Enzymes for second generation biofuels: Recent developments and future perspectives

Accepted Manuscript Enzymes for second generation biofuels: Recent developments and future perspectives

Parameswaran Binod, Edgard Gnansounou, Raveendran Sindhu, Ashok Pandey PII: DOI: Reference:

S2589-014X(18)30049-5 doi:10.1016/j.biteb.2018.06.005 BITEB 49

To appear in:

Bioresource Technology Reports

Received date: Revised date: Accepted date:

26 April 2018 14 June 2018 14 June 2018

Please cite this article as: Parameswaran Binod, Edgard Gnansounou, Raveendran Sindhu, Ashok Pandey , Enzymes for second generation biofuels: Recent developments and future perspectives. Biteb (2018), doi:10.1016/j.biteb.2018.06.005

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ACCEPTED MANUSCRIPT Enzymes for second generation biofuels: Recent developments and future perspectives

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Parameswaran Binoda*, Edgard Gnansounoub, Raveendran Sindhua and Ashok Pandeyc

Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and

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Technology (CSIR-NIIST), Trivandrum-695 019, India

Bioenergy and Energy Planning Research Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015

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Lausanne, Switzerland

CSIR - Indian Institute of Toxicology Research (CSIR-IITR), 31 MG Marg, Lucknow-226 001, India

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*Corresponding author. Tel 91-471- 2515361; Fax 91-471-2491712 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Even though the search for alternative fuels from lignocellulosic biomass started a few decades ago, still it is relatively immature mainly because of few challenges which are yet to be solved for making the process commercially viable. The main challenge is the higher

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production cost of lignocellulose degrading enzymes. There are many factors need to be considered on lignocellulosic biomass degrading enzymes for practical applications mainly

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due to the heterogeneity of the biomass. A single enzyme will not degrade the biomass

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efficiently hence it needs to consider for a cocktail of enzymes. Similarly, one enzyme cocktail will not work on every biomass types due to the compositional variability. All these

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factors need to be addressed which makes the research tiresome. Also, there are several gaps

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in enzyme technology, especially for biofuel applications. The present review addresses the lacuna in research on lignocellulose degrading enzymes for biofuel production and possible

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solutions.

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Keywords: Biomass; Lignocellulose; Enzymes; Hydrolysis; Biofuel; Pretreatment.

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ACCEPTED MANUSCRIPT 1. Introduction Currently, renewable energy has become increasingly popular and widely accepted by the people due to exhaustible and polluted nature of the oil and coal resources. Solar, wind, geothermal and hydroelectric powers are more preferred sources of renewable energy. This form of energy can replace conventional and more-pollution energy sources with new less-

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polluting sources and hence reduce greenhouse gas emissions which in turn reduce global

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warming (Owusu and Asumadu-Sarkodie, 2016). It will also help in reducing the oil imports

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and improve energy independence of net oil import countries. The major challenge is how to replace non-renewable fuels like petroleum based gasoline with a renewable one. Bioethanol

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serves as a substitute for gasoline which can be produced from biomass including agricultural residues (Saini et al., 2015). This thought has created a lot of research on bioethanol

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production from sugar and starch biomass– known as first generation bioethanol which is

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now fully matured commercialized successfully.

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Results indicate that the use of first generation bioethanol increase the usage of crop lands for bioethanol which in turn increase greenhouse gases (GHG) emission (Timothy et al., 2008). Though corn based ethanol showed a 20% savings, but the greenhouse gases doubled over

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the past three decades. Cultivation of switch grass for biofuel production showed that the

biofuels.

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GHG emissions increased by 50%. All these reports raise concerns of the first generation

A greenhouse gas emission depends directly on the source of the biomass and its land usage. Carbon emissions cannot be reduced by replacing fossil fuels with biofuels since the CO2 released from tailpipes and smokestacks is same per unit of energy irrespective of the source (Searchinger et al., 2009). So the choice of biomass for biofuel is very critical. The biofuel must be derived from a feedstock without any competition with food or animal feed. This should also produce lower GHG emissions compared to fossil fuels. 3

ACCEPTED MANUSCRIPT The lignocellulosic biomass is complex in nature and is composed of cellulose, hemicellulose, and lignin. The cellulose and hemicellulose are polysaccharides made up of monomeric sugars while lignin is a complex aromatic polymer of polyphenols. Upon fractionation, cellulose and hemicellulose yield monomeric sugars which can be used for the production of bioethanol or other commodity chemicals through microbial fermentation

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methods. Lignin form a cementing substance with hemicellulose and cellulose, serves as a

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barrier for the saccharification of cellulose and hemicellulose, hence harsh pretreatment is

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necessary for the accessibility of the carbohydrate polymers.

Several treatment strategies are currently available for the hydrolysis of lignocellulosic

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biomass for the generation of fermentable sugars among these enzymatic methods is the most preferred and environmentally friendly method. The present review discusses the current

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developments in the research on lignocellulosic biomass hydrolysing enzymes. 2. Importance of second generation biofuels

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The fossil resources are a non-sustainable form of energy. The burning of fossil fuels

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contributes a tremendous increase in the atmospheric CO2 which leads to global warming. Increase in consumption of fossil fuels and depletion of fossil fuels as well as increase in

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GHG emissions leads to search for alternative strategies of energy (Owusu and AsumaduSarkodie, 2016). The search for renewable, sustainable and environmentally friendly source

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of energy created an interest in the production of fuels from a biological source. Adopting strategies for renewable sources of fuels helps in the reduction of CO2 production due to the burning of fossil fuels (Ibeto and Okpara, 2010). Adopting strategies to produce biofuels from lignocellulosic biomass in turn reduce the world’s dependence on crude oil as well as CO2 production. ‘First generation’ biofuels offers several advantages like CO2 benefits and energy security.

