Microbial Mediated Valorization

of Lignocellulose: A Green Technology 3

for Bioethanol Production

Viabhav Kumar Upadhayay, Amir Khan, Jyoti Singh, and

Ajay Veer Singh

Abstract

In the modern world, the attention is raised for the development of newer
technologies for the transformation of biological wastes into biofuels as an
alternative option of exhaustible petroleum or other sources. The organic parts
of agricultural wastes, forest residues, food wastes, and municipal and industrial
wastes contain an unlimited source of lignocellulosic biomass which could
potentially be used for generating second-generation biofuels such as
“bioethanol.” Microorganisms play an important role in all probable steps
intended for lignocelluloses hydrolysis. The greener technological approach for
green fuel production through application of microorganisms is a sustainable and
renewable approach which is carried out in three steps such as (a) hydrolysis of
lignin; (b) hydrolysis of cellulose and hemicelluloses; (c) fermentation of glucose
to ethanol. The high production of ethanol is the need of the cotemporary world
and therefore it becomes necessary to explore different microorganisms having a
high potential for ethanol yield. Moreover, introducing metabolic engineering
techniques is the current advancement for development of modified microbial
cells for enhanced production of ethanol from lignocellulosic biomass. The
present chapter focuses on the valorization of lignocelluloses waste through
microorganisms and their mechanisms required for bioethanol synthesis from
lignocellulosic biomass.

Keywords
Lignocellulose · Bioethanol · Valorization · Fermentation · Hemicellulose

V. K. Upadhayay · A. Khan · J. Singh · A. V. Singh (*)

Department of Microbiology, College of Basic Sciences and Humanities, Govind Ballabh Pant
University of Agriculture and Technology, Pantnagar, Uttarakhand, India

# Springer Nature Singapore Pte Ltd. 2021 53

S. Shah et al. (eds.), Bio-valorization of Waste, Environmental and Microbial
Biotechnology, https://doi.org/10.1007/978-981-15-9696-4_3
54 V. K. Upadhayay et al.

3.1 Introduction

From the past few years, the global researches endeavored to find alternative sources
of energy. Main reasons behind searching sustainable energy supply are (1) increase
of atmospheric CO2 and concern for global climate change (Venkatramanan et al.
2020; 2021a), (2) depletion of non-renewable energy source, (3) rising energy
demand, (4) energy security, (5) rural economic development, (6) rapid urbanization,
(7) development of power driven technology, and (8) transportation (Baños et al.
2011; De Bhowmick et al. 2018; Prasad et al. 2019; Shah et al. 2019). It has been
anticipated that in near future (approximately by 2025) around 50% increment in the
energy demand will be appeared from a number of both developed and developing
countries (Tong et al. 2012). Therefore, harvesting energy from plant biomass
through sustainable, environmentally friendly and cost-effective approach is an
important substitute of petroleum and non-renewable energy source (Prasad et al.
2021; Venkatramanan et al. 2021b). Plants and related waste materials contain
cellulosic properties so they can be utilized to produce bioethanol (Prasad et al.
2019). In present world, the liquid biofuel in the form of “bioethanol” is being used
having various benefits over fossil fuels. It can diminish the emission of greenhouse
gases and reduce the particulate materials in the atmosphere (up to 50%) (Riccio
et al. 2017; Donato et al. 2019). Various food crops such as “maize,” “sugarcane,”
and “sugar beet” have become prominent source of carrying out fermentation
process for bioethanol production, and such type of production is also described as
“first-generation technology” which is anticipated to attain a level of approx. 100 bil-
lion liters (in 2022) (Saini et al. 2015). The maize and sugarcane are rich source of
starch and sugars (sucrose) as raw materials and exhibit inadequacy to meet emer-
gent requirement of bioethanol. Moreover, the cultivation of these crops for energy
production has shown negative impact on issue of biodiversity and food chain, and
considered as probable means for deforestation as huge farmland would be needed.
Keeping such issues about risks associated with first-generation bioethanol, the
research focus has been moved towards “second-generation technologies,” where
the exploitation of non-food-based crops (with no-food parts) and wastes originated
from wood or food-based industries represent most plentiful renewable organic
constituents in the biosphere (Zucaro et al. 2016; Donato et al. 2019). Therefore,
the second-generation bioethanol is derived from “lignocellulosic biomass” which is
generated by agricultural practices, wood-based industries, municipal solid wastes,
and dedicated energy crops cultivating on trivial lands (Nair et al. 2017). The
biomass in form of lignocelluloses represents an economically feasible and renew-
able/inexhaustible reservoir for the production of eminent fuel in form of
“bioethanol” (Donato et al. 2019; Prasad et al. 2019). “The lignocellulosic crop
residues have huge potential to be used as feedstock for biofuel production”
(Venkatramanan et al. 2021c). Although the fixing of lignocellulosic material into
bioethanol production has been attributed to give numerous advantages in terms of
environmental impact and sustainability, the “2G” or second-generation technology
for bioethanol production is under infancy and all the concerning researches are
going on all aspects (from biomass treatment to hydrolysis and fermentation). In this
3 Microbial Mediated Valorization of Lignocellulose: A Green Technology for. . . 55

