified for “bio-methane” enrichment (93–96 % of CH4, 4–7 % of CO2
and <20 ppm of H2S) (Alves et al. 2013). It is investigated that, after these three
sequential steps, the probability of CH4 content higher than 95 % could be very less
using economically feasible treatment procedures (Effendi et al. 2005; Kolbitsch
et al. 2008; Ohkubo et al. 2010; Lau et al. 2011).
Prior to the reforming process, pretreatment of biogas is required for the removal
of corrosive or poisonous species present in biogas stream that can be conducted by
two different methods: (i) physicochemical treatment (including absorption in
aqueous solutions, chemical adsorption of H2S on solid adsorbents such as zeolites,
modified zeolites, activated carbon, and specific metal surfaces with the formation
of metal sulfide and scrubbing with specific solvents); and (ii) biological treatment
(removal of impurities by living microorganisms such as species of chemotrophic
fungi and bacteria which used to act as oxidants in the bio-trickling filter, sulfur
bio-filter, and bioaugmentation unit that can convert impurities into lighter and less
harmful forms) (Holladay et al. 2009; Alves et al. 2013).
Generally, reforming processes for H2 production are performed under low
pressure (mostly below 1 atm) in tubular fixed-bed or fluidized-bed reactors, in a
wide range of temperature 600–1000 °C (endothermic and reversible reactions)
(Goransson et al. 2011; Alves et al. 2013). It also involves predominant catalytic
reactions that are mostly combined (see Table 3).
These reforming processes mainly use natural gas as a raw material (methane
source). However, the biogas also can be used as a methane source in various
reforming processes (based on its composition and purity level). That is why most
of the researches involving methane reforming can also be adapted or modified for
biogas reforming (Galvagno et al. 2013). Name of chemical reactions are cited and
their respective enthalpy of reaction at 298 K (25 °C) has also been shown in
Table 3.
Table 3 Chemical reaction involved in reforming processes (Alves et al. 2013)

Reaction Name of Type of reaction ΔH298 K
Eqs. reaction (kJ/mol)
Equation 1 SR CH4 + H2O → CO + 3H2 206.2
Equation 2 Gas-shift CO + H2O → CO2 + H2 −41.2
reaction
Equation 3 Combined CH4 + 2H2O → CO2 + 4H2 165
reaction
Equation 4 Methane CH4 → C + 2H2 74.9
cracking
Equation 5 Boudouard 2CO → C + CO2 −172.4
reaction
Equation 6 Reduction of CO + H2 → C + H2O −131.3
CO
Equation 7 POR CH4 + ½ O2 → CO + 2H2 −35.6
Equation 8 Complete CH4 + 2O2 → CO2 + 2H2O −801.7
oxidation
Equation 9 ATR CH4 + ½ x O2 + yCO2 + (1 − x − y) H2O → (y + 1) ≈0
CO + (3 − x − y) H2
Equation 10 DR CH4 + CO2 → 2CO + 2H2 247.4
Equation 11 DOR CH4 + βCO2 + (1 − β)/2 O2 → (1 + β)CO + 2H2 (285β − 38)
0≤β≤1
3 Overview of Different Conventional Reforming

Processes
3.1 Steam Reforming (SR)
In SR, methane reacts with water vapors in the presence of a suitable catalyst that
produces CO and H2 (Eq. 1, Table 3). The reaction is highly endothermic in nature
and requires temperatures in the range of 650–850 °C, to achieve maximum H2
yield (between 60 and 70 %). SR requires substantial energy expenditure and
despite that it is the most used industrial methods to produce H2. In SR, the H2/CO
ratio is three that results in higher productivity of hydrogen. In order to remove CO,
the water-gas shift reaction (also known as “Shift reaction”) is employed (Eq. 2,
Table 3) that requires temperature in the order of 300–450 °C and specific catalysts
based on Cu, Fe, Mo, and Fe–Pd alloys that increases the production of additional
hydrogen (Effendi et al. 2005; Kolbitsch et al. 2008). The reaction of methane SR
associated with the shift reaction (Combined reaction) has been shown in Eq. 3
(Table 3).
These severe conditions required for SR used to promote the parallel carbon
formation reactions such as methane decomposition reaction (Eqs. 4, Table 3),
Boudouard reaction (Eq. 5, Table 3), and disproportionation reduction reaction of
CO (Eq. 6, Table 3). It also promotes the carbon deposition on the catalyst surface
causing deactivation of it. If the carbon deposited on catalyst surface is in the form
of nanotubes then that is advantageous since that lowers the carbon dispersed over
the catalyst surface and preserves the catalyst activity for a longer duration. The
catalysts typically employed in the SR process could be made of either transition
metals (such as Pt, Ni) or noble metals (such as Rh, Pd). Among these, the Ni has
the lower cost advantage. On the contrary, Ni has the greater deactivation sus-
ceptibility by the coke formation (due to the high temperature processing) that
makes the Pd and Pt catalysts more fascinating with regards to the stability of the
catalyst. In case of limited mass transfer, Rh is employed as it has a better catalytic
activity which is much better than that of Ni and offers lower coke formation
(Iulianelli et al. 2010; Zhai et al. 2011). Basic supports containing promoter ele-
ments such as Mg, Ca, and K can also be utilized to minimize the carbon deposition
on the catalyst surface, since they promote the gasification of carbon species by the
carbon–water steam reaction (reverse of Eq. 6, Table 3), due to the increased
adsorption of water. The referred metals (Ni, Pt, Rh, Ru, etc.) and matrices (such as
SiO2, ZrO2, Al2O3, etc.) can be used for the preparation of active sites in support
catalysts (Amin et al. 2012; Roh et al. 2012; Alves et al. 2013).
To obtain highly pure H2 gas, the CO2 and CO formed in SR process are
required to be effectively separated from the syngas. Similarly, these species (CO2
and/or CO) are required to be separated for other processes as well (POR, ATR,
DR, DOR). Typically, the H2 production unit of the SR process comprises of three
integrated sections namely reformer (2 or more), conversion reactor (shift reaction)
and a separator.
Some of the studies show significant improvement in the separation process with
the application of selective membrane filters or reactors (Mahecha-Botero et al.
2009). The membrane reactor permits all reactions to occur in a single vessel. In the
membrane system, SR methane reaction and shift reaction (Eqs. 1 and 2 respec-
tively, Table 3) occur simultaneously within the reactor that contains a catalyst bed.
In addition, membrane provides better ability to shift the chemical equilibrium of
the reaction that increases the production of hydrogen. The shifting of chemical
equilibrium enables the reforming reaction to be carried out at much lower tem-
peratures (below 500 °C) without any specific conversion loss (Lin et al. 2003; Lu
et al. 2007; Sato et al. 2010).
3.2 Partial Oxidation Reforming (POR)
Partial oxidation reforming (POR) is one of the best alternative processes to pro-
duce H2 with reduced energy costs due to its moderately exothermic nature (Eq. 7,
Table 3). This is contrary to the SR process which is highly endothermic in nature.
In POR process, methane is partially oxidized with O2 at the atmospheric pressure
and CO and H2 (collectively called “syngas or synthesis gas”) are produced. It
requires temperatures in the range of 700–900 °C that reduces the coke formation
and ensures the complete conversion of methane (H2/CO ratio is two). In case of
slight decrease in CO selectivity, the methane reacts with oxygen and produces CO2
(Eq. 8, Table 3) that promotes the complete combustion and results in a sudden
increase in temperature. That causes the formation of hot spots in the reactor bed
and deposit coke on the catalyst surface (Alves et al. 2013).
Some researchers have focused on the development of highly active and stable
catalyst for biogas POR. Some catalysts such as solid solutions of Ca–Sr–Ti–Ni,
NiO–MgO, Ni–Mg–Cr–La–O, and mixed metal oxides are reported to be highly
selective as well as active catalysts at elevated temperature ranges (above 800 °C)
with lower coke formation (Ruckenstein and Hu 1999; Corbo and Migliardini 2007;
Rogatis et al. 2009).
3.3 Auto-Thermal Reforming (ATR)
ATR is an intensive endothermic process. That is why it requires internal heating of

the reactor, as compared to the external heating for efficient and economical
hydrogen production. Although, the POR of CH4 has the advantage over ATR since
it is exothermic (Eq. 7, Table 3) in nature, it generally offers lower H2/CO ratio than
that offered by ATR. ATR is the combination of already discussed reforming
technologies (SR and POR) that results in the sum of reactions described by Eqs. 3,
7 and 8 in Table 3 (Souza and Schmal 2005; Cai et al. 2006; Simeone et al. 2008). It
also takes place in the presence of carbon dioxide as shown in Eq. 9 in Table 3
(Araki et al. 2009, 2010). ATR offers several advantages such as it speeds up the
processing of reactor, increases H2/O2 ratio in comparison to the POR. Moreover,
the combination of SR and POR improves the control over temperature during the
processing and consequently the formation of hot-spots is also reduced that inhibits
the catalyst deactivation.
In ATR process, methane partial oxidation reaction occurs in parallel with the
methane steam reforming that makes the overall process self-sustainable and less
energy expensive. That is why most of the researchers favor adiabatic SR of biogas
under auto-thermal condition and in combination with the partial oxidation reaction.
It produces syngas with a H2/CO ratio between 2.0 and 3.5, using O2/CH4 and
H2O/CH4 ratios between 0.25–0.55 and 1.0–2.5, respectively (Cai et al. 2006;
Mosayebi et al. 2012). It is also observed that the temperature of the different
oxidation zones can affect the product composition selectivity. In this process, the
total oxidation reaction (Eq. 8, Table 3) is favored by reducing the temperature,
whereas partial oxidation reaction (Eq. 7, Table 3) is favored by increase in tem-
perature (Galvagno et al. 2013).
3.4 Dry Reforming (DR)
DR is a process in which CH4 present in biogas reacts with CO2 and produce H2
and CO (Eq. 10, Table 3). This reaction is very appealing as per environmental
views, since it utilizes CH4 and CO2 as feed (which are potent greenhouse gases).
However, the researchers observed that due to the endothermic nature of the
reaction involved in the DR process, it requires a large amount of energy input in
the form of heat, and production of that required heat increases the CO2 emissions
(due to the burning of other fossil fuels). For industrial application of biogas DR, it
must have to satisfy the requirements of several manufacturing processes of liquid
hydrocarbons and oxygenated compounds to efficiently produce syngas yielding a
H2/CO ratio *1 (Eltejaei et al. 2012).
Based on earlier findings, the major reaction (Eq. 10, Table 3) can be completed
by competing with the other parallel reactions that shift the equilibrium conversion
of CO2 in CH4. These reactions are reverse gas–water shift (Eq. 2 reverse),
Boudouard reaction (decomposition of carbon monoxide, Eq. 5 in Table 3), and
decomposition of CH4 (Eq. 4, Table 3). DR process uses a CH4/CO2 ratio between
1 and 1.5 and it mainly occurs at temperatures between 700 and 900 °C (Alves et al.
2013). This process gives around 50 % H2 yield (Serrano-Lotina et al. 2012;
Eltejaei et al. 2012; Lucredio et al. 2012).
In DR process, if the carbon removal is slower than the methane decomposition
(Eq. 4, Table 3) rate then a serious problem of coke formation arises that results in
the deactivation of the catalyst and blockage of the reactor and pipes due to the coke
accumulation. Low temperature favors the Boudouard reaction (decomposition of
CO, Eq. 5 in Table 3) and it produces carbon. Since, this reaction is exothermic in
nature, it promotes the methane cracking reaction also (Eq. 4, Table 3), which
produces an additional amount of carbons. Therefore, the coke formation step is
highlighted as the major drawback of DR process and it also affects the active
surface of the catalyst. Therefore, several studies have been carried out for the
development of suitable catalysts that can prevent the carbon deposition, be highly
selective in nature and thermally stable during the production of H2 (Barrai et al.
2007; Eltejaei et al. 2012).
Several metals as the most active catalysts have been reported in the recent
studies that belong to the groups 8, 9, and 10 of the Periodic Table, such as Rh, Ru,
and Pt. However, these catalysts are not suitable for industrial applications as they
are very expensive and limited in nature. Therefore, other catalysts have been
developed such as Co and Ni catalysts with lower price and greater availability that
have higher coke susceptibility as well (San-Jose-Alonso et al. 2009; Xu et al.
2009; Bereketidou and Goula 2012).
3.5 Dry Oxidation Reforming (DOR)
DOR is a specific method to control the carbon deposition on the surface of the
catalysts. It is the combination of dry reforming and partial oxidation reforming,
using a parallel feeding of oxygen with methane and CO2. DOR offers some
additional advantages such as improved methane conversion, reduced total energy
involved in the process, increased deactivation resistance of the catalyst, enhanced
catalyst stability, and increased hydrogen yield at lower temperature. The H2/CO
ratio could be controlled with the modulation of oxygen to meet the flow
requirements (Lau et al. 2011). CH4–DOR reaction has been shown in Eq. 11
(Table 3), where β is the stoichiometric fraction of CO2 fed with traditional DR. It is
clearly understood that the theoretical H2/CO ratio, given by 2/(1 + β), is greater for
DOR process for β < 1 than that for non-oxidative DR process (β = 1) (Alves et al.
2013).
In CH4–DOR process, changing of the process variables such as reaction tem-
perature and/or relative concentration of oxygen feed helps to control the H2/CO
ratio and the nature of the reaction (endothermic or exothermic). Basically, the
exothermic nature is increased with the increase in feed O2 concentration at a given
temperature. Because the addition of oxygen determines the oxidation process
(complete oxidation occurs when β = 0, Eqs. 7 and 8; or traditional DR reaction
occurs when β = 1, Eq. 10, Table 3) and accordingly, the amount of heat consumed
or released. It may be noted that, POR process (Eq. 7, Table 3) is exothermic,
whereas traditional DR process is endothermic (Eq. 10, Table 3) in nature. Hence,
DOR process could be utilized as a better alternative over traditional DR process
using different degrees of oxygen input. Consequently, biogas can be converted to
syngas at higher energy efficiency using less external energy (Effendi et al. 2002;
Avraam et al. 2010).
Studies of different catalysts and their association with other metals such as
bimetallic catalyst, have been used to enhance the stability of the catalysts (Xu et al.
2009; Lau et al. 2011). In this process, the addition of oxygen reduces the coke
formation on the catalyst surface due to the better control over the partial oxidation
of the novel carbon species, CH4 and CO2 for CO formation (Lucredio et al. 2012;
Alves et al. 2013). Different H2 production routes and reaction conditions using
methane, biogas, or real biogas as a major substrate are summarized in Table 4.
The above mentioned reforming technologies have proven their effectiveness in
the form of better conversion efficiency of biogas/methane (XCH4 ) into a useful
hydrogen gas. The technical comparisons of these biogas reforming methods are
summarized in Table 5.
4 Current Status of Biogas Reforming Technologies
Biogas, being a renewable and abundant resource, has attracted a large number of
researchers to study the particular field and utilize this resource better for the
production and development of clean fuel such as hydrogen. Their investigations
are primarily focused on the efficient removal of H2S which upgrades the biogas for
further processing (Tippayawong and Thanompongchart 2010; Micoli et al. 2014;
Sisani et al. 2014; Díaz et al. 2015; Liu et al. 2015).
Xu et al. (2004) developed a model for polymer electrolyte fuel cell (PEFC)
usable H2 gas production from desulfurized biogas produced through anaerobic
Table 4 A summary of studies based on H2 production from methane or biogas

Process Reactor T (°C) Catalyst H2/CO XCH4 (%) References
SRa Fixed-bed 700 Ni/Al2O3 – 85 Maluf and Assaf 2009
SRa Fixed-bed 525 Rh/CeαZr1−αO2 – 79 Halabi et al. 2010
SRb Fluidized-bed 850 Ni/Al2O3 2.1 98 Effendi et al. 2005
SRb Fixed-bed 715 Ru/Al2O3 2.7 90 Avraam et al. 2010
SRb Fixed-bed 750 Ni/CaO–Al2O3 2.5 95 Kolbitsch et al. 2008
SRb Fixed-bed 750 Ni/Al2O3 2.0 85 Effendi et al. 2002
SRb Fluidized-bed 750 Ni/Al2O3 2.2 96 Effendi et al. 2002
PORa Fixed-bed 800 Pt/CeO2 2.0 85 Corbo and Migliardini
2007
PORa Fixed-bed 850 NiO/MgO 2.0 87 Ruckenstein and Hu
1999
PORa Fixed-bed 700 Ni/Al2O3 2.0 100 Rogatis et al. 2009
ATRa Fixed-bed 700 Ni/MgAl2O4 3.2 92 Mosayebi et al. 2012
ATRa Fixed-bed 800 Pt/ZrO2/Al2O3 2.0 100 Souza and Schmal
et al. 2005
ATRa Fixed-bed 700 Rh/Al2O3 3.5 95 Simeone et al. 2008
ATRa Fixed-bed 750 Ni/Cu5Zr10Ce20Al65O8 3.9 100 Cai et al. 2006
ATRc Fixed-bed 750 Ni/Cordierite 2.6 90 Araki et al. 2010
ATRc Fixed-bed 850 Ni/Insulating (Si, Mg, 2.8 95 Araki et al. 2009
Al)
ATRb Fixed-bed 800 Ni/SBA-15 (molecular 1.4 92 Barrai et al. 2007
sieve)
DRb Fixed-bed 860 Ni/CeO2–Al2O3 1.3 90 Bereketidou and
Goula 2012
DRb Fixed-bed 750 Rh/NiLa/γ–Al2O3 0.9 70 Lucredio et al. 2012
DRb Fixed-bed 900 Ni–Co/La2O3/Al2O3 1.01 88 Xu et al. 2010
DRb Fixed-bed 800 Reformax®-250 0.98 67 Chattanathan et al.
2014
DRb Fixed-bed 850 Ni/Y (zeolite) 0.9 97 Sharifi et al. 2014
DORb Fixed-bed 750 Rh-NiLa/γ–Al2O3 1.0 86 Lucredio et al. 2012
DORb Fluidized-bed 900 Pt–Rh/Ce–ZrO2– 1.0 100 Lau et al. 2011
Al2O3
a
Methane
b
Synthetic biogas
c
Real biogas
—Not reported
digestion. They used a gas producer, consisting of a steam reformer, two water-gas
shift reactors at high and low reaction temperatures, respectively, and a selective
CO oxidizer. The authors obtained that H2-rich gas contained usually 70 % H2,
30 % CO2, and residual CH4.
Purwanto and Akiyama (2006) studied H2 production using hot slag in a packed
bed reactor. They used continuous flow of CH4 and CO2 (biogas) into the packed
bed of hot slag at a constant flow-rate and atmospheric pressure. The authors
Table 5 Comparison of biogas reforming methods (Yang et al. 2014)

Methods Advantages Challenges Remarks
SR Produces highly pure Needs to remove H2S and add It is widely
hydrogen; low carbon oxidizing agents; high used for
formation operating temperature and hydrogen
energy demand; catalyst can production
be expensive
POR High energy efficiency; May completely oxidize It can be
relatively low operating methane to CO2 and H2O; combined with
temperature limited industrial application other
reforming
methods
ATR Produces high-purity Complex process control; It can use both
hydrogen fuel; high energy needs multiple catalysts; CH4 and CO2
efficiency relatively unstable in biogas
DR High conversion efficiency Carbon formation; moderate It can also use
selectivity; side reaction CH4 and CO2
consumes hydrogen; high in biogas
operating temperature and
energy demand; catalyst can
be expensive
DOR Less carbon deposition; Complex process control; It is the
reduced overall energy produce higher CO2 combination of
requirement; better DR and POR
conversion efficiency
observed that slag acted as not only a thermal media, but also a good catalyst, for
better decomposition. The product gases were mainly H2 and CO with/without solid
C deposition on the surface of slag that depends on the reaction temperature.
Increased temperature led to the large hydrogen generation with the higher methane
conversion (about 96 %).
Chun et al. (2008) examined the reforming characteristics and optimum oper-
ating conditions for the hydrogen production through the plasmatron-assisted CH4-
reforming process. The authors also conducted some studies related to methane
conversion rate and parametric screening to increase the productivity. They
obtained the optimal methane conversion rate as 99.2 %, H2/CO ratio as 6.6,
thermal efficiency of the reformer as 63.6 %, and the H2 yield as 78.8 %.
Bensaid et al. (2010) investigated the applicability of molten carbonate fuel cells
(MCFCs) for power generation from landfill biogas, which was also coupled with
the in situ production of hydrogen. They had performed the system modeling of the
plant in steady state conditions, with the aim of assessing the overall power effi-
ciency. Due to the exothermic nature of MCFCs (working temperature was around
650 °C), it showed compatibility to the steam reforming for the production of
syngas. Moreover, they utilized the high temperature flue gases from the MCFC for
power generation through the turbine. The authors reported that hydrogen was
produced through a pressure swing adsorption system, whose feed could be from
either the MCFC anode outlet or a split of the reformate before the anode inlet. The
overall net power efficiency of the system was found almost similar for both
mechanisms, 56 and 55 %, respectively.
Lin et al. (2012) proposed a distinct method to produce hydrogen from biogas
reforming using mesoporous Ni2xCe1−xO2 catalysts. These catalysts were prepared
through reverse precipitation method. The authors obtained the higher H2/CO ratio
and the lower H2/CO2 ratio than commercial catalyst in the steam reforming
reaction with temperature range 500–900 °C. They also observed that the methane
conversion was increased with increase in nickel content in all experiments.
Rueangjitt et al. (2012) assessed the possibility of upgradation of biogas through
multi-staged AC (Alternating current) gliding arc system that could be further
utilized for production of H2 gas. They found that sudden increment in the stage
numbers of plasma reactors would increase both CH4 and CO2 conversions. The
combination of the plasma reforming and partial oxidation provided an exceptional
improvement to the overall process performance achieved by the authors.
Meyer et al. (2014) proposed a novel sorption enhanced reforming
(SER) technology which could also capture CO2. This technology has the potential
to produce renewable hydrogen and also provide bio-CO2 that can be used for the
production of biomethane through the Sabatier process. The authors designed and
simulated a small SER prototype using a dual bubbling fluidized-bed reactor system
with the help of advanced modeling and simulation tools.
Italiano et al. (2015) studied the synthesis of nanocrystalline Ni/CeO2 catalysts
for the biogas oxy-steam-reforming (OSR) used for the production of hydrogen.
The study was mainly focused on the characterization of newly synthesized cata-
lyst. In addition, they also observed that the OSR of biogas at 800 °C with the
newly synthesized catalyst offered high H2 yield (above 85 %).
There are also many biological processes that have been studied for the
hydrogen production such as enzymatic hydrogen production and bio-hydrogen
production through bio-abundant polysaccharides (Kovács et al. 2004; Redwood
et al. 2009; Chang et al. 2012; Esquivel-Elizondo et al. 2014). Fermentation is the
integral part of biohydrogen production that can be easily operated and the process
is eco-friendly too (Wongtanet et al. 2007). However, low bio-H2 production yield
and slow processing rate have been reported as its main drawbacks. To overcome
these limitations several strategies have been followed in the past such as genetic
modifications and adjusting the reaction conditions. Zhang et al. (2007) proposed
an unnatural enzymatic pathway which could produce 12 mol of hydrogen per mole
of glucose unit of polysaccharides and water. In this process, the energy required
for splitting of the water molecule was obtained from carbohydrates.
5 Future Research Directions
For the broader hydrogen production through biogas reforming technologies, pro-
gressive efforts are needed to a greater extent. Based on a substantial literature
survey and recommendation or suggestion of several researchers, the following
directions for potential research routes may be recommended:
• Proper design and development of process and reactor used for biogas reforming
provides the efficient production of hydrogen with less overall energy expen-
diture. A scale-up study of the advanced processes is required to estimate the
return on the investment and operating costs of an industrial-scale production
plant.
• Operating parameters such as catalyst characteristics and throughput, optimum
temperature, presence of impurities and its concentration have been found to
determine the limit to which an effective hydrogen production is achieved.
• CFD modeling studies of advanced and combined biogas reforming technolo-
gies are still open areas of research that could be utilized to validate the existing
experimental results.
• Some researchers favor biological methods of hydrogen production over
chemical methods because of the possibility to use sunlight, CO2, and organic
wastes as substrates for environmentally benign conversions under moderate
conditions that require more attention.
6 Conclusion
Biogas reforming is a promising process to produce green hydrogen as well as to