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ACCEPTED MANUSCRIPT Major concerns and drawbacks of first generation biofuels is the food - versus - fuel concerns which indirectly contributes a rise in food price (Laursen, 2006). Production and use of some first generation biofuels can be the expensive option for reducing the GHG emissions and improving energy security. However, that issue is controversial. International Energy Agency (IEA) reported that the GHG mitigation from biodiesel as well as from corn - based ethanol

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depends on the nation and the strategy adopted and it mostly exceeds UD$ 250 / t CO2

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avoided (IEA 2008). The competition with food crops will be a major concern since first

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generation biofuels dominate total biofuel production (IEA, 2008).

Biofuels produced from lignocellulosic biomass serves as second generation biofuels.

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Lignocellulosic biomass serves as a economically viable feed stock for the production of biofuels. Several challenges associated with the first generation fuels could be addressed by

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the second generation fuels. One major attraction of second generation fuels is that there is no competition with food. A comparison of petroleum derived, first generation and second

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generation fuels are given in Table 1. Even though second generation fuels offer much

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advantageous, it has several limitations also. The major bottleneck in the second generation fuels is the lack of a mature technology for its production. Several technical barriers exist that

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make its production uneconomical. Several research and developmental activities are going on throughout the world to make the process economically viable. In addition to agricultural

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residues, crops and forest residues, wastes from wood processing industries can also be used as a feed stock for biofuel production. The production of biofuels, especially bioethanol, from lignocellulosic materials involves a complex process. There are two different processing routes that are available, which includes: 1. Biochemical: In this strategy either microorganisms or enzymes will be used for the saccharification of lignocellulosic biomass to produce sugars before fermenting to ethanol. The present review considers the biochemical routes only. 5

ACCEPTED MANUSCRIPT 2. Thermo-chemical: In this strategy either gasification/ pyrolysis is adopted for the generation of syngas composed of carbon monoxide and hydrogen. This can be used for the production of synthetic diesel or aviation fuels. Bioethanol production from lignocellulosic biomass involves three unit operations like pretreatment, enzymatic saccharification and fermentation. Pretreatment involves removal of

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hemicelluloses and lignin from the lignocellulosic biomass, so that cellulose can be easily

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hydrolysed to sugars. The pretreatment should be carried out in such a way that there will be

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minimal or without any inhibitor generation which will affect subsequent fermentation. The pretreatment step produces a solid substrate in which the cellulose is further converted to

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glucose by cellulolytic enzymes, and the hemicellulose fraction can be converted to bioethanol or other products/chemicals by C5 utilising microbes. Strategies currently in

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practice for cellulose hydrolysis include dilute acid hydrolysis, concentrated acid hydrolysis and enzymatic hydrolysis. Dilute acid hydrolysis is the most common method adopted in

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most of the pilot plants. 3. Why we need enzymes?

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The cell wall of lignocellulosic plant biomass consists of three layers, middle lamella, primary cell wall and secondary cell wall. Each layer has its own unique structure and

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function and the chemical composition of these layers differ on the genera and species of the plant. The middle lamella is made up of pectic substances which cement the cell walls of two adjoining cells together. The primary cell wall is the polysaccharide rich, thin and flexible layer surrounds the plant cells. The major polysaccharides present in the primary cell wall are the following. Cellulose – a polysaccharide composed on 1, 4-linked -D-glucose residues.

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ACCEPTED MANUSCRIPT Hemicellulose – is a heterogeneous polymer composed mainly of xylan (Saha, 2003). Other hemicelluloses include glucuronoxylan, arabinoxylan, glucomannan and galactomannan found in primary and secondary walls. Pectin is a complex polysaccharides composed of 1, 4-linked α-D-galacturonic acid. Three classes of pectic polysaccharides are homogalacturonan, rhamno-galacturonan, and

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substituted galacturonans. Composition of primary cell wall changes during plant growth and

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development.

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The secondary cell wall is formed within the primary cell wall when the cell is fully grown. It is mainly composed of cellulose, lignin, and hemicellulose (xylan, glucuronoxylan,

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arabinoxylan, or glucomannan). Cellulose is sealed in hemicelluloses and lignin, and act as a

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barrier for enzymatic saccharification. Fig.1. depicts intra and inter polymer linkages between different components of lignocellulosic biomass.

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Lignocellulosic plant cell wall is very complex, heterogeneous, recalcitrant and resistant to

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degradation. Conversion of complex polysaccharide substances to simple monomeric form is the major challenge in cellulose to ethanol technology. Different physical, chemical,

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biological or integrated strategies are currently in practice. Enzymatic process is a green process with high specificity, eco-friendly and low energy requirement. Cellulase and

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xylanases converts cellulose and hemicelluloses into sugars which can be fermented by yeast or other microorganisms. Glucose tolerant enzymes to be used for improving the hydrolysis yield since they are resistant to feed back inhibition to a certain extent (Sweeney and Xu, 2012). 3.1.