Fig. 3.1 Schematic representation for valorization of lignocellulosic biomass

context, microorganisms (such as bacteria and fungi) and their enzymes have
provided reasonable and cost-competitive strategy for switching the lignocellulosic
biomass into bioethanol (Prasad et al. 2019). After pretreatment of raw materials
(lignocellulosic biomass), the next step includes hydrolysis of biopolymers (cellu-
lose and hemicelluloses) through hydrolytic enzymes into simpler sugars and their
use in process of fermentation for bioethanol production (Fig. 3.1). Present chapter
outlined the concise introduction of role of microorganisms and their enzymes in
valorization of lignocellulosic materials for production of second-generation biofuel
in more economically feasible and sustainable manner with considering the
associated facts of less detrimental impacts on the environment.

3.2 Source of Lignocellulosic Biomass

Plant and agricultural residues (such as barley straw, corn stover, wheat, rice, husk of
coconut, sugarcane bagasse, wood, sorghum stalks), forest residues, and municipal
organic wastes are the key sources for the lignocellulosic biomasses (Shah and
Venkatramanan 2019) (Fig. 3.2). Most of the countries produce considerable num-
ber of sources for deriving lignocellulosic material, for example corn stover is
produced at high level by the USA; however, wood and large quantity of residues
(agricultural and forest residues) produced by New Zealand and China (Zhu and Pan
2010). India, after China supplies approximately 0.2 billion tons of agriculture-based
residues annually (De Bhowmick et al. 2018). Additionally, overall huge amount
(approximately 180 million tons) of cellulosic biomass each year is derived from
agricultural resources (Kurian et al. 2013; De Bhowmick et al. 2018). Lignocellu-
losic material in the form of either as crop or residues is chiefly produced from
perennial herbaceous plants and woody plants, and such plant materials are abun-
dantly presented on earth. Besides agricultural and forest residues, the municipal
organic wastes are another main source of lignocelluloses (FitzPatrick et al. 2010;
De Bhowmick et al. 2018). Structurally the “lignocellulosic biomass” is comprised
of three most important biopolymers which are widely referred as cellulose,
56 V. K. Upadhayay et al.

Fig. 3.2 Important sources and constituents of lignocellulosic biomass

hemicellulose, and lignin. Other constituents in small quantity (such as acetyl

groups, phenolic substituents, and minerals) are also present in lignocellulosic
biomass (Fig. 3.2). Biopolymers involved in synthesis of lignocellulosic biomass
are organized in intricate and inhomogeneous three-dimensional structures to pro-
vide varying degrees of relative composition depending on type of lignocelluloses.

3.3 Importance of Pretreatment Technologies

The three important biological materials such as cellulose, lignin, and hemicelluloses
participate in the formation of lignocellulosic biomass, where cellulose and lignin as
a matrix are bounded with chains of hemicelluloses. The main motto of the
pretreatment process is to breakdown the lignocellulosic material which results in
reduced crystallinity of cellulose and also augments the part of amorphous form of
cellulose. Such cellulose form is actually exerting suitability for enzymatic activity
(Sánchez and Cardona 2008). Moreover, the pretreatment is necessary to make
lignocellulosic waste liable for fast hydrolysis with augmented monomeric sugars
(Mosier et al. 2005), and features of pretreatment must be proficient and effective for
the production of biofuel (Lu and Mosier 2008; Saxena et al. 2009; Gupta and Verma
3 Microbial Mediated Valorization of Lignocellulose: A Green Technology for. . . 57