reduce the overburden on natural gas. The major problems raised in the biogas
reforming processes, reviewed by most of the researchers, are related to coke
formation on the catalyst surface and the poisoning due to sulfur, which may cause
the deactivation of the catalyst and reduce the H2 production. Mostly, Ni-based
catalysts are used in different reforming processes. A large amount of research
works is focused on the evaluation of effectiveness of the catalyst support and the
addition of promoter elements to encounter the coke formation issues. Several
studies show the importance of biogas purification to eliminate or reduce the cor-
rosive species such as H2S. This step plays a vital role in the maximization of
productivity and durability of the process and reduces the maintenance cost. The
physicochemical methods including chemical adsorption and absorption processes
have been used substantially, and several routes involving biological degradation
with the help of living microorganisms (that consume contaminants) such as bac-
teria and fungi are also highlighted.
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doi:10.1371/journal.pone.0000456
Modifications in Improved Cookstove
for Efficient Design
Amit Ranjan Verma, Rajendra Prasad, Virendra Kumar Vijay

and Ratnesh Tiwari
Abstract In India, majority of the population (around 85 %) living in rural areas

still use traditional biomass fuels in form of wood, agriculture residues, animal
dung, etc., for meeting their energy needs. These fuels are burnt in mud (traditional)
cookstoves for cooking and other thermal applications. Inefficient burning of bio-
mass in traditional cookstoves poses serious health hazards. Experiments have been
carried out on low cost, improved forced draft cookstove, and modifications were
made to increase the thermal efficiency and to lower smoke emissions by varying
parameters such as height of grate from base, variations in air flow rate, height
between top plate and vessel, etc., without making major changes in the structure of
cookstove such as diameter and air inlet points. Modification in air flow rate leads
to increased thermal efficiency with low smoke emissions. All measurements were
made according to the Bureau of Indian Standards (IS 13152: Part 1-Portable Solid
Bio-Mass Cookstove (Chulha).

Keywords Cookstove Improved cookstove Thermal efficiency Forced draft

cookstove Bureau of Indian Standards
1 Introduction
Biomass has been a key resource for human being in meeting the fuel requirement
for cooking and space heating since ancient time. Though the gaseous fuels, i.e.,
LPG and natural gas have replaced biomass in developed countries and most of
urban homes in the developing world, half of the world’s population and about
90 % of rural households in developing countries are primarily dependent on coal
or biomass, such as wood, crop residues, cattle dung, and charcoal for their cooking
and heating needs (WRI 1998–1999). According to National Census 2011, 67 % of
A.R. Verma (&) Rajendra Prasad V.K. Vijay Ratnesh Tiwari

Improved Biomass Cookstove Laboratory, Centre for Rural Development and Technology,
Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
e-mail: arviitd@gmail.com

DOI 10.1007/978-81-322-2773-1_18
246 A.R. Verma et al.
all households in India used solid fuels, mostly biomass. Firewood, crop residue,
and cow dung cake (all biomass fuels) have a penetration of 62.5, 12.3, and 10.9 %,
respectively. LPG penetration in rural households stands at 11.4 % followed by
kerosene, coal/charcoal, and electricity at 0.7, 2.9, and 0.1 %, respectively (Indian
Census 2011).
The majority of household energy requirement is in various cooking needs.
Traditional cookstoves or chulhas, having overall thermal efficiencies less than
10 % are generally practiced in rural households in the developing countries, are
main sources of large quantities of pollutants resulting various health hazards.
Large quantity of biomass residues such as agricultural residues, animal dung,
forest waste, firewood, etc., is available in India. The biomass is considered as
sustainable and carbon-neutral source of energy (Kothari et al. 2008). Moreover, the
use of biomass feedstocks in an inefficient traditional cookstoves leads to genera-
tion of increased indoor air pollution and wastage of stored chemical energy in
biomass. These less efficient traditional cookstoves consume large amount of
fuelwood and create pressure on the region’s forest cover (Bhattacharya and Leon
2005). To increase the performance of cookstove several modifications in cook-
stove have been carried out by many researchers. Galitsky (2006) made several
design modifications in existing design of Technology and Action for Rural
Advancement (TARA) cookstove to improve stove performance in windy weather
operating condition. These modifications included a windshield around the upper
stove body and addition of a metal ring to prevent direct air flow through the stove
body and to provide stability; stakes were also incorporated in the design. These
modifications were incorporated in a prototype of cookstove called as
Berkeley-Darfur Stove (BDS). BDS cookstove was found to consume less than
60 % of the fuelwood with thermal efficiency of 43 % and produced noticeably
lower smoke compared to the three-stone fire. The time needed for cooking using
the BDS cookstove was about the same as that needed over the three-stone fire.
They had also modified locally available mud-and-dung “ITDG” cookstove, called
as the modified stove “Avi”. They also incorporated cast iron grate placed over the
opening cut out of the bottom, which improved the combustion efficiency and
reduced smoke level. The new design also included vertical ventilation channels
carved into the inner walls of the stove and three mud knobs were added to the top
to permit enhanced combustion air flow even when a tight-fitting large pot or a flat
metal plate is being used for cooking. However, the experimental results were not
found good enough, as the cookstove was consuming about 90 % of the fuel
compared to the three-stone fire cookstove, and smoke emission was found sig-
nificantly higher. Furthermore, the cookstove took more time to cook the food than
the three-stone fire. Guillaume and LaRue (1995) experimentally verified that the
swirl effect improved the combustion of volatiles by increasing air–fuel mixing and
the residence time of the reactant gasses. Bhandari et al. (1988) incorporated four
different types of swirling device in the throat area of cookstove combustion
chamber and found that the strong swirling effect had increased the cookstove’s
efficiency to about 42 %.
Modifications in Improved Cookstove for Efficient Design 247
This paper presents the studies on the parameters of existing force draft biomass
cookstove and necessary modifications required in design variables which were
improved the performance of biomass cookstove in terms of power output, thermal
efficiency, and emission level.
2.1 Parametric Studies on Existing Improved Biomass

Cookstove
Parametric studies were conducted in existing low cost improved biomass cook-
stove (IMPMUD model). Parameters of studies were those which affect the per-
formance of cookstove, namely burning rate of fuel wood, air supply required for
combustion, and power supplied to the fan using standard methods/protocols
(Verma et al. 2014).
2.2 Selection of Suitable Material for Cookstoves
Through several past studies it was concluded that ceramics are superior for longer
cooking periods. If the choice is between metal and clay, the latter is preferred for
cooking times’ longer than 15 min Krishna Prasad and Sangen (1983). On the basis
of heat transfer calculations, preference was given to ceramics as a construction
material. However, there are important drawbacks of ceramics, i.e., lack of suitable
clays and the fragility of the final product. Ceramics are not strong enough for some
cooking jobs. This has led to total rejection of ceramics in many cases and the
choice is between metal and mud.
3 Dimensioning the IMPMUD Stove Elements
3.1 The Combustion Chamber
The combustion chamber can be thought of as being built-up from two separate
parts with height Hcc1 and Hcc2. The first is occupied by the fuel bed where
charcoal burns and the pyrolysis takes place; the second part is occupied by the
flames. The fuel bed volume (Hcc1 × Acc) depends on the power output which is
required, the wood mass charged, the packing density (χ), and the burning char-
acteristics of the wood Bussmann PJT (1988). The maximum power density can be
defined as the maximum power per m2 fuel bed area can be obtained from a steadily
burning fire. A power density of 180 kW/m2 was obtained experimentally in the
literature from free-burning fires of varying diameter, without grate, using oven-dry
woodblocks of 15 × 15 × 50 mm3. The power density could be increased by 50 %
when using a grate. In the design work, the power density could be chosen as
200 kW/m2. The cross-sectional area of the combustion chamber and the com-
bustion chamber height are then given by
ACC ¼ PDesign =200

Hcc1 ¼ DMf =ðv:qf ÞðAcc Þ
where, Acc is the cross-sectional area of combustion chamber in m2; PDesin is the
design power of cookstove in kW; Hcc1 is the height of the fuel bed combustion
chamber in m; ΔMf is the mass of wood charged in kg; and χ is packing density of
fuel in combustion chamber.
3.2 The Fuel Loading Opening
A closed combustion chamber facilitates the control of incoming combustion air.

However, the demands put forward by stove users often do not allow for such a
configuration: the stove must accept long pieces of fuelwood. Consequently, the
aeration of the fuel bed becomes a problem. The least that can be done is to keep the
size of the fuel loading opening small. The criteria as shown in Table 1 could be
used. Performance can be improved with a metal flap which closes the combustion
chamber when fuel wood does not project out of the stove.
3.3 Primary and Secondary Air Holes
The aeration of the combustion chamber in a closed stove fire is very effective. The
inlet air holes form the main flow resistance. The aeration of the fuel bed is a
problem for cookstove having an open combustion chamber. The only measure to
be taken into consideration is to keep the total area of both primary and secondary
air holes to equal (including fuel loading opening).
Table 1 Criteria used for dimensioning the fuel loading opening

Height Smaller than the combustion chamber height larger than two stick diameters:
2 × 30 mm
Base Smaller than the combustion chamber diameter larger than three stick diameters:
3 × 30 mm
3.4 The Grate
The purpose of the grate is twofold. First, it is used to concentrate the fire during
flaming combustion. Second, the grate is used to promote the combustion of
charcoal formed during pyrolysis. For both reasons it is necessary that the flow
resistances of the grate and other air entrance passages are of the same order of
magnitude. The grate can easily regain its function if its porosity and the number of
primary air holes underneath are increased. In optimizing the above stove it had
been suggested, first, to increase the number and size of the primary air holes to
equal the total cross-sectional area of the fuel loading opening; and second, to
increase the porosity of the grate from 5 to 10 % to the required 50 %.
3.5 Flow Path Resistances
The experiments conducted by various researchers clearly showed the importance

of reducing the air flow. Preferably this has to be done at the air inlets which make it
possible to control primary and secondary air separately. For stoves having a large
fuel loading opening this is not possible. The flow resistance must be created
downstream of the combustion chamber. Each stove can concentrate flow resistance
in the annular gap between pan and shield.
3.6 Design Description of IMPMUD
Dimensions of cookstove are as follows; (1) inner diameter of combustion cham-

ber = 13.0 cm, (2) outer diameter of combustion chamber = 15.0 cm, (3) height of
combustion chamber = 18.0 cm, and (4) pan support height = 3.0 cm. Figure 1
shows the top and front view of the cookstove. The computer aided design of
domestic IMPMUD cookstove is shown in Fig. 2.
3.7 Parametric Study
Various factors which affect the performance of cookstove have been experimen-
tally studied in this section.
(1) Determination of optimum burning rate: cookstove was experimentally tested
with different burning rate and it was found that burning rate of 1.7 kg fuel/h
is optimum for this IMPMUD cookstove.
(2) Determination of optimum air flow through primary and secondary holes for
complete combustion of fuel: In all forced draft cookstove optimum air is
Fig. 1 Top and front view of IMPMUD stove (L to R, respectively)
Fig. 2 Computer aided design of domestic IMPMUD cookstove
suppled through an external exhaust fan, but it is difficult to find a suitable fan
which can supply optimum air in combustion chamber for clear combustion.
Experimentally fans with different input power had been used for suitable air
flow and fan of power 3.6 was suitable to provide air for complete combustion.
(3) Determination of optimum pan support: Pan support height or gap between top
plate of cookstove and pot bottom. The height of the support has a consid-
erable effect on the efficiency of the cookstove, and hence needs optimization.
Stove with small support height although have high efficiency, but they also
reduce the amount of gas that can flow through the gap and thus limit the
firepower. In case of larger fire either smoke will pour out the stove door, or
else the fire will be choked and suffer poor combustion or simply not build up
to desire power. Experimentally it had been found pan support of height
2.5 cm is optimum.
4 Results
4.1 Performance and Emissions Results
The power output, thermal efficiency, and emissions, including both particulate
matter (PM) and carbon monoxide (CO), performance of cookstove are given in
Table 2. Furthermore, the thermal efficiency and emission performances for forced
and natural draft cookstoves as specified by the Bureau of Indian Standards (BIS
2013) are given Table 3 for performance comparison analysis.
As per the results obtained from performance test, the cookstove performance
could not meet the desirable limit of thermal efficiency and emissions. Thermal
efficiency was low either because of low combustion efficiency or due to poor heat
transfer between cookstove top plate and pot (pan) bottom. Emissions were too high
because of poor combustion of fuel inside combustion chamber which is either
because of insufficient amount of air was blown in side of combustion chamber
through blower or excess air was supplied through blower than the required amount
of air for complete combustion. Hence, it has been decided to perform parametric
study to find out parameters which are affecting the cookstove performance and
Table 2 Performance of IMPMUD cookstove (without modification)

S. No. Power output (kW) Thermal efficiency (%) PM (mg/MJd) CO (g/MJd)
1 1.04 21.3 600.5 7.1
2 1.16 23.3 578.9 7.0
Average 1.1 22.3 589.7 7.05
Table 3 BIS limits for Parameters Forced draft Natural draft

forced and natural draft
cookstoves Thermal efficiency ≥35 % ≥25 %
PM in mg/MJd ≤150 ≤350
CO in g/MJd ≤5 ≤5
Table 4 Performance of IMPMUD forced draft cookstove (with incorporated modifications)

S. No. Power output (kW) Thermal efficiency (%) PM (mg/MJd) CO (g/MJd)
1 2.96 35.67 101.11 1.48
2 3.01 36.29 97.46 1.36
3 2.94 35.47 101.13 1.07
4 3.02 36.49 106.42 1.69
5 3.01 36.36 96.40 1.45
6 3.04 37.11 81.55 1.22
Average 2.99 36.23 97.34 1.38
further made necessary modify in existing parametric design so that it could meet
the BIS requirement.
Modifications in IMPMUD forced draft cookstove have been done according to
the results of parametric study and modified IMPMUD was again tested with BIS
protocol. The results of modified IMPMUD forced draft biomass cookstove are
presented in Table 4. It is clearly evident that the average thermal efficiency of the
cookstove was found as 36.23 % which is significantly higher than the BIS limit
(≥35 %). The emission of particulate matter (PM) and carbon monoxide (CO) was
found to be 97.34 mg/MJd and 1.38 g/MJd, respectively. The emissions were lower
than the BIS limits as given in Table 3.
5 Conclusions
The experimental study had revealed that the modifications can be done in existing
cookstoves for enhanced thermal efficiency and lower emissions by varying
parameters such as fuel burning rate, air supply and power supply of fan, and height
between top plate and vessel without making major changes in the structure of
existing cookstoves (such as diameter and secondary air inlet points, etc.). With
above modifications cookstove efficiency was increased up to 63 % and there was
decrease in CO emission up to 80 % and PM emission up to 84 %.
References
Bhandari S, Gopi S, Date AW (1988) Investigation of CTARA wood-burning stove. Part 1.

Experimental investigation. Sadhana 13(4):271–293
Bhattacharya SC, Leon MA (2005) Prospects for biomass gasifiers for cooking applications in
Asia’. http://www.retsasia.ait.ac.th/Publications/WRERC2005/AIT-gasifierstoveforcooking-
final.pdf
Bureau of Indian Standard (BIS) on Solid Biomass Chulha (2013) Specification CIS 1315Z (Part
1): 1991, First revision (2013)
Bussmann PJT (1988) Woodstoves Theory and applications in developing countries’, Ph. D.
thesis, Eindhoven, University of Technology, Eindhoven, pp 1–174
Census of India (2011) “Fuel Used for Cooking” http://censusindia.gov.in/Census_Data_2011/
Census_data_finder/HH_Series/Fuel_used_for_cooking.htm
Galitsky C, Gadgil A, Jacobs M, Lee Y (2006) Fuel efficient stoves for Darfur IDP camps: report
of field trip to North and South Darfur. Lawrence Berkeley National Laboratory Report
LBNL-59540
Guillaume DW, LaRue JC (1995) Combustion enhancement using induced swirl. Exp Fluids
20:59–60
Kothari DP, Singal KC, Ranjan R (2008) Biomass energy, renewable energy sources and emerging
technologies, pp 284–307. P.H.I. Learning India
Krishna Prasad K, Sangen E (1983) Technical aspects of wood burning cookstoves, a report from
the wood burning Stove Group, Eindhoven University of Technology Eindhoven, Netherlands
Verma AR, Prasad R, Vijay VK, Tiwari R, Mal R (2014) Modifications in traditional Indian
cookstove and improved cookstove for efficient design. 2014-ETHOS conference, Kirkland,
Washinton, DC
WRI (World Resources Institute) (1998–1999) UNEP, UNDP, World Bank. World resources: a
guide to global environment. Oxford University Press, Oxford, UK
Part IV
Biomass Management
Thermochemical Characterization of Pine
Needles as a Potential Source of Energy
Anil Kumar Varma and Prasenjit Mondal
Abstract Present paper deals with the thermochemical characterization of pine

needles to assess its suitability as an energy feedstock. Proximate and ultimate
analyses revealed that pine needles contain low moisture, ash, nitrogen, and sulfur
content, as well as high fixed carbon and volatile matter content. The higher heating
value (HHV) is determined as 19.22 MJ kg−1 using bomb calorimeter as well as the
lower heating value (LHV) is 17.66 by using HHV value. The empirical formula of
pine needle is found to be CH1.83O0.74N0.008 on the basis of ultimate analysis.
Fourier transform infrared (FTIR) spectra is developed to identify the basic func-
tional groups in pine needles. Thermal degradation analysis has been done by using
a thermogravimetric analyzer from 32 to 1000 °C in a nitrogen atmosphere at a
heating rate 10 °C min−1. In addition, the thermal decomposition behavior of pine
needles has been determined through TGA/DTG/DTA analysis. The chemical
characteristics and thermal behavior show that pine needles have good energy
potential for exploitation through pyrolysis.
Keywords Biomass Pine needles Pyrolysis FTIR Thermogravimetric

analysis
1 Introduction
The world’s energy market is mainly dependent on the fossil fuels such as coal,
petroleum, and natural gas as a prime source of energy. Since millions of years are
required to form fossil fuels in the earth, their reserves are finite and it is predicted
that these sources of energy will deplete within the next 40–50 years (Saidur et al.
2011). Further, due to continuous degradation in qualities these fossil fuels are
A.K. Varma Prasenjit Mondal (&)

Department of Chemical Engineering, Indian Institute of Technology Roorkee,
Roorkee 247667, Uttarakhand, India
e-mail: mondal2001@gmail.com

DOI 10.1007/978-81-322-2773-1_19
258 A.K. Varma and Prasenjit Mondal
creating environmental problems such as global warming and acid rain. Thus,
applications of renewable energy sources are being investigated around the world to
meet the energy security and environmental sustainability. Biomass is a clean fuel
because it has a very low content of sulfur, nitrogen, and ash, which gives lower
emissions of SOx, NOx, and soot than conventional fossil fuels. Moreover, biomass
itself is carbon neutral and its application in an engineered way may add carbon
credit.
In India, pine needles are available in large quantities in the Himalayan region of
Uttarakhand. Himachal Pradesh, etc. Around 340,000 hectares of pine forests cover
is available in Uttarakhand (Bisht et al. 2014), which produces around 2.06 million
tons of pine needle annually (Web 1). Pine needles are needle-shaped leaves falling
from tree mainly in the summer season in the month of March to July. Due to their
highly inflammable nature, it is a serious threat to forest fires. These fires create
environmental pollution, damage the fertile top layer of soil, and destroy grazing
grounds for cattle. So, their collection and disposal are very important in envi-
ronmental point of view. Normally, collection of pine needles is done by local
people and it creates opportunity for earning to the common mass of the hilly
region. Due to creation of awareness among the people, collection of pine needle is
increasing gradually for use as an energy source. In 2012 nearly 600 tons of pine
needle was collected in Hamirpur district of Himachal Pradesh, which is around 12
times more than the corresponding collection in 2011 (Web 2). Further, in com-
parison to other biomass such as sugar cane bagasse, semi-dried banana leaves, rice
husk, elephant grass (Fernandes et al. 2013), etc., this biomass has better potential
as a feedstock for bioenergy through suitable thermochemical conversion process
such as pyrolysis to fulfill the energy requirement of the hilly region. Pyrolysis is a
thermochemical conversion process to convert biomass into volatile fraction
composed of condensable organic vapors (bio-oil), carbon-rich solid waste (char),
and noncondensable gasses (Fernandes et al. 2013). This process is very attractive
option considering the current initiatives of seeking new renewable sources for
clean energy production and may be applied for this feedstock. However, to
understand the requirement of operating conditions, reactor configuration/design,
etc., the thermochemical properties of the biomass are essential (Ceylan and Topçu
2014). In the previous literatures, there is not much evidence available regarding
thermal characterization of pine needles.
The aim of this study is to characterize the pine needle through proximate and
ultimate analyses, find the empirical formula for pine needles from ultimate analysis
data and determine its heating values. It also includes FTIR analysis to find out the
functional groups in the sample as well as thermal analysis such as (TG/DTG/DTA)
to evaluate the thermochemical conversion of pine needles. Suitablity of this bio-
mass as a feedstock for pyrolysis has also been assured.
Thermochemical Characterization of Pine Needles … 259
2.1 Materials
Pine needles were collected from the campus of Indian Institute of Technology
Roorkee, India. The collected pine needles were sun dried for 2 days, the dried
biomass were stored in polyethylene bags. 1 kg of this biomass was further oven
dried at 105 °C for 2 h, ground in Wiley grinder and powdery sample having
particle size 250–500 µm was prepared by using IS sieves. Figure 1 shows the
photograph of pine needles before grinding and after grinding.
2.2 Methods
Proximate analysis was done as per procedure laid in the Bureau of Indian Standards
code IS: 1350 (PART 1)-1984 to determine the moisture (M), volatile matter (VM),
ash (As), and fixed carbon (FC) of the sample. The fixed carbon was obtained by
subtracting the percentage of volatile matter, moisture, and ash from 100.
The ultimate analysis was used to find out the elemental composition such as
carbon, hydrogen, nitrogen, sulfur, and oxygen in the biomass sample and analysis
was done in Elementar Vario EL III model. The higher heating value (HHV) of the
pine needles was determined in a Parr 6300 Bomb calorimeter and this experi-
mental value of HHV was compared with the theoretical value determined using the
following Eq. (1) (Channiwala and Parikh 2001).

