Hydrolysis of Cellulosic and Hemicellulosic Biomass

C H A P T E R

19 Hydrolysis of Cellulosic and Hemicellulosic Biomass *

Parameswaran Binod*, Raveendran Sindhu*, Kanakambaran Usha Janu*, Ashok Pandey† Centre for Biofuels, Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, India † Centre for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow, India

19.1 INTRODUCTION The recovery of fermentable sugars from lignocellulosic biomass seems very attractive and provides a renewable and sustainable way for the production of various chemicals and other products. Lignocellulosic biomass are ubiquitous in nature and its noncompetitiveness with food crops makes it attractive raw material. Reduction in green house gas emission in compared to fossil fuels is another significant factor which adds value as well as importance to lignocellulosic ethanol when used a transportation fuel. Even though extensive studies have been carried out to meet the future challenges of bioenergy generation, there is no self-sufficient process or technology available to convert the lignocellulosic biomass to bioethanol. The process in the conversion of lignocellulosic biomass to bioethanol involves three major steps: pretreatment, hydrolysis, and fermentation. Pretreatment is necessary to remove the lignin and also makes the biomass more amenable to enzyme attach. Hydrolysis is the crucial step where the cellulose is converted into sugars. Different methods are available for the generation of sugars from lignocellulosic biomass, of which the chemical and enzymatic methods are proven to be more successful.

Biofuels: Alternative Feedstocks and Conversion Processes for the Production of Liquid and Gaseous biofuels https://doi.org/10.1016/B978-0-12-816856-1.00019-1

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19.2 CHEMICAL HYDROLYSIS In chemical hydrolysis, acids are used for the hydrolysis of cellulose for the generation of sugars. Chemical hydrolysis is usually done by using acids. Concentrated mineral acids such as H2SO4 and HCl are commonly used. The main drawbacks of this process are reactor corrosion, product separation, poor catalyst recyclability as well as there is a need for proper treatment of effluent generated during the process [1]. The use of ionic liquids (ILs) are also becoming popular nowadays where the process is less hazardous. Recently several studies revealed that sulfonated carbonaceous-based acids, magnetic solid acids, and polymer-based acids serve as a good agent for cellulose hydrolysis [1]. Although the rate of acid hydrolysis is much faster than enzymatic hydrolysis, the major drawback is glucose also degrades rapidly under acidic conditions [2].

19.2.1 Acid Hydrolysis During acid hydrolysis, acids penetrates into the biomass and breaks lignin and ultimately breaking down the cellulose and hemicellulose polymers into individual sugar molecules. Different acids like H2SO4, HCl, HNO3, etc. are used in this process. The hydrolysis of native cellulose in cotton by concentrated sulfuric acid was reported in the literature as early as 1883 [3]. Most of the research on concentrated acid hydrolysis processes has been done using corncobs. Researchers at the US Department of Agriculture (USDA) proposed a process scheme for the production of sugars and other products from corn cobs based on a two-stage process where the biomass is treated with dilute acid to remove the hemicellulose in the first stage followed by decrystallization and hydrolysis of the cellulose fraction using concentrated acid in the second stage [4]. A concentrated sulfuric acid hydrolysis process has been commercialized in Japan where 80% of the acids were recovered back [5, 6]. A strategy for acid hydrolysis of hemicellulosic fraction in the potato peel was reported by Lenihan et al. [7]. In order to make the process more environmental friendly and economically feasible instead of using concentrated acid, dilute acid hydrolysis followed by enzymatic hydrolysis is followed. Sulfuric acid concentration below 4% is usually used since it is comparatively inexpensive and helps in achieving high reaction rates. Since sugar decomposition takes place at moderate temperature, this process requires high temperature and neutralization of pH is also necessary for the downstream enzymatic hydrolysis or fermentation process. Apart from this, to make the process economically feasible, these acids must be recovered from the reaction mixture after hydrolysis.