2015). However, the important goals of pretreatment methods can be summarized in

brief as (a) production of sugars through the hydrolysis, (b) avoiding the degradation
of sugars, (c) avoiding the maximum formation of products having inhibitory
properties, (d) to lessen the energy demand, and (e) decreasing the costs. The
basic structure of plants such as “cell wall” hampers the entry of various pathogens.
Number of pathogens actually produced certain hydrolytic enzymes which disrupt
the internal parts of plants, but tough cell wall restricts the enzymatic activity to
perform its action of degradation (Kim 2013). For the effective production of sugars
required for fermentation from the cellulosic material, there is the necessity to further
modify the physical and chemical characteristics of the cell wall structure of the
plants. The factors involved in increment of pretreatment step are: (a) very less
amount of lignocellulosic biomass (less than 20%) undergoes incomplete/partial
digestion in its native state; (b) complex or mixed composition; (c) recalcitrant
nature of the cellulose; (c) elevated crystallinity of cellulose fiber; and
(d) enhancement in the accessibility of the enzymes (Kim 2013; De Bhowmick
et al. 2018). Furthermore, it is also apparent that the preferable pretreatment pro-
cesses have revealed an incredible impact on the physicochemical properties of the
treated lignocellulosic biomass. Such properties influence the downstream processes
including preconditioning, selection of microbes, utilization of by-products, and
waste management along with the recuperation of the aimed product, concentration
and purification of the product (da Costa Sousa et al. 2009). In addition, grasses and
woods (both soft and hardwood) present the wide arrays of lignocellulosic material
bearing different chemical and physical properties which necessitates various
approaches. As a result, the suitable pretreatment processing means should be used
for a particular substrate, and such aspects of interdependence between pretreatment
processing and substrates make the pretreatment step as fundamental unit opera-
tional division in “lignocellulosic biorefinery” (De Bhowmick et al. 2018).

3.4 Pretreatment of Lignocellulosic Substrates

The pretreatment is an important process in valorization of lignocellulosic material

and the production of second-generation biofuel, namely “bioethanol.” There are
various ways of pretreatment such as physical or physio-chemical or chemical or
biological or combinations of all these (Fig. 3.3). The critical step for pretreatment of
biomass results the alteration of complex lignocellulosic material into amorphous
and crystalline cellulose and such form of cellulose exhibits suitability for its further
digestion (Saini et al. 2015; Furusato et al. 2018). Thus, it is a noteworthy step to
attain elevated yield of ethanol from lignocellulosic material. One of the critical
ways of pretreatment is “physical treatment” which involves certain important steps
such as fragmentation, grinding, milling/shearing of the biomaterial/biomass. These
all steps assist in lessening the level of polymerization and particle size, and on
another side provide lignocellulosic material with increased bulk density, and
surface area (Maurya et al. 2015; Amin et al. 2017). Physical treatment is considered
as an ordinary step for enzymatic accessibility and effective bioconversion
58 V. K. Upadhayay et al.

Fig. 3.3 Schematic representation of various methods used in pretreatment process of lignocellu-
losic biomass

competence to the distorted particles (Barakat et al. 2014; Kumar and Sharma 2017).
Pyrolysis, sonication, and irradiation (particularly with gamma radiation) are other
methods of physical treatments (Isikgor and Becer 2015). Physio-chemical treatment
is another important way of pretreatment method which involves chemical reactions
for the distortion of the structure of lignocellulosic material. Physio-chemical treat-
ment involves (a) steam explosion (also referred as hydrolysis), (b) CO2 explosion,
(c) ammonia fiber explosion, (d) steam explosion with addition of sulfur dioxide
(SO2), (e) liquid hot water-based pretreatment, and (f) microwave-chemical
pretreatment (Brodeur et al. 2011; Isikgor and Becer 2015). Chemical treatment
also played a significant role in process of pretreatment and there are number of
foremost chemical treatment methods such as acidic treatment and alkaline treat-
ment. Besides these ionic liquids (also known as green solvents), sulfite pretreatment
and wet oxidation are other important methods of chemical-based pretreatment
(Bensah and Mensah 2013; Amin et al. 2017). Next method of treatment is widely
known as “biological treatment” process which has been illustrated as microbial
mediated step to treat the biological material. As compared to other two methods of
pretreatments (physical and chemical), the biological way of pretreatment is deter-
mined as an inexpensive and eco-friendly approach for the valorization of
lignocelluloses (Wan and Li 2012; Maurya et al. 2015). In biological pretreatment
process, the enzymes secreted by microorganisms (both bacteria and fungi) involve
in degradation of the substrate. A range of bacteria such as “Actinomycetes” have
been determined to produce lignocellulose degrading enzymes, and these enzymes
are prominently efficient in degradation of grasses (as grasses possess huge cellu-
losic biomass) (Amin et al. 2017). However, biological pretreatment process of
lignocelluloses is relatively economically feasible and proficient. Moreover, it is
an eco-friendly source of wide arrays of enzymes for degrading complex biomass,
and in industrial application enzymes hold huge potential.
3 Microbial Mediated Valorization of Lignocellulose: A Green Technology for. . . 59