HHV MJ kg1 ¼ 0:3941C þ 1:1783H þ 0:1005S 0:1034O 0:0151N
0:0211As ð1Þ
Fig. 1 Photograph of pine needles. a Before grinding. b After grinding

where C, H, S, O, N, and As represent the carbon, hydrogen, sulfur, oxygen,

nitrogen, and ash content of the sample in wt. (%).
The lower heating value (LHV) of pine needles was calculated by Eq. (2) (Basu
2010).

LHV MJ kg1 ¼ HHV MJ kg1 hg ð9H=100 þ M=100Þ ð2Þ
where LHV, HHV, H, M, and hg are the lower heating value, higher heating value,
hydrogen percentage, moisture percent, and latent heat of steam (2.26 MJ kg−1),
respectively.
FTIR analysis of the pine needles sample was done in FTIR spectrometer
(Thermo Nicollet, Model Magna 760). Pellet technique was used for this analysis,
where dried samples were embedded in KBr pellets with a ratio of 1:10. The spectra
were recorded in the wavenumber range of 4000–500 cm−1.
The thermal decomposition behavior of pine needles was determined by using a
thermal analyzer (Model–SII 6300 EXSTAR). The thermal analysis, such as TG
(thermogravimetric), DTG (derivative thermo-gravimetric) and DTA (differential
thermal analysis) were done simultaneously. The sample was analyzed in an Al2O3
crucible at a heating rate of 10 °C min−1 from 32 to 1000 °C in nitrogen (N2)
atmosphere with a flow rate of 200 ml min−1 as sweeping gas. The mass of sample
for the experiment was taken about 10 mg.
3.1 Characteristics of Pine Needles
The result from proximate analysis, ultimate analysis, and heating values of the
present pine needles sample are shown in Table 1. As evident in Table 1 the moisture
content of pine needle is 5.03 wt%, which is less than 10 wt%, the upper limit of
moisture content for pyrolysis preferably (Asadullah et al. 2007). High moisture
content in biomass requires prior drying to remove the moisture and reduces the
energy efficiency of the system. Further, high moisture content also increases water
content in the bio-oil and reduces its heating value. From Table 1 it is also evident that
the volatile matter content in pine needle sample is high (77.55 wt%). High amount of
volatile matter facilitates the thermal decomposition and such biomass is better for
the pyrolysis. The result shows that the fixed carbon content is 13.77 wt%, which is
comparable to the other biomass such as semi-dried banana leaves (12.5 wt%),
rice husk (14.6 wt%), elephant grass 15 wt%, and briquette 14.2 wt% (Fernandes
et al. 2013; Wilson et al. 2011; García et al. 2012; Mesa-Perez et al. 2005). The ash
content of above biomass samples falls in the range of 5.9–11.7 %, which is higher
than that of the present biomass, i.e., 3.38 wt%. High ash content in the biomass has a
negative effect on the thermochemical conversion processes. Ash content in the
biomass affects the handling and processing costs in thermochemical conversion
Table 1 Proximate and Proximate analysis (wt%)

ultimate analyses of pine
Moisture 5.03
needles
Volatile matter 77.55
Fixed carbona 13.77
Ash content 3.38
Ultimate analysis (wt%)
Carbon 46.38
Hydrogen 7.08
Oxygena 46.02
Nitrogen 0.45
Sulfur 0.07
Empirical formula CH1.83O0.74N0.008
H/C molar ratio 1.83
O/C molar ratio 0.74
HHV (MJ kg−1) 19.22
HHV by correlation (MJ kg−1) 19.70
LHV (MJ kg−1) 17.66
a
By difference
process. Other problems associated with the high ash content are reactor fouling,
sintering, corrosion, and slagging in pyrolysis process (Fernandes et al. 2013). Low
ash and high volatile content in the present biomass make it good feedstock for
pyrolysis. Ultimate analysis of pine needle shows carbon, hydrogen, oxygen,
nitrogen, and sulfur content are 46.38, 7.08, 46.02, 0.45, and 0.07 wt%, respectively.
This result indicates that the sulfur and nitrogen content in pine needle is very low, so
the emission of toxic gasses like Sox and Nox is expected to be very less during
thermochemical conversion process. The empirical formula for the present pine
needle is found to be CH1.83O0.74 N0.008. The H/C and O/C molar ratios as calculated
from the ultimate analysis are 1.83 and 0.74, respectively. The HHV of the pine
needles is found to be 19.22 MJ kg−1 and calculated HHV from Eq. (1) is
19.70 MJ kg−1. The difference between experimental and calculated values of HHV
is *0.48 MJ kg−1. The HHV of pine needles is close to other biomass such as
switchgrass 19.6 MJ kg−1 (Masnadi et al. 2014), semi-dried banana leaves
19.8 MJ kg−1 (Fernandes et al. 2013), sugar cane bagasse 19.2 MJ kg−1 (Asadullah
et al. 2007), cedar wood 19.26 MJ kg−1 (Asadullah et al. 2004), etc. The lower
heating value (LHV) of the pine needle calculated from Eq. (2) is 17.66 MJ kg−1.
Low moisture and ash content, as well as high volatile content and HHV
(19.22 MJ kg−1) suggest pine needles as a good potential for production of bio-oil
through pyrolysis.
The FTIR analysis is used to identify the characteristic functional groups and
chemical constituents present in the sample, which are important for determining
the composition and distribution of pyrolysis products (Nyakuma et al. 2014).
Presence of hemicelluloses, cellulose, and lignin in some biomass such as nonedible
3450
Absorbance (%)
1626
1384
1109
615
Wavenumber (cm-1)
Fig. 2 FTIR spectra of pine needle
oil seeds as mahua, karanja, niger, and linseed (Shadangi and Mohanty 2014),
empty fruit bunch briquettes (Nyakuma et al. 2014), orange waste (Lopez-
Velazquez et al. 2013), mesua ferrea L.deoiled cake (Chutia et al. 2013), date palm
(Sait et al. 2012), have recently been confirmed by FTIR analysis. For the present
case Fig. 2 shows the FTIR spectra of pine needle in the wavenumber range of
4000–500 cm−1. The first strong peak at 3450 cm−1 as shown in Fig. 2 denotes the
O–H stretching vibration due to the presence of carbohydrates and lignin (Chutia
et al. 2013). The peak around 1626 cm−1 is related to the C=C stretching vibration
of aromatic ring, which corresponds to lignin (Asadieraghi and Wan Daud 2014).
The peak at 1384 cm−1 is due to the presence of C–H stretching (Chutia et al.
2013). The peaks between the regions from 1200 to 900 cm−1 are due to the
presence of C–O, C–C, C–O–C, and C–O–P stretching vibrations of polysaccha-
rides, which are related to hemicelluloses and cellulose components (Asadieraghi
and Wan Daud 2014). The FTIR analysis of pine needles shows the presence of
functional groups associated with the cellulose, hemicelluloses, and lignin.
3.2 Thermogravimetric Analysis of Pine Needles
Figure 3 shows the TG (thermogravimetric), DTG (derivative thermogravimetric),

and DTA (differential thermal analysis) plots of pine needles, where TG curve
shows the mass loss profile as function of temperature, DTG curve denotes the rate
of mass loss as function of temperature, and DTA curve denotes the differential
thermal analysis of sample, it shows the energy change with temperature during
thermal analysis of sample (Doshi et al. 2014). The entire pyrolysis process of
present pine needles can be divided into four stages. The first stage starts at 32 °C
DTG
DTA
TG
Fig. 3 TG/DTG/DTA curves of pine needle
and ends at 200 °C. As evident from TG curve the mass loss in this stage is *5 %
due to the removal of moisture and very light volatile components. (Shadangi and
Mohanty 2014), which is also represented in the DTG curve (weak peak). Very
small deviation in the DTA peak (absorption of heat) and proximate analysis is also
in agreement with this fact. Second stage starts at 200 °C and ends at 340 °C with
mass loss of *53 % as evident from TG curve. At this stage DTG curve shows the
maximum mass loss rate of *1.42 mg min−1 at 317 °C, it is due to the degradation
of hemicellulose and cellulose (Jeguirim and Trouvé 2009). This stage is stated as
active pyrolysis stage. The third stage starts at 340 °C and finishes at 500 °C with
mass loss of *38 % from TG curve. This stage is also denoted in DTG curve with a
mass loss rate of 0.36 mg min−1 at 451 °C. The DTA curve gives the energy change
of *47.4 µv, *49.2 µv at 338 and 456 °C, respectively, and energy release during
this stage is 4.48 J mg−1 from DTA curve. This stage, also denoted as an active
pyrolysis stage, is due to the degradation of cellulose. The fourth stage starts at
500 °C and continues up to 1000 °C, the mass loss occured in this stage is
only *2 % and there is very less variation in DTG and DTA curve. This stage is
referred as passive pyrolysis. The mass loss observed in the second, third, and
fourth stage can also be explained by hemicellulose, cellulose, and lignin decom-
position. From the above discussion it is evident that around 91 % of mass loss
takes place within the temperature range of 500 °C, which indicates the suitability
of the sample for application as pyrolysis feedstcok.
4 Conclusions
Low moisture and ash content, as well as high volatile content and HHV
(19.22 MJ kg−1) suggest pine needles as a good potential for production of bio-oil
through pyrolysis. Low content of nitrogen and sulfur reduces threat of toxic gasses
(NOx and SOx) emission. An empirical formula originated from ultimate analysis
for pine needle is given as CH1.83O0.74N0.008. The FTIR result shows the presence
of hemicellulose, cellulose, and lignin in the biomass sample. The results of ther-
mogravimetric analysis show the active pyrolysis in the temperature range of
200–500 °C, so the results obtained in thermochemical characterization of the pine
needles indicate that it has good potential for energy generation through pyrolysis
process.
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Biomass Supply Chain Management:
Perspectives and Challenges
Yogender Singh Yadav and Y.K. Yadav
Abstract In India, biomass is abundant renewable energy source available in terms

of about 686 MT gross agricultural residues annually with surplus of 234 MT for
bioenergy production. Biomass-based bioenergy is having major emphasis for
potential solution in renewable energy programmes across India. The bioenergy and
biofuel produced from biomass is reliable which provide eco-friendly energy source
under clean development mechanism but it also depends on many reasons which
also includes primarily as biomass assessment and supply chain management.
Biomass supply chain is the association of material between the initial source and the
end-user. Characteristic of biomass supply chain is typically comprised of some
distinct processes as harvesting, collection, transportation, pretreatment, storage and
end use, which is crucial for success of bioenergy production. There are several
issues in this aspect especially for transportation and storage such as seasonal
availability, production cost and efficient conversion technologies. Research and
development is essential to optimize the biomass supply chain management for
maximise the utilization capacity with minimizing the inputs. The information
presented in this paper would be useful for biomass supply chain management-based
energy programmes in India. In this facet, multi-biomass approach is sustainable in
present scenario for more advantageous with relatively available renewable energy
alternative.
Keywords Biomass Supply chain Bioenergy Biofuels Renewable energy
Y.S. Yadav (&) Y.K. Yadav

Sardar Swaran Singh National Institute of Bio-Energy,
Kapurthala 144601, Punjab, India
e-mail: yogender784@yahoo.co.in

DOI 10.1007/978-81-322-2773-1_20
268 Y.S. Yadav and Y.K. Yadav
1 Introduction
The increased economic and industrial development results are having severe con-
text for available energy segment in India. Although India is known as rapid growing
economy in the world but basic energy requirement for the people is yet to fulfil
(Hiloidhari et al. 2014). The demand of energy in commercial sector is increasing
with the same speed as it is produced. India is an agriculture dominant nation having
more strength to frame biomass-based bioenergy programmes to meet the present
energy requirement. Although, agriculture is regarded as the backbone of Indian
economy and it contributes only 17 % to country GDP and it is also a source of
subsistence for nearly 60 % of its population. About 60 % of land area of the country
is under various agricultural practices (http://data.worldbank.org). Biomass is
extensively used in various traditional and rural enterprises (Kishore et al. 2004) and
the advanced utilization of biomass takes the advantages of modern biomass con-
version technologies (combustion, pyrolysis, gasification, fermentation and anaer-
obic digestion) for production of heat and electricity, liquid and gaseous
transportation fuel, biogas, etc.
Many researchers studied the aspects of biomass supply chain management and
estimated that the biomass used for production of energy in domestic and industrial
is yet not fully exploited in biomass harvesting, transport and processing (Nielsen
et al. 2007). In this context, the logistics and transport sector accounts about
one-third of the total energy consumption which is almost fully dependent on fossil
fuels and this consumption of energy from biomass would significantly lead to
energy independency (Nielsen et al. 2007). Biomass supply chain having certain
important barriers for biomass utilization for generation of bioenergy/biofuel such
as energy production cost and the technology used. Hence it is an essential need to
pretend and optimize biomass supply chain for better management applications
with significant reduced cost and efficient logistics operations.
2 Biomass to Bioenergy
The various biomasses available can be used for potential bioenergy production
with several bioenergy applications in India. In this context, agricultural residue
obtained during many agricultural operations is being considered as potential
biomass feed stock for bioenergy. Agricultural residue comprises of organic
materials as biomass produced from tillage to post-harvest operations (Singh and
Gu 2010). This biomass potential can be demarcated as gross residue potential as
the total amount of residue produced while surplus residue potential is the residue
left after any competing uses (such as feed, animal bedding, heating and cooking
fuel and organic fertilizer). The surplus fraction can be used for bioenergy gener-
ation. Gross residue potential of a particular crop depends upon three parameters
such as area covered by the crop, yield of the crop and residue to product ratio
Biomass Supply Chain Management: Perspectives and Challenges 269
(RPR) of crop (Hiloidhari and Baruah 2011). There is some significant availability
of biomass which is observed apart from agricultural residues such process
industries, vegetable market places, road side sweepings and plantations which can
be considered as potential bioenergy generation source.
In this perspective, biomass utilization (including, energy crops, biosolids, crop
and agricultural residues, municipal waste and industrial by-products and wastes)
has an emerging scope among other available resources and is reflected as a viable
alternative for energy production, including various potential conversion processes
(Parikka 2004; Yamamoto et al. 2001). The most relevant processes available and
commonly used are thermal, biochemical and chemical conversions. The thermal
conversion processes mainly comprise of direct combustion, pyrolysis and gasifi-
cation, the biochemical conversion is primarily based on fermentation and anaer-
obic digestion with the end product ethanol and biogas, chemical conversion
consists of transesterification and other significant processes which convert plant
and vegetable oils to biodiesel and biofuel.
3 Biomass Supply Chain
Typically biomass supply chain can be defined as movement of material or goods

between the source and end-user. It mainly comprises of four commercial entities
such as raw material supplier, distribution centre, manufacturer and the customer
(Beamon 1998). Biomass supply chain management mainly emphasis on integra-
tion of all such entities so that end product is to be produced and distributed in the
right quantity at right time to right location with providing preferred quality and
service at minimum cost (Simchi-Levi et al. 2003). The biomass supply chain of
crop residue essentially consists of the biomass supplier, storage, pretreatment,
transportation and conversion of biomass to bioenergy (Becher and Kaltschmitt
1994; Vlachos et al. 2008). The complex biomass supply chain involves two
interdependent and interconnected processes as production and control processes
and distribution and logistical processes. The production planning and control
processes consist of planting, harvesting, baling and pretreatment of biomass,
whereas distribution and logistical processes mainly contains storage and trans-
portation (Fiedler et al. 2007). The mutual integration of both the processes of
biomass supply chain plays a crucial role for competing bioenergy industry.
The biomass supply chain also deals with biomass supply and availability
uncertainty to determine commercially economic viability for the industry. Because,
any bioenergy sector industry needs cost efficient year-around uniform and reliable
supply of desired biomass feedstock. Also, there are numerous sources of variability
in any biomass supply chain, i.e. weather conditions, seasonal availability, biomass
characteristics, suppliers, transportation and distribution and government policies
(Iakovou et al. 2010; Cundiff et al. 1997; Gold and Seuring 2011).
Generally, biomass supply chain models concentrate on the objective to mini-
mize the production cost along with providing a better supply chain structure
(De-Mol et al. 1997). This can help to insight potential future production need of
bioenergy from biomass for planning purpose (Kim et al. 2011; Akgul et al. 2010).
Conventionally, biomass was used for energy production through direct combustion
in areas which are close to its production sites. The increased attentiveness and
complexity of supply chain prescript the need to develop planned and optimized
biomass supply chain management system (Meixell and Gargeya 2005; Min and
Zhou 2002; Sarmiento and Nagi 1999; Vidal and Goetschalckx 1997).
4 Indian Scenario of Biomass Supply Chain Management
In India, agriculture is being considered as main source of livelihood among major

population, hence biomass of agro residues and other sources is readily available to
overcome the regional limitations for bioenergy production as in case of other
renewable resources. In present scenario of climate change, India confronts to
severe energy shortages with energy insecurity and also limits its ability to adapt to
these impacts. The present renewable energy installed capacity of the country
accounts to nearly 32,000 MW with large contribution is of wind power and solar.
Further, biomass considered as third resource in present pattern in which conversion
of biomass for heat and power generation is the most common produced form of
bioenergy.
According to MNRE’s Renewable Energy Plan 2022, the target allocated is of
1,75,000 MW out of which 99,533 MW (*57 %) for solar, 60,000 MW (*34 %)
for wind energy, 5000 MW (*3 %) for small hydro and 10,000 MW (*6 %) for
biomass energy. Efficient biomass supply chain management is crucial step for
success of any biofuels generation system, as the biofuel price is considered as an
important factor for its economic viability. The performance of the any supply chain
also depends on integration and coordination among all entities along with positive
flow of information and its products.
5 Typical Layout of Biomass Supply Chain
A typical biomass supply chain includes several distinct processes, i.e. harvesting,
handling, storage, transportation and utilization for bioenergy production (Fig. 1).
The selection of these discrete processes for bioenergy production are critical and
strategic decisions that many researchers have studied for specific biomass as raw
materials, i.e. switchgrass (Cundiff and Marsh 1996), cotton plant residues (Gemtos
and Tsiricoglou 1992) corn stover (Shinners et al. 2007). The relevant research
reviewed for different biomass concludes that there is a need to further optimization
of each step of the process in the biomass supply chain for efficient planning to
make it sustainable.
Fig. 1 Layout of biomass Collection/Storage/Pre-treatment processes

supply chain
Harvesting/collection of biomass
Storage
Loading and unloading
Transportation
Pre-treatment/processing
The availability of biomass is varying seasonally due to specific harvesting time

but the requirement at the bioenergy generation is continuous and year-round basis
which results the necessity to store the biomass at peak season. The biomass storage
is crucial linkage in respective supply chain to choose low-cost storage solutions in
the present situation. Several researchers stated that on-field biomass storage has the
advantage of low cost (Huisman et al. 1997; Sokhansanj et al. 2006) but on the other
way there will be significant material loss due to uncontrolled climatic condition, thus
leading to potential problems in the biomass power plant processes. Additionally,
health and safety issues also exist with danger of spores and fungus formation
(Nilsson 1999). Hence, on-farm storage of the biomass may not be allowed to the
farmers exceeding the significant storage time period (Sokhansanj et al. 2006).
Logistics and transportation of biomass mainly depends upon type of biomass,
available form, quantity, proposed customer and destination distance. It can be
further characterized in two steps as from field to farm or temporary storage and
from farm to power generation plant in which main mode of transport system is
road as it offers great flexibility between distances traveled (Mitchell et al. 1999).
Further processing of biomass is essential to enhance its efficiency that can be take
place at any stage of biomass supply chain but often preferred before transportation.
6 Factors Influencing the Biomass Supply Chain
The biomass supply chain represents several distinctive characteristics and also is
dependent on many variables, hence it is important to consider the significant
factors that affect the optimal biomass chain. These factors can be described as
follows.
6.1 Seasonal Availability
The time period available for different biomass types is limited which can be
determined through harvesting period and replanting time of the fields (Skoulou and
Zabaniotou 2007). Hence for year-round operation of the bioenergy generation
plant there is a need of storing significant amount of biomass, this problem may be
avoided if year-round biomass to be used or by using multi-biomass approach to
overcome these problems.
6.2 Weather
Weather is somewhat impossible to predict in long term but possible to planning

carefully for year-round process to have less effect from it. There is a great influence
on bioenergy generation indirectly because it reduces the yield of the biomass and
quality significantly at the time of harvesting due to bad working conditions of field.
6.3 Collection and Handling Equipment
Almost all types of biomass materials require personalized harvesting, collection

and handling equipment which lead to a typical structure of the supply chain. The
procurement of biomass from field in available form generally determines the
production and operational costs of the respective bioenergy generation system in
any biomass supply chain.
6.4 Low-Density Material
Biomass primarily deals with low density due to occupancy of larger volume which
results increased transportation and handling cost and more storage space
requirement. This can be enhanced through low heating value which is partly due to
the increased moisture of types biomass obtained in the respective biomass supply
chain (Allen et al. 1998).
6.5 Communication Issues Between Industry and Suppliers
The lack in communication among industry and supplier can lead to failure of any
biomass generation system with less efficient biomass supply chain. This is also due
to deficiency in real-time knowledge processes. The use of a common database and

information technologies of suppliers and producers help to make it up to date. The
process of information sharing and communication with state-of-art keep can
provide today’s difference between success and failure of any system.
6.6 Sustainability
Sustainability of any operation is critical and important must be considered during

