Final Integrated Lignocellulosic Biorefinery Gateway For Production of Second Generation Ethanol and Value Added Products

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Integrated lignocellulosic biorefinery: Gateway for production of

second generation ethanol and value added products
Amisha Patel, Amita R. Shah∗
Post Graduate Department of Biosciences, Sardar Patel University, Satellite Campus, Vadtal Road, Bakrol-388315, Gujarat, India
a r t i c l e i n f o a b s t r a c t
Keywords: An increasing demand for energy and depleting petroleum sources has elevated the need for pro-
Lignocellulosic biomass ducing alternative renewable resources. Owing to the prominence of lignocellulosic biomass as
Biorefinery bio-renewable and the most abundant resource on Earth, this critical review provides perceptions
Second generation (2G) ethanol
into the potential of lignocellulosic biomass for production of second generation (2G) ethanol and
Value added products
value added products in a biorefinery manner. The efficient utilization of all three components
of lignocellulosic biomass (i.e., cellulose, hemicellulose and lignin) would play a significant role
in the economic viability of cellulosic ethanol. The pretreatment method is the key to the success
of bioconversion processes and greatly influences the economics of biorefinery process. Biotech-
nology tools and process engineering play pivotal roles in development of integrated processes
for production of biofuels, biochemicals and biomaterials from lignocellulosic biomass. Although,
lignocellulosic biorefinery has ample scopes, commercial production of biofuels and chemicals is
still challenging. In this context, this review entails concept of lignocellulose biorefinery, latest
developments in 2G ethanol production process, importance and market potential of 2G ethanol
as renewable fuel and value added chemicals, integration of processes, challenges for integrated
production of fuel together with value added chemicals and future directions.
1. Introduction
Lignocellulosic biomass has a potential as a renewable and alternative energy source to mitigate global climate change. Utilization
of biomass residues for production of high value compounds can prevent adverse effects on the soil quality and negative impact of
burning on the field. On the other hand, it can bring benefits to rural economies and promote energy security. However, cost effective
conversion of lignocellulosic biomass into biofuel and value added products is highly challenging because of highly complex structure
and recalcitrance of lignocellulose.
Valorization of lignocellulosic biomass residues generated after industrial, agricultural and forestry activities for production of
biofuels and chemicals in a biorefinery manner is becoming one of the most important field of study. The economic value of the
biomass can be maximized by converting into useful biomaterials and/or energy carriers in an integrated manner which also reduces
the waste streams produced (Thomsen, 2005). Biorefining of lignocellulosic biomass into second generation (2G) ethanol and value
added product is not only primarily linked to pollution prevention but also offers sustainable development. It involves the three
pillars of sustainability: people, planet and profit, i.e., social, environmental and economic elements. For the development of global
bioeconomy, three major components of lignocellulosic biomass i.e., cellulose, hemicellulose and lignin are the promising stakeholders
∗
Corresponding author: Professor in Microbiology, Post Graduate Department of Biosciences, Sardar Patel University, Satellite Campus, Vadtal
Road, Bakrol-388315 Gujrat, India.
E-mail address: [email protected] (A.R. Shah).
https://doi.org/10.1016/j.jobab.2021.02.001
Received 18 September 2020; Received in revised form 15 November 2020; Accepted 23 November 2020
Available online xxx
2369-9698/© 2021 The Author(s). Published by Nanjing Forestry University. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Please cite this article as: A. Patel and A.R. Shah, Integrated lignocellulosic biorefinery: Gateway for production of second generation
ethanol and value added products, Journal of Bioresources and Bioproducts, https://doi.org/10.1016/j.jobab.2021.02.001
JID: JOBAB
A. Patel and A.R. Shah Journal of Bioresources and Bioproducts xxx (xxxx) xxx
Fig. 1. Integrated biorefinery scheme.
under the biorefinery regime (Chandel et al., 2018). From lignocellulosic biorefinery, more than 200 value-added biochemicals can
be produced (Isikgor and Becer, 2015).
The overarching goal of this review is to address the challenges and possibilities of integrated production of 2G ethanol with
other value added products for bioeconomy-based biorefinery. This article intends to provide insights and perspectives on the recent
developments in 2G ethanol research and the commercialization status of 2G ethanol production as well.
2. Biorefinery: An integrated approach
Biorefinery concept corresponds to integrated production of energy, chemicals and biomaterials (Fig. 1). In literatures, three dif-
ferent types of biorefineries have been defined: phase I biorefinery (i.e., single feedstock, single process and single product) phase
II biorefinery (i.e., single feedstock, multiple processes and multiple products) and phase III biorefinery (i.e., multiple feedstocks,
multiple processes and multiple products) (Kamm and Kamm, 2004; Fernando et al., 2006). Phase III biorefineries are the most
advanced and developed type of biorefinery. This phase of biorefinery includes not only production of energy and chemicals like
phase II biorefineries, but also the utilization of several types of feedstocks and processing technologies to create a variety of indus-
trial products that have demands in society. Currently, there are four phase III biorefinery systems being pursued in research and
development which include: lignocellulosic feedstock biorefinery, the whole crop biorefinery, green biorefinery and two-platform
concept biorefinery. In lignocellulosic feedstock biorefinery, lignocellulosic biomass is fractionated through array of processes and
converted into energy and value added products (Clark and Deswarte, 2008). A demonstration plant based on lignocellulosic feedstock
biorefinery is currently running in The Icelandic Biomass Company and is processing 20 000 t per year of lignocellulosic biomass,
including hay, lupine straw and barley straw (Kamm and Kamm, 2004). The plant can produce a variety of chemical products from
lignin and the various side streams in addition to 7 × 106 L of ethanol per year. An imminent lignocellulosic feedstock biorefinery,
Processum in Sweden is another example, which links cluster of industries converting wood into energy, chemicals and materials. A
whole crop biorefinery will make use of cereals and convert the entire plant into energy, chemicals and materials. Green biorefinery
involves the bioconversion of natural green biomass into energy, chemicals and materials. Two-platform concept biorefinery has been
recently defined by the National Renewable Energy Laboratory (NREL). The feedstock is divided into a ‘sugar platform’ (biochemical)
and a ‘syngas platform’ (thermochemical). Both platforms can make use of the entire feedstock(s) by offering energy, chemicals and
materials (Clark and Deswarte, 2008).
3. Lignocellulosic biomass: Potential source for phase III biorefinery
Lignocellulosic biomass can be grouped into energy crops (perennial grasses), forest materials (hard wood, softwood, sawdust,
pruning and bark thinning residues), agricultural residues (cereal straws, bagasse and stovers), aquatic plants (water hyacinth) and
organic portion of municipal solid wastes (Zabed et al., 2017). The key constituents of lignocellulosic biomass are cellulose (35%–
50%), hemicelluloses (20%–35%), lignin (5%–30%) and other extracted substances (1%–10%) (Menon and Rao, 2012) (Table 1).
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Table 1
Cellulose, hemicellulose and lignin contents in some important lignocellulosic biomass.
Biomass Cellulose (%) Hemicellulose (%) Lignin (%) Reference
Corn cob 42–45 35–39 14–15 Prasad et al., 2007