3.5 Microbial Mediated Biological Pretreatment

In the modern era, the production of high-quality biofuel (such as ethanol) from least
useful biomass through fermentation has given a new trend (Mohd Azhar et al.
2017). The bioethanol production is the green synthesis of renewable biofuels and
may assist in reducing the need of precious fossils fuels. Moreover, it will be
attributed to sustain future generation in respect of fuel-based energy. After
illustrating few modern various pretreatment strategies in previous section, the
greener approach in form of biological pretreatment has been assigned as most
effective and eco-friendly approach causing lesser pollution. Biological approach
for the pretreatment involves numerous enzymes which indirectly exhibit the role of
microorganisms producing the particular enzyme. Conventional approach or the
physio-chemical method for lignocelluloses degradation needs huge energy input
and also determined as an important factor to cause pollution. Therefore, biological
based pretreatment process of lignocelluloses could be an excellent instance of
environment friendly and inexpensive strategy (Maurya et al. 2015). The conversion
or transformation of the biomass/raw materials to the biofuel by using the preemi-
nent microorganism could provide better productivity in most efficient way with less
investment of money. The conversion of raw biomass might be improved by having
appropriate understanding of the microorganisms participated in different steps of
pretreatment. Biological pretreatment is essential because it enhances fermentation
rate. This approach particularly uses the cellulose or hemicelluloses degrading
microorganism for carrying out pretreatment of substrate such as lignocelluloses.
Earlier studies reported the vital role of bacteria including Bacillus to degrade
organic materials (Poszytek et al. 2016), and such organisms have important place
in the biological pretreatments of raw materials. Bacteria are profoundly beneficial
for secreting enzymes (both industrial and biotechnological important enzymes)
(Singh et al. 2012). The combination of more than two microorganisms (also
known as microbial consortia) aids in enhanced degradation of complex
biomaterials. Microbial consortia comprising of cellulolytic bacteria (Bacillus and
Streptomyces), and fungi (Candida and Aspergillus) showed wide-spectrum biodeg-
radation (Nikiema et al. 2017). Biomolecules with complex structure such as the
polysaccharides are degraded to the simpler sugars through the involvement of wide
arrays of enzymes like amylase, cellobiase, cellulase, and xylanase. Moreover,
protease plays a pivotal role for the degradation of protein into the amino acids
and lipase breaks the lipids into two subsequent main products (such as glycerol and
long-chain fatty acids) (Indrasith et al. 1988; Lass et al. 2011). However, the lignin
shows extremely resistive nature against degradation, but few fungi degrade lignin
too. Modification in conventional steps also required for improving the bioethanol
synthesis from the biomass, and it is also reported that simultaneous “saccharifica-
tion” and “fermentation” through the association of fungi can improve bioethanol
productivity (Cheng et al. 2017). White-rot fungi were examined to being an
effective candidate to bring out pretreatment process of most of the available
lignocellulosic biomass (Kumar and Wyman 2009). Numerous white-rot fungi
(Ceriporia lacerate, Cyathus stercolerus, P. chrysosporium, Pleurotus ostreatus,
60 V. K. Upadhayay et al.

Phanerochaete chrysosporium, and Pycnoporus cinnabarinus) have the trait to

produce lignin peroxidases (lignin-degrading enzymes) and manganese- dependent
peroxidases, and these enzymes were reported to exhibit higher delignification
efficacy on different lignocellulosic biomasses (Shi et al. 2008; Kumar and
Wyman 2009; Maurya et al. 2015; Ummalyma et al. 2019). An effectual
delignification of different biomass was reported by fungus, namely Ceriporiopsis
subvermispora in the mutual action of two enzymes such as laccase and manganese
peroxidase (Wan and Li 2012). Assessment of mild alkali and also the steam
pretreatment of “wet-milled corn fiber” are done with using fungi, namely
Gloeophyllum trabeum, P. chrysosporium, and Trichoderma reesei, which resulted
into the instant hydrolysates fermentation to ethanol. This phenomenon illustrates
that the yields of ethanol are 75% superior as compared to a commercially accessible
cellulase enzyme utilized in instantaneous saccharification and fermentation process
(Brahmachari et al. 2016). Microorganisms which had been isolated from diverse
ecological niches or regions (such as soil, manure/compost, agriculture-based
residues, and rumen of animals) are potential consortia having capacity for carrying
out efficient degradation process of lignocelluloses (Poszytek et al. 2016). It became
important to comprehend the specific microorganism involved in making a particular
microbial consortium for the relevant lignocellulosic biomass to be treated, and this
understanding could direct to an insightful modification in the eminent production
rate of bioethanol. Consortia (mixture of pure strains of yeast and cellulolytic
bacteria) screened from natural environment were also employed for successful
pretreatment of lignocellulosic matter in process of biological pretreatment (Zhang
et al. 2011).

