1.1 Introduction - Shodhganga : a reservoir of Indian...

Chapter 1 General Introduction …………

- 2 -

1.1 Introduction One of the greatest challenges for society in the 21st century is to meet the increasing

demand of fuel energy for transportation and industrial processes in a sustainable

way. Global energy consumption continues to increase while fossil-fuel reserves are

diminishing. To prevent a looming energy crisis, the development of renewable

energy sources is becoming a priority. Bioethanol is the most widely used biofuel for

transportation worldwide. Bioethanol from lignocellulosic biomass including

agricultural, forestry residues and woody crops appears promising, as the raw material

are ubiquitous, abundant; their non-competitiveness with food crops. Lignocellulosic

materials consist mainly of cellulose (35-50%), hemicellulose (20-35%) and lignin

(10-25%) (Sun and Cheng, 2002). The chemical properties of components of

lignocellulose make them a substrate of enormous biotechnological value. Thus,

extensive research has been carried out on ethanol production from lignocellulosic

materials because it is a cleaner fuel in addition to being a renewable alternative to

petroleum (Cadoche et al., 1989; Yang and Wyman, 2008; Binod et al., 2010).

Currently, it is a hotspot in the bioenergy research field.

1.2 Cellulose occurrence, structure and composition

Cellulose is the most abundant and common renewable organic biopolymer on earth,

representing about 1.5 x 1012 tons (in the tropics) of the total annual biomass

production through photosynthesis. It is considered to be an inexhaustible source of

raw material for different products. Cellulose is a crystalline biopolymer, where

cellulosic chains in the crystals are stiffened by inter- and intra- hydrogen bonds and

the adjacent sheets are held together by weak Van-der Waals forces. An important

feature of this crystalline array is the relative impermeability of large molecules such

as water. In this polymeric structure there are both crystalline and amorphous regions

in addition to several types of surface irregularities. This heterogeneity makes the

fibers capable of swelling when partially hydrated, resulting in the enlargement of

micropores and cavities that allow the penetration of large molecules including

enzymes.


- 3 -

The cellulose is embedded in a matrix of hemicellulose, pectin and lignin (Eriksson

and Bermek, 2009). Lignin and hemicellulose are found in the spaces between

cellulose micro fibrils in primary and secondary cell walls, as well as the middle

lamellae (Eriksson and Bermek, 2009). Fig 1.1 demonstrates a simplified model to

illustrate the cross linking of cellulose microfibril and hemicellulose in lignocellulosic

biomass.

Fig. 1.1 A simplified model to illustrate the cross linking of cellulose microfibril and hemicellulose in lignocellulosic biomass

At the molecular level, cellulose is a linear polymer of glucose composed of

anhydrous units coupled to each other by β-1-4 glycosidic bonds. The number of

glucose units in the cellulose molecules varies and degree of polymerization ranges

from 250 to well over 10,000 units depending on the biomass. Fig 1.2 shows the

schematic representation of a cellulosic chain.

Fig. 1.2 Schematic representation of a cellulosic chain


- 4 -

Hemicellulose is more varied in structure and composition than cellulose and includes

xylan, mannan, galactan and arabinan polymers (Beg et al., 2001). The most abundant

hemicellulose in nature is xylan, containing mainly β-D-xylopyranosyl residues

linked by β-1,4-glycosidic bonds (Koukiekolo et al., 2005). In plants, the xylan forms

an overlying layer through hydrogen bonding with the cellulose, while covalently

linked with lignin which forms an outside sheath to protect the plant. Xylan forms an

important part of the plant cell walls, forming 30–35% of total dry weight, although

the exact abundance of the xylan may differ between plants (Beg et al., 2001).

Lignins are highly branched polymeric molecules consisting of phenyl-propane based

monomeric units linked together by different bonds, including alkyl-aryl, alkyl-alkyl

and aryl-aryl ether bonds and is very resistant to degradation (Hendriks and Zeeman,

2009). Lignin composition is variable between hardwoods and softwoods, although

the specific three dimensional structure of lignin is unknown (Eriksson and Bermek,

2009). Older woody plants contain higher levels of lignin deposited in cell walls to

give rigidity and strength, making cell walls water proof, providing effective

protection against pathogens and resistance to microbial attack (Raven et al., 1999;

Saha, 2003). Table 1.1 shows the composition of lignocellulosic biomass of different

agricultural residues.