Corn stover 38–40 24–26 7–19 Saini et al., 2015
Sugarcane bagasse 42–48 19–25 20–42 Kim and Day, 2011
Rice straw 28–36 23–28 12–14 Saini et al., 2015
Wheat straw 33–38 26–32 17–19 Saini et al., 2015
Barley straw 31–45 27–38 14–19 Saini et al., 2015
Sweet sorghum bagasse 34–45 34–45 14–21 Saini et al., 2015
Cotton 85–95 5–15 0 Menon and Rao, 2012
Depending on the type of biomass, lignocellulosic biomass contains varying amounts of cellulose, hemicellulose and lignin. The
agricultural residues most frequently used include wastes such as wheat straw, barley hull, barley straw, rice straw, rice husks, oat
straw, corn cobs, corn stalks, sugarcane bagasse and sorghum straw. In India, 5 × 108 t of agricultural residues are generated as waste
(Sarsaiya et al., 2019). Covalent and hydrogen bonds connect cellulose and hemicellulose to each other and both further link to lignin
forming complex lignocellulosic matrix in native biomass, which is highly recalcitrant to depolymerisation (Zabed et al., 2016). The
cellulose fragment is composed of linear chain of glucose units linked by 1,4–𝛽 glycosidic bonds (Saxena et al., 2009). Crystalline
network is formed by cross linking of various hydroxyl groups within hydrogen bonds within the cellulose molecules (Limayem and
Ricke, 2012). Hemicellulose is a heteropolymer consisting of branch chain of different monomers including hexoses (𝛽–D–glucose, 𝛼–
D–galactose and 𝛽–D–mannose), pentoses (𝛽–D–xylose and 𝛼–L–arabinose) (Limayem and Ricke, 2012) and is attached by functional
groups like acetyl, methyl, glucuronic, galactouronic and cinnamic acids (Brandt et al., 2013). Lignin is a water insoluble and aromatic
polymer providing the structural reinforcement and resilience to the plant tissue (Menon and Rao, 2012; Brandt et al., 2013). The
monomers of a lignin polymer are three phenolic compounds such as coumaryl, coniferyl and sinapyl alcohol (Limayem and Ricke,
2012).
Major steps involved in bioconversion of lignocellulosic biomass are pretreatment and fractionation of biomass, production of
suitable cellulolytic/hemicellulolytic enzymes, enzymatic hydrolysis of pretreated biomass, fermentation of enzymatic hydrolysate
by suitable microorganisms and downstream processing.
4. Pretreatment of lignocellulosic biomass
Pretreatment is an indispensable step for breakdown of tightly interwoven matrix of recalcitrant lignocellulose. Removal of lignin,
partial or total hydrolysis of hemicellulose, reduction of crystalline fraction of (relative to amorphous) cellulose and reduction in
degree of polymerization are necessary to make the biomass more amenable for the subsequent hydrolytic processes to release fer-
mentable sugars. Various chemical, physical, physicochemical and biological pretreatments are available which increase the lignocel-
lulose digestibility in different manners (Alvarado-Morales et al., 2009). Generation of easily hydrolysable cellulose-rich residue is the
prerequisite criteria for selecting pretreatment process in 2G biorefinery. Selection of pretreatment method for enzymatic hydrolysis
can be done by the assessment of cellulose hydrolystability by cellulases. Yang and Wyman (2008) suggested that the pretreatment
method should be such that more than 90% of cellulose should be digested from pretreated biomass in less than three days using
10 FPU/g of enzyme loading. In addition, many considerations like chemical composition of biomass, loss of substrate, generation
of fermentation and enzyme inhibitors, consumption of water, degradation of sugars, easy recovery of lignin and other valuables,
economic viability, environment safety etc. are important while choosing a pretreatment process.
One major concern during pretreatment process is eneration of enzyme inhibitors that interferes with subsequent saccharification
step and fermentation. The fermentation inhibitors can be divided in two main categories: 1) process-derived inhibitors (such as
furan derivatives), and 2) feedstock-inherited inhibitors (such as acetic acid and phenolic compounds) (Verardi et al., 2020). The
formation of various inhibitors as a result of different pretreatments is outlined in Fig. 2. The acid treatment of woody biomass gen-
erated phenolic compounds like vanillin, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, dihydroconiferyl alcohol, syringaldehyde,
coniferyl aldehyde, syringic acid and Hibbert’s ketones. Phenolic acids like ferulic acid and p-coumaric acid are also common products
during acid treatment of annual plants. Some phenolic compounds also originate from extractives of lignocellulosic biomass. Non-
phenolic aromatic compounds like cinnamic acid, 3,4-dimethoxy-cinnamic acid, cinnamaldehyde, benzoic acid, benzyl alcohol, para-
and ortho-toluic acid have also been reported during acid treatment. Under alkaline conditions, saccharinic acids, formic acid, minor
amount of lactic acid, different dicarboxylic and dihydroxy acids are released. Moreover, acetic acid is formed due to saponification of
acetyl group during alkaline pretreatment. During oxidative treatment xylonic acid is formed as a conversion of decarboxylation and
oxidation of glucuronic acid, which is released due to demethylation of 4–O–methylglucuronic acid. Phenolic acids, such as vanillic,
4-hydroxyphenolic and syringic acids have also been reported during oxidative treatment (Jönsson and Martín, 2016).
In a lignocellulosic biorefinery, biomass is generally fractionated into its structural constituents, i.e., cellulose, hemicellulose and
lignin. This process is very crucial for subsequent valorization of each polymer. Fractionation process in an integrated biorefinery
should not only be able to separate each component but should also facilitate recovery processes of the fractions with minimal
purification steps. Some of the successful fractionation processes are dilute acid, alkali, hydrothermal, steam and organosolv. In an
integrated 2G ethanol biorefinery, pre-extraction and isolation of hemicelluloses and lignin followed by the production of value-
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Fig. 2. Inhibitors generated during pretreatment of lignocellulosic biomass