3.6 Hydrolysis: A Process Involves Microbial Enzymes

Both celluloses and hemicelluloses undergo the enzymatic hydrolysis which is

regulated by numerous factors such as temperature, pH, quality of substrate, incuba-
tion period, and ratio of enzyme-substrate (Achinas and Euverink 2016). Though,
the use of either diluted or concentrated acid such as sulfuric acid for the acid
hydrolysis is a common practice to degrade the celluloses. But, to hydrolyze the
cellulosic polymers through the “acid hydrolysis” has limitations and shows unsuit-
ability for efficient ethanol fermentation due to synthesis of toxic components such
as phenols (Sun and Cheng 2002; Moe et al. 2012; Achinas and Euverink 2016).
Moreover, this method of acid hydrolysis is not economically feasible as it involves
high consumption of acids (Moe et al. 2012) and requires specialized reactors
because of higher degree of corrosion and high toxicity rate (Wijaya et al. 2014).
Therefore, it is required to use microbial based enzymes for solving the purpose of
hydrolysis of celluloses and hemicelluloses in more effective manner. Plenty of
researches have been performed on microbes (both bacteria and fungi) bearing
cellulolytic/lignocellulolytic nature and the respective hydrolytic enzymes for effi-
cient hydrolysis of sugars and their conversion into the ethanol (Jessen et al. 2015;
Prasad et al. 2019).
3 Microbial Mediated Valorization of Lignocellulose: A Green Technology for. . . 61

Each step in the hydrolysis of polysaccharide matrix of plant cell wall is a

complex phenomenon and needs a suitable treatment. The method of pretreatment
of lignocellulosic material as substrate is connected with enzymatic hydrolysis, and
such practices further help in enhanced porosity and enzyme accessibility to the
substrate (lignocellulosic biomass) (Limayem and Ricke 2012; Prasad et al. 2019).
In pretreatment process, the separation of lignin moiety from the lignocellulosic
material is necessary as it interferes with the hydrolysis step through blocking the
access of cellulose degrading enzyme “cellulases.” Therefore, the separation of the
lignin can dramatically result into the increased hydrolysis rate of celluloses
(McMillan 1994). Enzymes mediated hydrolysis have exhibited benefits over acid-
based hydrolysis, as the method of enzyme hydrolysis is very mild process and
potentially provides high yields with low cost. Moreover, it doesn’t have corrosion
problems so it can be proposed as the preferable method for “wood-to-ethanol
processes” in future (Menon and Rao 2012).

3.6.1 Cellulases

Enzymes hydrolysis coupled with activities of various kinds of hydrolytic enzymes

which converts complex carbohydrate molecules into the simple monomeric sugars.
In comparison with acid hydrolysis, the enzymatic hydrolysis needs less input of
energy and mild conditions (Ferreira et al. 2009). Cellulase is the most significant
enzyme present in various cellulolytic bacteria (Acetovibrio, Bacillus, Bacteroides,
Cellulomonas, Clostridium, Erwinia, Ruminococcus, Streptomyces,
Thermomonospora) and cellulolytic fungi (Fusarium, Penicillium, Phanerochaete,
Schizophyllum sp., and Trichoderma). Cellulases possess the ability to convert
cellulose into simplest sugars (e.g., glucose or galactose monomer) (Gupta and
Verma 2015). Cellulase enzymes are comprised of a catalytic unit and a
non-catalytic carbohydrate-binding unit and also associated with other accessory
domains (Herve et al. 2010; Chatterjee et al. 2015). The enzyme “cellulases” belong
to glycoside hydrolases family with three different classes of enzymes: (a) Endo-1,4-
β-endoglucanase (cleave the glucosidic linkages randomly on the complex molecule
of polysaccharide), (b) Exo-1,4- β-exoglucanase (binds to crystalline region of the
cellulose and randomly cleaves the cellulose molecules), and (c) β-glucosidase or
cellobiase (these enzymes specifically cleave the cellobiose molecule) (Willis et al.
2010; Chatterjee et al. 2015). Cellulose degrading microorganisms are widely
known as cellulolytic microorganisms and possess the capability to degrade recalci-
trant plant cell wall. The cellulolytic microorganisms, for instances thermophilic and
mesophilic anaerobes, fungi, and bacteria are robustly capable to hydrolyze
extremely crystalline insoluble cellulose (Shaw et al. 2008; Himmel et al. 2010).
Lamed and Bayer (1988) stated that there is huge attention towards thermophiles as
these microorganisms have the ability to secrete “thermo-stable cellulose” mainly
under higher temperature (more than 90  C temperature too). In case of anaerobic
bacteria, the degradation of cellulose is carried out by a particular multienzyme
complex, termed as “cellulosomes,” which either found in free or associated to the
62 V. K. Upadhayay et al.