Enzymes for lignocellulosic biomass hydrolysis

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ACCEPTED MANUSCRIPT Due to the complex nature of lignocellulosic biomass, a single enzyme cannot do the complete biomass hydrolysis. There is a need for a sequential action of a series of enzymes. The major enzymes involved in the hydrolysis of lignocellulosic biomass are described below. 3.1.1. Cellulases Cellulases are enzymes which hydrolyse cellulose. Based on the sequence homology and

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hydrophobic cluster analysis, cellulases are classified into glycosyl hydrolase families

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(Carbohydrate Active Enzymes). Complete degradation of cellulose to glucose takes place by

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the synergistic action of three enzymes- endoglucanases, cellobiohydrolases and βglucosidases.

Endoglucanases (EG) or endocellulases (EC 3.2.1.4), which hydrolyse internal

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3.1.1.1.

β-1, 4-glucosidic linkages randomly at amorphous sites in the cellulose chain. Cellobiohydrolases or exocellulases (CBH, also known as exoglucanases) (EC

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3.1.1.2.

3.2.1.91), which cleaves the long chain oligosaccharides produced by the action of

β - Glucosidases or cellobiases (BG, also known as β–glucoside

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3.1.1.3.

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endoglucanases to short chain oligosaccharides.

glucohydrolases) (EC 3.2.1.21), which hydrolyse the glycosidic bonds of β-D-

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glucosides and oligosaccharides and produce glucose. Fungi are the potential source for cellulolytic enzymes. Trichoderma reesei produces two

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CBHs, five EGs and two BGs. Most of these enzymes act synergistically. Hydrolysis of cellulose occurs in two stages, via, primary hydrolysis and secondary hydrolysis. Enzymes responsible for primary hydrolysis are endoglucanases and exoglucanases. These enzymes act on the solid substrate and release sugars with a degree of polymerization (DP) up to 6 in the liquid phase. Hence the rate limiting step for cellulose hydrolysis process is depolymerisation. Second hydrolysis takes place by the action of β-glucosidase which converts cellobiose to glucose.

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ACCEPTED MANUSCRIPT Endoglucanases and cellobiohydrolases have two domains- a cellulose binding domain (CBD) and a catalytic domain (CD). CBD ensure perfect orientation of the substrate with the catalytic module of enzyme. CBD has been divided into several families based on the amino acid sequence similarities. With the help of a glycosylated flexible linker CBD is connected to catalytic domain and it helps in the degradation of crystalline cellulose. For

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cellobiohydrolases CBDs can move laterally along the cellulose chain whereas the catalytic

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domain cleaves off the cellobiose units. Till date not many studies were carried out to reveal

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the mechanism of how the aromatic residues interact with the crystalline cellulose as well as the mechanism of desorption and reattachment to the substrate. Degradation of lignocellulose

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does not follow classical Michaelis-Menton kinetics (Binod et al., 2011). The heterogeneous nature of lignocellulosic biomass makes it difficult to understand the mechanism of

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hydrolysis. 3.1.2. Xylanases

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Xylan is another component of lignocellulosic biomass. It is the main component in hemicelluloses. It is a polysaccharide made up of xylose, a pentose sugar. Xylanases are the enzymes which help in the hydrolysis of xylan. Removal of hemicellulosic components from

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the lignocellulosic biomass using xylanases is an eco-friendly strategy for increasing the

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enzymatic saccharification of cellulose. Since xylan is not a tightly packed crystalline structure like cellulose it is more susceptible for enzymatic saccharification than cellulose. Complete hydrolysis of xylan requires the action of xylanolytic enzymes which contains either a single domain – either catalytic or non-catalytic domains. Most of the commercially available xylanases are produced from Trichoderma reesei, Humicola insolens or Bacillus and their optimum temperature ranges from 40-60C. Xylanase complex consists of a series of enzymes which acts synergistically to produce sugars from xylan.

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ACCEPTED MANUSCRIPT Due to heterogeneous nature of xylan, the complete breakdown takes place by the action of several hydrolytic enzymes which includes endo-1,4-β-xylanase, β-xylosidase, αarabinofuranosidases and esterases. All these enzymes act synergistically to convert xylan to constituent sugars. Endo-1, 4-β-xylanase (1, 4-β-D-xylanxylanohydrolases, EC 3.2.1.8) is the

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3.1.2.1.

most important xylan degrading enzyme. It cleaves the glycosidic bonds in the

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xylan backbone releasing xylo-oligosaccharides, β- D-xylopyranosyl oligomers.

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Mono, di as well as tri saccharides of β-D-xylanopyranosyl are produced at a later stage of hydrolysis. Based on the end products it is subdivided into non-

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debranching and debranching enzymes. Non-debranching enzymes do not

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hydrolyse 1, 3-α- L-arabinofuranosyl part of arabinoxylan and hence won’t liberate arabinose. Debranching enzymes hydrolyse the 1, 3-α-L-arabinofuranosyl parts of acetoxylan and liberate arabinose.

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β -xylosidase (1,4-β-D-xylanxylohydrolase, EC 3.2.1.37) acts upon the small

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3.1.2.2.

oligosaccharides and cellobiose, generating β-D-xylopyranosyl residues from the non-reducing terminus.

β-D-xylosidases

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variety of

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microorganisms including bacteria and fungi. These enzymes hydrolyse

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xylooligosaccharides and xylobiose to xylose. The best substrate is xylobiose and the affinity of oligosaccharides is inversely proportional to the degree of polymerization. 3.1.2.3.