19.2.2 Biomass Fractionation by ILs The ILs are salts that are liquid at room temperatures and contain organic cation, usually quaternized aromatic or aliphatic ammonium ions [8]. A concept for dissolving cellulose in molten organic salts was earlier proposed by Charles Graenacher [9]. This method dissolves cellulose in N-alkyl- or N-arylpyridinium chlorides in the presence of nitrogen-containing bases. Later, Robin Rogers with his research team at the University of Alabama applied ILs for the dissolution of cellulose [10]. The IL-based pretreatment of lignocellulosic biomass

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offer an environmentally friendly approach for the recovery of cellulose from lignocellulosic biomass. The ability of 1-ethylpyridiniumchloride, [C2pyr]Cl to dissolve cellulose is known for some time [9]. Recently, it has been demonstrated that a range of ILs with 1,3dialkylimidazolium cations are also effective in cellulose dissolution [10]. A considerable number of studies have been conducted on the solubility of cellulose on IL in the past few years. The solubility of cellulose depends on the IL and the condition. The dissolved cellulose can be regenerated either by adding water or using mixtures of water with organic solvents like acetone or ethanol. It has been observed that the anion in IL plays an important role in determining an IL’s ability to dissolve cellulose [10]. The IL identified to date contains anions that can form strong hydrogen bonds with hydroxyl groups. The dissolving power of the ILs has been typically attributed to strong hydrogen-bonding interactions between the anions and equatorial hydroxyl groups on the cellulose. In comparison to traditional solvents, ILs exhibit very interesting properties such as reasonable chemical inertness, production of no toxic or explosive gases during reaction, good thermal stability, low volatility, negligible vapor pressures, and unique solvation abilities that makes it an important candidate for lignocellulosic treatment. The combination of anion and cation affects their physical and chemical properties such as melting points, viscosity, hydrophobicity, and hydrolysis stability. Therefore, optimal ILs for certain applications can be designed. Another interesting point regarding ILs is their low volatility which permits distillation of the volatile substances, thereby making IL recovery feasible. The cost of production of IL is still high and the recovery and efficient reuse of ILs are continuing as a major challenge.

19.3 ENZYMATIC HYDROLYSIS Unlike chemical hydrolysis, enzymatic hydrolysis is conducted under mild conditions at a pH 4.8 and temperature 45–50°C which is optimum for the cellulase enzyme. The main advantage of enzymatic hydrolysis over chemical hydrolysis is that it does not create a corrosion problem [11]. The time taken for enzymatic hydrolysis is more than that of chemical hydrolysis. Moreover, the final product of enzymatic hydrolysis inhibits the enzyme and ultimately affects the process unless they are removed immediately after they are formed. Apart from this, a major bottle neck in lignocellulosic ethanol production, at present, is the cost of the enzymes.

19.3.1 Enzymes Involved in the Hydrolysis of Lignocellulosic Biomass Considering the complexity and heterogeneity of the lignocellulosic biomass, a single enzyme will not hydrolyze the entire biomass and development of a cocktail of enzymes such as cellulases, hemicellulases, and other accessory enzymes is required for the complete hydrolysis. Following are the enzymes involved in the hydrolysis of lignocellulosic biomass.