C, cellulose; HC, hemicellulose; L, lignin.
added products such as ethanol, sugar-based polyesters, other chemicals and biopolymers offer a potential opportunity (Lin and
Luque, 2014). Quantitative comparison of some important pretreatment methods is shown in Table 2.
5. Enzymatic hydrolysis: Key bottleneck for lignocellulosic biorefinery
Saccharification of cellulose and hemicellulose polymers of pretreated biomass is the most crucial step affecting the economics
of the bioconversion process significantly. Saccharification by enzymatic hydrolysis is considered as effective, specific, viable and
eco-friendly approach over non-enzymatic hydrolysis. Furthermore, hydrolysis of cellulosic feedstock mediated by cellulases does not
generate inhibitors. Even though stepwise improvements in pretreatment have resulted in several fold reduction of enzyme loading
for hydrolysis, the efficient enzymatic conversion of cellulose and hemicellulose is still a major bottleneck in utilization of biomass
for biofuel and value added products.
Cellulases, mainly derived from fungi are mixture of at least three different enzymes: i) Endoglucanase or 𝛽–1,4–D–glucan–4–
glucanohydrolases (EC 3.2.1.4), cut the internal glycosidic linkages in amorphous cellulose randomly and produce oligosaccharides
of various chain lengths and open new chain ends. (ii) Exoglucanases including 𝛽–1,4–D–glucan glucanohydrolases (EC 3.2.1.74) and
𝛽–1,4–D–glucan cellobiohydrolases (EC 3.2.1.91), act in a processive manner on the reducing or nonreducing ends of cellulosic poly-
mer chains, releasing either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase). (iii) 𝛽–D–Glucosidases or 𝛽–D–glucoside
glucohydrolases (EC 3.2.1.21), act on cellobiose and cellodextrins and release D–glucose units (Shah et al., 2017). A combination of
enzymes (endoglucanase, exoglucanse and 𝛽–glucosidase) is required for complete hydrolysis of cellulose to glucose which works in
a synergistic manner for hydrolysis of both native and modified cellulose (Saini et al., 2015).
Recent discoveries have revealed that auxiliary enzymes like lytic polysaccharide monooxygenases (LPMO) and cellobiose de-
hydrogenases (CDH) act synergistically with cellulases during cellulose hydrolysis, improving the hydrolysis efficiency (Barbosa et
al., 2020). The LPMO, a copper-dependent enzyme can act through an oxidative mechanism. In the presence of an external elec-
tron donor, the copper ion reduces oxygen. Then, the reduced oxygen abducts a single hydrogen from the substrate and splitting
the 𝛽–1,4 glycosidic bond. The LPMOs produce an entry point for cellulases by oxidizing the glycosidic bond and thus improving
biomass degradation (Valenzuela et al., 2017). The active site of the LPMO is composed of N- and C-terminal histidines complied
with copper. In this reaction, the CDH and ascorbic acid were shown to act as electron donors (Courtade et al., 2017). The CDH
are glycosylated extracellular proteins, known to produce hydrogen peroxide, reduce Fe3+ levels, and donate electrons to the LPMO.
This auxiliary action is essential for the LPMO to perform the redox reactions for lignocellulosic hydrolysis (Barbosa et al., 2020).
Additionally, ‘non-hydrolytic’ accessory protein like swollenin is also involved in cellulose hydrolysis. It was found to raise cellulase
activity synergistically but did not exhibit any enzymatic activity on cellulose. Swollenin is known to reduce cellulose crystallinity
by disrupting hydrogen bonds and thereby increasing cellulase accessibility (Shah et al., 2017). In addition to cellulases, hemicellu-
lases are also essential as they increase the cellulose accessibility by solubilizing the structure of hemicelluloses that act as a barrier
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Table 2
Quantitative comparison of important pretreatment processes.
Pretreatment
method Efficiency Cost Developmental stage
Mechanical Release of sugars after enzymatic hydrolysis of Expensive in terms of Commercial (Park et al.,
pretreatment rice straw: 89% (Hideno et al., 2009) energy requirement and 2016)
operating costs (Barakat et
al., 2014)
Irradiation Enhanced enzymatic hydrolysis rate of pretreated Expensive in terms of Commercial (Kumar et al.,
corn cob up to 80% (Zheng et al., 2017) energy requirement 2020)
Acid treatment Delignification rate of wheat straw: 21% (Jaisamut High cost of acid (Rajan Commercial (Kumar et al.,
et al., 2016), release of sugars after enzymatic and Carrier, 2014) 2020)
hydrolysis of corn stover: 83%–92.5%, Glucan
conversion of corn stover: 97%, Xylan conversion
of corn stover: 93% (Bhutto et al., 2017)
Alkaline treatment Delignification rate of rice straw: 64.51% (Rezania Highly costly (Rezania et Commercial (Kumar et al.,
et al., 2020), release of sugars after enzymatic al., 2020) 2020)
hydrolysis of sugarcane bagasse: 97.6%
(Nosratpour et al., 2018), glucan conversion of
corn stover: 94%, xylan conversion of corn stover:
76% (da Costa Sousa et al., 2009)
Ozonolysis Delignification rate of maize stover: 78%, release Costly due to high energy Laboratory-scale (Travaini
of sugars after enzymatic hydrolysis of maize demand (Travaini et al., et al., 2016)
stover: 80% (Li et al., 2015) 2016)
Ionic liquids Release of sugars after enzymatic hydrolysis of Highly expensive (Zhao et Exploratory stage (Biswas
triticale straw: 81% (Smuga-Kogut et al., 2019), al., 2016) et al., 2015), pilot scale
Delignification rate of corn straw: 81.73% (Liu et (Kumar et al., 2020)
al., 2018b), glucan conversion of wheat straw:
54.8% (Li et al., 2009)
Organosolv Delignification rate of wheat straw: 84.5%, release Highly costly (Bhutto et Laboratory-scale (Rostagno
of sugars after enzymatic hydrolysis of wheat al., 2017) et al., 2015)
straw: 99% (Smit and Huijgen, 2017)
Steam explosion Delignification rate of barely straw: 85% (Oliva et Lower cost (Maurya et al., Commercial (Kumar et al.,
al., 2017), release of sugars after enzymatic 2015) 2020)
hydrolysis of corn stover: 84.7% (Katsimpouras et
al., 2017), glucan conversion of corn stover: 87%,
xylan conversion of corn stover: 78% (da Costa
Sousa et al., 2009)
Liquid hot water Delignification rate of sugarcane bagasse: 77.0% Energy requirement and Pilot scale (Biswas et al.,
(Gurgel et al., 2016), recovery of sugar up to 95% water consumptions are 2015), Commercial (Kumar
with increased enzyme efficiency of loblolly pine high (Zhuang et al., 2016) et al., 2020)
(Biswas et al., 2015), glucan conversion of corn
stover: 91%, xylan conversion of corn stover: 81%
(da Costa Sousa et al., 2009)
Ammonia fiber Delignification rate of corn stover: 24% (Zhao et High cost of equipment Method adopted to
explosion (AFEX) al., 2014), glucan conversion of corn stover: 96%, and ammonia (Sun et al., DuPont’s cellulosic ethanol
xylan conversion of corn stover: 91% (da Costa 2016) biorefinery, US (Sheridan,
Sousa et al., 2009) 2013), commercial (Kumar
et al., 2020)
Wet oxidation Delignification rate of rape straw: 68% (Arvaniti et Moderate capital cost BioGasol demonstration
al., 2012), release of sugars after enzymatic (Biswas et al., 2015) plant in Denmark (Zhuang
hydrolysis of softwood: 79% (Palonen et al., 2004), et al., 2016)
glucan conversion of sugarcane bagasse: 67%,
xylan conversion of sugarcane bagasse: 44%
(Martín and Thomsen, 2007)
Microwave assisted Delignification rate of cotton stalks: 81% (Dimos Very costly (Rezania et al., Pilot scale (Kumar et al.,
chemical treatment et al., 2019), release of sugars after enzymatic 2020) 2020)
hydrolysis of wheat straw: 68.71% (Patel et al.,
2017)
Biological Delignification rate of paddy straw: 71.34% (Arora Costly due to long Laboratory scale
pretreatment et al., 2016), release of sugars after enzymatic pretreatment time
hydrolysis of rice straw: 24.95% (Sreemahadevan (Rezania et al., 2020)
et al., 2018)
for cellulose. Hemicellulases can be characterized as main-chain-degrading enzymes (xylanase, mannanase, arabinose, 𝛽–xylosidase,
𝛽–mannosidase) and side-chain-cleaving enzymes (esterases, 𝛼–L–arabinofuranosidase).
Apart from holocellulolytic enzymes, ligninolytic enzymes also play a vital role in the removal of lignin and thereby facilitation
enzymatic saccharification of biomass. Laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) are the key enzymes in
lignin degradation (Chukwuma et al., 2020). Laccases oxidize aromatic amines and phenolic compounds using molecular oxygen
as a terminal electron acceptor. The MnP and LiP are capable of oxidizing lignin at the non-phenolic and phenolic aryl-ether posi-
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Table 3
List of commercial cellulases by various industries and their properties.
Product name Company Source pH Temperature ( °C) Reference
Celluclast 1.5 L Novo Nordisk Trichoderma reesei 5.0 50 Saini et al., 2015
(Danbury, CT)
Cellic CTec 2 Novozymes (Bagsvaerd, – 5.0–5.5 45–50 Novozymes, 2019
Denmark)
Cellic CTec 3 Novozymes (Bagsvaerd, – 4.75–5.25 50–55 Novozymes, 2019
Denmark)
Spezyme Genencor (USA) Trichoderma reesei 4.0 50 Saini et al., 2015
Accellerase®1500 Genencor (USA) Trichoderma reesei 4.0–5.0 50–65 Dupont Accellerase®, 2019
(genetically modified)
Accellerase®1000 Genencor (USA) Trichoderma reesei 4.0–5.0 50 Dupont Accellerase®, 2019
(genetically modified)
Cellulase TAP10 Amano Enzyme (Troy, Trichoderma viride 5.0 50 Saini et al., 2015
VA)
Cellulase AP30 K Amano Enzyme (Troy, Aspergillus niger 4.5 60 Saini et al., 2015
VA)
tions, respectively (Lopes et al., 2018). Recent studies have stimulated lots of interest in utilization of laccase-cellulase cocktails for
simultaneous pretreatment and saccharification of lignocellulosic biomass (Masran et al., 2020).
Development of efficient and low-cost enzyme cocktails still remains a challenge in biomass processing. In view of obtaining
complete hydrolysis of carbohydrate polymers of pretreated biomass, it is clearly established that a multienzyme complex is necessary.
The hydrolytic efficiency depends both on the ratio of individual enzymes in the multi-enzyme cocktail and properties of individual
enzymes (Gusakov et al., 2007). Moreover, a single enzyme cocktail does not show optimal performance to each type of biomass
due to compositional variability. The rate and extent of enzymatic hydrolysis of lignocellulosic biomass depends on enzyme-related
factors and substrate-related factors. Some of the crucial factors are enzyme loading, hydrolysis periods, temperature and pH as well
as structural features of biomass resulting from pretreatments (Banerjee et al., 2010). It is also evident that enzyme loading and types
of enzymes required depend on severity of pretreatment process. Substrates prepreated with milder processes may need high doses
of enzymes to achieve high yield of sugars. Some of the structural features altered during pretreatments like cellulose crystallinity,
degree of polymerization, surface area, porosity of substrate, hemicellulose and lignin content etc. have profound effect on enzymatic
hydrolysis (Alvira et al., 2010). In the past decades, researchers have been focussed on enzymatic hydrolysis at high substrate loading
in such a way that the process can generate a concentrated sugar solution. As a result, downstream processing in ethanol fermentation
can be less energy intensive and cost effective. However, such a process lacking free water at high biomass concentration (above 20%
(w/w)) is highly challenging because of the problems of mixing and mass transfer. Such a system is obviously prone to substrate and
end product inhibition too (Penner and Liaw, 1994). Several researches and developmental activities are going on for the development
of tailor-made enzymes for improved hydrolysis of lignocellulosic biomass.
The ideal cellulase complex must be highly active on the intended biomass feedstock, operate well at mildly acidic pH, withstand
process stress, withstand high concentrations of end products and be cost-effective (Knauf and Moniruzzaman, 2004). The On-site
enzyme production is proposed a potential step to reduce the operation cost as it is produced on low cost abundantly available
substrates, avoids enzyme and transportation storage and also dismisses highly sophisticated downstream processes. If microorganism
is exposed to the same lignocellulosic biomass for cellulase production which is utilised for saccharification process it may produce
an isoform with higher affinity (Siqueira et al., 2020). Several research groups are working on thermostable cellulases to increase the
efficiency of cellulose hydrolysis at elevated temperatures. Thermostable cellulases from bacteria (Bacillus, Geobacillus, Caldibacillus,
Acidothermus, Caldocellum and Clostridium), fungal (Chaetomium, Talaromyces and Thermoascus) and even from metagenome would be
useful (Patel et al., 2019b).
The development of cellulase, or more correctly, cellulolytic enzyme blends is a crucial factor for biorefinery. Enzymes can
constitute up to 30% of the cost structure for producing biofuel from cellulosic sugar. In order to achieve economic feasibility, the
cost of enzyme for biomass depolymerization should not exceed US$ 0.10 per L of ethanol product (Champreda et al., 2019). Globally,
there are two prominent players known for cellulases used in biomass conversion—‘Novozyme’ and ‘Genencor’. An increased enzyme
activity and 30-fold cost reduction have been achieved by Genencor and Novozymes (Saini et al., 2015). Recently, Genencor has
launched Accelerase®1500, a cellulase mixture specifically for lignocellulosic industries. It is claimed to be more efficient and cost-
effective than its predecessor-Accelerase® 1000 for 2G ethanol industries. It ensures almost complete conversion of cellobiose to
glucose as it contains higher levels of 𝛽–glucosidase activity than other commercial cellulases available today. For enhancement of
both xylan (C5) and glucan (C6) conversion, Genencor has also launched Accellerase® XY, an accessory xylanase enzyme complex and
Accellerase® XC, an accessory xylanase/cellulase enzyme mixture. Another Accellerase® BG, an accessory 𝛽–glucosidase enzyme is
also launched for enhancement of glucan (C6) conversion (Dupont Accellerase®, 2019). Novozymes developed enzyme Cellic CTec3
which is a mixture of cellulase and hemicellulase complex. As compared to other cellulase or enzyme complex available in the market
for cellulosic ethanol production, Cellic CTec3 allows the most cost-effective conversion of pretreated lignocellulosic materials to
fermentable sugars (Saini et al., 2015). The cellulases manufactured by various industries are enlisted in Table 3.
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Fig. 3. Block diagram of various second generation (2G) ethanol production process integration from lignocellulosic biomass: SHF, separate hy-
drolysis and fermentation; SSF, simultaneous saccharification and fermentation; CF, co-fermentation; SSCF, simultaneous saccharification and co-
fermentation; CBP, consolidated bioprocessing; CBS, consolidated bio-saccharification (Sánchez and Montoya, 2013).
6. Fermentation process for 2G ethanol
Various fermentation processes are available for production of 2G ethanol from lignocellulosic biomass which includes: Sepa-
rate hydrolysis and Fermentation (SHF), Simultaneous Saccharification and Fermentation (SSF), Co-fermenation (CF), Simultaneous
Saccharification and Co-fermentation (SSCF) and Consolidated Bioprocessing (CBP). The SHF process ensures optimal conditions for
enzymatic saccharification and fermentation by ethanologen. However, inhibition of glucose and cellobiose on activity of cellulase
is a major drawback with SHF process. The SSF process offers a more economic process with high ethanol productivities as both
enzymatic hydrolysis and fermentation happen simultaneously wherein problems of feedback inhibition of cellulases can be over-
come. However, optimal temperature of enzyme reaction is much higher than that of the ethanologens for fermentation and hence
enzymatic hydrolysis becomes a rate limiting step in SSF process (Patel et al., 2019a; Singh et al., 2018). The SSF process at high
solid loading offers significant economic advantage as well as allows high concentration of ethanol which is preferable for distillation
(Hoyer et al., 2013). However, high solid loading results in high viscosity of the medium, higher levels of inhibiting compounds and
severe mixing problems (López-Linares et al., 2014). Hence, pre-hydrolysis and simultaneous saccharification and co-fermentation
were projected within the integrated biorefienry in order to overcome these drawbacks (Chen and Fu, 2016). The SSCF is analogous
to SSF, involving the fermentation of both hexoses and pentoses thereby increasing the ethanol yield. The advantage of the SSCF in
contrast with the SHF and CF processes is that it involves the simultaneous fermentation of the released glucose and minimizes the
inhibition caused by the end product. Moreover, it also increases xylose to glucose ratio which permits fermenting microorganism to
consume xylose (Toor et al., 2020). The CBP has been considered as a promising approach by using engineered micro-organisms that
produce cellulases and ethanol in a single step and reduce the cost of entire bioconversion process. For high efficiency CBP, Clostridium
thermocellum and Clostridium cellulolyticum are being tested using gene transfer studies and organism development strategies (Lynd
et al., 2005). Recently, a new CBP derived strategy, Consolidated Bio-Saccharification (CBS) is proposed for lignocellulose biocon-
version wherein enzyme production is integrated with hydrolysis steps but separated from fermentation process. Consequently, the
cellulolytic capability can be maximized and fermentation would not be limited by hydrolysis conditions as well (Liu et al., 2020).
The schematic outline of all these fermentation processes is shown in Fig. 3.
Even after remarkable research and developmental activities in the last several decades, production of 2G ethanol from lignocel-
lulosic biomass is still challenging with regard to utilization of pentoses. Saccharomyces cerevisiae is a well-established ethanologen
which ferments hexoses to ethanol very efficiently. Generally, this yeast has been recognized as unable to metabolize xylose naturally.
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United States Brazil European Union