designing, planning and executing the biomass supply chain for bioenergy pro-
duction. Researchers have studied and represented the biomass generation system
and investigate the resulting profiles of bioenergy supply chains (Frombo et al.
2009; Linton et al. 2007). Elghali et al. (2007) also propose a sustainable frame-
work to assess the bioenergy systems to provide better practical advice for
policy-makers, planners commercially using multi-criteria decision-based analysis.
7 Supply Chain Design and Planning
Design and planning of any supply chain decides the optimal use of available
resources and infrastructure for better bioenergy production practices in order to
demand and supply forecasting of any system. This has been recognized earlier also
in management and science to coordinate the planning and management with
economic viability of supply chain (Fig. 2). The need of different approaches to be
explored for focusing the supply chain management has been identified as under.
7.1 Modelling Approach
In modelling approach of any biomass supply chain mathematical modelling is

common to identify the prescribed problem. The mathematical models are set of
equations which describe real-world phenomena in more efficient manner to opti-
mize the given set of variables (Keramati and Eldabi 2011). The available models
can be categorized mainly as stochastic, deterministic, hybrid and
information-driven models. The stochastic models, also called as probabilistic
models, having uncertain and random parameters, whereas deterministic models are
fixed with certain parameters. The hybrid models are comprised of both deter-
ministic and stochastic model elements but IT-driven models integrate and coor-
dinate various phases of supply chain (Christensen and Koppenjan 2010).
In present situation of technology advancement special simulation and algorithm
approach should be developed to overcome the real challenges. Optimization
through simulation makes the process fast and less expensive to assess large
Fig. 2 Factor considered for developing modelling approach for biomass supply chain (Source
Sharma et al. 2013)
number of dependent variables to provide the more optimal decision. This tech-
nique also consists of unique ‘What-if’ analysis which is very popular among the
industry and corporate sector to identify the uncertainty of the process (Sharma
et al. 2013).
Since, biomass supply chain is complex and difficult to optimize through the
mathematical modelling due to incapable to identify the uncertain parameters and
dynamics of the supply chain through the pure mathematical optimization models.
While combining the simulation may provide the appropriate solution of the issues
related to the biomass supply chain modelling. Consequently, there is a need to
develop techniques that result both in handing of complexity of problems along
with providing solution for economic viable, environmental and social aspects to
the biomass supply chain system (Fig. 2).
Many researchers have studied about optimisation-based decision algorithm of
supply chain to manage the complex problems starting facility selection to product
distribution (Brown et al. 1987; Timpe and Kallrath 2000; Neiro and Pinto 2004;
Camm et al. 1997; Voudouris 1996; Pinto et al. 2000; Karimi et al. 2005). They
also focused the process operation issues through combining integer programming,
network optimisation and geographical information systems (GIS) to improve
efficiency and responsiveness of supply chain system. The novel feature of any
mathematical model can accommodate different timescales and distribution of

variable length for production with better resolution before planning of the supply
chain system for efficient logistics and production operations to enhance its
performance.
7.2 Multi-biomass Approach
The concept of multi-biomass approach is not much familiar to the researcher till
now and research carried out is also limited even with many direct and indirect
advantages. This method covers simultaneous use of different types of available
biomass (Nilsson and Hansson 2001). Researchers also reached to the conclusion
that adopting this technique leads to decrease in system operation cost up to 20 % as
compared to use of single biomass (Papadopoulos and Katsigiannis 2002). There
will be significant saving in the product cost with the smooth running of biomass
power generation system round the year and additional reduction in requirement of
equipment and labour facilities. The major technical challenge involved in this
approach is efficient biomass to energy conversion technology to use multi-biomass
with distinct fuel characteristics according to the seasonal availability. The storage
and logistics may become complex as dissimilarities in availability, handling,
storage and transportation and processing of biomass sources (Faaij et al. 1997).
Now available optimisation and simulation algorithms are capable to resolve the
issues of farm and industrial relevance such as process representation, process
integration, modelling tools, model nonlinearities and performance measures.
8 Biomass Supply Chain Management
Supply chain management includes the processes for efficient utilization of

resources through coordinating and integrating suppliers and manufacturers to
produce and distribute the products to satisfy the service level requirements
(Simchi-Levi et al. 1999). The coordination and integration of multiple functions at
a time is complex in supply chain context. But technological advancement and
applicability of information technology especially software applications are greatly
reducing the strategic costs and planning of biomass supply chains. It has been
recognized through information sharing, process automation and relationship
management to facilitate the supply chain management system. The increasing use
of information technology in present business context further improved the avail-
able supply chain systems through real-time association, accessibility and 24/7
availability global market (Lancioni et al. 2003) which helps the business entities to
manage with uncontrollable factors affecting the demand in terms of prices, lack of
resources, and production.
9 Issues Related to Biomass Supply Chain Management
There are several issues related to biomass supply chain management to keep it
competitive and continuous operation, which is basically categorised in terms of
feedstock costs and supply, harvesting and collection, storage and transportation,
pretreatment, conversion process, infrastructure, economic feasibility, regulations
and policies, design and planning, etc. (Fig. 3).
9.1 Harvesting and Postharvest Operations
The literature suggested that most relevant harvesting and postharvest operations of
biomass carried out manual or mechanical drying and bailing (Rauch 2007). Bailing
is a specific technology in harvesting process for agricultural and crop residues to
improve density of biomass and makes it convenient for handling, transport and
storage through bailer equipment. Researchers suggest that advancement in harvest
technologies should be primarily focused by considering the need and the sus-
tainability of bioenergy production system, further limited harvesting period and
seasonal availability causes unutilised machinery and resources with additional
Fig. 3 Facilitating biomass flow along the bioenergy chain (Source Gold and Seuring 2011)
increase in production cost (Forsberg 2000; Hess et al. 2007; Madlener and
Bachhiesl 2007; Hamelinck et al. 2005; Dunnett et al. 2007).
9.2 Storage Throughout the Bioenergy Chain
The storage of biomass feedstock is essential concern due to round the year
operation of the biomass power generation plant. In which, less time available for
harvesting with large geographical distributions resulting the need of convenient
storage facilities can ensure the constant supply of feedstock bioenergy power
plants (Sims and Venturi 2004; Hess et al. 2007; Uslu et al. 2008; Gronalt and
Rauch 2007). The risk involved for storage of biomass feedstock is significant
weight loss and quality of stored biomass which mainly depends on the climate
conditions and biomass procession stage.
9.3 Transport in the Biomass Supply Chain
Transport issues related to the biomass supply chain can be categorised as legal and
infrastructural framework but up to some extent social and environmental factor are
also significant impact of transport process such as low density of biomass fuels as
compared to the fossil fuels (Mayfield et al. 2007). Usually, the conversion of
biomass to bioenergy is also not identical therefore, there is a need of environmental
and social controls required in the transport determinations (Kumar et al. 2006).
9.4 Pretreatment Analysis
Pretreatment analysis for the available standard techniques may be considered

according to purpose and need of the process such as drying, pelletization, tor-
refaction and pyrolysis etc. (Dunnett et al. 2007; Uslu et al. 2008).
9.5 Design of the Bioenergy Production System
The supply chain is the core issue for any bioenergy generation system which can
be described through available tools and strategies for enhancing the performance
of the system. The bioenergy power generation systems exist in various sizes and
production capacity to consider as decisive parameter for bioenergy production
system designing which are generally small as compared to fossil fuel power plants
due to requirement of massive feed stock of relatively low calorific value (Elghali
et al. 2007; Caputo et al. 2005). These systems deal with high complexity of various
biomass resources, different market segments, conversion methods and different
end use applications (Heaton et al. 2004; Gronalt and Rauch 2007).
Management of biomass supply chain may be available and can be further
extended with use of more advance management approaches. The efficiency of the
supply chain network can be better assessed by launching the suitable and optimal
performance measures (Beamon 1998). This can be used to design development of
a system to enhance performance of supply chain in a highly competitive envi-
ronment with improved decisions such as number, size and location of bioenergy
production system, production planning and scheduling, network connectivity,
management and replenishment policies.
10 Conclusion
The overview of the biomass supply chain management suggested that knowledge
gap exists to get an optimal biomass supply chain considering technical, societal,
business, economic and environmental aspects. Supply chain management in any
industrial organization implicates combination of the activities from raw material to
end use, whereas coordination of all aspects of the supply chain is paramount to
success. As evident from the above considerations, India has huge potential to
explore the possibilities to convert the surplus biomass to bioenergy and in this
aspect multi-agricultural biomass approach can be an attractive option for biomass
based power plants for bioenergy generation. The factors considered for sustainable
management and through combining optimal processes and technologies can lead to
an organized and integrative biomass supply chain.
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Part V
Electrochemical Processes
Enhanced Power Generation in Microbial
Fuel Cell Using MnO2-Catalyzed Cathode
Treating Fish Market Wastewater
Md.T. Noori, M.M. Ghangrekar, A. Mitra and C.K. Mukherjee
Abstract Development of cost effective metal-based catalyzed cathode for

enhancing oxygen reduction reaction (ORR) is necessary to produce higher power
in microbial fuel cell (MFC). In this study, γ-MnO2 was synthesized using chemical
co-precipitation method and tested in MFC as cathode catalyst. Two single chamber
MFCs, one with γ-MnO2 catalyzed cathode (MFC-M) coated with carbon sup-
ported MnO2 (0.5 mg cm−2) on cathode and other control MFC (MFC-C) coated
without catalyst in cathode, were fabricated using clayware cylinders. Maximum
power density of 1.66 and 0.5 W m−3 with a coulombic efficiency (CE) of
9 ± 0.65 % and 4.6 ± 0.26 % was obtained in MFC-M and MFC-C, respectively.
The average organic matter removal efficiencies in terms of chemical oxygen
demand (COD) were found to be 84.4 ± 3.4 % and 75 ± 1.4 % and total nitrogen
removal efficiencies of 66.5 ± 2.56 % and 52.8 ± 3.17 % were achieved in MFC-M
and MFC-C, respectively. MFC-M demonstrated enhanced ORR and subsequent
increase in electrochemical activity on cathode, thereby improving power genera-
tion and substrate degradation. The present study shows the applicability of
cost-effective metal-based (γ-MnO2) catalyst for scaling-up of air-cathode MFCs to
treat fish market wastewater, which can also be used for treatment of other organic
matter containing wastewater.
Keywords Air-cathode Catalysis Fish market wastewater Microbial fuel

cell γ-MnO2 catalyst
Md.T. Noori A. Mitra C.K. Mukherjee

Department of Agricultural and Food Engineering,
Indian Institute of Technology Kharagpur, Kharagpur 721302, India
M.M. Ghangrekar (&)
Department of Civil Engineering, Indian Institute of Technology Kharagpur,
Kharagpur 721302, India
e-mail: ghangrekar@civil.iitkgp.ernet.in

DOI 10.1007/978-81-322-2773-1_21
286 Md.T. Noori et al.
1 Introduction
India is the second largest inland fish producer in the world after China (FAO 2012).
The major fish consuming states in India are West Bengal, Kerala, Goa, Tripura, and
Orissa (Anon 2001). Wastewater generated from fish market contains organic matter
measured in terms of chemical oxygen demand (COD), protein, and fats of similar
concentration as in fish processing industry wastewater (González 1996).
Consequently it is necessary to treat this wastewater before discharging into the
water body. On the other hand, global energy shortage and environmental pollution
are affecting human population in wide extent. Hence, the problem needs to be
addressed urgently. Many researchers reported the suitability of microbial fuel cell
(MFC) to solve these two problems simultaneously (Logan 2008; Behera et al.
2010a). MFC is a type of fuel cell in which bacteria acts as biocatalyst, oxidize
organic and inorganic matter, and produces electricity (Gabr et al. 2009; Rahimnejad
et al. 2011). A typical MFC consists of an anode and a cathode chamber separated by
proton exchange membrane (Ieropoulos et al. 2008; Behera et al. 2010b).
Performance of MFC depends on several factors such as microorganism, the
cathode catalyst, and the architecture (Liu and Logan 2004; Aelterman et al. 2006b;
Oh and Logan 2006). Most commonly used catalyst in fuel cells is Platinum (Pt).
However, the high cost of Pt makes it unfeasible for large-scale commercial
application of MFC. Therefore, finding or development of alternative low cost
catalyst to Pt is highly necessary (Rosen and Dincer 1999). Extensive efforts have
been made to find/develop low cost transition metal-based electro-catalyst, metal
oxides, and organic polymers composite transition metal oxides (Bashyam and
Zelenay 2006; Meng et al. 2013; Ghoreishi et al. 2014).
Manganese metal-based oxide (MnOx) cathode catalyst is getting greater con-
cern among researchers because of low cost, abandoned availability, and envi-
ronmental friendly (Mao et al. 2003). Zhang et al. (2009) reported that Pt could be
replaced by MnO2 catalyst in MFC. MnO2-treated graphite cathode showed higher
power generation as compared with non-treated graphite in MFC (Clauwaert et al.
2007). There are five distinct crystallographic phases as α, δ, γ, λ, β of MnO2 as
reported in the literature; whereas the catalytic activity in presence of air follows β-
< λ- < γ- < δ- = α-MnO2 (Cao et al. 2003). Synthesis of different phases of MnO2
using hydrothermal, solo-gel, and chemical co-precipitation method is widely
reported (Chu and Zhang 2009; Xiao et al. 2009). The chemical co-precipitation
method is simple, high yield, and the reaction condition is easy to control among
other methods.
In view of high catalytic activity and simple synthesis method, γ-MnO2 was
synthesized in this study using chemical co-precipitation method, and tested in
single chamber air-cathode MFC made with ceramic cylinder. Fish market
wastewater was used as substrate. Performance of MFC having γ-MnO2 as cathode
catalyst was evaluated on the basis of operating voltage (OV), power generation,
organic matter removal, total nitrogen removal, and coulombic efficiency (CE).
In addition, results were also compared with control MFC (zero catalyst).
Enhanced Power Generation in Microbial Fuel Cell … 287
2.1 Synthesis of MnO2
Potassium permanganate (KMnO4) and manganese sulfate (MnSO4) were used

without further purification. MnO2 was synthesized using chemical co-precipitation
route reported elsewhere (Chu and Zhang 2009). In brief, 0.2 M potassium per-
manganate solution was added slowly in 0.3 M manganese sulfate solution in
stirring condition at room temperature. Resultant precipitates were filtered and then
air dried at room temperature for 24 h. Collected solids were washed several times
with deionized water to remove soluble materials and then dried at 80 °C in air for
12 h.
2.2 Characterization of as-synthesized MnO2
X-ray diffraction (XRD) was used to characterize the crystal structure of MnO2.
Diffractograms were obtained in 2θ range of 20°–70° using Diffraktometer D8
(Bruker Axs, Germany) diffractometer with Cu Kα radiation (λ = 0.1541 nm). FTIR
analysis was performed on spectrophotometer (Spectrum Rx, PerkinElmer, Inc.,
USA). Morphological structure was observed using surface electron microscopy
(SEM, ZEISS EVO 60, Carl ZEISS SMT, Germany) equipped with tungsten fila-
ment and Oxford EDS detector. Images were taken by applying electron beam
having an acceleration voltage of 20.00 kV.
2.3 Test MFC
Two single chamber MFCs, viz., catalyzed cathode MFC (MFC-M) and control
MFC (MFC-C) were constructed using ceramic cylinder (Behera et al. 2010a). Both
MFCs had a similar working volume and cathode surface area of 900 ml and
370 cm2, respectively. Cathodes were coated on the outer surface of earthen cylinder
using ink-based carbon solution containing MnO2 and carbon loading of
0.5 mg cm−2, polyvinyl alcohol (5 % w/v) and 20 ml acetone. For performance
comparison, cathode of MFC-C was made similarly without having MnO2 in the
solution. Stainless steel mesh having total projected surface area of 350 cm2 was
used as anode in both MFCs. MFCs were inoculated with anaerobic septic tank
sludge and fish market wastewater was used as substrate. MFCs were operated under
batch mode of 4 days batch cycle and fresh feed having COD of 2512 ± 69 mg l−1
was added at the beginning of each batch cycle.
2.4 Measurement and Calculation
Electrical parameters such as operating voltage (OV) and open-circuit voltage

(OCV) were measured using data logger (Agilent Technology, Malaysia).
Polarization studies were performed using a variable resistance box by varying
resistance from 10,000 to 5 Ω and corresponding voltage (E) was recorded using a
data logger connected with personal computer. Power density was calculated
according to P (W m−3) = E2/RV, where E is voltage, R is resistance, and V is total
volume of anode chamber. Chemical parameters such as COD and total nitrogen
were determined using standard protocols (APHA 1998). CE was calculated
according to CE, % = (Ch/Cth) * 100, where Ch = total harvested coulombs and
Cth = theoretical coulombs available in the amount of COD consumed by
microorganisms during each batch cycle (Logan et al. 2006).
3 Result and Discussions
3.1 Characterization of as Synthesizes MnO2
Structural characterization of as synthesized MnO2 was done using XRD. Peaks of

diffractogram (Fig. 1a) appeared at 2θ = 37.14o, 42.48o, 56.30o, and 67.8o. The
indexable broad peaks of 131, 300, 160, 003 as shown in Fig. 1a, confirm the plane
of γ-MnO2 phase of synthesized MnO2 (JCPDF file no. 14-0644) having high
crystalline structure.
In FTIR spectra (Fig. 1b), major peaks appeared at wave number 3400 cm−1
–654 cm−1, which are associated to the absorbed water molecule and carbon
dioxide because of high surface-to-volume ratio of MnO2 (Zhang et al. 2005). Other
peaks at 535 and 1125 cm−1 are associated to the vibration of Mn–O and Mn–OH
Fig. 1 Characteristics plot of MnO2 synthesized using co-precipitation method. a XRD diffraction
pattern. b FTIR spectra
Fig. 2 SEM image of as

synthesized γ-MnO2
functional group, respectively. Figure 2 shows surface morphology of as synthe-

sized MnO2, which confirms growth of crystalline round shape MnO2.
3.2 Electricity Production
MFCs were started giving voltage just after inoculation and OV across 100 Ω
external resistance was found to be 10 and 54 mV in MFC-C and MFC-M,
respectively. The OV increased with days of operation and became stable after two
sequential fed-batch cycles (Fig. 3). The average OV after getting stable perfor-
mance was found to be 215 ± 8.3 mV and 410 ± 5.49 mV, respectively, in MFC-C
and MFC-M. The enhanced voltage generation in MnO2 loaded MFC (MFC-M) as
compared to control MFC (MFC-C) is because of higher oxygen reduction reaction
(ORR) and enhanced electrochemical kinetics due to catalytic action of MnO2 (Mao
et al. 2003).
Polarization studies were performed after getting stable performance of MFCs.
The voltages were recorded at external resistances varying from 10,000 to 5 Ω, and
the corresponding polarization curves of MFCs are shown in Fig. 4. Higher voltage
(604 mV) and power density (1.64 W m−3 at current density of 113 mA m−2) was
observed in MFC having MnO2 as catalyst in cathode (MFC-M) than that of control
MFC (without catalyst) with voltage and power density of 455 mV and
0.491 W m−3 (73 mA m−2), respectively. Internal resistances of MFCs were cal-
culated by the slope of voltage versus current graph (Logan et al. 2006). Lower
internal resistance of 71 Ω in MFC-M as compared to MFC-C (93 Ω) showed less
Fig. 3 Operating voltage

generated from MFC-M and
MFC-C during entire
experiment
Fig. 4 Polarization and

power density plot of MFC-M
and MFC-C with respect to
current density at higher to
lower external resistance.
Arrow shows the
corresponding Y-axes of
power density and cell
potential
ORR overpotential loss and superior electrochemical activity in MnO2 catalyzed

cathode to produce higher power.
Moreover, power production in MFC-M was found higher than reported
(0.77 mW m−3) by Liu et al. (2010), which is due to improved crystal structure of
MnO2 (γ-phase). However, Khilari et al. (2013) reported 2.85 times higher power
(4.68 W m−3) than present work using graphene supported α-MnO2 nanotube in
cathode, which is possibly due to the superior electrical conductivity of graphene
and higher ORR kinetics of α-MnO2.
3.3 Wastewater Treatment and Coulombic Efficiency
MFC could be used as an effective wastewater treatment system with simultaneous

generation of electrical energy (Aelterman et al. 2006a). The organic matter
removal efficiency in terms of COD and total nitrogen (Fig. 5a) were calculated to
observe wastewater treatment efficiency of MFCs, whereas CE (Fig. 5b) was
estimated to evaluate electricity harvesting efficiency. The average influent COD
concentration was 2512 ± 69 mg l−1 during entire experiment. COD and total
nitrogen removal efficiencies were always higher in MFC-M as compared to
MFC-C in all batch cycles. Average COD and total nitrogen removal efficiencies
were found to be 84.4 ± 3.4 % and 66.5 ± 2.56 %, respectively, in MFC-M, whereas
in MFC-C, the respective efficiencies were 75 ± 1.4 % and 52.88 ± 3.17 %.
Enhanced organic matter removal efficiency in MFC-M is due to the higher ORR
and electrochemical activity which tended the microorganism to utilize more sub-
strate by maintaining pH balance in anodic chamber of MFC (Ghasemi et al. 2011).
The average CE of 9 ± 0.65 % and 4.6 ± 0.26 % was estimated for MFC-M and
MFC-C, respectively. The enhanced CE of MFC-M than that of MFC-C is in
agreement with the power generation (Fig. 4). The result shows the effectiveness of
MnO2 as a catalyst for cathode in MFC for enhancing power and wastewater
treatment and its potential ability to replace costly catalyst for large commercial
applications. Figure 5b shows the CE of MFCs during different batch cycles, where
CE of MFC-M was found to be better in each batch cycle in terms of Coulombs
recovery than MFC-C.
Figure 6 shows the overall biochemical degradation pathway of macromolecules
to micro-molecules and finally to electrons and protons in anode using bacteria as
biocatalyst (Noori et al. 2016); and cathodic oxygen reduction using MnO2 catalyst.
During substrate degradation, electrochemically active microorganisms convert
chemical energy associated with the organic matter into electrons and protons.
Fig. 5 Performance of MFCs in terms of organic matter removal and coulombic efficiency during
each fed batch cycle: a COD removal and total nitrogen removal, and b coulombic efficiency
Fig. 6 A schematic diagram of overall working mechanism of MFC
Electrons pass through the external circuit and protons migrate from the proton
exchange membrane to the cathode. In cathode, reduction reaction occurs in pres-
ence of terminal electron acceptor. The ORR in presence of MnO2 catalysts favors
four electron oxygen reduction pathways using the following reaction sequence:
2MnðIVÞ O2 + 2H2 O + 2e ! 2MnðIIIÞ OOH + 2OH ð1Þ

2MnðIIIÞ OOH + O2 + 2e ! 2 MnðIVÞ OOHO ð2Þ

2 MnðIVÞ OOHO ! 2MnðIVÞ O2 + 2OH ð3Þ
Overall reaction is as follows:
O2 + 2H2 O + 4e ! 4OH ð4Þ
4 Conclusions
Effectiveness of low cost MnO2, synthesized using chemical co-precipitation

method was evaluated as cathode catalyst in single chamber MFC to treat fish
market wastewater and simultaneous generation of electricity. The results showed
significant increase in power generation up to 330 % in MFC-M having MnO2 as
catalyst in cathode as compared to control MFC-C. Moreover, COD removal, total

nitrogen removal and CE was higher in MFC-M than that of MFC-C. The low cost
and easy to synthesis γ-MnO2 demonstrated greater potential to be used as cathode
catalyst in MFC to treat fish market wastewater and valuable energy recovery.
Acknowledgments The grant received from Department of Science and Technology, Govt. of
India (File No. DST/TSG/NTS/2010/61) to undertake this work is duly acknowledged.
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Bioelectricity Generation from Marine
Algae Chaetoceros Using Microbial Fuel
Cell
P.P. Rajesh and M.M. Ghangrekar
Abstract Marine algae Chaetoceros can be effectively used both as an electron

donor and methanogen inhibitor in MFCs due to its high organic matter content and
methanogen inhibition properties. Hexadecatrienoic acid present in Chaetoceros
inhibits the growth of gram-positive methanogenic archaea via adsorption and
disruption of cell membrane. Performance evaluation of two dual-chambered MFCs
without any pretreatment of anaerobic sludge used as anodic inoculum was com-
pared using Chaetoceros and acetate as substrate. MFC fed with Chaetoceros
having a COD concentration of 2000 mg l−1, could produce a maximum operating
voltage of 495 mV and sustainable power density (PD) of 12.25 W m−3 after a
period of 12 days from start-up. A maximum PD of 15.13 W m−3 and Coulombic
efficiency (CE) of 47.25 % could be obtained in MFC fed with Chaetoceros;
whereas MFC fed with acetate (2000 mg COD l−1) produced a maximum PD of
3.64 W m−3 and a CE of 19.65 %. An average total chemical oxygen demand,
carbohydrate and protein removal efficiencies of 73.57 ± 2.1 %, 75.77 ± 1.1 %, and
43.11 ± 1.4 % were obtained in MFC using Chaetoceros as substrate. Cyclic
voltammetry study reveals a higher electron discharge on the anode surface of the
MFC fed with Chaetoceros.