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19.3.1.1 Cellulases Cellulases are glycoside hydrolases (GHs) that decompose cellulose into short-chain polysaccharides such as cellodextrin, cellobiose, and glucose. They commonly have a catalytic domain (CD) that cleaves the glycosidic bond; carbohydrate-binding module (CBM) that targets the CD to the polysaccharide substrate; and, in many cases, additional types of ancillary modules such as FN3-like modules [12]. Cellulose is enzymatically degraded to glucose by the synergistic action of three distinct classes of enzymes. Endoglucanases (EG) (EC 3.2.1.4), which hydrolyze internal β-1,4-glucosidic linkages randomly in the cellulose chain. Cellobiohydrolases (CBH, also known as exoglucanases) (EC 3.2.1.91), which progresses along the cellulose and cleave off cellobiose units from the ends. β-glucosidases (BG also known as β–glucoside glucohydrolases) (EC 3.2.1.21), which hydrolyze cellobiose to glucose and also cleave off glucose units from cello-oligosaccharides. Several of these apparently redundant enzymes have been shown to exhibit synergy by either hydrolyzing different ends of the cellulose chain or exhibiting different affinities for different sites of attack. The whole hydrolysis process can be divided into two steps: primary hydrolysis and secondary hydrolysis. Primary hydrolysis involves endoglucanases and exoglucanases and occurs on the surface of solid substrate releasing soluble sugars with a degree of polymerization (DP) up to six into the liquid phase. This depolymerization step is the rate-limiting step for the whole cellulose hydrolysis process. Secondary hydrolysis occurs in the liquid phase involving primarily the hydrolysis of cellobiose to glucose by β-glucosidases. A schematic diagram of mechanism of cellulase action is shown in Fig. 19.1. The CBHs and EGs have a CD and a cellulose-binding domain (CBD). The function of the CBD is to bring the enzyme catalytic module in close contact with the substrate and ensure correct orientation. The removal of the CBD from the enzyme significantly impairs the hydrolysis of crystalline cellulose, demonstrating its importance. The CBD is connected to the CD with a glycosylated flexible linker, which help them to dock with and degrade crystalline cellulose. The CBDs of cellobiohydrolases are able to move laterally along the cellulose chain while the CD cleaves off cellobiose units. Only little is known about how the aromatic Cellulose

Oligosaccharides Endo-glucanase Exo-glucanase β-glucosidase Cellobiose

Glucose

FIG. 19.1

Mechanism of action of cellulase.

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residues of the CBD interact with the cellulose crystal structure and how they desorb from the substrate and reattach. Because of the insoluble nature of native cellulose and anchoring of CBDs, cellulases primarily work in a two-dimensional environment with the unidirectional movement of cellobiohydrolases along the cellulose chain. Hence the synergistic degradation of lignocellulose does not follow classic Michaelis-Menten kinetics. Moreover, factors like the heterogeneous nature of lignocellulose makes the understanding of hydrolysis mechanisms more complicated. 19.3.1.2 Xylanases Another major component present in lignocellulosic biomass is xylan, which is the main carbohydrate present in hemicelluloses. These are polysaccharides made of xylose, a pentose sugar. Hydrolysis of xylan is carried out by a group of enzymes called xylanases. The removal of xylan from lignocelluloses using xylanases increases the accessibility of cellulose to enzymatic hydrolysis. Xylan does not form tightly packed crystalline structures like cellulose and are more susceptible to enzymatic hydrolysis. The complete hydrolysis of xylan requires the action of multiple xylanases with overlapping but different specificities and action. The complete degradation of xylan requires the cooperative action of the following enzymes. Endo-1, 4-β-xylanase (1, 4-β-D-xylan xylanohydrolases, EC 3.2.1.8) cleaves the glycosidic bonds in the xylan backbone releasing xylo-oligosaccharides. β-xylosidase (1,4-β-D-xylan xylohydrolase, EC 3.2.1.37) acts upon the small oligosaccharides and cellobiose, generating β-D-xylopyranosyl residues from the nonreducing terminus. α-arabinofuranosidase (EC 3.2.1.55) and α-glucuronidase (EC 3.2.1.139) remove the arabinose and 4-O-methyl glucuronic acid substituents, respectively, from the xylan backbone. Esterases act upon the ester linkages between xylose units of the xylan and acetic acid (acetyl xylan esterase, EC 3.1.1.72) or between arabinose side chain residues and phenolic acids such as ferulic acid (ferulic acid esterase, EC 3.2.1.73) and p-coumaric acid (p-coumaric acid esterase). The mechanism of action of xylanase enzyme complex is schematically represented in Fig. 19.2. 19.3.1.3 Peroxidases Peroxidases are a group of enzymes involved in the degradation of lignin which is tightly bound to cellulose making it inaccessible to the cellulase enzyme. Lignin peroxidase [also called ligninase (LiP), EC 1.11.1.7] and manganese peroxidase [also called Mn-dependent peroxidase (MnP), EC 1.11.1.7] are the two major components of the lignolytic enzyme system. These are heme-containing glycoproteins which require hydrogen peroxide as oxidant. These enzymes were discovered in Phanerochaete chrysosporium and are called true ligninases due to their high redox potential. The LiP degrades nonphenolic lignin units (up to 90% of the polymer). The LiP isozymes are glycoproteins of 38–46 kDa, with pI values of 3.2–4.0. It has a distinctive property of an unusually low pH optimum near pH 3. The enzyme contains 1 mol of iron protoporphyrin IX per mole of protein. The LiP oxidizes nonphenolic lignin substructures by abstracting one electron and generating cation radicals which are then decomposed chemically. Schoemaker and Piontek [13] described the mechanism of interaction of lignin peroxidase (LiP) with lignin polymer. Veratryl alcohol (valc), which is a secondary metabolite of white rot fungi, acts as a cofactor for the enzyme. It was observed that, in the depolymerization with