China Canada India
Thailand Argentina Rest of World
120,000
100,000
Ethanol ( 106 L)
80,000
60,000
40,000
20,000
0
2014 2015 2016 2017 2018 2019
Year
Fig. 4. Global trends for ethanol production (Renewable Fuel Association, 2020).
However, some reports are there showing ability of S. cerevisiae strains to grow on xylose at slow rates owing to the presence of genes
related to xylose metabolism (Patiño et al., 2019). Lots of research has been directed towards the expression of heterologous genes
through recombinant DNA technology to enable it to ferment xylose (Jin and Jeffries, 2004; Ruohonen et al., 2006; Chu and Lee,
2007). However, a significant yield was not obtained using recombinant strains and they do not possess robustness required in context
to industry as well as they are specific towards particular type of biomass. Various bacterial (Thermus thermophilus, Clostridium ther-
mosaccharolyticum) and yeast strains (Pichia stipitis, Candida shehatae, Pachysolen tannophilus, Debaryomyces hansenii) were reported
to utilize pentoses but with lower ethanol yield (Avanthi et al., 2017). Xylose-metabolizing yeast can metabolize xylose to xylulose
through the two-step oxidation/reduction pathway catalyzed by xylitol dehydrogenase (XDH) and xylose reductase (XR) (Liu et al.,
2019). Recently, Scheffersomyces stipitis has been gaining interest due to its ability to metabolize both hexose and pentose sugars
(Ceccato-Antonini et al., 2017). Enormous efforts have been done in evolutionary engineering and metabolic engineering to create
robust strains able to convert hexoses and pentoses with a higher efficiency and capability to resist toxic compounds (Yamakawa et
al., 2018).
6.1. Bioethanol as second generation (2G) biofuel
Gasoline can be blended with 10%–25% (E10–E25) of 2G ethanol to operate an increasing number of cars. New flex-fuel vehicles
are capable of burning any level of blended 2G ethanol, including 100% hydrous 2G ethanol (E100) (Martin, 2010). A normal E10
blend which achieve 2% reduction in GHG emissions, 3% reduction in fossil energy use and 6%–6.6% reduction in petroleum use
(Chen and Fu, 2016). The 2G ethanol has a lower energy density than gasoline; 65%–70% of the energy (∼21 MJ/L) can be produced
from a liter of 2G ethanol which is equivalent to a liter of gasoline (∼32 MJ/L) (Clark et al., 2012).
The combustion efficiency of the 2G ethanol is higher (15%) than that of gasoline as it contains 34.7% oxygen. Ethanol also
keeps down the emission of particulate nitrogen oxides. Hence, the 2G ethanol is considered as an eco-friendly oxygenated fuel (Kar
and Deveci, 2006). The emission of sulfur is also reduced by the use of 2G ethanol as it contains a negligible amount of sulfur as
compared with gasoline (Pickett et al., 2008). Ethanol is a safer substitute to methyl tertiary butyl ether (MTBE) which is added to
gasoline to reduce the combustion of carbon monoxide (CO) and carbon dioxide (CO2 ) and to enhance octane rating (McCarthy and
Tiemann, 2006). United States, Brazil and China are the topmost nations on the list of global 2G ethanol producers. The global trends
for ethanol production are shown in Fig. 4.
The ethanol yield reported in certain literature from some potential lignocellulosic biomass is shown in Table 4. Production of the
2G ethanol from lignocellulosic biomass has been greatly improved after remarkable research and developmental activities. However,
there are still several challenges which block industrial progress for cost effective and commercial production. Key challenges are
related to pretreatment, enzymatic saccharification, fermentation process, limitations of ethanologens and waste utilization. Sum-
maries of industrial problems and overcomes during the 2G ethanol production process are outlined in Fig. 5. Table 5 summarizes
the status of the major 2G ethanol producers from lignocellulosic biomass worldwide.
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Table 4
Ethanol yield from different lignocellulosic biomass.
Biomass Ethanol yield (L/t) Reference
Sugarcane 70–90 Zabed et al., 2017