α -arabinofuranosidase (EC 3.2.1.55) removes the arabinose and 4-O-methyl

glucuronic acid substituent respectively from the xylan backbone. Arabinases hydrolyse arabinose, arabinoxylan and arabinogalactan. These enzymes act on the non-reducing α-L-arabinofuranosyl groups of arabinose. Arabinases are classified into two groups- Exo-acting α-L-arabinofuranidases (E.C.3.2.1.55) and Endo- 1, 5, 10

ACCEPTED MANUSCRIPT α-L-arabinofuranosidases (E.C.3.2.1.99). Most of the reported arabinose is Exoacting α-L-arabinofuranosidases. 3.1.2.4.

α- Glucuronidase (E.C.3.2.1.131) - These enzymes hydrolyse α-1, 2 -

glycosidic linkages between xylose or D-glucuronic acid or its 4-O-methylether. Several bacteria and fungi are reported to produce α-glucuronidase. Esterases – These are enzymes which act on the ester linkages between xylose

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3.1.2.5.

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units of xylan and acetic acid (Acetoxylan esterase E.C. 3.1.1.72). This enzyme

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removes O-acetyl groups from β-D-pyranosyl residues of acetyl xylan. Other esterases include Ferulic acid esterase (E.C.3.1.1.73) and p-coumaric acid esterases.

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Ferulic acid esterases cleave between arabinose and ferulic acid side groups. pcoumaric acid esterases cleaves between arabinose and p-coumaric acid.

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The mode of action of xylanolytic enzymes is depicted in Fig. 2. 3.1.3. Peroxidases

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Lignin degrading enzymes are oxido-reductive enzymes which includes lignin peroxidase

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enzyme (LiP, E.C. 1.11.1.7) and manganese –dependent lignin peroxidase (MnP, E.C. 1.11.1.7). LiP acts on both phenolic as well as nonphenolic aromatics residues and produce

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cation radicals (Schoemaker and Piontek, 1996). MnP catalyse the oxidation of Mn2+ to Mn3+ which in turn oxidises phenolic substrates. The mode of action of peroxidases is given below.

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Lip + H2O2 → H2O + LiPI

LiPI + valc → LiPI+ 2H+

valc+ + LiPII

→ valc+ + H2O + LiP

2Mn (II) +2H+ +H2O2 → 2Mn (III) +2H2O 3.1.4. Laccases Laccases (E.C.1.10.3.2) are copper containing oxidising enzymes. It is widely distributed in plants, insects and microorganisms including bacteria and fungi. These enzymes play an

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ACCEPTED MANUSCRIPT important role in lignin degradation and are abundantly present in white rot fungi. It can act on a broad spectrum of substrates like diphenols, polyphenols, aliphatic and aromatic amines. Mediators like 3-hydroxyanthranilic acid (3-HAA), 2, 2 P-azino-bis-(3-ethylthiazoline-6sulfonate (ABTS) were reported to increase the degradation of lignocellulose (Eggert et al., 1996). Laccase holoenzyme consists of dimer or tetramer glycoprotein containing four copper

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atoms per monomer bound to the redox sites – type 1, type 2 and type 3 Cu pair (Binod et al.,

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3.1.5. Accessory or helper proteins in hydrolysis

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2011).

During enzymatic saccharification of lignocellulosic biomass some proteins seems to increase

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the activity of cellulase without causing significant hydrolysis of cellulose. These proteins are called accessory or helper proteins. These proteins help in inducing structural modifications in

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cellulose. Swollenin is a kind of helper protein isolated from Trichoderm reesei. This helps in loosening of cellulose network (Saloheimo et al., 2002). T. reesei displays several swollenin

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like activities which differs in the mode of action and helps in the efficient hydrolysis of plant

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polysaccharides (Binod et al., 2011).

3.1.6. Microbial glycosyl hydrolases

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Glycoside hydrolases (GH) are enzymes which help in the hydrolysis of biomass and these

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enzymes are reported to be produced by a wide variety of microorganisms including archaea, bacteria, fungi, protists, plants, and animals. These enzymes form cellulosome thus makes them promising industrial biocatalysts for biomass hydrolysis. More than hundreds of GH families are there (Henrissat and Davies, 1997; Henrissat and Bairoch, 1993; Henrissat, 1991). These enzymes hydrolyse the carbon–oxygen–carbon bonds that link the sugar residues in cellulose and hemicelluloses polymers of lignocellulosic biomass (Lundell et al., 2010; Sanchez, 2009). Glycoside hydrolases play a critical role in the degradation of main chains of cellulose and hemicelluloses. Development of glycoside hydrolases with improved 12

ACCEPTED MANUSCRIPT properties as well as better cocktails for biomass hydrolysis is two major strategies for reducing the cost of bioconversion (Murphy, 2011). 3.1.7. Glycosyl hydrolase of fungal origin Several fungi are reported to produce extracellular cell wall degrading enzymes. Murphy et al

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[14] catalogued 453 glycoside hydrolases from 131 different fungal species. This includes 46 different GH activities and almost 44 of the 115 CAZy GH families. The study revealed that

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nearly 27% of the GH comprises cellulases and these represents family GH5. GH6 and GH7

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are the two families of cellobiohydrolase. GH1 and GH3 are the two families of βglucosidase. GH families 10 and 11 constitute xylanases. GH43 and GH93 constitute 3-endo-

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arabinases and 2-exoarabinases. 17 α-arabinofuranosidases were reported and act on arbinans

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and arabinosyl side chains found attached to other polysaccharides. β-mannanases belongs to family GH5 and GH26. While the glycoside hydrolases active on the main chains of pectins

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are from GH 28 showed exo- polygalacturonase activity.