cell surface (Chatterjee et al. 2015). Mitchell (1998) illustrated the cellulolytic
activity of Clostridium (a thermophilic anaerobe bacteria) for the degradation of
cellulosic plant materials and also showed adaptable fermentable ability. For the last
some years, T. reesei based cellulases have drawn attraction for the research, and are
extensively employed in the laboratory and pilot-scale study for ethanol application
(Gray et al. 2006). Cellulases from two prominent fungi such as Aspergillus niger
and Trichoderma viride are also used for the hydrolysis of biomass (Passos et al.
2009). The majority of commercially available enzymes for hydrolysis of biomass
are in fact blends of cellulases from fungi (Aspergillus or Trichoderma)
supplemented with β-glucosidases. Other potent cellulases producing
microorganisms are Cellulomonas sp., Clostridium sp., Thermomonospora sp.,
Aspergillus sp., and Trichoderma sp. (Kuhad et al. 2011).

3.6.2 Hemicellulases

“Hemicellulase” is a unique factor for plant biomass degradation and particularly

acts on hemicelluloses. This enzyme is main constituent for carbon flow in nature.
The main substrate of this enzyme is hemicelluloses which can be represented as an
assemblage of branched and linear polysaccharides connected through hydrogen
bonds to the cellulose microfibrils. Hemicellulose is comprised of a combination of
glucose and sugar monomers (Ummalyma et al. 2019). Xylan is the most copious
hemicelluloses which contain pentose sugars (such as xylose), and the enzyme
namely “xylanase” catalyzes the hydrolysis of xylan. The catalytic or functional
unit of hemicellulases can be described either as glycosidic hydrolases (which
hydrolyze glycosidic bonds) or carbohydrate esterases (which catalyze the degrada-
tion of ferulic acid and acetate). Multiple xylanases with varied specificities and
functions perform the action of xylan hydrolysis. There are numerous
microorganisms (A. niger, Bacillus sp., Humicola insolens, and T. reesei) from
which xylanases are produced on a commercial basis. The action of endoxylanases
and exoxylanases commence the process of hydrolysis of hemicelluloses (Binod
et al. 2011; Ummalyma et al. 2019). The redundant by-product of hemicelluloses
hydrolysis is L-arabinitol which affects the diminution of D-xylose to xylitol. How-
ever, xylose reductase has the ability to reduce the L-arabinose to “L-arabinitol.” Nair
and Zhao (2010) engineered a strain of Escherichia coli with a “xylose reductase
mutant” which resulted into elimination of L-arabinitol production to synthesize
xylitol from a combination of hemicelluloses sugars (such as L-arabinose, D-glucose,
and D-xylose). Sakamoto et al. (2012) and his group designed Saccharomyces
cerevisiae through genetic engineering intervention which showed the ability to
degrade hemicelluloses through co-presenting the enzymes from different
microorganisms such as endoxylanase (from Trichoderma reesei), β-xylosidase
(from Aspergillus oryzae), β-glucosidase (from Aspergillus aculeatus), expression
of xylulokinase (from S. cerevisiae) and xylose reductase and xylitol dehydrogenase
(from Pichia stipitis) with the inclusion of xylose. The genetically engineered
microorganisms also have the ability to produce bioethanol using rice straw, as the
3 Microbial Mediated Valorization of Lignocellulose: A Green Technology for. . . 63

rice straw also provide suitable hemicelluloses (cellooligosaccharides,

xylooligosaccharides, and xylan) substrate. Su et al. (2015) engineered a bacterial
strain, namely E. coli W3110 to secrete xylitol to display xylose reductase from
Neurospora crassa at elevated temperature without inclusion bodies. The genes of
xylose isomerase (“xylA”) and xylulose kinase (“xylB”) liable for “D-xylose catab-
olism” were eradicated. This engineered bacterial strain can abolish catabolite
repression, therefore permits the simultaneous uptaking of sugars including glucose
and xylose, which is reliant on “phosphoenolpyruvate-dependent glucose
phosphotransferase system (ptsG).”