Sorghum bagasse 250 Zhao et al., 2009
Corn stover 450 Barros-Rios et al., 2015
Corn cob 510 Kreith and Krumdieck, 2013
Wheat straw 490 Kreith and Krumdieck, 2013
Fig. 5. Summary of industrial problems and their remedies during 2G ethanol production process. SHF, separate hydrolysis and fermentation; SSF,
simultaneous saccharification and fermentation; SSCF, simultaneous saccharification and co-fermentation.
6.2. Scope of 2G ethanol biorefinery in India
India is the fourth leading consumer of petroleum across the world and also the fourth biggest emitter of greenhouse gasses (GHG).
The country heavily depends on the import of petroleum products due to the requirement of about 95% of transportation fuel through
petroleum products. Hence there is prerequisite to replace this major portion of transportation fuel with alternative renewable energy
notably through 2G ethanol. Moreover, the country generates about 6.50 × 108 t of agro-residues, so huge quantities of lignocellulosic
biomass are available although major part of these is utilised for other purposes (Sukumaran et al., 2017). In 2018, the National
Policy on Biofuels, India announced to accelerate promotion of biofuels with indicative targets of achieving 20% blending of ethanol
in petrol by 2030. As per the BIS specifications, Oil Marketing Companies (OMCs) are marketing Ethanol Blended Petrol (EBP) with
10% to achieve a target by 2022. About 450 crore litres of ethanol would be required in order to meet 10% ethanol blending target
with a projected demand for petrol in 2021–2022. In India, the estimated production of ethanol is around 300 crore litres, majority
of which is required for chemical industries and potable alcohol and the rest is used for EBP program. The ethanol procurement
from first generation route may not be sufficient to achieve 10% ethanol blending during 2021–2022 although new alternatives
for enhancing ethanol procurement by OMCs has been opened by the government of India. Therefore, there is an urgent need to
explore other options/routes for enhancing ethanol production in the country for the purpose of blending with petrol. In December
2014, the government had already allowed procurement of ethanol produced from other non-food feedstocks other than molasses
to meet the relevant BIS standards. In India, lignocellulosic surplus biomass availability is around 12 – 16 crore tons per annum.
The ethanol yield can be enhanced up to 2500–3000 crore litres per annum if available biomass can be exploited, which reduces
India’s dependence on imported crude oil considerably. Apart from producing cellulosic ethanol, 2G ethanol biorefineries can also
produce pellets (from surplus lignin); biogas (which can be upgraded to Bio CNG); liquid CO2 per dry ice and assured quality compost.
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Table 5
Major 2G ethanol producers from lignocellulosic biomass and technological implementation in bioethanol
production worldwide (Chandel et al., 2010).
Industry Raw material used Technology
Iogen Ottawa, ON, Canada Agro residues, switch State-of-the-art enzyme

grass, miscanthus and advanced
(elephant grass) fermentation technology
Novozymes, Bagsvaerd, Denmark Corn stover, sugarcane In-house cellulases
bagasse and wood chips production and
simultaneous conversion
into ethanol
Genencor, Palo Alto, CA, USA Paper pulp, starchy Developed Accellerase®
materials derived from product line
wheat and corn
Verenium Corporation Cambridge, USA Sugarcane bagasse, Pretreatment, novel
grasses, softwoods enzyme development,
fermentation
Celunol Dedham, MA, USA Cellulosic biomass Use of recombinant
Escherichia coli to ferment
all types of sugars released
from cellulose
Abengoa Bioenergy MO, USA Corn stover, grains, dried Enzymatic conversion of
distillers grain, wheat biomass, fermentation
straw
SunOpta, Little Falls, MN, USA Wood chips Steam explosion
pretreatment, enzymatic
degradation and
fermentation
Range Fuels GA, USA Wood waste Invented a two-step
thermo-chemical process
(solid biomass to gas and
subsequently to liquid
ethanol)
SweTree Umea, Sweden Agricultural and forest Functional genomics
waste program to develop robust
kind of fungi for higher
cellulase and ethanol titres
TMO Biotec Guilford, UK Corn Developing thermophilic
microorganisms to replace
yeast in ethanol
fermentation
Direvo Cologne, Germany Corn stover Improving
cost-effectiveness of
ethanol production
Praj Industries, Pune, India Raw materials (sorghum, Fermentation by Hi-Ferm
cane juice/grains, cassava) systems, molecular sieve
dehydration for ethanol
distillation
Reliance Biofuels Pvt. Ltd, Navi Mumbai, India Cellulosic biomass Enzyme assisted
saccharification of
cellulosic biomass,
development of yeast for
higher titre of ethanol
COFOL, Beijing, China Cellulosic biomass Enzyme based hydrolysis
of agroresidues
Petrobras, Rio de Janeiro, Brazil Sugarcane juice, bagasse Enzymatic hydrolysis and
and leaves ethanol fermentation
NA: not available.
Utilization of lignocellulosic biomass residues can certainly reduce GHG emissions as well as soil and water pollution which results
from landfilling. So, setting up of 2G ethanol biorefineries will help in improving citizen health. Lignocellulosic ethanol biorefineries
also present a potential to manufacture high value by-products such as xylitol, furfural, high fructose syrup, l-arabinose, etc. in
addition to ethanol, which may increase profitability of bioconversion process. With this regard, the central government of India has
announced a scheme “Pradhan Mantri Jaiv Indhan- Vatavaran Anukool fasal awashesh Nivaran (JI-VAN) Yojana” through Ministry
of Petroleum & Natural Gas on 4th August 2017 to provide financial support for 2G biorefineries. This scheme envisages the setting
up of 12 commercial scale 2G ethanol projects and 10 demonstration scale 2G ethanol projects based on non-food biomass feedstocks
and other renewable feedstocks (Ministry of Petroleum and Natural Gas, 2019).
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Table 6
Lactic acid production from different lignocellulosic biomass.
Organism Fermentation Lactic acid Lactic acid Productivity

Substrate Pretreatment used time (h) (g/L) yield (g/g) (g/(L•h)) Reference
Wheat straw Wet oxidation LactoBacillus 24 4.7 0.61 0.20∗ Garde et al., 2002
brevis
Lactobacillus 6.7 0.90 0.28∗
pentosus
Sugarcane bagasse Acid hydrolysis Lactococcus 64 10.85 – 0.17∗ Laopaiboon et al., 2010
lactis IO-1
Sugarcane bagasse Acid treatment Bacillus 49 55.99 0.87 1.7 Alves de Oliveira et al., 2019
coagulans
Sugarcane bagasse Acid treatment Lactobacillus 60 35.81 0.93 0.60 Alves de Oliveira et al., 2018
plantarum
CCT 3751
Sugarcane bagasse Acid treatment Lactobacillus 33 32.5 – 0.98∗ Wischral et al., 2019
pentosus
ATCC 8041
Corn cobs Acid treatment Lactobacillus – 24.7 0.53 0.34 Moldes et al., 2006
pentosus
CECT-4023T
Wheat straw Steam Lactobacillus 48 6.61 0.32 0.14 Cubas‐Cano et al., 2019
explosion pentosus
CECT 4023 T
Note: ∗ indicates calculated from data.
7. Hemicellulose-based value added products
Hemicelluloses are prone to degradation during pretreatments like acid hydrolysis, steam explosion and organosolv treatment.
Therefore, hemicellulosic portion is considered as the most underutilized component during the bioconversion of lignocellulosic
biomass to ethanol. Potential value of this relatively vulnerable biomass component can be achieved by pre-extraction of biomass
hemicelluloses prior to subsequent biomass processing into ethanol (Zhang et al., 2011). Major value added products produced from
hemicellulosic hydrolysates or extracted hemicellulose are as follows.
7.1. Lactic acid
In recent years, lactic acid (LA) is a very popular industrially relevant organic acid. The LA is recently being used for the preparation
of various high value products, particularly pharmaceuticals, cosmetics, chemicals and biopolymers like poly-lactic acid (PLA) (Abdel-
Rahman and Sonomoto, 2016). The PLA is considered as a suitable alternative to petrochemical-based plastics for packaging, as well
as in fabrication of prosthetic devices and drug delivery systems because of the high biodegradability and the biocompatible features
(Cubas‐Cano et al., 2019). In the present scenario, the LA is mainly produced by fermentation of glucose because of very low xylose
fermentation rates (Patel et al., 2004) and the need of detoxification from hemicellulosic sugar-rich hydrolysates (Moldes et al., 2006).
To valorize the lignocellulosic prehydrolysate (hemicellulosic extract) for the LA production, the use of hetero-fermentative lactic
acid bacteria (LAB) that can metabolize xylose and sugar mixtures is an appealing option. Table 6 summarizes the yield of lactic acid
produced from hemicellulosic hydrolysate of different lignocellulosic biomass that has been recently reported.
7.2. Xylitol
Xylitol (C5 H12 O5 ), another commercial product manufactured predominantly from hemicellulose, is a polyol that has a hydroxyl
group attached to each carbon atom in its chain. Xylitol is a low-calorie sweetener popularized by its use in toothpastes, chewing
gums, products for diabetics and as a preventive agent against dental caries. Due to its low caloric and anti-carcinogenic properties, it
is used as a sugar substitute in the food industry (Irmak et al., 2017). Currently, Danisco is a world’s largest producer of xylitol, which
manufactures via catalysis using xylose obtained from hardwood sources. As biological route is more sustainable, environmentally
friendly, has lower energy requirements and hence the bioconversion of xylose-to-xylitol by yeasts is considered as an attractive alter-
native to the currently large-scale commercial chemical process (Mussatto, 2012; Cortez et al., 2016). The process steps involved in
biological production of xylitol are: acid/enzymatic hydrolysis of lignocellulosic biomass, detoxification of hydrolysate, fermentation
of hydrolysate to xylitol and xylitol purification. Xylitol can be biologically produced using yeasts, fungi and bacteria. Among them,
the highest yield of xylitol can be achieved by yeast mainly Candida sp. Yeast bioconversion occurs via two steps: reduction and
oxidation. d-xylose is firstly reduced to xylitol by a xylose reductase (XR) and then oxidized to xylulose by xylitol dehydrogenase
(XDH) before phosphorylation into xylulose-5-phosphate, catalyzed by xylulokinase (XK). The two enzymes i.e., XR and XDH are
key catalyst for bioconversion of xylose to xylitol (Mohamad et al., 2015). Table 7 summarizes the yield of xylitol from different
lignocellulosic biomass reported by several researchers.
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Table 7
Xylitol production using different lignocellulosic biomass.
Xylitol yield Productivity