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Glycosyl hydrolase from Trichoderma reesei is one of the most potent biomass hydrolysing enzymes commercially available today. Glycosyl hydrolase of T. reesei is composed of five

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β-1,4-endoglucanases (EG I–EG V), two β-1,4-exoglucanases (cellobiohydrolase [CBH] I and CBH II), two xylanases (XYN I and XYN II), a β-D-glucosidase, an α-L-

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arbinofluranosidase, an acetyl xylan esterase, a β-mannanase, and an α-glucuronidase (Vinzant et al., 2001). Till date the most potent biomass hydrolysing enzyme is produced by T. reesei.

3.1.8. Glycosyl hydrolase of bacterial origin There are several technical challenges in the construction of enzyme expression system for fungal species when compared to bacterial enzymes. Though many cellulases are commercially available, several studies are going on for the isolation of new cellulases with

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ACCEPTED MANUSCRIPT improved properties like higher activity and stable at extreme conditions of temperature, pH etc which in turn improves the overall process economics. Several bacterial genera such as Cellulomonas, Clostridium, Bacillus, Thermomonospora, Ruminococcus, Bacteriodes, Erwinia, Acetovibrio, Microbispora and Streptomyces are known to produce cellulase and other biomass hydrolysing enzymes (Maki et al., 2009; Wilson, 2011). Can we avoid pretreatment?

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3.2.

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The cell wall model of lignocellulosic biomass shows cellulose microfibrils enveloped by a

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matrix of hemicellulose, pectin and lignin. The cellulose microfibrils are made of polymers of -1, 4-linked glucose is packed by hydrogen bonding. Hydrogen bonds are present between

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unbranched hemicelluloses and the surface of cellulose microfibrils, while ester linkages are

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present between branched hemicellulose and phenolic acids in lignin (Chundawat et al., 2011; Sjostrom, 1993). These cellulose, hemicelluloses, pectin and lignin, result in the formation of

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macrofibrils. Due to this structural integrity, the cellulose is not exposed to the cell wall

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surface and hence it is resistant to microbial as well as enzymatic digestion. Complete degradation of a lignocellulosic biomass to its individual monomeric forms, by the sequential action of a repertoire of enzymes with high specific activity. Chemical digestion with strong

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acids or alkali is an easy method, but this involves several environmental as well as

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ecological issues and more over there is a chance for the formation of several other inhibitory components by the degradation of sugars. Due to complex nature of lignocellulosic biomass, a complex of enzyme system is needed for its complete degradation and many microorganisms are devoid of enzyme systems necessary for efficient degradation of lignocellulosic material completely. Hence a pretreatment process is to be carried out for the removal of hemicelluloses and lignin so that cellulose can be effectively hydrolysed to monomeric sugars.

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ACCEPTED MANUSCRIPT Several pretreatment strategies are currently available. This includes physical, chemical, biological and combined processes for effective removal of hemicelluloses and lignin. Most of these techniques increase the accessibility of enzymes to cellulosic fibres (Mosier et al., 2005). Acid pretreatment is carried out with dilute mineral acids like HCl, H2SO4, HNO3 etc. In this process the hemicelluloses component is removed. Alkali pretreatment is normally

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carried out with NaOH and KOH. In this process, delignification and uronic acid

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substitutions on hemicelluloses which lowers the enzyme accessibility to hemicelluloses and

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cellulose.

Several operation conditions are needed to be maintained during hydrothermal treatments.

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Based on the pretreatment strategies adopted, the treatment time varies from seconds to

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hours. Steam explosion removes hemicelluloses which in turn improves the saccharification rate. It is carried out at high pressure and high temperature (160- 260°C). SO2 assisted steam explosions helps in the recovery of both hemicelluloses and cellulose fractions (Chandra et

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al., 2015).

Ozonolysis pretreatment of lignocellulosic biomass seems attractive due to its efficiency and mild operating conditions (Travaini et al., 2016). It specifically reacts with lignin than

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carbohydrates and helps in effective delignification. Microwave pretreatment is also a green

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and efficient way to pretreat biomass. Microwave assisted pretreatment using acid and alkali were reported for better removal of hemicelluloses and lignin. Biological pretreatment is another strategy for improvement of enzymatic saccharification rate. Several brown as well as white rot fungi are known for effective delignification. Whiterot fungi such as Trametes versicolor, Phanerochaete chrysosporium, Pleurotus ostreatus and Ceriporiopsis subvermispora are reported for effective degradation of lignin from lignocellulosic biomass. The main advantages of biological pretreatment are no inhibitor

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ACCEPTED MANUSCRIPT generation and less energy requirement. The main drawback of this strategy is time consuming and sugar consumption by the microbes (Sindhu et al., 2016). Torrefaction is a relatively mild thermo-chemical process which is carried out at temperature, 200-300 °C in an inert gas atmosphere to produce homogeneous solid fuels with higher hydrophobicity and lower oxygen content relative to the feed biomass (Park et al., 2013).

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Selection of an effective pretreatment strategy is a critical factor which determines effective

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hemicelluloses and lignin removal based on the composition of lignocellulosic biomass. The

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whole process economics is to be considered while selecting a pretreatment method. Pretreatment is one of the energy intensive operations in the lignocellulose to ethanol

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technology. Hence pretreatment is to be carried out in such a way to minimise inhibitor generation. Cost effective corrosion resistant bioreactors should be developed which can

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withstand reasonable operating pressures. It is beneficial if the pretreatment strategies without inhibitor generation as well as neutralization were selected will minimize or eliminate

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neutralization step as well as detoxification which could substantially reduce the overall

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process economics. By products of lignin should be recovered and to be used for the production of value added products.