Keywords Chaetoceros Coulombic efficiency Marine algae Methanogenesis
Microbial fuel cell
P.P. Rajesh
PK Sinha Centre for Bioenergy, Indian Institute of Technology Kharagpur,
M.M. Ghangrekar (&)

DOI 10.1007/978-81-322-2773-1_22
296 P.P. Rajesh and M.M. Ghangrekar
1 Introduction
Increasing energy demand and the escalating fuel cost have encouraged the search
for alternative source of energy. Bioelectrical systems such as microbial fuel cell
(MFC) offers a sustainable means of energy generation from organic matter. The
use of algae biomass as a substrate in MFCs can provide a cost-effective means of
electricity generation due to its low energy requirement for cultivation. In the anode
of MFC, microbes oxidize the substrate and electrons are donated to the solid
anode. These electrons move through an external circuit toward the cathode and
combine with an electron acceptor to generate electricity. Various genera of bacteria
present in the activated sludge such as Alcaligenes faecalis, Enterococcus galli-
narum, Pseudomonas aeruginosa, and Shewanella sp. can degrade algae biomass
and transfer electrons to the anode surface (Naim et al. 2013). It is possible that
MFCs can be used as an energy efficient biological treatment process for algal
organic matter degradation (Velasquez-Orta et al. 2009).
Methanogenesis is often considered as the greatest challenge in achieving
on-field applications of MFCs due to the reduction in Coulombic efficiency (CE).
The similar growth conditions as that of anode-respiring electrogens and ability to
grow in planktonic form favors the growth of methanogenic archaea in MFCs.
Inhibition of growth of methanogens in MFCs can lead to a reduction in energy loss
to methanogenesis and the consequent improvement in CE of the system. Various
pretreatment methods such as 2-bromoethanesulfonate dosing (Zhuang et al. 2012),
heat and ultra-sonication treatment (Kim et al. 2005; More and Ghangrekar 2010);
oxygen stress condition (Chae et al. 2010) and lauric acid dosing (Rajesh et al.
2014) have been tried in MFCs previously.
The marine algae Chaetoceros can be effectively used both as an electron donor
and inhibitor for methanogens in MFCs due to its high organic matter content and
methanogen inhibition properties. Long chain saturated fatty acid (SFA), hexade-
catrienoic acid present in Chaetoceros inhibits the growth of methanogenic archaea
via adsorption and disruption of cell membrane. Gram-negative bacteria are
inhibited to a limited extent at higher fatty acid concentrations (Galbraith and Miller
1973). Utilization of Chaetoceros as a methanogen inhibitor enhances the perfor-
mance of MFC (Rajesh et al. 2015).
The main goal of present study was to assess the potential of Chaetoceros to be
used as substrate in MFC for producing bioelectricity. The effect of different
concentration of Chaetoceros on the performance was evaluated. Performance of
MFC fed with Chaetoceros was compared with control MFC fed with acetate as
substrate. Cyclic voltammetry was performed to find the change in the redox
activity at the anode surface, while using acetate and Chaetoceros as substrates.
Bioelectricity Generation from Marine Algae Chaetoceros … 297
2.1 MFC Construction and Operation
Experiments were performed in two dual chambered aqueous cathode MFCs with
an anodic liquid volume of 250 ml. Baked clayware cylinders served as the anodic
chambers of these MFCs and the 8 mm thick wall material of the cylinder acted as a
separator between anodic and cathodic chambers as well as the cation exchange
membrane (Behera et al. 2010). The anode and cathode electrodes of both MFCs
were made up of carbon felt with a projected surface area of 192 and 260 cm2,
respectively. Concealed copper wire was used to connect both the electrodes
through an external resistance of 100 Ω.
Mixed anaerobic sludge collected from the bottom of a septic tank without any
pretreatment was used as the inoculum in the anodic chamber. Pure culture of
Chaetoceros maintained in a commercial shrimp hatchery was collected for the
present study by sieving 100 l of algae grown in a batch culture system using F/2
medium (Guillard and Ryther 1962). The marine algae were then sundried and
provided as ground preparation. Dry algae biomass was pretreated using ultra-
sonication method to release the content of the cell and provided in the anodic
chamber with a concentrated solution of 200 ml so as to result in anolyte total COD
concentration of 512, 942, 2000, and 2870 mg l−1 after mixing. As a control another
identical MFC system was maintained with acetate as the substrate with other
chemicals as reported by Behera et al. (2010). These MFCs were operated under
controlled temperature varying from 33 to 37 °C in batch mode with a feeding
interval of 4 days. At the end of each batch cycle, 200 ml of anodic fluid was
replaced with 200 ml of fresh concentrated feed having pre-decided COD con-
centration so as to maintain the same concentration of substrate in MFC fed with
algae and acetate in every batch operation.
2.2 Analysis and Calculations
Performance of MFC was evaluated in terms of voltage (V) and current

(I) measured using a data acquisition unit (Agilent Technologies, Malaysia) and
converted to power according to P = V * I, where P = power (W), I = current (A),
and V = voltage (V). The electrode potentials of both anode and cathode were
measured with an Ag/AgCl (+197 mV vs. SHE, Bioanalytical Systems Inc., USA)
reference electrode. Open-circuit voltage (OCV) was measured under no current
flow condition of the circuit. Power density (PD) and power per unit volume were
calculated by normalizing power to the anode surface area and net liquid volume of
anodic chamber, respectively. Polarization studies were carried out after attaining a
stable cell potential by changing the external resistances from 20,000 to 5 Ω using
the resistance box (GEC 05 R Decade Resistance Box). Cyclic voltammetry
(CV) was performed using Autolab PGSTAT 302 N potentiostat (Metrohm, The
Netherland) and NOVA 1.9 software.
Total and soluble COD concentrations of the anolyte in MFCs were measured
for total and soluble fractions of the bulk liquid by closed reflux colorimetric
method as mentioned in standard methods (APHA 1998). Carbohydrate estimation
was carried out by colorimetric method based on anthrone reagent (Gerhardt et al.
1994). Total and soluble protein was estimated using Bradford method (Kruger
1994). For the measurement of soluble fraction, the anolyte was pre-filtered using
GF/C filter paper. The CE was calculated as the fraction of total coulombs actually
transferred to the anode against that theoretically present in substrate for current
generation over the time period (Logan et al. 2006).
3.1 Organic Matter Removal in MFC Fed with Chaetoceros
Algal organic matter (AOM) is composed primarily of carbohydrate, protein, and

lipid. The fermentable macromolecules in algal organic matter such as protein and
carbohydrate can be removed by syntrophic interaction between acidogenic bacteria
and electrogenic species. A total COD removal of 73.57 ± 2.1 % and soluble COD
removal of 85.25 ± 1.3 % could be achieved while using Chaetoceros (as
2000 mg l−1 COD concentration) as substrate in MFC (Fig. 1). Total COD removal
was found to be less since it requires the degradation of particulate organic matter
into soluble substrates by fermentative bacteria. Carbohydrates were removed to a
greater extent as compared to protein. A total carbohydrate removal of
75.77 ± 1.1 % was observed in MFC, whereas total protein removal was limited to
43.11 ± 1.4 %. Both the particulate and soluble carbohydrate removal was found to
be higher than that of particulate and soluble protein removal which suggests that
Fig. 1 Organic matter 100

removal in MFC fed with 90 Total Soluble
algae as substrate at COD
concentration of 2000 mg l−1 80
Removal Efficiency (%)
70
60
50
40
30
20
10
0
COD Carbohydrate Protein
carbohydrate was properly used by microorganisms as compared to protein which

has a more complex molecular structure. This observation indicates that microbial
selection occurred in MFCs for carbohydrate and protein metabolism. The reason
for the higher removal of carbohydrate might be due to the acclimatization of
carbohydrate degrading microbial strains in MFCs.
3.2 Electricity Generation in MFCs Fed with Algae

and Acetate as Substrate
A repeatable cycle of power generation was obtained by Chaetoceros as a substrate

after three batch cycles. The long time incurred to stabilize the system suggests the
longer period required for the fermentative and electrogenic bacteria to get accli-
matize in MFC fed with algae. MFC fed with Chaetoceros (at 2000 mg l−1 COD
concentration) generated a maximum operating voltage of 495 mV, whereas the
voltage generation in MFC fed with acetate was limited to 280 mV. A sustainable
PD of 128 mW m−2 could be obtained in MFC fed with algae, whereas the MFC
fed with acetate could only produce a PD of 44.26 mW m−2. Anode potential of the
MFC fed with algae (−413 ± 8 mV; versus. Ag/AgCl) was found to be lower as
compared to MFC fed with acetate (−336 ± 12 mV; vs. Ag/AgCl). This might be
due to the enhanced utilization of substrate by electrogenic species in MFC fed with
algae; whereas, the methanogens present in MFC fed with acetate accounted for
substrate loss and consequent decrease in performance. Rajesh et al. (2015)
reported that Chaetoceros dosing can enhance the electrogenesis in MFCs, while
limiting the substrate loss to methanogens.
MFC fed with Chaetoceros produced an average current of 4.9 ± 0.8 mA after
reaching a stabilized state of operation. A maximum CE of 47.25 % could be
obtained in MFC using algae as substrate (Table 1). This result indicates that the
algal organic matter present in Chaetoceros can be effectively used for electricity
generation while reducing the substrate loss to methanogenesis. A maximum CE of
Table 1 Overall performance of the MFCs

Parameters MFCacetate MFCalgae
Feed COD 2000 512 942 2000 2870
concentration (mg l−1)
Sustainable power 3.64 3.20 7.22 12.25 11.52
density (W m−3)
Cell voltage (mV) 270 ± 10 370 ± 14 420 ± 11 480 ± 14 465 ± 12
Total COD removal 88.31 ± 1.9 78.21 ± 2.7 75.21 ± 1.9 73.57 ± 2.1 68.91 ± 3.1
efficiency (%)
Soluble COD removal 89.32 ± 1.7 86.14 ± 2.1 85.25 ± 1.3 82.13 ± 2.9
efficiency (%)
Maximum CE (%) 19.65 66.39 65.97 47.25 35.24
19.65 % could only be obtained in MFC fed with acetate. The CE obtained in this
study using Chaetoceros as substrate was higher than that obtained with Chlorella
vulgaris (28 %) and Ulva lactuca powder (23 %) as MFC feed stock
(Velasquez-Orta et al. 2009).
3.3 Polarization of MFCs
Polarization was conducted by changing the external circuit load from 20,000 to
5 Ω once stabilized performance of MFC was observed. The MFC fed with
Chaetoceros with COD concentration of 2000 mg l−1 showed a maximum power
density of 159 mW m−2 and volumetric power density of 15.13 W m−3 (Fig. 2). The
maximum power density in acetate fed MFC was lesser (36.14 mW m−2). This
might be due to the fact that part of the substrate was utilized for methanogenesis in
MFC fed with acetate; whereas in MFC fed with algae, enhanced utilization of
substrate by electrogens occurred. The maximum volumetric power density
obtained in this study was considerably higher than the reported value using
Scenedesmus (8.67 W m−3) and Chlorella vulgaris (3.7 W m−3) (Cui et al. 2014;
Wang et al. 2012).
3.4 Cyclic Voltammetry
Bioelectrochemical behavior of the MFCs fed with acetate and algae was studied
in situ by cyclic voltammetry. Voltammograms were recorded by applying a
potential ramp of +1 to −1 V at a slow scan rate of 1 mV/s. The CV analysis was
Fig. 2 Polarization curves of 800 250

the MFCs fed with algae and
700
Power density (mW.m-2)
acetate at a COD 200

concentration of 2000 mg l−1 600
Voltage (mV)
500 150
400
300 100
200
50
100
0 0
0 200 400 600 800 1000
-2
Current density (mA.m )
OV of MFCacetate OV of MFCalgae
PD of MFCacetate PD of MFCalgae
Fig. 3 CV of MFCs fed with 0.18

algae and acetate at a COD 0.16
MFCAlgae
concentration of 2000 mg l−1 0.14
0.12 MFCAcetate
0.10
Current (A)
0.08
0.06
0.04
0.02
0.00
-0.02
-0.04
-0.06
-0.08
-0.5 0.0 0.5 1.0
Applied potential (V)
performed for anodic half cell with carbon felt as a working electrode, Ag/AgCl as a
reference electrode (+197 vs. SHE) and graphite rod as counter electrode. MFC fed
with algae (2000 mg COD l−1) has noted higher oxidative current (150 mA) as
compared to the MFC using acetate (2000 mg COD l−1) as substrate (oxidative
current of 30 mA) (Fig. 3). Higher current observed on the voltammograms can be
correlated to higher electron discharge by the biofilm developed on the anode
surface of MFC fed with algae. Higher electrogenic activity observed in MFC fed
with algae than MFC using acetate can be correlated to the enhanced utilization of
substrate by electrogens while limiting the energy loss to methanogenic species.
3.5 Effect of Chaetoceros Concentration on MFC

Performance
The performance of MFC in terms of maximum power generation at different

substrate (algae) concentration (512, 942, 2000 and 2870 mg COD l−1) was eval-
uated. The cell voltage increased with the increase in COD concentration of marine
algae from 512 mg l−1 (370 ± 14 mV) to 2000 mg l−1 (480 ± 14 mV) and remained
more or less stable (465 ± 12 mV) at a COD concentration of 2870 mg l−1 (Fig. 4).
The bioelectrochemical reaction and the subsequent electron transfer to the anode
surface vary with the concentration of organic matter available for the microbes to
degrade. A maximum sustainable PD of 12.25 W m−3 was obtained at a COD
concentration of 2000 mg l−1 of Chaetoceros. The performance of MFC did not
improve further and almost remained stable at COD concentration of 2000–
2870 mg l−1. Similar trend of effect of varying concentration of substrate on
electricity generation was reported by Cui et al. (2014) with the microalgae
Scenedesmus as feed. A maximum COD removal efficiency of 78.21 ± 2.7 % could
be obtained with algal feed at COD concentration of 512 mg l−1. With increase in
concentration of the algae the COD removal efficiency was found to be decreasing.
Fig. 4 Effect of Chaetoceros 20 100

concentration in maximum 18 y = 79.3 - 0.0034x 90
power density and COD R 2 = 0.8980
16 80
Power density (W/m 3)

removal efficiency
COD removal (%)

14 70
12 60
10 50
8 y = 12.05 - 12.65e- 0.0017x 40
R 2 = 0.8909
6 30
4 20
Power density
2 COD removal 10
0 0
500 1000 1500 2000 2500 3000
Substrate Concentration (mg COD l -1)
The COD removal efficiency at the concentration of 2870 mg l−1 was found to be
lesser (68.91 ± 3.1 %). These results indicate that maximum utilization of substrate
for electrogenesis occurred at a COD concentration of 2000 mg l−1 and reached to
an optimum performance of MFC.
4 Conclusion
Utilization of marine algae Chaetoceros as an electron donor and methanogenesis

inhibitor in MFC system was explored. The use of microalgae biomass as a sub-
strate showed higher coulombic efficiency than acetate as substrate. Polarization
study and CV analysis showed a higher electrogenic activity on the anode surface
using marine algae as substrate. Carbohydrate contained in Chaetoceros was
degraded more than the protein present. Maximum power production was found to
be at COD concentration of 2000 mg l−1 of Chaetoceros. A maximum coulombic
efficiency of 47.25 % could be obtained using Chaetoceros as a substrate. This
study reveals that the marine algae Chaetoceros can be used as an efficient and
sustainable biofuel feed stock for electricity generation in MFCs.
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Technol 180:66–71
Velasquez-Orta SB, Curtis TP, Logan BE (2009) Energy from algae using microbial fuel cells.
Biotechnol and Bioeng 103(6):1068–1076
Wang H, Liu D, Lu L, Zhao Z, Xu Y, Cui F (2012) Degradation of algal organic matter using
microbial fuel cells and its association with trihalomethane precursor removal. Bioresour
Technol 116:80–85
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Selective Enrichment of Electrochemically
Active Bacteria in Microbial Fuel Cell
By Pre-treatment of Mixed Anaerobic
Sludge to Be Used as Inoculum
B.R. Tiwari and M.M. Ghangrekar
Abstract Pre-treatment methods for seed sludge which are capable of selectively
enriching the electrogenic bacteria in a microbial fuel cell (MFC) need to be
employed in order to enhance the electricity production. Effect of combined
pre-treatments such as acid-ultrasonication pre-treatment (AU), acid-heat
pre-treatment (AH), and ultrasonication-heat pre-treatment (UH) on anaerobic
mixed sludge to be used as inoculum in a dual chambered MFC was evaluated. All
pre-treatments were found to have an incremental effect on the power generation
and Coulombic efficiency. MFC inoculated with acid-ultrasonication pre-treated
sludge achieved an increment of 520 % in power density (2.91 W m−3) and 276 %
in Coulombic efficiency (6.2 %) than that produced by control MFC operated with
untreated sludge as inoculum. Acid-ultrasonication pre-treatment was found to be
the most effective inoculum pre-treatment, followed by acid-heat pre-treatment and
ultrasonication-heat pre-treatment.

Keywords Inoculum pre-treatment Methanogenesis inhibition Microbial fuel

cell Power density Wastewater treatment
1 Introduction
The huge reserves of fossil fuels on which the world energy needs depend will be
consumed in about a century. Again, the alternate sources of energy which will cater
to the energy needs must not add to the already elevated levels of carbon dioxide in
the atmosphere. Hence, we need to look out for an option which not only provides
sufficient energy but also should be carbon neutral. In the present world, organic load
in wastewater is no longer seen as waste, but instead as a valuable energy resource.
The simultaneous production of energy and the degradation of contaminants in
B.R. Tiwari M.M. Ghangrekar (&)


DOI 10.1007/978-81-322-2773-1_23
306 B.R. Tiwari and M.M. Ghangrekar
wastewater by microbial fuel cells (MFCs) can provide both economic and envi-
ronmental benefits (Logan and Regan 2006; Liu and Logan 2004).
MFC is a bioreactor in which biodegradable substrates are oxidized through
catalytic reaction of a group of microorganism capable of harvesting electricity
from degraded organic matter without external energy input (Logan et al. 2006). It
consists of an anaerobic anode compartment and an aerobic cathode compartment
separated by a proton exchange membrane. The cathode chamber is free from
microorganisms and is filled with a catholyte, usually water, which is continuously
aerated. The anode compartment is inoculated with anaerobic sludge which harbors
bacteria which are electrogenic in origin. These bacteria get attached to the anode
and oxidize the organic matter present in the wastewater into carbon dioxide,
protons, and electrons. The anode acts as the electron acceptor and the electrons are
transported to the cathode across an external circuit. The protons migrate across the
proton exchange membrane into the cathode chamber (Behera et al. 2010). The
protons and electrons combine with oxygen to produce water.
Anode: CH3 COOH þ 2H2 O ! 2CO2 þ 8H þ þ 8e DE ¼ 0:300 V