3.6.3 Ligninases

Lignin is considered as second major abundant organic polymer which provides a

rigidity to plant cell wall structure and also inhibits hydrolysis of hemicelluloses and
celluloses. The valorization of huge biomass such as “lignocelluloses” is performed
for producing green fuel “bioethanol” (Kawaguchi et al. 2016; Ragauskas et al.
2014), but degradation of lignin is prime task for efficient utilization of biomass in
biorefineries. Structurally lignin is determined as a cross-linked polymer of “4-
hydroxyphenylpropanoid monomers/monolignols” having various carbon(C)-car-
bon(C) and ether bonds. p-hydroxyphenyl, guaiacyl, and syringyl groups are the
phenolic moieties of monomeric units and their proportion varied with the plant
species. Generally, the most common linkages present in lignin are β-β, β-0-4, and
β-5 bonds (Vanholme et al. 2010). Highly lignin selective enzyme “ligninases” is the
current demand for lignin degradation. There are few fungi which produce
ligninases, and among these specifically white-rot fungi synthesize some particular
enzymes including MnP (Mn peroxidases), LiP (lignin peroxidases), and laccases
which all arrive in category of “ligninases.” Various microorganisms produce
different combinations of lignin-degrading enzymes displaying varying mechanisms
of lignin degradation (Sahadevan et al. 2013). The term “enzymatic combustion” has
been described in case of degradation of lignin by lignin-degrading microorganisms,
where the oxidizing potential of hydrogen peroxide/molecular oxygen by two
enzymes, namely “ligninolytic peroxidase” or “laccase” are subjected to oxidize
aromatic units (Kirk and Farrell 1987; Bugg and Rahmanpour 2015). White-rot
fungi Phanerochaete chrysosporium has been extensively studied to produce extra-
cellular enzymes (Mn peroxidases, lignin peroxidases, and laccases) for biodegrada-
tion of lignin (Bugg and Rahmanpour 2015). Several researchers have reported
MnPs production from wide range of microorganisms (bacteria, fungi, and algae)
(Zhang et al. 2018; Bugg and Rahmanpour 2015). MnPs are the broadly distributed
extracellular and potential peroxidases produced by fungi, especially white-rot fungi
(C. subvermispora, Dichomeris squalens, P. sordida, P. chrysosporium, P. radiate,
and P. rivulosu) (Hakala et al. 2006). Laccases and LiPs also show vibrant role in the
course of lignin de-polymerization (Hammel and Cullen 2008; Bugg and
Rahmanpour 2015). Besides aforementioned three important enzymes (cellulases,
hemicellulases, and ligninases), some other enzymes including “xyloglucanase”
64 V. K. Upadhayay et al.

have been employed for degradation of those secondary polysaccharides which are
unable to be transformed into simple sugars through the action of “cellulases”
(Stickel et al. 2014). The process of enzymatic hydrolysis carried out at elevated
solid loadings is considered to be inexpensive approach due to the accumulation of
higher concentration of sugar at the end phase of hydrolysis. And this plentiful
amount of sugar is converted into elevated level of ethanol which exhibits
low-priced approach with less energy requirement for distillation process
(Modenbach and Nokes 2013). Another saccharification method is termed as simul-
taneous saccharification and fermentation (SSF) in which fermentative microbes are
used for simultaneous SSF of hemicelluloses and celluloses (Mosier et al. 2005).

3.7 Fermentation

After hydrolysis, the next imperative step is “fermentation,” where the molecules of
sugar are taken up by the enzymes synthesized by bacteria or yeasts for producing
variety of organic acids and alcohols (Mussatto and Teixeira 2010; Bhagchandanii
et al. 2020). The efficiency of the fermentation depends upon two main factors:
(1) effective hydrolysis and (2) selection of correct microbial strains to diminish the
formation of inhibitory toxic compounds to attain elevated yield of ethanol (Achinas
and Euverink 2016). “SHF (Separated Hydrolysis and Fermentation)” is determined
as the conventional method in which the process of hydrolysis is performed at
earliest to produce monosaccharide sugar as the fermentation proceeds (Dahnum
et al. 2015; Devarapalli and Atiyeh 2015; Prasad et al. 2019). One more and
important method of hydrolysis and fermentation is known as “SSF (Simultaneous
Saccharification and Fermentation)” where the process of cellulose hydrolysis and
the process of fermentation of hexose take place in a same reactor by using yeast and
enzyme together, so glucose is quickly transformed into ethanol (Cantarella et al.
2004; Dahnum et al. 2015; Prasad et al. 2019). Wyman et al. (1992) described SSF
as the better process for providing high ethanol yield in comparison of SHF. Besides
the better ethanol yields, the SSF process helps in elimination of end product
inhibition, and eradicates the requirement for separate reactors. Saccharomyces
cerevisiae is the common yeast which plentifully used in the ethanol fermentation.
Moreover, Saccharomyces is also used as food additive and “generally recognized as
safe (GRAS),” and as a result it became best candidate for manufacturing alcoholic
beverages. Generally, S. cerevisiae has been characterized to carry out glucose
fermentation to ethanol very effectively. But on contrary, the fermentation of xylose
is exigent as very few conventional ethanol-producing microorganisms depict the
ability to readily ferment xylose, although a lot of microorganisms consume
“xylose” as a carbon (“C”) source (Lin and Tanaka 2006). Biofuel-based industries
use different biomass or substrate and specific microbial strain for ethanol produc-
tion, and are seeking various approaches for the modifications for huge level
production of green fuel in more economical manner. In sugar-based and corn-
based biofuel industries, the extensive preference has been given to Saccharomyces
cerevisiae for carrying out fermentation (Achinas and Euverink 2016; Prasad et al.
3 Microbial Mediated Valorization of Lignocellulose: A Green Technology for. . . 65