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Arabinoxylan

Smaller polysaccharide

Endo-1,4-β-xylanase Feruloyl esterase α-Arabinofuranosidase

Ferulate Arabinose

β-xylosidase

Xylose

FIG. 19.2

Mechanism of action of xylanases.

fungal cultures, both the presence of LiP and veratryl alcohol stimulated the degradation of lignin. Lip + H2 O2 ! H2 O + LiPI LiPI + valc ! valc + + LiPII LiPI + 2H + ! valc + + H2 O + LiP In this process LiP oxidizes the first molecule of veratryl alcohol to the corresponding radical cation (valc+), which is liberated from the active site. Subsequently, the second substrate molecule is oxidized by LiPII to form a second valc+. In the process, LiPII is converted to native enzyme. The MnP generates Mn3+, which acts as a diffusible oxidizer on phenolic or nonphenolic lignin units through lipid peroxidation reactions. It oxidizes Mn(II) to Mn(III) which then oxidizes phenol rings to phenoxy radicals which lead to the decomposition of compounds. 2Mn ðIIÞ + 2H + + H2 O2 ! 2Mn ðIIIÞ + 2H2 O:

19.3.1.4 Laccases Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a copper-containing enzyme that belongs to the small group of enzymes called the blue copper proteins or the blue copper oxidases. These enzymes are also involved in the degradation of lignin. Laccase, alone or together with lignin peroxidase and manganese peroxidase, has been demonstrated in a wide variety of white rot fungi and can completely mineralize this substrate. The presence of laccase in nonlignolytic fungi also has been demonstrated. Laccases may be constitutive or

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inducible enzymes. Several compounds like phenolic compounds, strictly related to lignin or lignin derivatives have been shown to induce and improve laccase formation. However, nonlignin compounds and extracts from different origins are also found to be effective inducers of laccase production. Laccases catalyze the oxidation of phenolic units in lignin and a number of phenolic compounds and aromatic amines to radicals, with molecular oxygen as the electron acceptor, is reduced to water. Although lignocelluloses is common substrates, they show a considerable diversity in molecular weight, pH optimum, and other properties. It has been shown that the ability of laccases to break down lignocellulose is increased by certain phenolic compounds (2, 2 P-azino-bis-(3-ethylthiazoline-6-sulfonate (ABTS) or 3-hydroxyanthranilic acid (3-HAA) which act as mediators [14]. A mediator is a small molecule that acts as an “electron shuttle.” Once it is oxidized by the enzyme, generating a strongly oxidizing intermediate, the comediator (oxidized mediator), it diffuses away from the enzymatic pocket and in turn oxidizes any substrate that due to its size could not directly enter into the active site. Due to this specificity for phenolic subunits in lignin and its restricted access to lignin in the fiber wall, laccase has a limited effect without these redox mediators. In an active holoenzyme form, the laccase molecule is a dimeric or tetrameric glycoprotein usually containing four copper atoms per monomer, bound to three redox sites (type 1, type 2, and type 3 Cu pair).