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During pretreatment the amorphous components like hemicelluloses and lignin remove and it will increase the crystallinity index and develop numerous pores which in turn have a positive

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impact on enzymatic hydrolysis. Hence a pretreatment is necessary for lignocellulosic biomass for getting maximum hexose sugars after removing the lignin and hemicelluloses. But the kind of pretreatment is depends on the composition of biomass and the end use of the enzymatic hydrolysate. 3.3.

Challenges in enzyme development

Development of a cost-effective cocktail is one of the major challenges in biomass hydrolysis. Several industries have made significant progress in the development of cost 16

ACCEPTED MANUSCRIPT effective tailor - made enzymes with higher specific activities. However, techno-economic analyses revealed that most of the currently available strategies are not commercially viable. The different approaches for enzyme development include developing novel multifunctional enzymes which can hydrolyse a wide variety of polysaccharide linkages, identifying novel enzymes with high titres, use of genetic as well as metabolic engineering approaches to

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improve the enzyme property and stability, directed evolution, expression of enzymes in

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plants etc. Another approach for reducing the cost of products in lignocellulosic biorefinery is

3.4. Factors affecting enzymatic hydrolysis

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the use of in-house enzymes.

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Heterogeneous nature of the lignocellulosic biomass and due to several other reasons related to the enzymes, the efficiency of hydrolysis of the biomass varies. The lignin is regarded as

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an inhibitory substance of enzymatic hydrolysis as lignin irreversibly binds with cellulose. Another factor affecting enzymatic hydrolysis is the effectiveness of pretreatment. Cellulose

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crystallinity plays a significant role in enzymatic saccharification rate. In a novel process

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engineering approach, the biomass was incubated for 24 hours and the sugars were removed and fermented separately, while the un-hydrolysed residues were hydrolysed along with fresh

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2012).

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pretreated substrate. This strategy reduces the biomass to sugar processing time (Jin et al.,

Another main challenge in enzymatic hydrolysis is the adsorption of cellulase by lignin. Hence the presence of lignin plays an important role in enzymatic hydrolysis. The adsorption of enzymes takes place by irreversible binding of cellulase to lignin by hydrophobic interaction which in turn leads to loss of enzyme activity. The factors affecting enzymatic hydrolysis of lignocellulosic biomass is classified into two groups- enzyme related factors and substrate related factors. 3.4.1. Enzyme related factors 17

ACCEPTED MANUSCRIPT Enzyme related factors affecting enzymatic hydrolysis of lignocellulosic feed-stocks include factors like concentration of enzyme, enzyme adsorption onto lignin, synergism, end-product inhibition, mechanical deactivation as well as thermal inactivation. The main factor affecting hydrolysis is the synergistic action of enzymes. 3.4.1.1. Temperature

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Incubation temperature is one of the significant parameter affecting enzymatic hydrolysis of

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lignocellulosic biomass. Temperature plays a critical role in cellulase adsorption. Normally

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cellulases works better at temperature below 60 °C and during this period there occurs enhanced adsorption and saccharification of cellulosic substrate which is not happening at

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temperature above 60°C. The decrease in adsorption beyond 60°C may be due to denaturation of enzyme. The optimum temperature of most of the fungal cellulases and β- glucosidase were

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reported as 50 °C and pH in the range of 4.5 – 5.0 (Taherzadeh and Karimi, 2007). Increase in temperature to 60°C leads to loss of 60 % activity while at 80 °C it leads to loss of almost

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complete activity (Gautam et al., 2010). Several studies revealed that enzyme activity depends

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on the hydrolysis duration as well as on the source of the enzymes (Tengborg et al., 2001). 3.4.1.2. Surfactants

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Surfactants have a positive role in enzymatic hydrolysis. It is reported that the hydrolysis time as well as the enzyme dosage is reduced by the addition of surfactants (Helle et al., 1993). The

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positive effect of surfactant on hydrolysis is either by surface modification or disruption of the lignocellulosic biomass or by preventing non-productive adsorption of enzyme or by acting as enzyme stabilizer (Kim et al., 1982). Surfactant has a positive effect especially with biomass containing high lignin content. Addition of surfactant seems to double the yield in such case (Li et al., 2016). This is due to the hydrophobic interaction between lignin and surfactants. The surfactants enhance the availability of reaction areas, through surface disruption which in turn increase the hydrolysis

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ACCEPTED MANUSCRIPT rate (Kaar and Holtzapple, 1998). Studies revealed that non-ionic surfactants are more effective which includes Tween-80, Tween-20 and polyethylene glycol (PEG). This is brought about by the protrusion of the surfactants into the aqueous solution thereby preventing the nonproductive adsorption of cellulase. Bovine serum albumin (BSA) also has a similar effect like non-ionic surfactants.

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3.4.1.3. Inhibitors in enzymatic hydrolysis

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Pretreatment is to be carried out in an economically viable way to minimise formation of

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fermentation inhibitors which are produced by the degradation of carbohydrates. The common inhibitors formed during pretreatment includes organic acids like acetic acid, formic acid, and

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levulinic acid, sugar degradation products like furfural, 5-hydroxymethyl furfural (5-HMF), uronic acid like galacturonic acid, glucuronic acids and 4-o-methyl glucuronic acid and lignin

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degradation products like vanillin, syringaldehyde and 4-hydroxybenzaldehyde (Jonsson and Martin, 2016). Ethanol is also found inhibitory to cellulase (Chen and Jin, 2006).