Substrate Pretreatment Organism used Xylitol (g/L) (g/g) (g/(L•h)) Reference
Sugarcane bagasse Steam Candida tropicalis JA2 109.5 0.86 2.81 Morais Junior et al., 2019
explosion
Rape seed straw Acid treatment Debaryomyces hansenii 27.5 0.78 1.15 López-Linares et al., 2018
Corn cob Autohydrolysis Saccharomyces cerevisiae PE-2 148.5 0.95 1.16 Baptista et al., 2018
Corn stover Vapor-phase Pichia stipitis NRRL Y-30,785 12.5 0.61 0.18 Rodrigues et al., 2011
diethyl oxalate
pretreatment
Wood sawdust Acid treatment Candida tropicalis BCRC 20,520 41.4 0.70 0.43 Ko et al., 2008
7.3. Xylooligosaccharides
Xylooligosaccharides (XOS) are non-digestible, soluble dietary fibres constituted of oligomers of xylose units linked through 𝛽–1,
4–glyosidic bonds (Carvalho et al., 2013). The XOS have been recognized by their potential prebiotic activity as well as many other
health benefits to human beings and animals. A prebiotic is defined as a non-digestible food ingredient that benefits the host by
selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and consequently improving
the host’s health (Gibson and Roberfroid, 1995).
The biological activity of the XOS will depend on their degree of polymerization that can vary from 2 to 12. It is known that
proliferation of beneficial bacteria in the human intestinal tract like bifidobacteria can be proliferated by XOS with < 4 monomeric
units. Short chain fatty acids (SCFA) which is related to the prevention of colon cancer can be produced by bifidobactreia as a result
of the XOS utilization for their growth (Carvalho et al., 2013). The XOS that contain uronic acids as branch are known to present
antioxidant and anti-allergic properties (Jain et al., 2015). Because of the ability of the XOS to control body weight, glucose and lipid
homeostasis and insulin sensitivity, the XOS can present a beneficial effect in metabolic conditions of diabetes. The XOS have positive
effects in animal health similar to the antibiotics and hence can be used as substitutes of antibiotics in animal feed. The XOS can be
used for the production of drugs used to the control of obesity and in treatment of gastrointestinal infections in the pharmaceutical
industry. In agriculture, the XOS can be used for the development of growth stimulants (Vázquez et al., 2000; Moure et al., 2006). In
addition to presenting acceptable properties, the XOS has potentials as food ingredient because the XOS exhibit acidity (pH 2.5–8)
and temperature (up to 100 °C) stability in a higher range as compared to inulin and fructooligosaccharides (FOS) (Aragon et al.,
2013; Mano et al., 2018).
The XOS are being costlier than other prebiotics particularly because of commercial xylan substrate, which is expensive and
difficult to obtain. Hence, production of the XOS from lignocellulosic biomass is an attractive. The XOS can be produced from
lignocellulosic biomass using chemical, autohydrolytic, enzymatic or combination of processes (Carvalho et al., 2013; Kumar and
Satyanarayana, 2015). Chemical or auto-hydrolytic processes have several disadvantages include contamination by-products such
as furfural and hydroxymethylfurfural (HMF) (Bian et al., 2014); generate XOS with a wide range of polymerization (from DP2 to
DP20) (Li et al., 2018), while enzymatic processes generate mostly low-DP XOS (from DP2 to DP4) which are required for prebiotic
application (Carvalho et al., 2015; Sukri and Mimi Sakinah, 2018). Higher growth of Bifidobacterium and Lactobacillus strains was
particularly reported using xylobiose (X2) and hence found to present the strongest prebiotic activity among the other xylose oligomers
(Palframan et al., 2003; Moura et al., 2007). As per recommended dose, 1.4–2.8 g/d, the XOS are required in a lower quantity to
achieve prebiotic effects. Hence in terms of price per recommended dose, the XOS are considered as the most competitively priced
prebiotic ingredient (Finegold et al., 2014). The XOS from lignocellulosic biomass are generally produced by a combination of two
methods: 1) extraction of xylan, and 2) the XOS production using endoxylanase (Chapla et al., 2012). Table 8 summarizes the
yield of XOS produced (mg) per gram of xylan, YXOS/xylan (mg/g), that have been recently reported using enzymatic hydrolysis
of lignocellulosic biomass. Prebiotic effect of XOS produced from various lignocellulosic biomass was evaluated in vitro by their
utilization using probiotic strains like Bifidobacter adolescentis, Bifidobacter bifidum (Chapla et al., 2012), Lactobacillus acidophillus
(Pedraza et al., 2014), Lactobacillus fermentum and Lactobacillus plantarum (Jagtap et al., 2017). Various in vivo intervention trials
have been established on animals/humans to prove that the XOS have a strong bifidogenic activity. For example, prebiotic effects
of the XOS produced from corn cobs were tested in humans (Kisoet al., 2005; Chung et al., 2007), rats (Hsu et al., 2004) and mice
(Santos et al., 2006). Longlive, Kangwei, Hfsugar are the topmost three XOS manufacturers in the world (Amorim et al., 2019).
7.4. Polyhydroxyalkanoates
Polyhydroxyalkanoates (PHA) are compostable biobased polyesters that are valuable for potential integrated biorefinery (Snell
and Peoples, 2009). The PHA are sustainable alternatives for the growing global polymer market as their physical properties are
comparable to those of common petro-polymers. The industrial production of the PHA is still expensive and the cost of raw material
is one the major cost contributing factor for the PHA production (Dietrich et al., 2017). The production of the PHA from hemicellulosic
hydrolysate is becoming a promising approach to make integrated biorefinery more economic feasible and also to reduce the cost of
the PHA. Table 9 summarizes the PHA yield from hemicellulosic hydrolysate reported by several researchers.
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Table 8
Xylooligosaccharides production using enzymatic hydrolysis.
Substrate Pretreatment Enzyme source Time (h) Yxos/xylan (mg/g) DP Reference
Pretreated corn cob Ultra-high Streptomyces 18 106.6 X2–X4 Seesuriyachan et al., 2017
pressure thermovulgaris
TISTR1984 xylanase
Sugarcane bagasse Aqueous 𝛽–xylosidase-free 30 670.0 X2–X4 Reddy and Krishnan, 2016
ammonia xylanase of B. subtilis
KCX006
Rice straw Milled at Magnetic cross-linked 1 841 X2, X3, X5/X6 Purohit et al., 2017
particle size < xylanase aggregate
2 mm developed from
Acinetobacter pittii
MASK 25 xylanase
Corn cob 691
Corn cob Steam Paenibacillus 12 750 X2–X4 Liu et al., 2018a
explosion barengoltzii
(PbXyn10A) xylanase
Rice husk Milled Aspergillus nidulans 24 690 X3–X6 da Silva Menezes et al., 2018
XynC A773
Wheat straw Alkaline Bacillus halodurans S7 7 397.7 X2, X3 Faryar et al., 2015
extraction endoxylanase A
mutated at K80R
Wheat straw Alkaline Aspergillus foetidus 10 114 X2–X5 Chapla et al., 2013
extraction MTCC 4898
endoxylanase
Corn cob Alkaline Aspergillus foetidus 8 107.04 X2–X5 Chapla et al., 2012
extraction MTCC 4898
endoxylanase
Note: Y, Yield.
Table 9
Polyhydroxyalkanoates production using different lignocellulosic biomass.
Substrate Pretreatment Organism used PHA (g/L) Yield (g/g) Productivity (g/(L•h)) Reference
Sugarcane bagasse Dilute acid C. necator 6.27 – – Yu and Stahl, 2008

Sugarcane bagasse Dilute acid Burkholderia sp. F24 12.25 – 0.28 Lopes et al., 2014
Rice Straw Dilute acid B. firmus 1.70 Sindhu et al., 2013
Sugar maple wood chips Dilute acid B. cepacia 8.72 0.19 0.09 Pan et al., 2012
Miscanthus Dilute acid Ralstonia eutropha 5119 2.0 0.14 – Bhatia et al., 2019
Barley 1.8 0.11 –
Pine 1.7 0.12 –
Note: PHA, Polyhydroxyalkanoates.
Table 10
Production of furfural from hemicellulose in raw biomass.
Substrate Production condition Yield (%) References
Wheat straw H2 SO4 treatment, 160 °C 36.2 Carvalho et al., 2015