19.3.2 Other Helper Proteins in Hydrolysis In the process of enzymatic hydrolysis of lignocellulosic materials, some proteins have been identified that are capable of nonhydrolytically loosen the packaging of cellulose fibril network; a process called amorphogenesis. These proteins act synergistically along with cellulases thereby increasing the accessibility of cellulose to the enzymes. Hence these helper proteins are called amorphogenesis-inducing agents. Swollenin is an example for such helper proteins which is isolated from Trichoderma reesei. It comes under the category of expansinlike proteins which are proteins that have “loosening” effect on the cellulosic network within plant cell walls during growth. Swollenin contains an amino terminal fungal-type cellulosebinding module linked to the plant expansin homologous module. It shows sequence similar to the fibronectin (Fn) III-type repeats of mammalian titin proteins which have been shown to be able to unfold and refold easily, allowing the protein to stretch. Swollenin has been reported to disrupt the structure of cotton fibers without revealing any hydrolytic activity and formation of reducing sugars [15]. This indicates that the protein is involved in the swelling of the cellulosic network within the cell walls and is not active against the β-1,4-glycosidic bonds in cellulose. The protein increases the access of cellulases to cellulose chains by promoting the dispersion of cellulose aggregations and exposing individual cellulose chains to the enzyme. This ability makes it an important component in the enzyme mixture use for the hydrolysis of lignocellulosic biomass. There are several swollenin-like activities displayed by T. reesei, which differ in their modes of action but contribute synergistically for the efficient hydrolysis of the plant polysaccharides. Gourlay et al. [16] reported that swollenin aids in the amorphogenesis step during enzymatic saccharification of lignocellulosic biomass. This is achieved by enhancing access to the hemicelluloses fraction that limits accessibility of cellulose component of lignocellulosic biomass.

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The production of a recombinant swollenin from Trichoderma harzianum in Escherichia coli and its potential synergistic role in biomass degradation were demonstrated by Santos et al. [17]. The study revealed the potential of recombinant swollenin as a potential additive for use in enzyme cocktails to improve the overall performance of the biomass hydrolysis.

19.3.3 Tailor-Made Enzyme Mixtures for Biomass Hydrolysis The major components of lignocellulosic biomass like cellulose, hemicelluloses, and lignin differs in soft wood and hard wood depending on the species. Hence various types of biomass require a minimal set of enzymes which has to be tailor-made for improving the hydrolysis efficiency. Most of the tailor-made biomass hydrolyzing enzymes is composed of high levels of cellulases, in combination with low levels of other enzymes which attacks noncellulosic components like hemicelluloses and lignin [18]. Study conducted by Wilkinsen et al. [19] revealed that supplementation of additional enzymes like ferulic acid esterases, acetyl xylan esterases, and xylanases as well as cofactors like ascorbate and copper could significantly improve glucose yields.

19.4 FACTORS AFFECTING ENZYMATIC HYDROLYSIS There are several enzyme-related and biomass-related factors that affect the enzymatic hydrolysis. The composition of biomass plays a major role in determining the effectiveness of enzymatic hydrolysis. During enzymatic hydrolysis, cellulases tend to irreversibly bind to lignin through hydrophobic interactions that cause loss in enzyme activity. Hence, the amount and composition of lignin in the biomass used critically affects the formation of soluble sugars during enzymatic hydrolysis. Along with this, the type of pretreatment employed, enzyme dosage and its efficiency for saccharification, etc. also have a great influence on biomass digestibility. Even though the individual impacts of these factors in determining the efficiency of enzymatic hydrolysis has not been fully resolved, many of these factors are found to be interrelated during the saccharification process. The main factors that influence the enzymatic hydrolysis of lignocellulosic feedstocks can be divided into two groups: enzyme-related and substrate-related factors.