Cathode: 4H þ þ 4e þ O2 ! 2H2 O DE ¼ 0:805 V
MFCs are an environment friendly technology because they reduce the load on
the environment by consuming the organic matter present in the waste and in turn
produce a green and clean energy. No intermediate steps are involved in the con-
version of the substrates to electricity, so the conversion efficiency is also high (Ren
et al. 2015). MFCs are anaerobic systems; thereby the estimated sludge production
is less. However, power outputs from MFCs are insufficient to meet the day-to-day
energy needs. So, there is a need to modify the present fuel cell architecture,
electrode materials, and inoculum in order to maximize the current production
(Malvankar et al. 2012).
The inoculum is the most essential component of a MFC as it is the only biotic
factor influencing the power generation. Types of communities residing in the
anode biofilm are responsible for maximum current output from MFCs. Mixed
consortia from anaerobic sludge which are commonly used for inoculating MFCs
are metabolically diverse (Du et al. 2007; Logan 2009) and consist of fermentative
bacteria, methanogens, and sulfate reducers along with electrogenic bacteria
(Angenent et al. 2002; Dollhopf et al. 2001; Wheatley 1990). Microscopic and PCR
analysis have confirmed the presence of methanogens along with electrogens in the
anode compartment of MFCs. As the growth conditions and feed dependency of
electrogens and methanogens are similar, both groups of bacteria have been found
to inhabit the anode compartment of MFCs (Chae et al. 2010; Zhuang et al. 2012).
Utilization of a part of the substrate by methanogens is a direct loss in Coulombs to
the MFC (Rabaey et al. 2010). The methanogens may also compete for the space on
the anode, thus decreasing the density of electrogenic population on the anode. This
will lead to decrease in the power generation from the MFCs. So, it would be worth
Selective Enrichment of Electrochemically Active Bacteria … 307
finding ways to selectively enrich the anodic communities with bacteria which are
electrogenic in origin and restrict the growth of methanogens (Kim et al. 2005).
Different methods have been adopted to enhance the performances of electro-
gens and limit the growth of methanogens in bioelectrochemical systems such as
ultrasonication pre-treatment to inoculum (More and Ghangrekar 2010),
2-bromoethanesulfonic acid application (Chae et al. 2010), methanogenesis control
by electrolytic oxygen production (Tice and Kim 2014), use of a ferric oxide-coated
electrode (Kim et al. 2005), temperature stress (Liu et al. 2005), and oxygen stress
(Chae et al. 2010). Although the above-mentioned methods were found to be
effective to some extent, still each of them had some drawbacks.
2-bromoethanesulfonic is expensive and large quantities need to be administered
throughout the operation of MFC when implemented for large scale (Chae et al.
2010).
In the present study, physiological differences between the methanogens and
electrogens have been exploited to device a combination of three different physical
pre-treatments, i.e., acid pre-treatment (pH 5.3), ultrasonication pre-treatment
(40 kHz, 5 min), and heat pre-treatment (100 °C, 15 min). Methanogens are easily
affected by variations in the pH even when slightly away from neutral pH (van
Haandel and Lettinga 1994). Methane production rate decreases at pH below 6.3 or
above 7.8 (van Haandel and Lettinga 1994). Thus, exposure of anaerobic sludge to
pH below 6.0 would be effective in inhibiting methanogens. Ultrasonication was
used as pre-treatment for inoculum in MFC, and this MFC performed better as
compared to MFC inoculated with untreated sludge in terms of the power as well as
organic matter removal (More and Ghangrekar 2010). Exposure to heat can lead to
suppression of methanogens because of their inability to form protective spores
(Zhu and Beland 2006). Heating inoculum at 100 °C for 15 min results in sup-
pressing methanogens (Ghangrekar and Shinde 2007). This study was conducted to
improve the performance of MFCs by inhibition of methanogenesis in MFCs under
the application of combination of pre-treatments to the inoculum.
2.1 MFC Construction and Operation
Four identical earthen pots with wall thickness of about 6 mm were used as anodic
chambers of four different MFCs inoculated with differently pre-treated sludge. The
anodic chambers had an effective volume of 1.3 l and it was placed in the plastic jar
of volume 10 l, where tap water was used as catholyte. The catholyte was con-
tinuously aerated by an aquarium air pump. Non-transparent polyacrylic sheet was
used to seal the top of anode chamber. Electrode spacing of 20 mm was maintained
RESISTANCE
REFERENCE
ELECTRODE e- AIR PUMP
e-
CATHODE
e-
e-
ANODE
H+
H+
CH 3 COO + 2H 2 0
4H+ + O 2 + 4e -
2H2 O
2CO 2 +7H + +8e - PEM
Fig. 1 Schematic diagram of the experimental setup
(Fig. 1). Stainless steel mesh of total surface area of 354.75 cm2 and graphite plates
having a surface area of 305 cm2 were used as anode and cathode materials,
respectively. Electrodes were connected externally with concealed copper wires
across a resistance of 100 Ω. Acetate as a source of carbon having chemical oxygen
demand (COD) of about 3000 mg l−1 was fed at an interval of 4 days and MFCs
were operated in batch mode. Synthetic wastewater was prepared as per the com-
position given by Jadhav and Ghangrekar (2009).
2.2 Inoculation and Pre-treatment
Mixed anaerobic sludge having VSS of 16 g l−1, collected from septic tank bottom,
was used for inoculating the MFCs. The inoculum sludge was sieved through 1 mm
opening sieve. Performance of three MFCs inoculated with different pre-treatments
given to the mixed anaerobic sludge was evaluated along with a control MFC-WT,
which was inoculated with mixed anaerobic sludge without any pre-treatment.
Pre-treatments such as acid-ultrasonication pre-treatment (AU), acid-heat
pre-treatment (AH), and ultrasonication-heat pre-treatment (UH) were applied.
Acid pre-treatment was administered by addition of 0.01N H2SO4 drop by drop
to the inoculum sludge till it attained a final pH of 5.3. Heat pre-treatment was
provided by heating the inoculum sludge at 100 °C for 15 min. Sonication
pre-treatment was provided for a duration of 5 min using ultrasonic processor
(Piezo-U-Sonic) having constant supplied power of 120 W and frequency of
40 kHz. In all the MFCs, 200 ml of sludge was added along with synthetic
wastewater. Cathodic chamber did not receive any microbial addition.
2.3 Analysis and Calculation
Operating voltage (V) and current (I) were measured using a data acquisition unit
(Agilent Technologies, Malaysia). Power density was calculated by normalizing
power to net liquid volume of anode chamber. Polarization studies were carried out
during stable power generation phase by changing the external resistances in steps
from 20,000 to 1 Ω by using the resistance box (GEC 05 R Decade Resistance Box,
Renown Systems, Kolkata, India). A time interval of 30 min was provided after
each change of resistance. Cathode and anode potentials were measured during
polarization using Ag/AgCl reference electrode (Bioanalytical Systems Inc., West
Lafayette, Indiana, USA).
Exchange current density and charge transfer resistance values were calculated
from Tafel curves plotted using the equation shown below:
ln j ¼ ln j0 þ bFgðRT Þ1 ð1Þ
where j0 is the exchange current density (mA m−2), j is the electrode current density
(mA m−2), β is the electron transfer coefficient, R is the ideal gas constant
(8.31 J mol−1 K−1), F is the Faraday constant (96,485 C mol−1 e−1), T is the
absolute temperature (K), and finally η is the activation over potential.
Coulombic efficiency (CE) of MFCs operated under batch mode, evaluated over
a period of time t, was calculated as given by Logan et al. (2008).
Rt
M 0 Idt
CE ¼ ð2Þ
FbVDCOD
Where M is the molecular weight (32) of oxygen, F is Faraday’s constant, b is the

number of electrons exchanged (4) per mole of oxygen, V is the volume of liquid in
the anodic chamber, and ΔCOD is the change in COD over time t.
Volatile suspended solids of the inoculum and wastewater COD were measured
according to APHA standard methods (APHA 1998). The procedure described by
Bhunia and Ghangrekar (2007) was adopted to calculate specific methanogenic
activities of the sludge after receiving different pre-treatments. Methane gas pro-
duction was measured by a liquid displacement system which was filled with 5 %
NaOH solution. The pH was monitored using water quality bench meter (Cyber
Scan, PCD 6500, Eutech Instruments, Ayer Rajah Crescent, Singapore).
3.1 Effect of Pre-treatment of Inoculum Sludge

on Electricity Generation
During the stable stage, the operating voltage outputs from these MFCs were
535 ± 18 mV (MFC-AU), 492 ± 20 mV (MFC-AH), and 301 ± 11 mV (MFC-UH).
The MFC inoculated without any treatment to the sludge (MFC-WT) recorded
working voltage of 236 ± 15 mV. The operating voltages denote that all the MFCs
inoculated with pre-treated sludge were capable of generating current higher than
the MFC-WT. Highest operating voltage was recorded in the MFC-AU followed by
MFC-AH and MFC-UH. More and Ghangrekar (2010) reported that ultrasonication
pre-treatment limits the activity of gram positive methanogens and it was ineffective
against gram negative bacteria. Most of the electrochemically active bacteria, i.e.,
Geobactor metallireducens (Lovley et al. 1993), Shewanella oneidensis,
Shewanella putrefaciens, Desulfuromonas acetoxidans (Debabov 2008), and
Geobactor sulfurreducens (Caccavo et al. 1994) are gram negative bacteria.
Researchers have found that acidophilic conditions (pH 5.5) help to suppress the
activity of methanogens in a MFC (Venkata Mohan et al. 2008). So when acid and
ultrasonication pre-treatment were used in a combination, the voltage generated was
the highest among all pre-treatments.
3.2 Polarization of MFCs
When current is passed across the electrodes in a MFC, then the respective electrode
potential registers a shift from their equilibrium potentials, which is denoted by the
term polarization (Raman and Lan 2012). The polarization study was conducted to
visualize the difference in electron discharge process with respect to different
inoculum pre-treatments. MFCs recorded a maximum power density of
2.915 W m−3 (V = 389 mV, R = 40 Ω) for MFC-AU, 2.339 W m−3 (V = 427 mV, R
= 60 Ω) for MFC-AH, 811 mW m−3 (V = 251 mV, R = 60 Ω) for MFC-UH, and
559 mWm−3 (V = 225 mV, R = 70 Ω) for MFC-WT, respectively (Fig. 2). MFC-AU
recorded the highest power density, which is 520 % higher than that demonstrated by
the MFC-WT. Power density obtained follows the following trend—
MFC-AU > MFC-AH > MFC-UH > MFC-WT. The trend establishes the fact that all
pre-treatments work in synergistic manner to improve the power generation from the
MFCs.
Since, methanogens are sensitive to changes in factors like temperature and pH,
exposure of the inoculum to acidic pH, high temperature, and ultrasonication can be
applied to suppress their activity and population. The different combinations of
pre-treatments were found favorable to the electrogenic species and may have lead
Fig. 2 Power density curves 3000

for MFCs inoculated with
different pre-treated sludge 2500
Power density (mW/m )

3
2000
1500
1000
500
0
0 2000 4000 6000 8000 10000 12000
3
Current density (mA/m )
MFC-WT MFC-UH MFC-AH MFC-AU
Fig. 3 Polarization curves for 1000

MFCs inoculated with
different pre-treated sludge
800
VOLTAGE (mV)
600
400
200
0
0 2 4 6 8 10 12 14
CURRENT (mA)
% (MFC-WT) % (MFC-UH) % (MFC-AH) % (MFC-AU)
to the inhibition of nonelectrogenic species, thereby recording higher power gen-

eration in MFCs with pre-treated sludge as inoculum.
Internal resistance was calculated from the polarization curves between the
recorded voltage and current values for MFCs. Internal resistance values were 36,
49, 72, and 89 Ω for MFC-AU, MFC-AH, MFC-UH, and MFC-WT, respectively
(Fig. 3). It is evident that the administration of pre-treatments has lead to the
decrease in the internal resistance values, thereby leading to an increase in the
power generated.
3.3 Electrode Kinetic Study of MFCs with Different

Inoculum Pre-treatments
Tafel plots, a plot between the logarithm of the current density and the overpotential
i.e., the difference between the potential under the load and without a load, were
drawn to study the electrocatalytic activity of the anodes in terms of exchange
current density (j0) and charge transfer resistance (Rct) (Table 1). Exchange current
density gives an idea about the rate of reaction taking place at the surface of
electrodes by taking into account the total number of electrons exchanged during
electro-oxidation and electro-reduction at equilibrium (Raman and Lan 2012). The
rate of oxidation at the anode is directly proportional to the number of electrons
exchanged from the electrochemically active bacteria to the anode (Raghavulu et al.
2013). The Tafel equation is as shown below:
h anFg ð1aÞnFg
i
j ¼ j0 e RT e RT ð3Þ
where j0 is exchange current density (mA m−2), j is the electrode current density
(mA m−2), a is the electron transfer coefficient, n is the number of electrons
transferred in Faradaic charge transfer process, η is the overpotential, R is the ideal
gas constant (8.314 J mol−1 K−1), T is the absolute temperature (K), and F is the
Faraday constant (96,485 C mol−1e−1) (Bard and Faulkner 2001).
Although all the MFCs were fabricated using the same electrode materials, dif-
ferent reaction kinetics were observed at the respective anodes. This implies that the
application of different pre-treatments for sludge inoculum has resulted in variation in
electrogenic activity at the anodes. The exchange current density values denote that
the reaction rates were in the order of MFC-AU > MFC-AH > MFC-UH > MFC-WT.
The electrochemical reactions at anode of MFC-AU were fastest, which clearly
indicates the effective growth of electrogenic bacterial biofilm on its anode. The
exchange current density values for MFC-WT were lowest among all, indicating that
pre-treatment to inoculum had a positive effect on enhancing the electrogenic activity
on anodes of other MFCs which contributed to higher voltage output.
Charge transfer resistance (Rct) represents the capability of system to resist the
transfer of charge from electrode against the potential difference. The Rct values
obtained for anode of MFCs were in the order of MFC-AU < MFC-
AH < MFC-UH < MFC-WT. In MFC-WT, non-electrochemically active bacteria
were higher in number, thereby decreasing the kinetics of electro-oxidation taking
Table 1 Exchange current density and charge transfer resistance obtained from Tafel plot for
anodes of MFCs inoculated with differently pre-treated sludge
Exchange current density (mA m−2) 2.33 5.64 7.31 11.6
Charge transfer resistance 2.77 1.14 0.88 0.55
(Ω m−2) × 10−3
place at the anode which resulted in increasing activation loss and thus increasing the
charge transfer resistance (Ramasamy et al. 2008). But in the case of MFCs inoc-
ulated with pre-treated sludge, electrogenesis was the dominating process which
facilitated a decrease in the activation energy required for initiating the electro-
chemical reaction and thus low charge transfer resistance values were obtained
(Clauwaert et al. 2008).
3.4 Effect of Pre-treatment of Sludge Inoculums

on Substrate Degradation
The pre-treatments applied act as a shock to the bacteria residing in the inoculum,
thereby inducing selective inhibition. As a result, the group of bacteria which were
not capable of surviving under those altered conditions perishes. So, there is a
decrease in the total population of substrate consumers immediately after the
application of the pre-treatments. This is reflected by the reduced COD removal in
the MFCs with pre-treated sludge as inoculum. As the shocks were applied tar-
geting the methanogens, the surviving population is expected to be mostly elec-
trogenic in origin. Once the performance of the MFCs was stable, COD removal
efficiencies of 71.6 ± 3.84, 76.2 ± 4.65, 79.2 ± 3.63, and 84.8 ± 2.49 % were
obtained for MFC-AU, MFC-AH, MFC-UH, and MFC-WT, respectively (Fig. 4).
COD removal efficiency was highest in MFC-WT as compared to MFCs inoc-
ulated with pre-treated sludge because the activity of microbial population con-
tributing to the COD removal in case of MFC inoculated with raw sludge is not
disturbed; whereas pre-treatment has resulted in decreasing the viable bacterial
population.
Fig. 4 COD removal 100

% COD REMOVAL
efficiencies in MFCs 85
inoculated with differently 79
80 76
pre-treated sludge 72
% COD REMOVAL
60
40
20
0
PRETREATMENTS
3.5 Effect of Pre-treatment of Sludge Inoculum

on Coulombic Efficiency
The oxidation of any substrate is accompanied by the removal of electrons, which

need to be recovered as much as possible in the form of current in order to make
MFC a feasible option for power generation. The percentage of electrons recovered
to the overall amount stored in the substrate is explained by the term ‘Coulombic
efficiency.’ More is the share of substrate consumption by the electrogenic bacteria
more will be the Coulombic efficiency of the system. Once the COD removal was
stable, CE values were found to be 6.23 ± 0.28, 5.43 ± 0.26, 3.20 ± 0.01, and
2.26 ± 0.09 % for MFC-AU, MFC-AH, MFC-UH, and MFC-WT, respectively
(Fig. 5). CE values are higher for the MFCs inoculated with pre-treated sludge as
inoculum which clearly indicates that the pre-treatments applied were effective in
suppressing the nonelectrogenic population. Hence, a higher percentage of the
substrate is consumed by the electrogenic bacteria and converted to current.
3.6 Methane Production Potential of Sludge with Different

Pre-treatments
Methane production was measured for sludge receiving different pre-treatments in

anaerobic batch reactor fed with synthetic wastewater of 3000 mg COD l−1 per day
having acetate as a carbon source for 4 days. The effect of pre-treatments on the
methane production is a function of the methanogenic population present in the
inoculum. It was found that sludge pre-treated with acid and ultrasonication
recorded the lowest methane production (Table 2) and the raw sludge produced
highest methane production in the reactor. This clearly denotes that all other
Fig. 5 Coulombic efficiency 7

COULOMBIC EFFICIENCY 6.23
of MFCs inoculated with
sludge after different 6
COULOMBIC EFFICIENCY
5.43
pre-treatments
5
4
3.2
3
2.26
2
0
MF-WT MFC-UH MFC-AH MFC-AU
PRETREATMENTS
Table 2 Specific Pre-treatment g VSS g CH4-COD/g

methanogenic activity of used VSS.day
sludge after different
pre-treatments Without 0.298 0.322
pre-treatment
Acid-ultrasonication 0.312 0.061
Acid-heat 0.280 0.070
Ultrasonication-heat 0.270 0.075
pre-treatments were effective in inhibiting methanogens. The specific methanogenic

activity of acid-ultrasonication pre-treated sludge was fivefold lower than untreated
sludge, indicating effective suppression of methanogens by this pre-treatment
method.
4 Conclusion
Avoiding methanogenesis in MFCs poses a challenge as it reduces the power

generation and Coulombic efficiency obtained from MFCs. The combined
pre-treatment studies performed on anaerobic mixed inoculum demonstrated the
feasibility of using combined pre-treatment for enrichment of electrochemically
active bacteria in MFCs. Combined pre-treatments such as acid-ultrasonication
pre-treatment, acid-heat pre-treatment, and ultrasonication-heat pre-treatment used
for selective enrichment of electrochemically active anaerobic consortia showed
considerable influence on the power production and substrate removal efficiency.
All pre-treatment methods showed positive influence on the overall power pro-
duction and Coulombic efficiency as compared to the control experiments.
Individually, acid-ultrasonication pre-treatment demonstrated highest power yield.
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Performance Analysis of Separators
in Dual-Chambered Microbial Fuel Cell
and Treatment of Combined Industrial
Effluent of South Gujarat
Purvi Zaveri, Tanvi Modi, Lipi Parekh, Aditi Patel,

Sameer Kureshi and Nasreen Munshi
Abstract India being the developing country faces lots of issues related to energy
crisis. Increased population rate leads to generation of various types of wastes and
associated treatment problems. Microbial Fuel Cells (MFCs) provide new oppor-
tunities for the sustainable production of energy from biodegradable, reduced
compounds present in wastewater. MFC is a bio-electro-chemical process leading
to generation of voltage difference between two electrodes by capable microbes,
carrying out oxidation of substrate. In this study, combined wastewater produced by
various industries of South Gujarat was investigated as an alternative to pure
substrates for generation of electricity. The bacterial culture, isolated from
wastewater collected from Common Effluent Treatment Plant (CETPs) of South
Gujarat region, identified as Escherichia coli, was used for oxidation of substrates
in MFC run under laboratory conditions. This study evaluated and compared the
performance of MFC with two different separators, salt bridge and proton exchange
membranes in terms of voltage production in dual-chambered system. The use of
proton exchange membrane as separator was found to be superior as compared to
salt bridge in terms of voltage generation but they were less cost effective. The use
of anionic and cationic exchange membrane resulted in production of power density
up to 119.4 and 200.6 mW/m2, respectively, in MFC with synthetic wastewater,
while the MFC connected with salt bridge produced 148.76 mW/m2 of consistent
power density. The organism could reduce COD by 35.72 % from effluent of
CETP, Pandesara, South Gujarat and could produce open-circuit voltage of 0.72 V
Purvi Zaveri Aditi Patel Sameer Kureshi Nasreen Munshi (&)

Nirma University, Sarkhej Gandhinagar Highway, Ahmedabad 382481, India
e-mail: nasreen.munshi@nirmauni.ac.in; nasreenhaque@hotmail.com
Tanvi Modi
Nootan Science and Commerce College, Visnagar, India
Lipi Parekh
Cadila Research Center, Ahmedabad, India

DOI 10.1007/978-81-322-2773-1_24
320 Purvi Zaveri et al.
and power density of 200.6 mW/m2 where salt bridge was used as separator. The
study indicates the potential of such MFC for treatment of CETP wastewater after
process optimization.
Keywords Microbial fuel cell Wastewater Proton exchange membrane Salt

bridge
1 Introduction
Importance of alternative sources of energy has gradually increased at global scale.