2019). The role of bacteria in fermentation cannot be avoided as it is very economi-

cally feasible and easier strategy for ethanol production (Senthilkumar and
Gunasekaran 2005). The common bacterial examples are Corynebacterium
glutamicum and Zymomonas mobilis, which are extensively exploited in industry
for ethanol production (Senthilkumar and Gunasekaran 2005; Tsuchida et al. 2007;
Kang et al. 2014). Enhancement in ethanol yield is the main task for the researchers
(Rai et al. 2010; Jessen et al. 2015), therefore the approach of genetic engineering
accepted challenges and resulted in high ethanol production through genetically
modified microorganisms. The application of first metabolic engineering surpris-
ingly resulted into the construction of E. coli strains which selectively produce
ethanol, and E. coli presents numerous benefits as a biocatalyst for the ethanol
production, as well as the capacity to ferment wide ranges of sugars with no need
of complex growth factors (Lin and Tanaka 2006).

3.8 Advancement in Ligocellulosic Valorization: A

Biotechnological Mediated Reform

Current development in biotechnology brought a boom in excellent solubility of

lignocelluloses.
Modification in genetic program depicted alterations in either microorganisms for
efficient production of cellulose degrading enzymes or developing plants having
nature of easy solubility of residues for improved fermentation practices. Biotech-
nological advances have been resulted into the development of genetically modified
microorganisms for synthesizing modified cellulosome (cellulose degrading
machinery). Cloned and over-expressed man5K gene in Clostridium cellulolyticum
confirmed 20-fold higher activities of altered/modified form of cellulosomes on
substrate “galactomannan” in comparison with control with promising cellulase
activities (Perret et al. 2004). Ethanol yields and its titer can be improved by
inhibition of by-products, and for accomplishing such task the three respective
genes, namely lactate dehydrogenase (ldh), hygromycin phosphotransferase, and
phosphotransacetylase (pta) in C. thermocellum were knocked out. Deleting only pta
gene did not increase ethanol yield, but knocking out of all three genes resulted into a
fourfold enhancement in production of ethanol (Argyros et al. 2011). Research on
trifunctional cellulosomal complex has represented cellulosome chimera amid
cellulases and hemicellulase from several microbes exhibited improved hydrolytic
action on complex substrates (Fierobe et al. 2005).

3.9 Conclusion

Valorization of widely available lignocellulosic biomass and the synthesis of

bioethanol is the prime need for the present world for lessening the dependency on
non-renewable sources such as fossil/petroleum-based fuels. Lignocellulosic mate-
rial is generated from different sources including plant materials, agricultural and
66 V. K. Upadhayay et al.

forest residues, and wastes originating from wood and food-based industries. How-
ever, the practice of microbial mediated lignocellulosic waste valorization gave new
trends for efficient pretreatment process for increasing the accessibility of cellulose-
hemicellulose matrix. Microorganisms particularly bacteria and fungi secrete wide
range of hydrolytic enzymes which assist in hydrolysis of large biopolymers such as
cellulose, hemicelluloses, and lignin which eventually results in formation of fer-
mentable sugars. Crucial step of fermentation requires activity of numerous
microorganisms for utilizing various sugars and their transformation into bioethanol.
The microbial mediated steps for lignocelluloses valorization is considered to be
economically feasible, and provide environment friendly hub for higher yield of
bioethanol. Strategies such as pretreatment and hydrolysis of lignocelluloses and
subsequent fermentation step are using microorganisms and their enzymes in current
era for the green production of “second-generation biofuel” at efficient level.

3.10 Future Prospects

Application of microorganisms in valorization of lignocellulosic waste is wider, but

existing challenges must be addressed to further improvement in generation of
second-generation biofuel. The future research is required to employ strategies for
elimination of inhibitory by-products with more efficiency. Construction of geneti-
cally and metabolically engineered microbial strains should be the prime topic for
research in scientific world for improving cellular machinery for many folds higher
production of bioethanol with less cost. Therefore, future research needs to be
intended for developing strategies through microbial strains which could reduce
the duration of pretreatment period and other steps required for bioethanol
production.

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