19.4.1 Enzyme-Related Factors Several factors associated with the nature of the cellulase enzyme system have been suggested to be influential during the hydrolysis process. These include enzyme concentration, enzyme adsorption, synergism, end-product inhibition, mechanical deactivation (fluid shear stress or gas-liquid interface), thermal inactivation, and irreversible (nonproductive) binding to lignin. In the process of enzymatic hydrolysis, the nature of the enzyme system used, the mode of action (endo- vs. exo-enzymes), and their stereochemical mechanism of hydrolysis (inverting vs. retaining) are interrelated. In addition, the synergism between the enzymes can be of significant benefit in increasing the hydrolysis rates of complex substrate. Synergism is also substrate-dependent, with some mixtures showing cooperative action on

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amorphous substrates, but not on microcrystalline cellulose. All this factors can collectively influence the enzyme efficiency. Temperature exerts a profound effect on enzyme action. Higher or lower temperature alter the enzyme structure and hence its efficiency will be less. The addition of amphiphilic compounds like surfactants improves the enzyme activity [20]. The addition of surfactants also facilitates efficient recycling of cellulases after saccharification, a process step that ideally needs to be considered to reduce the cost of lignocellulosic ethanol production. It is proposed that surfactants may cause a surface structure modification or disruption of the lignocellulose that increases enzyme accessibility to cellulose or surfactants may affect enzyme-substrate interaction by preventing nonproductive adsorption of enzymes. It has also been shown that surfactants act as enzyme stabilizers. They adsorb at the air-liquid interface and thus prevent enzyme denaturation during agitation in the hydrolysis mixture [21]. A number of surfactants have been examined for their ability to improve enzymatic hydrolysis. Nonionic surfactants are the most effective among them. Fatty acid esters of sorbitan polyethoxylates (tween80, tween20) and polyethylene glycol (PEG) are among the most effective surfactants reported for enhancing enzymatic hydrolysis. The inhibitory compounds such as organic acids, furfural, 5-hydroxymethylfurfural, vanillin, etc. produced during the pretreatment of lignocellulosic biomass also found to decrease the efficiency of enzymatic hydrolysis. Hydrolysis is also found to be affected due to the end-product inhibition of cellulases. However, when working with insoluble substrate and kinetics that do not follow the Michaelis-Menten model, it is difficult to determine the exact type of inhibition. The removal of end product is possible by using the simultaneous saccharification, and fermentation (SSF) strategy. But in this case inhibition of cellulases by fermentation products should also be considered. Ethanol is inhibitory to cellulases, although less compared to glucose [22]. Hence the effect of end products on cellulase has to be evaluated before selecting the hydrolysis method and fermentation strategy.

19.4.2 Substrate-Related Factors The rate of enzymatic hydrolysis of lignocellulose is profoundly affected by the structural features of cellulose [23] which include cellulose crystallinity, DP, available/accessible surface area, structural organization, that is, macrostructure (fiber) and microstructure (elementary microfibril), particle size and presence of associated materials such as hemicellulose and lignin. The effect of substrate crystallinity has been shown to play a major role in limiting hydrolysis in some studies. Substrate concentration is one of the main factors that affect the yield and initial rate of enzymatic hydrolysis of cellulose. Maintaining high solid concentrations throughout the conversion process from biomass to ethanol is important from an energy and economic viability viewpoint. High solids enzymatic hydrolysis takes place at solids levels where initially no significant amount of free water is present. This allows for a larger system capacity, less energy demand for heating and cooling of the slurry and also less effluent discharge. Regarding the overall economic feasibility of lignocellulosic ethanol production, a high substrate concentration allows for the production of a concentrated sugar solution, which in turn is beneficial for the subsequent fermentation. By increasing the solid loading, the resulting sugar