Corn stover H2 O treatment, 140 °C, AlCl3 as catalyst 26 Yi et al., 2013
Poplar THF/H2 O treatment, 160 °C, AlCl3 –NaCl as catalyst 64 Yang et al., 2012
Maple wood THF/H2O treatment, 170 °C, 1% H2 SO4 as catalyst 100 Cai et al., 2013
7.5. Furfural
Furfural is a natural dehydration product of a pentose sugar xylose. Furfural is heterocyclic and aromatic aldehyde composed of a
furan ring with an aldehyde side group (Mathew et al., 2018). Furfural has various applications like for making inks, plastics, antacids,
adhesives, nematicides, fungicides, fertilizers, and flavouring compounds (Raman and Gnansounou, 2015). Furfural acts as a platform
chemical for production of furfuryl alcohol, tetrahydrofuran (THF), and levulinic acid (LA). Furfuryl alcohol is a monomer for furan
resins and prepared by hydrogenation of furfural. The THF is obtained by decarbonylation of furfural to furan followed by catalytic
hydrogenation. For the LA production, furfural is firstly converted into furfuryl alcohol and which is then boiled in ethyl methyl ketone
in the presence of HCl, giving rise to levulinic acid with 90%–93% yield (Xiu and Shahbazi, 2012). Recently, production of pyrrole
and d-proline from furfural was also reported. Pyrrole is a key primary N-containing chemical widely used as precursors for drugs,
fine chemicals, materials and catalysts whereas L/D-proline is extensively utilised either directly or as a precursor in pharmaceuticals
(Song et al., 2020). Table 10 summarizes the yield of furfural from hemicellulose extracted from lignocellulosic biomass.
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8. Lignin based value added products
The lignin portion is considered as a low-value by-product or a cheap energy source during production of 2G ethanol. However,
the valorisation of lignin must integrate fully to pursuit more sustainable and competitive biorefineries (Schutyser et al., 2018).
It is possible to produce different industrial commodities like fuels, aromatics, carbon fibres, adhesives, resins and dispersant either
directly or indirectly based on the properties of lignin (Azadi et al., 2013). Nonetheless, due to complex chemical linkages, conversion
of lignin into value-added chemicals remains a challenging task.
Overall economics of the biomass processing industries may be improved by production of vanillin from lignin. Vanillin can be
produced by depolymerization of lignin by treating an aqueous solution of lignin with oxidants at alkaline pH, high temperature and
pressure. A reaction mechanism is extremely challenging due to complexity and heterogeneity of lignin (Fache et al., 2016). Vanillin
acts as flavouring agent in food and pharmaceutical industries, especially in ice cream production. Vanillin can also be used for the
production of several classes of polymers like polyvanillin, condensation terpolymer, hydrogels, polyester epoxide, and polyethylene-
like polymers with improved antimicrobial activity and enhanced thermal stability. In addition to vanillin polymerization, vanillin
is oxidized under harsh conditions which yield vanillic acid that can be utilised to produce a variety of polyesters. Additionally,
epoxides and resins can also be produced from vanillic acid. Vanillin alcohol is another interesting product produced via reduction of
lignin other than vanillin and vanillic acid. Furthermore, vanillin alcohol may be used to produce polyesters and plastics (Ahmad and
Pant, 2018). In addition to vanilllin, benzoquinones and carboxylic acids can also be produced by oxidation of lignin. Benzoquinones
can be utilised as intermediates in dye production, key structural units in several biologically active compounds and electrolytes in
supercapacitors, redox flow batteries whereas dicarboxylic acids has been widely used as precursors in food, pharmaceutical and
the polymer industries. Lignin can be converted into alkoxyphenols, benzyl polyols, phenol, alkylphenols and catechol via reductive
depolymerisation process. Additionally, bio-oil and syngas can also be produced from lignin by employing thermal depolymerisation
process (Wong et al., 2020).
9. Potential market size and value of platform chemicals
In 2018, the global LA market size was valued at 2.64 billion dollar (USD). It is expected to grow at an estimated compound
annual growth rate (CAGR) of 18.7% from 2019 to 2025. By 2025, the global lactic acid market size is anticipated to reach 8.77
billion dollar (USD). In 2018, around 40.0% of the global revenue was held by sugarcane sourced lactic acid as a dominant segment
in U.S. North America leads the global market followed by Asia Pacific. Around 25.6% of the revenue share was accounted by Asia
Pacific in 2018. In 2018, the PLA is emerged as a dominant application and accounts for 28.3% of the revenue share (Grand View
Research, 2019a; 2019b). By revenue, the global PLA market valued at 698.27 million dollar (USD) in 2017. By 2023, it is poised
to reach 2091.29 million dollar (USD) at an estimated CAGR of 20.06% over the forecast period. In 2017, the market is expected to
be 286.37 kg metric ton in terms of volume, and is poised to reach 830.1 kg metric ton by 2023, at an estimated CAGR of 19.48%.
Corn starch is expected to dominate the global market by raw material (Mordor Intelligence, 2019). The global xylitol market was
esteemed at 737.2 million dollar (USD) in 2015. From 2016 to 2025, it is likely to grow at a CAGR of 6.6% and is expected to reach
1.37 billion dollar (USD) by 2025. Xylitol market is dominated by Asia Pacific, accounting for around 41% share of the global market
revenue in 2015. In terms of application, global xylitol market is dominated by chewing gum segment. It is accounting for over 67%
of global volume in 2015 (Grand View Research, 2017b). By 2025, global XOS market size will reach to 130 Million dollar (USD) at
a CAGR of 4.1% during the forecast period which was 94 Million dollar (USD) in 2018 (Market Watch, 2019). The global prebiotic
ingredients market is projected at 4.07 billion dollar (USD) in 2017, and it is likely to reach a value of 7.37 billion dollar (USD) by
2023 with the Asia-Pacific (APAC) region including China, India and Japan, expecting the highest gains at over 9.5% (Amorim et
al., 2019). The Global PHA Market is anticipated to grow at a CAGR of around 6.3% to reach approximately 119.15 million dollar
(USD) by 2025. The market is categorized into PHA Monomers, PHA Terpolymers, and PHA Co-Polymers based on product type. The
PHA Monomers are separated into polyhydroxybutyrate, polyhydroxyvalerate and other PHA monomers. The PHA Terpolymers is
further divided into P (3hb-Co-3hv-Co-4hb) and other PHA Terpolymers. The PHA Co- Polymers are separated by P (3hb-Co-3hv), P
(4hb-Co-3hb) and other PHA Co-Polymers (Research and Markets, 2019). In 2013, the global furfural market demand was 300 kgtons.
It is projected to expand at a CAGR of 11.9% from 2014 to 2020. In 2013, furfuryl alcohol arose as the largest application segment. It
is accounted for over 85% of overall market. In 2013, Asia Pacific emerged as the leading region with China alone accounting for over
70% of total market volume. Due to high rates of export of furfuryl alcohol, growing demand for furan resins have a positive impact
on the Chinese furfural market (Grand View Research, 2015). In 2016, the global vanillin market was 18 653.9 t and is estimated to
grow at a CAGR of 6.2% from 2017 to 2025 and is possible to reach 724.5 million dollar (USD) by 2025. In 2016, food and beverage
emerged as the largest end-use segment. It is estimated to generate revenue worth of 448.5 million dollar (USD) by 2025 (Grand
View Research, 2017a). Table 11 summarizes the key players for value added chemicals and their market price.
10. Studies on integrated production of 2G ethanol and other value added products
Many attempts have been reported on production of 2G ethanol and other value added products in a biorefienry manner. Im-
portant findings of such useful research have been presented in Table 12. These kinds of reports have been utilised for a large scale
development of 2G ethanol refineries.
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Table 11
List of companies and market price for important value added chemicals.
Product Key companies Market price
Lactic acid Danimer Scientific LLC; Nature Works LLC; Hawkins, Inc.; 2 USD per kg (PHARMACOMPASS, 2019)
Spectrum Chemical Manufacturing Corp.
Polylactic acid (PLA) BASF SE; NatureWorks; Mitsubishi Chemical; DowDuPont; –
Eastman Chemicals; Bayer; Corbion; Danimer Scientific
Xylitol Dupont Danisco; DFI Corporation; Shandong Futaste Co., 5 USD per kg (PHARMACOMPASS, 2019)
Ltd.; Xylitol Canada, Inc.; Zuchem Inc.; Roquette Freres
Xylooligosaccharides Longlive; Kangwei; HFsugar; Henan Shengtai; YIBIN 25–50 USD per kg (Mhetras et al., 2019)
YATAI; HBTX; YuHua; ShunTian
Polyhydroxyalkanoate (PHA) Biomatera; Biomer; Bio-On SRL; Bluepha Co., Ltd.; Cardia 2.25–2.75 USD per lb (Kourmentza et al., 2017)
Bioplastics Ltd.; Dayglo Color Corp.; Kaneka Corporation;
Meredian Holdings Group, Inc.; Metabolix, Inc.; Newlight
Technologies, LLC; PHB Industrial S.A.; Polyferm Canada,
Inc.; Procter & Gamble Co.; Shenzhen Ecomann
Biotechnology Co., Ltd.; Tepha, Inc.; Tianan Biologic
Materials Co., Ltd.
Furfural Teiling; Central Romana; TransFuran; Illovo Suga; Xing Tai 1 USD per kg (PHARMACOMPASS, 2019)
Chunlei; Nutrafur Furfural Espanol; KRBL Ltd.
Vanillin Evolva S.A.; Solvay S.A.; International Flavors Fragrances, 14 USD per kg (PHARMACOMPASS, 2019)
Inc.; De Monchy Aromatics;Comax Flavors
Table 12
Reports on integrated production of 2G ethanol and other value added products.
Lignocellulosic biomass Pretreatment 2G ethanol production Co-products formation Reference
Wheat straw Hydrothermal 0.41 g ethanol per g Biohydrogen production: Kaparaju et al.,
treatment glucose 178.0 mL-H2 per g-sugars 2009
Methane production:
0.324 and 0.381 m3 /kg volatile solids from
bioethanol and biohydrogen processes,
respectively
Hemp H2 O2 alkaline 149 kg of ethanol per Succinic acid production: Kuglarz et al., 2016
treatment ton of dry hemp 115 kg of succinic acid per ton of dry hemp
Rapeseed straw Dilute acid treatment 122–125 kg of ethanol Succinic acid production: Kuglarz et al., 2018
per mg of rapeseed 48 kg of succinic acid per mg of rapeseed straw
straw
Wheat straw Steam explosion (21.3 ± 1.1) g/L of Bio-oil production: Tomás-Pejó et al.,
treatment ethanol lignin was transformed by fast-pyrolysis into 2017
bio-oil with 31.9% yield
Corn stover Non-isothermal 51.6 g/L of ethanol Co-production of oligomers: Buruiana et al.,
autohydrolysis 12.6 g per 100 raw material 2014
Barely straw Steam explosion 14 g of ethanol per XOS production: Álvarez et al., 2018
treatment 100 g of barley straw 11.1 g of XOS (with DP2-DP6) per 100 g of barley
straw
Corncob Alkaline treatment 32.32 g/L of ethanol XOS production: Boonchuay et al.,
0.15 g/g of KOH treated corncob 2018
Elephant grass Steam explosion 42.25 g/L ethanol Lactic acid production: Montipó et al., 2018
treatment 13.35 g/L of LA
Sugarcane bagasse Dilute acid and steam 366 mL of ethanol per Furfural production: Ghosh et al., 2015
treatment kg of biomass 149 g of furfural per kg of biomass
Electricity generation:
0.30 kW of electricity per kg of biomass
Sugarcane bagasse Acid treatment 56.1 g/L of ethanol Xylitol production: Unrean and Ketsub,
24.0 g/L of xylitol 2018
Sugarcane bagasse Dilute acid and steam 287.64 ± 0.25 g of Xylitol production: Dasgupta et al.,
treatment ethanol from 2 kg of (184.43±0.3) g of xylitol from 2 kg of sugarcane 2019
sugarcane bagasse bagasse
Palm fronds Alkaline and acid 42.6 ± 0.8 g Vanillin production: Messaoudi et al.,
treatment ethanol/100 g glucose 140 μg/mL 2019
Xylose production:
37 g per 100 g of hemicelluloses
isolated from palm fronds
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Fig. 6. Major bottlenecks and challenges in integrated biorefinery.
11. Commercialization of lignocellulosic biorefinery: Bottlenecks and challenges
The typical lignocellulose refinery scenario is based on the product streams that 1) all isolated sugars are used to produce cellulosic
ethanol or biobased products, and 2) all the residues, including lignin and residual cellulose, are used as burning fuels to generate
steam electricity or value added chemicals. High processing costs, huge capital investment and a narrow margin between feedstock
and product prices are the three large obstacles for profitable biomass biorefineries (Zhang, 2008). In spite of large number of reports
have been published showing encouraging results for lignocellulosic biorefineries, very few industries have commercialised this tech-
nology. Currently, development of biorefining technologies for the production of advanced biofuels, biochemicals and biomaterials
from lignocellulosic biomass is undertaken by three promising companies Lignol Energy Corporation, British Columbia; Verenium
Corporation, San Diego, California and Mascoma Corporation, Lebanon, New Hampshire (Isikgor and Becer, 2015). Major drivers for
the disposition of lignocellulosic biorefineries producing biofuels and high value bioproducts are i) sustainable and renewable energy
supply, ii) inclusive economic growth in turn saving foreign exchange reserves and less dependency on imported crude petroleum,(iii)
establishment of carbon neutral and circular economy, and iv) low carbon footprint and green environment (Valdivia et al., 2016;
Oh et al., 2018). On the other hand, many challenges are faced by industries. Availability of single type of biomass throughout the
year is the major obstacle. Hence, lignocellulosic biorefineries must be developed for processing multiple feedstocks. In lignocellulose
biorefinery, cost of the feedstock has more than 50% share in cellulosic ethanol production (Junqueira et al., 2016) and hence cost
of biomass has utmost importance during commercialization of lignocellulose biorefineries. Transportation of lignocellulosic biomass
from agricultural farm to factory and its management are two paramount cost contributing factors that can affect the overall economy
of the biorefinery. Another critical challenge is scale up from laboratory scale process to industrial large scale process (Chandel et
al., 2018). Various paramount factors like process integration, selection of products, clear-cut master plan, risk factors analysis, cost
sensitivity analysis, safety and regulatory norms and reproducible economic modeling play vital roles for the scaling-up of biorefiner-
ies (Sanford et al., 2016). The outline of major challenges/bottlenecks for commercialization of lignocellulosic biorefinery is given
in Fig. 6.
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12. Concluding remarks
Integrated lignocellulosic biorefinery has a high potential to provide 2G ethanol at competitive price to the end user by conjugating
bulk production of value added products like oligosaccharides, lactic acid, PHA. There are enormous potential opportunities to
develop commercially feasible routes to extract and covert biomass lignin and hemicelluloses for high-value products along with
2G ethanol production from cellulose to utilize the full value. However, there is an urgent need to overcome certain challenges for
successful commercialization of integrated biorefinery. Special efforts are needed to reduce the cost of feedstock, biomass processing
and saccharifying enzymes. There is also an urgent need to identify robust enzymes and organisms that can enhance efficiency of
bioconversion. At present, it is very clear that process integration for 2G ethanol and high value bioproducts in a lignocellulosic
biorefinery is a promising approach.
Declaration of Competing Interest
There are no conflicts to declare.
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ARTICLE IN PRESS [m3GeSsc;March 10, 2021;4:43]