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3.4.2. Substrate - related factors

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Structural features of cellulase play an important role affecting the rate of enzymatic hydrolysis. Other factors affecting enzymatic saccharification includes crystallinity of

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cellulose, degree of polymerization, available surface area, particle size as well as associated components like lignin and hemicelluloses (Fan et al., 1981).

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Most of the hydrolysis experiments, it is observed that there is a rapid initial rate of hydrolysis followed by slower and incomplete hydrolysis. This is due to rapid hydrolysis of amorphous cellulose during the initial stage and the consequent increase of crystallinity as the hydrolysis proceeds (Mansfield et al., 1991). Several reports indicate that the crystallinity of substrate serves as a limiting factor for hydrolysis (Fan et al., 1981; Fan et al., 1980). Contrary results were also observed that the degree of crystallinity of substrate has no effect on hydrolysis (Puri, 1984).

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ACCEPTED MANUSCRIPT Accessibility of substrate is another factor influencing enzymatic hydrolysis of lignocellulosic biomass. It is measured by Bennet-Emmit-Teller (BCT) method (Masamune and Smith, 1964). The main drawback of this method is that it will overestimate the specific surface area (SSA) (Grethlein et al., 1984). 3.4.3. Biomass loading

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Biomass loading is one of the critical factors affecting enzymatic hydrolysis. Most of the

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recent studies have focussed on carrying out enzymatic hydrolysis at high biomass loading.

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Conversion of lignocellulosic biomass will become economically viable only if enzymatic hydrolysis is carried out with high biomass loading. The process to be fine-tuned in such a way

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that there is very little or no free water in the slurry so that we will get a concentrated sugar solution with less effluent generation as well as less energy and costs associated with

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distillation. This in turn improves the overall process economics. Though there are several advantages of high biomass loading for enzymatic hydrolysis, few

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challenges are also there. One of the main challenges is the lack of available free water in the

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reactor at high biomass loading. Water is essential in hydrolysis for mass transfer and lubricity. Most of the studies revealed that biomass loading above 20% (w/w), the biomass gets fully

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absorbed leaving very less free water which in turn increases the viscosity and will affect mixing of biomass. The substrate inhibition depends on the enzyme to substrate ratio (Penner

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and Liaw, 1994).

Another challenge of enzymatic hydrolysis with high biomass loading is the end product inhibition of cellulolytic enzyme system. Most of the β- glucosidases are susceptible to end product inhibition by glucose which leads to accumulation of cellobiose and affect the function of cellobiohydrolases. This can be overcome by the supplementation of glucose tolerant βglucosidases. Several research and developmental activities are going on in this direction for the development of tailor made enzymes for improved hydrolysis of lignocellulosic biomass

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ACCEPTED MANUSCRIPT based on the composition. Available free water is essential for the enzyme function and transport mechanism throughout hydrolysis as well as for the transfer of intermediates and end products (Felby et al., 2008). Adopting fed-batch strategies can increase the final biomass loading with minimal inhibition. Several studies are going on in this direction and the studies revealed that fed batch addition of

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biomass as well as fresh enzyme will increase the glucose yield, replacing the enzyme which is

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non-productively bound to the lignin.

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3.5. Strategies to reduce the enzyme cost

The major concern today we are facing on the enzymes for biomass hydrolysis is the high

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production cost. To make the biofuel economical, the cost of enzymes should be reduced to 10 to 100 fold. Several research and developmental activities are going on for the production

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of cost –effective enzymes for biomass hydrolysis. This can be achieved by manipulation of organism or enzymes through modern techniques such as genetic engineering, metabolic

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engineering, strain improvement and other related approaches. Another strategy is value-

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addition of by-products of enzyme fermentation. Trichoderma reesei is currently considered as the most efficient cellulase producer. The RUT

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C30 strain has emerged as a result of extensive strain improvement methods. Further

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improvement of this strain could focus on tailor made enzymes for specific biomass or by genetic elimination of the secreted enzymes (Banerjee et al., 2010). Another strategy is protein engineering of the enzyme. The amino acid residues are modified so that the enzyme shows improved properties such as high specific activity and improved stability. Directed evolution is another approach which is gaining more attention in recent years. It is proved as an effective strategy for improving or altering the activity of biomolecules based on the application of interest. This method combines random mutagenesis of the target gene for a particular enzyme with screening and selection of the desired properties. Several strategies 21

ACCEPTED MANUSCRIPT are currently available for creating DNA libraries which includes error-prone PCR, combinatorial oligonucleotide mutagenesis, DNA shuffling, exon shuffling, random-priming recombination etc. (Marek and Hari, 2004). Preparations of enzymes from a single organism may not be highly efficient for the hydrolysis of different feed stocks. For example, in Trichoderma reesei the β-glucosidase

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content is low though it produces a high titre of endoglucanases and exoglucanases. This

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results in inefficient biomass hydrolysis. Hence it is necessary to find another organism, for

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e.g. Aspergillus, which produces sufficient quantities of -glucosidase. It has seen that endoglucanases and cellobiohydrolases work synergistically in such a way that

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endoglucanases produce new chain ends by leaving the cellulose chains, thus creating new starting points for cellobiohydrolases. Two cellobiohydrolases (CBHI and CBHII) have also

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reported to have synergy due to their different specificity for reducing and non-reducing ends (Fagerstam and Pettersson, 1980; Igarashi et al., 2011). -Glucosidase reduces inhibition by

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cellobiose and has a synergistic effect on the hydrolysis with cellobiohydrolases or endoglucanases (Gruno et al., 2004; Lamed et al., 1991).