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concentration and consequently ethanol concentration can be increased with significant effects on distillation. A sugar concentration of at least 8% (w/w) is required to achieve an ethanol yield of 4% (w/w) by which the energy required for the distillation can be significantly reduced. However, high substrate concentration can also cause substrate inhibition, which substantially lowers the rate of the hydrolysis. The extent of substrate inhibition depends on the ratio of total substrate to total enzyme [24]. The enzymatic conversion (percent of theoretical) is found to linearly decrease with increased solids concentration despite using a constant enzyme-tosubstrate ratio. It may be explained by mass transfer limitations or nonproductive adsorption of enzymes. However, the specific mechanism behind the decreased hydrolytic efficiency is not fully studied. The extent to which solid loading can be increased in hydrolysis varies with each lignocellulosic biomass. At both laboratory and industrial scale, 12%–20% total solids is often considered the upper limit at which pretreated biomass can be mixed and hydrolyzed in conventional stirred tank reactors. Fed-batch operations can be employed in order to increase the final solid loadings. Novel enzymatic hydrolysis reactors were developed by Jorgensen et al. [25] for enzymatic liquefaction of high solid loading. These reactors can handle up to 40% biomass loading and with low enzyme loading 7FPU/g dry matter.

19.5 RECYCLING OF ENZYMES During enzymatic hydrolysis process, enzymes can either remain bound to the solid residue or are freely suspended in liquid phase [26]. Because of their relatively high stability and natural affinity to cellulose, these free proteins can be recycled by re-adsorption onto the fresh substrate. Free cellulases in hydrolysate had been successfully recycled by the addition of fresh pretreated substrate by Tu et al. [27]. A simple method of recovering enzymes after hydrolysis by centrifugation was carried out by Moniruzzaman et al. [28]. Ultrafiltration is another method for enzyme recovery [29]. Mores et al. [30] reported cellulase recovery by a combined sedimentation and membrane filtration process where 75% of the cellulase enzymes recovery achieved. Another method for recycling enzymes is using amphiphilic lignin derivatives. The effect of amphiphilic lignin on cellulase recycling was investigated in a continuous multistage saccharification process of cellulosic materials using cellulase as catalyst. The results indicate that amphiphilic lignin is an excellent water-soluble polymeric carrier for immobilization of cellulase to preserve the hydrolytic activity for a long period [31]. The use of surfactant on enzyme recycling was reported by Tu and Saddler [32]. The economic analysis of enzyme cost versus surfactant cost suggests that a 66% reduction in total enzyme cost was achieved and Tween 80 was the most effective surfactant in enzyme recycling. Reusability of enzymes by immobilization was carried out by Tu et al. [33]. Their study evaluated the potential for immobilization of β-glucosidase on a methacrylamide polymer carrier, Eupergit C for lignocelluloses hydrolysis. The immobilization could facilitate enzyme recycling in sequential batchwise or semibatchwise saccharification process. Eupergit C-immobilized β-glucosidase was examined for six successive rounds of lignocellulosic hydrolysis and exhibited relative stability during the subsequent five cycles.

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19.6 METHODS FOR IMPROVING ENZYMATIC HYDROLYSIS One strategy for achieving improved efficiency of enzymatic hydrolysis is to improve the specific activities of cellulases by genetic engineering. For lignocellulosic substrates, the nonproductive binding and inactivation of enzymes by the lignin component are the important factors limiting catalytic efficiency. Understanding the effect of these factors allow engineering of cellulases with improved activities [34]. The studies proved that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Cellulases lacking CBDs have a high affinity for lignin which indicates the presence of lignin-binding sites on the CD. The evolution of the cellulosome complex has led to colocalization of synergistic combinations of hydrolytic enzymes. This architectural feature has led to innovative molecular engineering approaches for diverse research and industrial applications [35]. The modular structure of the T. reesei endoglucanase IV (EG IV) was reconstructed by Liu et al. [36] by fusing EG IV with an additional catalytic module (EGIVCM). The genes were obtained through RT-PCR and gene fusion and expressed in recombinant Pichia strains. The results indicated that modification of the EGIV structure with an additional catalytic module in the C terminus has improved the specific activity by about fourfold. Two strategies are used for improving the properties of individual cellulase components: (1) rational design and (2) directed evolution. Qian et al. [37] have developed high-efficient cellulase mixtures by genetically exploiting the potentials of T. reesei endogenous cellulases for biomass hydrolysis. The study showed that overexpression of endoglucanase 2 (EG2) resulted in a 46-fold increase in the transcription level and a 1.5-fold enhancement of total cellulase activity which resulted in a significant enhancement of saccharification efficiency. Wahlstrom and Suurnakki [38] reported improved enzymatic hydrolysis of lignocellulosic biomass in ILs. They demonstrated the applicability of an integrated process, in which IL treatment followed by enzymatic hydrolysis was carried out. The study proved the advantages of integrated enzymatic hydrolysis of polysaccharides in the presence of ILs.