Journal of Bioresources and Bioproducts xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Bioresources and Bioproducts

Integrated lignocellulosic bioreﬁnery: Gateway for production of

Fig. 1. Integrated bioreﬁnery scheme.

2. Bioreﬁnery: An integrated approach

3. Lignocellulosic biomass: Potential source for phase III bioreﬁnery

Biomass Cellulose (%) Hemicellulose (%) Lignin (%) Reference

Corn cob 42–45 35–39 14–15 Prasad et al., 2007

4. Pretreatment of lignocellulosic biomass

Fig. 2. Inhibitors generated during pretreatment of lignocellulosic biomass

5. Enzymatic hydrolysis: Key bottleneck for lignocellulosic bioreﬁnery

Product name Company Source pH Temperature ( °C) Reference

6. Fermentation process for 2G ethanol

United States Brazil European Union

6.1. Bioethanol as second generation (2G) biofuel

Biomass Ethanol yield (L/t) Reference

Sugarcane 70–90 Zabed et al., 2017

6.2. Scope of 2G ethanol bioreﬁnery in India

Industry Raw material used Technology

Iogen Ottawa, ON, Canada Agro residues, switch State-of-the-art enzyme

Organism Fermentation Lactic acid Lactic acid Productivity

7. Hemicellulose-based value added products

7.1. Lactic acid

Xylitol yield Productivity

Substrate Pretreatment Enzyme source Time (h) Yxos/xylan (mg/g) DP Reference

Sugarcane bagasse Dilute acid C. necator 6.27 – – Yu and Stahl, 2008

Note: PHA, Polyhydroxyalkanoates.

Substrate Production condition Yield (%) References

Wheat straw H2 SO4 treatment, 160 °C 36.2 Carvalho et al., 2015

8. Lignin based value added products

9. Potential market size and value of platform chemicals

Product Key companies Market price

Lignocellulosic biomass Pretreatment 2G ethanol production Co-products formation Reference

Fig. 6. Major bottlenecks and challenges in integrated bioreﬁnery.

11. Commercialization of lignocellulosic bioreﬁnery: Bottlenecks and challenges

12. Concluding remarks

Declaration of Competing Interest

There are no conﬂicts to declare.

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