Developing countries like India needs to develop sources of clean energy for ever
increasing population and to combat the foreseen energy crisis. Discovery of
electro-active organisms like bacteria and yeast has been reported before long back,
and this gave a novel source of energy production using prokaryotic biomass
(Potter 1911). Microbial Electrochemical Technologies (METs) utilize microbial
metabolism to catalyze reactions such as desalination and substrate oxidation and
has diverse applications (Logan and Rabaey 2012). Microbial fuel cells are elec-
trochemical devices governed by biological activity, which comprises two basic
elements, anode and cathode. Organisms in anode chamber oxidize substrate and
lead to release of electron which is captured by electrode directly in case of
mediator-less MFC or via a mediator in mediator requiring MFC. The captured
electron is then transferred to cathode via external circuit. Internally, two com-
partments may be connected via salt bridge or proton exchange membrane which
allows passage of proton (Jadhav and Ghangrekar 2009). Fundamentally, microbial
fuel cell functions on ability of organism to transport electrons out of cell mem-
brane; however, substrate concentration, pH, configuration of MFC, etc. affect
performance of the cell. Microbial fuel cell has shown promising results in gen-
eration of electricity from various sources including wastewater (Mathuriya and
Sharma 2010), salt water (Qu et al. 2013), etc., indicating its applicability to
function with complex substrates.
In addition to structural variations, with emergence of advances in field of
material science and electronics, many novel approaches have been developed for
enhancing the functioning of MFC including the use of nano-material and incor-
poration of DC converters for higher voltage production (Wua et al. 2011). With all
available knowledge, certain challenges like low electron recovery and unstable
system performance still need to be addressed. Among many components affecting
power density produced by microbial fuel cell, the separator plays major role in
proton migration between two electrodes. Available separators belong to three
major classes, ion exchange membrane (Anion exchange membrane (AEM), cation
exchange membrane (CEM), bipolar membrane (BPM)), size selective separators
(microfiltration membrane (MFM), ultrafiltration membrane (UFM), glass fibers,
porous fibers), and salt bridge (Wei et al. 2011). Among the ion exchange
Performance Analysis of Separators … 321
Table 1 List of substrates reported in MFC run

Type of substrate Concentration of Maximum COD removal Reference
organic substrate power efficiency (%)
(mg l−1) (mW/m2)
Acetate 1000 2400 N.D. Logan et al.
(2007)
Glucose 480 (TOC) 2160 93 Catal et al.
(2008)
Distillery wastewater 2100–6100 17.76 64 Hampannava
(COD) 28.15 61 et al. (2011)
Food wastewater 4900 ± 350 *556 N.D. Jia et al.
(2013)
Full-strength cyanide laden 16000 (COD), 1800 28 Kaewkannetra
wastewater (cassava mill 86 mg l−1 cyanide et al. (2011)
wastewater)
Brewery wastewater 1250 ± 100 830 91.7–95.7 Wen et al.
mW/m3 (2010)
Steroid drug industrial N.D. 2230 82 Liu et al.
effluent (2012)
Swine wastewater 8320 (COD) 261 27 Min et al.
(2005)
Domestic wastewater 54,000 (COD) 422 25.8 Ahn et al.
(2010)
Starch processing wastewater 4852 (COD) 239.4 98 Lu et al.
(diluted to 1/2) (2009)
Note N.D. Not determined
membranes, Nafion has been widely used in MFC setups and has been proven
successful (Rahimnejad et al. 2014).
Use of separator has been debated a lot by researchers working on microbial fuel
cells. The absence of separator leads to higher oxygen in the chamber and lower
coulombic efficiency which leads to lower activity of anodic organism, whereas use
of separator at the same time leads to delay in proton transfer which generates pH
gradient between anode and cathode which is undesirable. If in system proton
exchange membranes are being used it also adds to the cost of the functioning.
Thus, selection of right type of separator for required functioning and its optimum
utilization is very important (Li et al. 2011).
As mentioned in Table 1 other than pure substrates, researchers have also used
wastewaters from various industries to run MFC and thus showed decrease in
organic load of wastewater (Wen et al. 2010). Wastewater contains multitude of
organic matter that can serve as electron source for MFC. However, there is a
difference between the composition of municipal and industrial waste; both can
serve the purpose. In South Gujarat, industrial wastewaters generated by various
industries are taken to the combined effluent treatment plants for treatment followed
by discharging them to natural water bodies. Efficiency of wastewater treatment is
generally measured in terms of reduction of COD and BOD. MFC as indicated has
capacity to use such incoming mix effluents as source of electricity generation

(Table 1). In several cases it has been reported to reduce as high as 82–91 % of
COD from synthetic wastewater after MFC run using hypochlorite as terminal
electron acceptor (Jadhav et al. 2014).
Use of combined industrial effluent remains unexplored till date due to its
complex nature. The golden corridor of Gujarat comprises most active industrial
area of the country. Industries involved in manufacturing of varieties of products
lead to generation of effluent which we hypothesized to use as anodic substrate.
Complexity of effluent throws challenge for its treatment, where MFC may serve
purpose of electricity generation and organic load reduction. The objective of the
present study was to compare the efficiency of various separators like salt bridge
and proton exchange membrane in MFC using synthetic wastewater, in terms of
electricity generation. The efficiency of dual-chambered MFC was also investigated
for treatment of combined industrial effluent from South Gujarat region.
2.1 Wastewater Source
Pandesara effluent treatment plant is one of the five CETPs situated in district Surat,
South Gujarat. Incoming effluent in this CETP is combined wastewater discarded
by nearly 122 different industries (Zaveri et al. 2015). Wastewater sample was
collected from inlet site of the treatment plant by following APHA guidelines
(Eaton 2005). Onsite sample analysis was accomplished using PCSTester 35
(Eutech Instruments, India) multimeter with respect to parameters such as pH, TDS,
salinity, etc. Samples were stored properly during transportation.
2.2 MFC Configuration and Operation
The construction of microbial fuel cell prepared at laboratory scale is presented in

Fig. 1. Dual chamber “H”-type system was constructed using plastic containers.
Graphite electrode of dimension 14.9 cm × 3.1 cm × 0.5 cm was used as anode as
well as cathode (Graphicarb Industries Ltd., Ahmedabad, Gujarat). The working
volume of MFC system was 1200 ml. The setup was run in batch mode. Multimeter
was used to measure the voltage difference. Synthetic wastewater (Ghangrekar and
Shinde 2009) containing 1 g % sucrose was used for preparation of anolyte. But the
synthetic wastewater was diluted by 20 %, hence termed as ‘modified synthetic
wastewater.’ For MFC with industrial wastewater as substrate Pandesara CETP
inlet was used and reduction in organic load was investigated in terms of BOD and
COD parallel with electricity generation. 0.5 g % cysteine was used as an oxygen
scavenger to provide reducing atmosphere in the anodic chamber (Logan 2008),

whereas methylene blue (1 g %) was used as mediator in anode. PBS buffer pro-
vided uniform concentration of ions in the cathodic chamber. Oxygen was used as
terminal electron acceptor in cathode without sparging.
2.3 Separator
Two different types of separators were evaluated, viz., salt bridge and ion exchange
membrane. The digested slurry of NaCl with agar was used to prepare salt bridge as
described by Khan et al. (2012). The salt bridge was allowed to solidify at room
temperature. Anionic exchange and cationic exchange membranes (Membranes
International Inc., New Jersey) were procured and used in different MFCs as sep-
arators between anodic and cathodic chambers (Logan et al. 2006). Membranes
were preconditioned by emersion in 5 % NaCl solution at 40 °C for membrane
hydration and expansion as per manufacturer’s instructions.
2.4 Electrogenic Organism and Circuit Assembly
Gram-negative coccobacillus, which was found to have electrogenic properties in

lab, was used in MFC. The organism had partial sequence similarity with E.coli
when analyzed by 16S rDNA sequencing and the sequence was submitted to
GenBank (Accession no: KR149337). The pure culture of organism was maintained
by periodic transfer on nutrient agar slant and was preserved at low temperatures.
For activation of organism, the culture was inoculated in nutrient broth to provide
20 % inoculum for MFC system and was incubated at 30 ± 2 °C for 14–16 h at
80 rpm. Activated culture was then harvested at 7500 rpm for 10 min at room
temperature. Culture was washed twice with sterile normal saline to assure least
contamination with complex nutrients present in broth, resuspended in equal vol-
ume of modified synthetic wastewater for addition to anode of MFC. The final
inoculum size was 20 %. Two chambers were internally connected by salt bridge,
and externally the circuit was completed using multimeter where low resistance
copper wires were used for connection. Power density of the system was calculated
by the following formula:
2
ðcellÞ
Power Density ¼ Area anode EResistance (Logan et al. 2006)
ð Þ ðexternalÞ
Data of voltage output was recorded at regular time intervals of 30 min. After
initiating the setup, samples were withdrawn for BOD and COD analyses (Eaton
2005). The setup was monitored till voltage drop was observed in the system.
Fig. 1 Schematic representation of double-chambered microbial fuel cell construction. a Using

proton exchange membrane as separator. b Using salt bridge as separator
3.1 MFC Performance Using Synthetic Wastewater

as Anolyte
Substrate used in anode not only affects the power density and voltage production
but also the microbial community composition. Synthetic or chemically defined
wastewater has been used by researchers in MFC (Pant 2010). It gives an advantage
of consistency in components and thus facilitates ease of optimization for other
physicochemical parameters. For purpose of optimization, primarily the system
preparation was carried out using standard synthetic wastewater as described by
Ghangrekar and Shinde (2003). Since this MFC system displayed higher voltage
only after ≈25 days with high fluctuations (data not shown), the medium was
modified. Hence, system was set up using modified synthetic wastewater which was
synthetic wastewater diluted by 20 %, and salt bridge was used as separator.
Figure 2 depicts the open-circuit voltage (OCV) over a period of time
using modified synthetic wastewater as an anolyte.
0.9
0.8
0.7
Cell Voltage (V)
0.6
0.5
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60 70 80 90 100
Time (d)
Fig. 2 Voltage production using modified synthetic wastewater as anolyte

Use of modified synthetic wastewater resulted in 0.82 V output for 2 days which
was comparatively higher than the use of the standard synthetic wastewater
described by Ghanghrekar and Shinde (2003). Voltage output was nearly 0.6 V for
almost 3 months (90 days) and was consistent. Several media components used for
growth of organism contain substantial amount of redox material which may lead to
generation of potential difference initially. The initial jump of cell may be con-
tributed by traces of such material adhered to cell even after washing steps, which
was then stabilized. The system could produce 224.24 mW/m2 power density.
Using glucose as substrate, Hu (2008) could produce 161 mW/m2 power density
while adding anaerobic sludge as inoculum. In our study, all further systems of
MFC were conducted using modified synthetic wastewater as anolyte as it produced
consistent OCV unless otherwise mentioned.
3.2 Influence of Different Types of Separators on MFC

Performance
Anion exchange (AEM), cation exchange (CEM), proton exchange (PEM), and
bipolar membranes have been previously used in MFCs, to separate electrode
chambers and decrease oxygen diffusion into the anode chamber (Kim et al. 2007).
When used as separator, anion exchange membrane containing system (AEM–
MFC) produced the density up to 119.4 mW/m2 while CEM–MFC produced
200.6 mW/m2 (Fig. 3).
After stabilization of the voltage, CEM–MFC could display slightly higher
voltage as compared to AEM–MFC. The anionic exchange membranes exhibit
positive charge on the surface due to quaternary ammonium groups attached. This
may lead to leakage of intermediates such as acetate, butyrate, etc., and may
contribute to less potential difference (Pandit et al. 2012). Although both the sys-
tems produced consistent voltage output till 27 days of incubation, MFC with AEM
0.8
CEM-MFC
0.7
Cell Voltage (V)
0.6
AEM-MFC
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30
Time (d)
Fig. 3 Influence of different types of proton exchange membranes on voltage output in MFC
could produce least voltage among all three separators investigated. CEM–MFC
displayed higher voltage output and ease of operation to the researcher but may not
prove as cost effective as salt bridge.
3.3 Performance of MFC Using Combined Industrial

Effluent as Anolyte
When combined wastewater of inlet collected from Pandesara CETP, South Gujarat
was used in MFC as substrate, MFC system was found to be efficient with pro-
duction of 0.72 V till 60 days of incubation (Fig. 4). Hence, proposed designed
MFC was found to effectively work on actual combined industrial wastewater.
The power density obtained was 200.6 mW/m2. Hence, the efficiency of the
electrogenic bacteria in MFC system was found to be comparative whether syn-
thetic wastewater or actual combined industrial wastewaters were used in MFC as
substrates. As high as 0.7 V output were obtained consistently in CETP wastewater
containing MFC. Subsequent adaptation of the organism may lead to higher voltage
production in upcoming runs of MFCs. Researchers have used wastewater collected
from individual industries to run MFC and treat the waste. Brewery wastewater was
used by Wen et al. (2010) to generate electricity using sequential arrangement of
anode and cathode. With external resistance of 300 Ω and brewery waste as ano-
lyte, it was possible to achieve open-circuit voltage of only 0.434 V. Similarly, Liu
et al. (2011) used steroidal drug industrial waste for electricity generation using
bacteria. With no available reports on combined industrial effluents being used as
anolyte, the present study throws opportunity of such complex effluent to be used in
MFC, where salt bridge successfully can be used as separator.
1.2
0.8
Voltage (V)
PIW
0.6
MSW
0.4
0.2
0
0 10 20 30 40 50 60 70
Time (d)
Fig. 4 Electricity generation in MFC using inlet wastewater collected from Pandesara combined
effluent treatment plant of South Gujarat. Note: “MSW” indicates modified synthetic wastewater
and “PIW” indicates Pandesara inlet wastewater
3.4 Reduction of Organic Load
As one of the important parameters of organic load reduction, the samples from
MFC systems were analyzed for reduction of BOD and COD values. Table 2 shows
reduction of organic load in systems using various separators with modified syn-
thetic wastewater and actual combined wastewater as anolyte connected with salt
bridge.
In MFC with modified synthetic wastewater with 1 % sucrose, the BOD
reduction achieved varied between 43 and 53 %, while COD reduction values
varied widely resulting in the highest 34 % COD reduction in CEM–MFC. With
external resistance of 300 Ω and brewery waste as anolyte, it was possible to
achieve 91.7–95.7 % of COD reduction (Wen et al. 2010). This may be because the
brewery wastewater is expected to contain simple and easily assimilable com-
pounds. When comparing the values of salt bridge and CEM containing MFCs, the
BOD reduction was almost similar, while COD reduction was 30 % higher in case
of CEM containing MFC. Moreover, MFC containing CEM as separator was -
nearly 10 % more effective than AEM containing MFC for COD reduction. In case
of inlet wastewater of Pandesara CETP as anolyte, even with use of salt bridge as
separator, BOD and COD reductions achieved were significant; however, intense
experimental design in future may help in achieving better system, where use of ion
exchange membranes may be evaluated and compared for feasibility.
To conclude, the use of CEM in MFC was found to be the most effective in high
voltage production and reduction of BOD, COD, and with ease of operation, but it
adds to cost of processing. Salt bridge was found to be favorable in case of
laboratory-scale experiments but needs optimization with respect to design if it is to
be used at large scale; however, AEM–MFC could not produce significantly high
power density as compared to systems with CEM and salt bridge. “H”-type system
Table 2 Reduction in BOD and COD values after MFC run using various connectors and
combined industrial effluent
Type of BOD reduction COD reduction
separator Initial BOD Final % Reduction Initial COD Final % Reduction
mgl−1 BOD mgl−1 COD
mgl−1 mgl−1
Salt 7061 ± 504 3600 49.01 16,750 ± 500 16000 04.47
bridge-MSW
CEM 7061 ± 504 3350 52.55 16,751 ± 500 11000 34.00
AEM 7061 ± 504 4050 42.64 16,752 ± 500 13000 22.38
Salt 3100 ± 141.4 1350 56.45 12447 8000 35.72
bridge-CETP
Note: (1) “Salt bridge-MSW” refers to MFC with salt bridge as connector and modified synthetic
wastewater as anolyte, (2) “CEM” refers to MFC with Cationic exchange membrane, (3)“AEM” refers to
MFC with anionic exchange membrane (4) “Salt bridge-CETP” refers to MFC with salt bridge as connector
and Pandesara CETP inlet wastewater as anolyte. In the first three MFC systems, modified synthetic
wastewater was used as anolyte
using synthetic wastewater resulted in 0.8 V of OCV and 0.72 when CETP
wastewater was used and could sustain for quite long duration with more than 50 %
of BOD reduction. Further modifications in MFC system design and optimization to
achieve similar efficient decrease in COD for wastewater in short duration are still
required. Adaptation of culture to the wastewater in fed batch mode of operation
may help in improvement of performance parameters of MFC system.
Acknowledgments The authors express their sincere thanks to Mr. Abdul Basit, Graphicarb
Industries Ltd., Ahmedabad for their kind gift of carbon felt plates required for MFC setup
preparation. Authors would also like to thank In-charge, Pandesara effluent treatment plant, Surat,
South Gujarat for support and help for sample collection. We would like to thank Gujarat Pollution
Control Board for their support in the present study.
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Part VI
Integrated Systems
Role of Rhamnolipid: A Biosurfactant
in Methane Gas Hydrate Formation
Kinetics
Amit Arora, Swaranjit Singh Cameotra, Rajnish Kumar,

Anil Kumar Singh, Pushpendra Kumar, Chandrajit Balomajumder
and Sukumar Laik
Abstract Naturally occurring methane gas hydrate is a vast source of methane gas
which is trapped in crystalline ice-like structure present in permafrost regions and
under the sea in outer continental margins. It is purposed that total amount of
carbon in the form of methane hydrates is almost twice the carbon content in all the
fossil fuel reserves put together, and hence these are supposed to be the future
potential energy resource. This paper investigates the laboratory investigations on
effect of a biosurfactant rhamnolipid on methane hydrate formation kinetics.
Rhamnolipid was produced by Pseudomonas aeruginosa strain A11. The presence
of P. aeruginosa has been reported in Gulf of Mexico gas hydrate samples.
Biosurfactant reduced the surface tension of water from 72 to 36 mN/m with CMC
of 70 mg/L. The biosurfactant dose is studied at two different concentrations in the
solution at 100 and 1000 ppm. Kinetic of hydrate formation and growth is com-
pared at 0, 100, and 1000 ppm of rhamnolipid showing that rhamnolipid acts as a
Amit Arora (&) Chandrajit Balomajumder

Department of Chemical Engineering, Indian Institute of Technology Roorkee,
Roorkee 247667, India
e-mail: aroraamitlse@yahoo.com
S.S. Cameotra A.K. Singh
Institute of Microbial Technology, Chandigarh, India
Rajnish Kumar
Chemical Engineering and Process Development Division, National Chemical Laboratory,
Pune, India
Pushpendra Kumar
Keshav Dev Malviya Institute of Petroleum Exploration, Oil and Natural Gas Corporation,
Dehradun, India
Sukumar Laik
Department of Petroleum Engineering, Indian School of Mines, Dhanbad, India

DOI 10.1007/978-81-322-2773-1_25
334 Amit Arora et al.
hydrate promoter at these concentrations. Thus, small dosages of rhamnolipids

produced by P. aeruginosa strain A11 must clearly affect the gas hydrate formation
kinetics in natural sites (as in Gulf of Mexico).
Keywords Methane hydrate

Potential energy resource Rhamnolipid

Induction time Pseudomonas aeruginosa
1 Introduction
Methane hydrates are inclusion compounds formed by water and methane at high
pressure and low temperature (Sloan and Koh 2007). They are present in sedi-
mentary deposits in permafrost regions and beneath the sea in outer continental
margins (Kvenvolden 1993; Paul and Dillon 2000; Hester and Brewer 2009). One
volume of gas hydrates releases about 160 volumes of methane at standard tem-
perature and pressure (STP) (Kvenvolden 1993). It is estimated that carbon bound
in methane hydrates is twice the carbon content in fossil fuel reserve, and hence
these are supposed to be the future potential energy resource. The worldwide
assessment of (Kvenvolden 1998a) methane hydrate is equivalent to 250 trillion
cubic meters of methane gas. The amount of hydrate bound methane gas is
approximately 100 times as large as that of conventional natural gas resource. As
per the USA department of energy, if only very small amount of the methane stored
in these hydrates could be exploited, it would be almost twice the current domestic
supply of natural gas (Haq 1998; Holder et al. 1987). They are also seen as future
technology for storage and transportation of gas.
1.1 Energy Potential of Gas Hydrates
The amount of methane caught in gas hydrate is not certain. Various groups have
given various amounts of methane trapped in global gas hydrate deposit ranging
from *1017 ft3 or 105 trillion cubic feet (TCF) (Mciver 1981) to 108 TCF
(Trofimuk et al. 1977; Kvenvolden 1988, 1998b; Gornitz and Fung 1994; Harvey
and Huang 1995). Recently in review on the concerned subject by (Boswell and
Collett 2011), the amount of methane caught in gas hydrates has been estimated
around 105 TCF (Boswell and Collett 2011). As per the idea of USA department of
energy, even negligible amount of methane stored in hydrates can be exploited. It is
more than the current supply of natural gas in the country (Holder et al. 1987; Haq
and Boulder 1998). Methane hydrates can be assumed upcoming source of
hydrocarbon energy and will be a future fuel (Paul and Dillon 2000; Kvenvolden
and McMenamin 1980).
Role of Rhamnolipid: A Biosurfactant in Methane Gas … 335
2 Present Study
This paper investigates the effect of a biosurfactant rhamnolipid on methane hydrate

formation kinetics in laboratory. Rhamnolipid was obtained from P. aeruginosa
strain A11. The presence of P. aeruginosa has been reported in Gulf of Mexico gas
hydrate samples (Lanoil et al. 2001) which helped in generating rhamnolipids at the
site. Methane hydrate formation experiments were performed with 90 % saturation
of distilled water (and water–surfactant solution) in porous C-type silica gel (pore
volume 0.90 cm3 g−1). The study is carried out in high-pressure vessel maintained
at low temperature.
3 Experimental Setup, Materials, Procedure
3.1 Apparatus
Video hydrate cell was used to study the gas hydrate formation which is a
mercury-free cell. It can measure the induction time for formation of hydrates and
monitor the pressure drop as a function of time during hydrate formation. The
system consists of constant volume hydrate cell having capacity of 250 cm3 and
pressure rating up to 3000 psi. The temperature is controlled by the thermostatic
bath. A computer is attached for data acquisition of temperature and pressure versus
time. The diagram of the gas hydrate cell is shown in Fig. 1.
3.2 Materials
Pure methane with 99.99 % pure methane (Chemtron Science Laboratory, Navi
Mumbai, India), reverse osmosis water (Millipore SA, 67,120 Molshein, France),
C-type silica gel (Merk Merck Specialities Pvt. Ltd., Mumbai, India), and rham-
nolipid synthesized in laboratory from strain A11 were used for the experiment.
3.3 Procedure
First, 90 % saturated C-type silica gel was used for experiment; then 90 % saturated
C-type silica gel with 100 ppm rhamnolipid aqueous solution and then 1000 ppm
rhamnolipid aqueous solution was used for the experiment. The test sample was
placed in hydrate cell and constant temperature was maintained immersing the cell
into a temperature-controlled bath. A mixture of water and ethylene glycol (25 %)
was used as a liquid for bath. A vacuum pump was used to remove the air from the
Fig. 1 Experimental setup
cell before pressurizing methane gas in the cell. The cell was pressurized up to the
desired value with methane gas. Sudden pressure decline was observed for the
hydrate formation and online video picture was also seen for identifying hydrate
formation. When noticeable pressure drop is not observed, it signifies the com-
pletion of process of formation of hydrates. The induction time, moles of gas
consumed, and rate of hydrate formation were calculated from the obtained pressure
temperature date.
The results obtained in the present study are discussed as follows.
4.1 Induction Time
The induction time, ti, is the passed from the beginning of the experiment at ts to the
onset of hydrate formation to and it is one of the kinetic parameters of hydrate
Table 1 Methane hydrate formation parameters for various types of samples

Test sample Initial cell Nucleation Nucleation Induction
pressure temperature pressure time (min)
P (Mpa) T (K) P (Mpa)
C-type silica gel without 11.62 277.16 10.46 44.24
presence of rhamnolipids
C-type silica gel containing 11.62 277.37 10.42 36.91
100 ppm rhamnolipid
formation. The induction time for various experimental conditions is given in

Table 1, and the moles of methane consumed are shown in Fig. 1.
It can be concluded from Table 1 that the induction time in the presence of
100 ppm rhamnolipid has been reduced which states that rhamnolipid is acting as
promoter for the formation of methane gas hydrates.
4.2 Moles of Gas Consumed
While the hydrate is formed, the drop of pressure was observed, which is due to the
consumption of gas of methane. The methane takes over the unoccupied cavities of
water while hydrate formation. The amount of gas consumed during hydrate for-
mation can be calculated by real gas equation:

V Pi Pf
n ¼ ni nf ¼ ð1Þ
R zi Ti zf Tf
where n is the amount of gas consumed when hydrates form, V is the gas volume,
Pi, Ti and Pf, Tf are the pressure and temperature at initial and final conditions, and
R is the universal gas constant. Compressibility factors (zi, zf) are calculated at the
corresponding pressure and temperature, respectively. The pattern of gas consumed
during formation of hydrate is shown in Fig. 2.
As it is evident from Fig. 2 that maximum numbers of moles are consumed for
1000 ppm rhamnolipid, at lower dose, i.e., at 100 ppm, the number of moles of
methane gas consumed is lesser than without rhamnolipid but as the concentration
of rhamnolipid is increased than amount of methane gas consumed is more than that
of without rhamnolipid.
Figure 3 shows the consumption of gas and pressure drops as a function of time.
The growth and gas consumption regions show the two symmetric plots that could
be explained as conservation of mass during the hydrate growth. Pressure drop
shows the number of methane molecules leaving the gas phase to hydrate phase
drop, whereas gas consumption plot shows the number of methane molecules
entering into the hydrate cages. Mass remains conserved in an isochoric system,
Fig. 2 Moles of methane gas consumed
Fig. 3 Growth curve of methane hydrate formation in C-type silica gel in the presence of 100 ppm
rhamnolipid
which is shown by the symmetry of the two plots about an axis passing through the
intersection of the two plots and parallel to the time axis. As shown in Fig. 3, the
process is splitted into three regions. First region starts at time zero where the
consumption of the gas is less in the system and pressure slightly decreases because
of dissolution of gas. Second region is after the induction time where gas
consumption is increased; this happens because of the hydrate growth. In third

region as the hydrate is formed, gas consumption is as shown in Fig. 3.
4.3 The Rate of Methane Hydrate Formation
As seen from Fig. 4, the slope of the curves after nucleation indicates an expo-
nential behavior from which we can assume a first-order reaction. Equations 2 and 3
can be used for a first-order reaction which is defined as follows:
N ¼ No ekt ð2Þ
N
ln ¼ kt ð3Þ
No
where N is the total number of moles at time t, N0 is the initial number of moles, k is
the rate constant (min−1), and t is the time in minute. The rate constant (k) of hydrate
formation can be calculated from the slope of the curve of ln (N/N0) versus time. The
hydrate formation from region 2 onward Fig. 3 was divided into five zones of time as
shown in Fig. 5 (0–20, 20–40, 40–80, 80–160, 160–260 min) after nucleation. The
data of these five zones are used to determine the rate constant of hydrate formation
Fig. 4 N/N0 versus time plot for methane hydrate formation in the presence of C-type silica gel
containing 100 ppm rhamnolipid
Fig. 5 Semi-logarithmic plot of change of moles of gas while methane hydrate formation in the
presence of C-type silica gel containing 100 ppm rhamnolipid
in each zone. Hydrate formation rate is determined by putting the values of slopes,
i.e., rate constant k calculated from Fig. 4 in the following equation:
dN
¼ No kekt ð4Þ
dt
Table 2 The rate of methane gas hydrate formation for 100 ppm rhamnolipid
Type of sample Time Rate Formation rate % over all increased rate
zone constant (mole/min) with respect to Silica
(min) K (min−1)
C-type silica gel 0–20 0.00484 0.003241392 57.08272969
containing 100 PPM 20–40 9.82E−04 0.000645076
Rhamnolipid
40–80 1.53E−04 9.9753E−05
80–160 1.61E−04 0.000103977
160–260 1.65E−04 0.0001047
The values of rate constant and concerned rates obtained in present study in the
presence of 100 ppm rhamnolipid are shown in Table 2.
The above results indicate that rhamnolipid acts as promoter for the methane
hydrate formation. It is also observed that the induction time of hydrate formation is
reduced in the presence of rhamnolipid (Table 1). The rate of hydrate formation is
found to increase many time (Table 2).
The zeta potential of rhamnolipid synthesized in the present study from strain
A11 was found to be negative; hence, it is anionic surfactant and much of work in
literature on anionic surfactants such as SDS, etc. on gas hydrate formation has
been reported. These results are in agreement with various studies reported on
synthetic anionic surfactants like SDBS (Dai et al. 2014) ans LABS (Ganji 2007;
Fazlali et al. 2013; Kumar et al. 2014).
The rhamnolipid acts as a promoter as the biosurfactant spherical micelles are
formed by long-chain carbon alkyl groups which solubilize hydrocarbon gases
(MacKerell 1995).
The above results indicate that rhamnolipids act as promoter which can help in
synthesizing gas hydrates. Industries looking upon gas hydrates not because of their
energy potentials rather it is becoming a technology these days because of their
other applications such as transportation and storage of gas, desalination of water,
etc. The present study clarifies the role of rhamnolipid a biosurfactant as promoter
in natural sites and giving a green biodegradable promoter as much of work as
promoter of gas hydrates is reported on synthetic surfactants; however, the study
has given a replacement of synthetic surfactant by a green biosurfactant.
5 Conclusion
The above study clearly indicates the role of rhamnolipid in the formation of
methane gas hydrate formation as promoter; thus small dosages of rhamnolipids
produced by P. aeruginosa strain A11 must clearly enhance the gas hydrate for-
mation kinetics in natural sites (as in Gulf of Mexico). Rhamnolipid is a green
biodegradable promoter for gas hydrates formation, and is a green bio-surfactant

which can substitute the synthetic surfactant which is used presently as promoter for
the formation of gas hydrates.
Acknowledgments The supports from institutes namely National Chemical Laboratory (CSIR
Laboratory), Maharashtra, Pune, India, Institute of Microbial Technology (CSIR Laboratory), UT,
Chandigarh, India, Keshav Dev Malviya Institute of Petroleum Exploration, Oil and Natural Gas
Corporation (ONGC), Uttrakhand, Dehradun, India. Indian School of Mines, Jharkhand, Dhanbad,
India are highly acknowledged.
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Economic Analysis of Solar PV/
Wind/Diesel Generator/Battery Connected
Integrated Renewable Energy Systems
for Residential Applications
Suresh Muthusamy and Meenakumari Ramachandran
Abstract Nowadays, energy becomes the basic need of each and every human
being. Most of the energy demands are met by the conventional energy sources like
coal, oil, natural gas, etc., which are going to exhaust in a day and cause severe
energy demand in the future. Renewable energy sources become the alternative for
meeting the energy demand, among which solar and wind energy resources are
most commonly used. Because of the intermittent nature of solar and wind energy
sources, hybrid/integrated renewable energy systems are evolved in the recent
years. This paper discusses about the economic analysis and the optimal selection
of solar PV, wind, diesel generator, and battery connected hybrid energy systems
for residential applications. For the analysis purpose, a small middle class family in
Perundurai, Erode District, Tamil Nadu has been considered for the study. Also, a
suitable hybrid optimized model has been developed and the results have been
discussed based on their net present cost (NPC) and cost of energy (COE), green
house gas (GHG) emissions, etc.
Keywords Cost of energy Net present cost Integrated renewable energy

systems
1 Introduction
Energy demand is growing day by day due to the increase in population, urbanization,
and rapid industrialization. Most of the peoples rely on the conventional energy-based
electricity production for meeting their daily needs. These conventional energy
sources emit greenhouse gasses, when the fuels are being burnt. Since the oil crisis in
Suresh Muthusamy (&) Meenakumari Ramachandran

Department of Electrical and Electronics Engineering, School of Electrical Sciences,
Kongu Engineering College, Perundurai, Erode 638052, Tamil Nadu, India
e-mail: infostosuresh@gmail.com
Meenakumari Ramachandran
e-mail: oremkay@gmail.com

DOI 10.1007/978-81-322-2773-1_26
346 Suresh Muthusamy and Meenakumari Ramachandran
late 1970s, renewable energy has been considered as an alternative way of generating
electrical power for meeting their needs.
Various industrial sectors and even general public and government organizations
are also turning their attention toward renewable energy-based energy production
for meeting their energy demands. The Government of India is also trying to
maintain the energy balance between the energy demand and the source availability.
Nowadays, hybrid energy systems are being mostly preferred rather than depending
on single source for supplying the electric power in urban, rural, and remote areas
due to the intermittent nature of solar and wind resources. Due to the integration of
renewable energy-based systems, it results in high cost, complexity and several
factors have been considered for integration.
As on March 31, 2014 (Renewable Energy Status Report 2014), the installed
capacity of renewable energy has reached 32,269.6 MW (12.95 %) of the total
potential available (249,199 MW) in India. The Ministry of New and Renewable
Energy (MNRE) has set down a target of 41,400 MW, as the total installed capacity
by the end of 2017. Even though there is a considerable growth in power gener-
ation, the demand for the electric power has been growing at a rapid rate, whereas
the opportunities for electricity production are very high. The Government of India
is trying to bridge the gap between energy demand and power production by
providing the funds to the research projects and development of Ultra Mega Power
Projects (UMPPs) by making use of renewable energy systems.
The remaining section of the paper is organized as follows:
Section 2 deals with IRES concepts.
Section 3 deals with the scope of the proposed work and the existing works in
IRES-based systems.
Section 4 explains about the system modeling and the availability of resources.
Section 5 presents the simulation results and discussion.
Section 6 concludes with the findings of the proposed work.
2 Integrated Renewable Energy Systems
Usually, renewable energy sources are of single energy-based systems and multiple
energy-based systems or hybrid/integrated energy systems (Kanase Patil et al.
2011). In case of single energy-based systems, it uses only one source along with
energy storing devices and power electronic systems, whereas in case of multiple
energy-based systems, it utilizes two or more renewable energy sources along with
DG sets, storage devices, and power electronic devices. Several hybrid energy
system combinations can be used for generating power depending upon their
requirements. Hybrid energy systems are more advantageous compared to single
Economic Analysis of Solar PV/Wind/Diesel … 347
energy-based systems because they are highly reliable and more efficient. But, a
proper design and better solution has been considered, in order to find the economic
and most feasible solution, i.e., it should be neither oversized nor undersized.
This paper clearly discusses about the economic analysis of solar
PV/wind/DG/battery connected integrated renewable energy systems for residential
load applications. For the study purpose, a small middle class family and their
energy demand have been considered in Perundurai, Erode District, Tamil Nadu.
A Hybrid Optimization Model has been developed and a better optimal solution
with proper design has been found out from the several possible combinations
based on the energy calculations like net present cost (NPC), cost of energy (COE),
excess energy (EE), etc.
3 Scope of the Proposed Work
The concept of integrated renewable energy systems was first introduced by R.G.
Rama kumar in the 1980s, where he worked on evaluating the design scenarios of
IRES. Later, he formulated the designs for integrating the renewable energy sources
from the existing, freely available sources in which a remote village had been
chosen and the versatility of IRES–KB has been discussed (Ramakumar 1977;
Ramakumar and Hughes 1981). Later, in the late 1980s, a linear programming
approach had been developed for integrating the renewable with the aim of
minimizing/reducing the total annual cost using the concept of loss of power supply
probability, in terms of power and energy calculations. The main aim of the IRES
model proposed by Rama kumar in linear programming approach was to minimize
the total annual cost per year subject to the energy produced.
Many computer-based simulation models are available in order to design and
evaluate the techno-economic analysis of the renewable energy-based systems
(Akella et al. 2007; Sinha and Chandel 2014). This hybrid optimization model is
one of the powerful tool which is mostly used by the researchers for designing and
analyzing the hybrid power systems, which contain a mix of conventional gener-
ators, combined heat, and power, wind turbines, solar photovoltaic panels, etc.
4 System Components and Modelling
4.1 Study Area
Based on the village electrification report by MNRE released during May 2014, only
few of the villages were fully electrified. Most of the villages remain
unelectrified/partially electrified even though they are ready to pay for the electricity
Fig. 1 Geographical location of the study area
and having a plenty of energy availability. Among the various states, some of the
states like Meghalaya, Odisha, and Arunachal Pradesh are facing these problems.
Even in some other states, a few of the villages are located at a remote site, where the
electrical power transmission is highly complex and involves huge investments. In
those cases, IRES-based systems may provide the better solution for rural
electrification.
Figure 1 shows the geographical location of the study area on the map. The
study area has the latitude of 11.270000 and a longitude of 77.580000 and has the
DMS latitude and longitude of 11°16″12.0000″N and 77°34″48.0000″E, respec-
tively, as shown in Fig. 2, wherein their load demand profile is shown in Table 1.
4.2 Modelling of the Proposed System
A suitable system has been developed using the hybrid optimization model, which
consists of solar PV, wind turbine generator, diesel generator set, and battery along
with the power electronic conversion systems as shown Fig. 3.
Figure 4 shows the load profile data of the selected site location for a single day,
wherein there will be a maximum load of nearly 5 kW during the evening hours.
The D map shows the monthly average report of the selected site from January to
December and also the annual scaled report given at the last.
Fig. 2 Study area
Table 1 Load demand profile

S. No. Name of the Power rating No. of hours Load demand
appliances in W used in W
1 CFL lamp 25 10 250
2 CFL lamp 40 10 400
3 Television 250 8 2000
4 Fans 50 19 950
5 Computer 200 6 1200
6 Radio 20 3 60
7 Refrigerator 700 24 16,800
8 A.C. 3.516 k 10 35,160
9 Washing machine 500 2 1000
4.3 Availability of Solar Energy Resources
The monthly average values of the solar and wind speed data related to the case
study location are shown in Fig. 1. It is observed that the solar radiation of this city
reaches its minimum of 4.7 kWh/m2/day in the month of November and it is having
the maximum of 6.6 kWh/m2/day in the month of February. Also, the average of
daily radiation in the whole year is about 5.46 kWh/m2/day. Figure 5 shows the
solar and wind resources with the clearness index values along with the daily solar
radiation and wind speed for the selected study location.
Fig. 3 Proposed system model
Fig. 4 Monthly load profile data
4.4 Availability of Wind Energy Resources
Although the proposed hybrid optimization model has the capability to generate the
wind data, once if the user defines the four parameters which are Weibull parameter
value, autocorrelation factor, diurnal pattern strength, and hour of the peak wind
Fig. 5 Average solar radiation data over a year
speed. The average wind speed is calculated as 2.17 m/s. The availability of wind
energy sources is shown in Fig. 6.
4.5 Energy Storage Devices
Lead acid batteries are included in the proposed model for storing the energy. Super
capacitors and hydrogen cells may also be considered for the same purpose but, the
cost of electrolysers in hydrogen cell is very high.
4.6 Power Electronic Converters
A power electronic converter is used to maintain the flow of energy between the AC
and DC busses. A suitable converter design is essential in order to convert AC–DC,
DC–AC, DC–DC, and AC–AC voltages.
Fig. 6 Average wind speed data at a selected study location
5 Simulation Results and Discussions
For the single residential load of the selected study area, various combinations are
obtained from solar PV, WTG, fuel cell, PE converter, DG, and battery by using
this proposed optimization model as shown in Figs. 7 and 8. When the developed
model is simulated, the possible combinations of DGs are listed out and ranked
Fig. 7 Annual electricity production of diesel generator

Fig. 8 Optimization results
Fig. 9 State of charge of battery
according to their cost of energy (CoE) and net present cost (NPC). Sensitivity
analysis can also be done by the proposed system model by eliminating the
unfeasible model and ranking the feasible and economical combinations alone.
The most economic system in each category is chosen and can be used based on
their ranking order. From the simulation results, it is observed that there are eight
feasible combinations based on their NPC and CoE values.
From the simulation results, it is observed that the most feasible and economic
solution with a minimum net present cost of about $374,507 and the cost of energy
is about $1.386/kWh can be obtained by running the diesel generator.
From Fig. 9, it is found that the wind + diesel is the economically viable system
next to the diesel generator of 15 kW independently, with the net present cost of
$417,038 and a COE of $1.544/kWh.
From the discussion, it was inferred that for the load demand of 4300 W, the
economically feasible and technically viable solution is found to supply the load
either by the stand-alone diesel generator or the mix of wind + diesel generator,
whose rectifier outputs and cash flow summary are shown in Figs. 10 and 11.
Fig. 10 Rectifier and inverter outputs
Fig. 11 Cash flow summary
6 Conclusion
An optimization model consisting of solar, wind, battery, and a conventional diesel

generator has been developed and simulated for the selected study area. The model
developed has been found to be very useful for the Rural Electrification of
Perundurai in Erode District, as it offers ‘n’ number of possible combinations for
the study area. This proposed model optimizes the different renewable energy
systems based on their net present cost, renewable fraction, cost of energy, fuel
emissions, etc. In this work, a diesel generator has been considered in order to keep
the constant output in spite of the fluctuating power outputs from solar and wind
energy resources. Simulation results present the economic analysis of the integrated
renewable energy systems for the selected study area. It implies that the IRES-based
system has the advantages of long term availability and flexibility.
References
Akella AK, Sharma MP, Saini RP (2007) Optimum utilization of renewable energy sources in a
remote area. J Renew Sustain Energy Rev 11:894–908
Kanase Patil AB, Saini RP, Sharma MP (2011) Sizing of integrated renewable energy system
based on load profiles and reliability index for the state of Uttarakhand in India. J Renew
Energy 36:2809–2821
Ramakumar R (1977) Technical and socio-economic aspects of solar energy and rural
development in developing countries. J Solar Energy 19:643–649
Ramakumar R, Hughes WL (1981) Renewable energy sources and rural development in
developing countries. IEEE Trans Educ 24:242–251
Sinha S, Chandel SS (2014) Review of software tools for hybrid renewable energy systems.
J Renew Sustain Energy Rev 32:192–205
Author Index
A L
Abhinav Trivedi, 15 Lata, 23
Abhishek Sharma, 169 Lipi Parekh, 319
Aditi Patel, 319
Amit Arora, 333 M
Anju Arora, 23 Madhuri Narra, 3
Anuchaya Devi, 131 Meenakumari Ramachandran, 345
Mitra, A., 285
B Mohd. Zeeshan, 149
Balamurugan, P., 121 Mohit Vasudeva, 149
Benni, Sangeeta D., 111 Mohod, A.G., 63
Mukherjee, C.K., 285
C Munnolli, Ravindra S., 111
Cameotra, Swaranjit Singh, 333 Murugan, S., 169
Chandrajit Balomajumder, 333
N
D Nanda Kishore, 197
Dhanapati Deka, 131 Nasreen Munshi, 319
Noori, Md.T., 285
G
Ghangrekar, M.M., 285, 295, 305 P
Gollakota, Anjani R.K., 197 Parihar, Amit Kumar Singh, 213
Prasenjit Mondal, 257
H Purvi Zaveri, 319
Harmanjot Kaur, 43 Pushpendra Kumar, 333
Himadri Sahu, 97
Himansh Kumar, 179 R
Rai, Monika Prakash, 159
J Rajendra Prasad, 245
James, Jisha P., 3 Rajesh, P.P., 295
Rajnish Kumar, 333
K Raman Rao, 43
Karthickeyan, V., 121 Ram Chandra, 15
Katagi, Kariyappa S., 111 Rangan Banerjee, 213
Kaustubha Mohanty, 97 Ratnesh Tiwari, 245
Khandetod, Y.P., 63 Renuka Barman, 131
Kulkarni, Sneha S., 111 Richa Arora, 81
Kumar, Sachin, 43, 81 Rupali Mahajan, 43

DOI 10.1007/978-81-322-2773-1
358 Author Index
S T
Sai Gu, 197 Tanvi Modi, 319
Samanta, Sujoy Kumar, 227 Tiwari, B.R., 305
Sameer Kureshi, 319
Sarma, A.K., 149, 179 V
Senthil, R., 121 Varma, Anil Kumar, 257
Shalley Sharma, 23 Velmurugan Balasubramanian, 3
Sharma, Nilesh Kumar, 81 Verma, Amit Ranjan, 245
Shivani Gupta, 159 Verma, Priyanshu, 227
Shrirame, H.Y., 63 Vijay, Virendra Kumar, 15, 245
Shuvashish Behera, 81 Virendra Sethi, 213
Singh, Anil Kumar, 333
Sonia Sharma, 23 Y
Subramanyam, Malladi D., 197 Yadav, Y.K., 179, 267
Sukumar Laik, 333 Yadav, Yogender Singh, 267
Surender Singh, 23
Suresh Muthusamy, 345

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Springer Proceedings in Energy

Proceedings of the First

Samir Kumar Khanal

ISSN 2352-2534 ISSN 2352-2542 (electronic)

Library of Congress Control Number: 2016935576

© Springer India 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Kapurthala, India Sachin Kumar

Prof. Pranab Goswami, IIT Guwahati, India

Dr. V. Sivasubramanian, PERC Chennai, India

Kapurthala, India Sachin Kumar

International Advisory Committee

National Advisory Committee

Prof. M.M. Ghangrekar, IIT Kharagpur

Part I Biochemical Conversion

Part II Chemical Conversion

Assessment of the Seed Oils of Persea americana and Melia dubua

Part III Thermochemical Conversion

Part IV Biomass Management

Part V Electrochemical Processes

Part VI Integrated Systems

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Dr. Sachin Kumar is Deputy Director in the

Dr. Samir Kumar Khanal is Associate Professor of

Prof. Dr. Y.K. Yadav received his Ph.D. in Energy

The First International Conference on “Recent Advances in Bio-energy Research”

Madhuri Narra, Jisha P. James and Velmurugan Balasubramanian

Keywords Lignocellulosic biomass Deligniﬁcation Thermotolerant yeast

Madhuri Narra (&) J.P. James Velmurugan Balasubramanian

© Springer India 2016 3

2 Materials and Methods

2.1 Lignocellulosic Biomass, Media and Chemicals

2.2 Acid Pretreatment

2.3 Microorganisms and Culture Conditions

2.4 Yeast Inoculum Preparation

The Saccharomyces cerevisiae inoculum was grown for 12 h at 30 ± 2 °C in a

2.5 SHF of Cellulosic Residue

2.5.1 Cellulase Preparation

2.5.2 Enzymatic Hydrolysis of Deligniﬁed Cellulosic Residue

2.5.3 Ethanol Fermentation

2.7 Analytical Methods

Endoglucanase activity was assayed using 2 % carboxymethyl cellulose (CMC,

3 Results and Discussion

3.1 Biomass Composition Analysis

3.2 Dilute Acid Hydrolysis of Lignocellulosic Biomass

RS yielded maximum amount of reducing sugars (110 g l−1) followed by WS and

activated charcoal was used. Overliming followed by activated charcoal treatment

3.3 Deligniﬁcation of Lignocellulosic Biomass

3.4 Enzymatic Sacchariﬁcation of Deligniﬁed Cellulosic

When deligniﬁed cellulosic substrates were subjected to enzymatic sacchariﬁcation,

Fig. 1 Enzymatic hydrolysis 300

Reducing sugars (mg g -1

Fig. 2 Influence of Tween 80 350

on enzymatic hydrolysis of 300

9 FPU g−1 substrate, pH 4.8, 200

3.5 Fermentation of Hemicellulosic and Enzymatic

Table 1 Fermentation proﬁle of hemicellulosic hydrolysate of lignocellulosic biomass using

Table 2 Fermentation proﬁle of enzymatic hydrolysate of lignocellulosic biomass using

Fig. 3 SSF of RS, WS, and 25 Rice straw