19.7 KINETIC MODEL FOR ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSES There are three principal approaches to model cellulose hydrolysis [39, 40]. First approach is to fit the experimental data to linear/nonlinear regression model. This method is simple to construct, but it requires large sets of experiments. Second approach involves the formulation of mechanistic models which attempt to model some of the underlying phenomenon with simplifying assumptions [41]. Rate expressions are generally described using MichaelisMenten-type enzyme kinetics with/without incorporating the effects of enzyme adsorption, temperature, pH, substrate, and product inhibition. The model structure results in a set of ordinary differential equations and model parameters are often determined by fitting model predictions to the experimental data. While a more general set of conditions can be simulated by incorporating additional model terms this often results in the loss of physical significance

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of the model terms and leads to overparameterization issues. Stochastic molecular modeling (SMM) of the hydrolysis in which each hydrolysis event in translated into a discrete event is another approach that can be used for modeling cellulose hydrolysis that can capture dynamic enzyme-substrate interaction during hydrolysis. This approach relies on modeling enzymatic hydrolysis process at molecular and enzymatic levels. One of the main advantages of the SMM approach is that structural characteristics and enzyme characteristics can be separately determined and incorporated into the model. The first reported model for hydrolysis of insoluble polysaccharides using stochastic modeling approach was developed by Fenske et al. [42] and later similar approach was used by Asztalos et al. [43] to model cellulose hydrolysis which had reasonable accuracy in predicting the hydrolysis trends for endoglucanse and CBH enzymes.

19.8 CONCLUSIONS AND PERSPECTIVES Conversion of lignocellulosic biomass into fermentable sugars is the key step in lignocellulosic ethanol production. There occur several challenges involved in this process which need to be addressed in order to improve the process efficiency. Even though conventional method of lignocellulosic hydrolysis using concentrated acids is an efficient process, there are several issues related to environment which make it to think an alternative to replace this method with more environmentally friendly processes. The use of ILs for deriving cellulose from lignocellulosic material seems to be promising method, but there occurs several challenges to make this process a feasible one. Currently the cost of ILs is too high and it is necessary to develop a technology to make ILs in cheaper way. In addition the recovery and reuse of ILs need to be addressed. Another challenge is to recover lignin and hemicelluloses from the ILs after cellulose has been extracted. So there occurs an immense opportunities for R&D in the area of IL-based process for the production of lignocellulosic ethanol. Due to the heterogeneous nature of lignocellulosic materials, it is necessary to screen a large variety of IL to find a suitable one for a particular biomass. Moreover, there occur wide possibilities for designing ILs based on the nature of lignocellulosic materials. Hydrolysis using enzymes is an attractive and environmentally safe alternative; still a lot of research scopes exist to improve the enzymatic conversion efficiency of lignocellulosic biomass to fermentable sugars by protein engineering approaches.

Acknowledgment Raveendran Sindhu and Parameswaran Binod acknowledges Department of Science and Technology for sanctioning a project under DST WOS-B scheme (SR/WOS-B/740/2016) and under INNO-INDIGO Bioenergy scheme (grant no. DST/IMRCD/INNO-INDIGO/INDO-NORDEN/2017(G), respectively.

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