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Accessory enzymes play an important role in enzymatic hydrolysis. These enzymes improve the performance of enzymatic hydrolysis though they are not directly involved in the

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hydrolysis of cellulose. Addition of other auxiliary enzymes like xylanases, peroxidases etc. will help for an efficient hydrolysis of biomass. Hence, most of the multinational companies producing enzymes are now focussed on the production of cellulase cocktails by enzymes assembly (multienzyme mixtures) or to construct engineered microorganisms to express the desired mixtures (Mathew et al., 2008). Studies revealed that xylanase, feruloyl esterase and acetylxylanesterases addition has increased the release of glucose in addition to xylose (Hu et al., 2011; Kumar and Wyman, 2009; Murashima et al., 2003; Selig et al., 2008; Zhang et al., 2011). The accelerating effect of xylanases and mannanases has been reported by several 22

ACCEPTED MANUSCRIPT researchers (Varuni et al., 2011; Arantes and Saddler, 2010) and this may be due to removal of hemicelluloses as well as increase in porosity due to distortion of the biomass (Arantes and Saddler, 2010; Jong et al., 1997; Suurnakki et al., 1997). Enzyme mixtures often derive from the co-fermentation of several microorganisms (Ahamed and Vermitte, 2008; Berlin et al., 2007). A synergistic model for enzymatic degradation of lignocellulosic biomass is shown in

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Fig. 3.

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During the past few decades enzyme cost has been significantly reduced, still it is one of the

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important factors that affect the total economy in a lignocellulosic biomass process. Studies revealed that enzyme costs contribute 16% of the overall ethanol production costs for corn

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stover (Humbird et al., 2011). Decreased enzyme cost can be achieved by adopting the strategies described earlier. Use of cheaper as well as readily available raw materials for

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enzyme production and in-house production of enzymes are also possible alternative strategies to reduce the enzyme costs (Humbird et al., 2011). Efficient pretreatment method

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also contributes better enzymatic hydrolysis which in turn reduce the overall process

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economics. Other strategies like enzyme recycling and reuse are also an important approach for making the process economically viable. The main difficulties with enzyme recycling are

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enzyme inactivation after one or two cycles and binding of cellulases to lignin (Rahikainen et

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al., 2011; Tu et al., 2009). The lignin can be removed by efficient delignifying pretreatment methods such as alkali pretreatment or surfactant assisted pretreatment (Tu et al., 2007). 4.

Conclusions and future perspectives

The hydrolysis of lignocellulosic biomass with the help of enzymes is a promising and environmentally friendly approach for any lignocellulosic biomass based process. Even though several break-through happened in the commercial production as well as reducing the price of enzymes, still exists several challenges which is yet to be addressed. Lot of opportunities for research exists in this field starting from searching for new sources of 23

ACCEPTED MANUSCRIPT enzymes, process improvement, cost reduction, cocktail preparation suitable for biomass and final formulation. Use of enzymes for complete saccharification of biomass without any pretreatment will become a reality in the near future. Acknowledgements

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Parameswaran Binod and Raveendran Sindhu acknowledge Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland for visiting fellowship. Raveendran Sindhu acknowledges

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Department of Science and Technology, Government of India for sanctioning a project under

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DST WOS-B scheme.

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References

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Arantes, V., Saddler, J., 2010. Access to cellulose limits the efficiency of enzymatic

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Fig.1. Structure of the cell wall of lignocellulosic plant biomass

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Captions to figures

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Fig.2. Schematic representation of the mechanism of action of xylanases

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Fig.3. Synergistic model for enzymatic degradation of lignocellulosic biomass

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Problems

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Technology

Advantage

Second generation fuels

Sugar, starch, vegetable Plant waste biomass oils Economical Economical Currently not economical compared to petroleum derived fuels and first generation fuels CNG, LPG, Diesel, FAME (biodiesel), Bioethanol, biobutanol, gasoline, Kerosene, jet bioethanol, biobutanol bio-oil fuel Depletion of petroleum Limited availability Technical barriers for reserves resources are efficient production limited Food vs fuel problem Production costs are High GHG emission and Contribute to higher uncertain and vary with environmental pollution food prices due to the feedstock available competition with food Economics and crops There is no clear ecological problems candidate for "best Provide only limited technology pathway" Non-renewable form of GHG reduction benefits between the competing energy biochemical and thermoDo not meet their chemical routes claimed environmental benefits because the The development and biomass feedstock may monitoring of several not always be produced large-scale demonstration sustainably projects is essential to provide accurate comparative data Petroleum or oil has Environmentally Environmentally friendly high density friendly Economic and social The average 1kg of Economic and social security burnt oil can generate security 10,000 kcal No competition with food Extracting oil is easy

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Crude petroleum

Feed stock

Products

derived First generation fuels

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Petroleum fuels

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Table 1: Comparison of petroleum derived, first generation and second generation fuels

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Low cost of extraction

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Strategies for cost reduction of biomass hydrolysing enzymes.

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High biomass loading and low incubation time makes process economically viable.

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Figure 1

Figure 2

Figure 3