Table 1.1 Composition of lignocellulosic biomass of different agricultural residues (J.S. Van Dyk and B.J Pletschke, 2012) Biomass Cellulose (%) Hemicellulose (%) Lignin (%)

Bermuda grass 47.8 13.3 19.4

Corn cobs 35-39 38-42 4.5-6.6

45.0 35.0 15.0

Corn stover 39.0 19.1 15.1

Wheat straw

36.6 24.8 14.5

44.0 29.6 10.4

33.0 23.0 17.0

Rice straw

41.0 21.5 9.9

39.0 15.0 10.0

32.1 24.0 18.0

Bagasse 38.1 26.9 18.4

39.3 27.2 12.2

Switchgrass 31.0 22.0 18.0


- 5 -

1.2.1 Cellulase(s) system

Cellulases belonging to a large family of glycosyl hydrolases (GHs) and are classified

into 132 glycoside hydrolase families (http://afmb.cnrs-mrs.fr/CAZY/index.html).

Cellulases mostly have a small independently folded carbohydrate binding module

(CBM) which is connected to the catalytic domain by a flexible linker (Fig 1.3). The

CBMs are responsible for binding the substrate for enzyme activity (Bayer et al.,

1998). Based on the presence and absence of CBMs, the cellulase systems of

microbes can be generally regarded as complexed or non-complexed.

Fig. 1.3 Schematic representation of a CBM with linker

Utilization of insoluble cellulose requires a cellulolytic enzyme system consisting of

either secreted or cell associated cellulases belonging to different classes which are

categorized based on their mode of action and structural properties. The three major

type of cellulase activities recognized are: i) endoglucanase / 1-4-β-D-

gluconohydrolases /EG-(EC3.2.1.4); ii) exoglucanases/1-4-β-D-

glucanglucanohydrolases/cellobiohydrolases/CBH-(EC 3.2.1.91); and iii) β-

glucosidase/BG/BGL/β-glucosdisase glucohydrolases-(EC 3.2.1.21).

Endoglucanases (EGs) are also referred to as carboxymethylcellulases (CMCase),

named after the artificial substrate, carboxymethyl-cellulose (CMC), which is

generally used to measure the enzyme activity. Endoglucanases cut randomly at

internal amorphous sites in the cellulose chain generating oligosaccharides and new

chain ends. This has shown by the effect of the enzyme on CMC and amorphous


- 6 -

cellulose. Fungal EGs are generally monomers with no or low glycosylation and have

an open binding cleft. There are several reports on the existence of multiple EGs in

fungi. In Trichoderma reesei strain at least 5 EGs are present (EG1/Cel7B,

EGII/Cel5A, EGIII/Cel12A, EGIV/Cel61A and EGV/Cel45A). Among these EGI,

EGII, EGIV and EGV possess CBM domain while EGIII does not contain the CBM

(Sandgren et al., 2005). Cellobiohydrolases (CBHs) act on both the reducing and

non reducing ends of the cellulose chains liberating glucose, cellobiose or

cellooligosaccharides as major products, this in turn increases the synergy between

opposite acting enzymes. For example, Trichoderma reesei has two CBHs acting

from non reducing (CBHII/Cel6A) and reducing (CBHI/Cel7A) ends, which results in

a more efficient cellulolytic degrader. Moreover, both CBHI and CBHII of

Trichoderma reesei have CBM at the carboxy terminus or at the amino terminus of

the catalytic module. Similar to EGs, CBHs are monomers with no or low

glycosylation. Cellobiose, the end product of CBHs, act as a competitive inhibitor,

which can limit the ability of the enzymes to degrade all of cellulose molecules in a

system (Divne et al., 1998; Sandgren et al., 2005). β-glucosidases (BGLs) hydrolyze

soluble cellodextrins and cellobiose to glucose. BGLs have been placed mainly in

families 1 and 3 of GH based on their amino acid sequence (Henrissat et al., 1991).

Family 3 includes BGLs from fungi, bacteria and plants, whereas family 1 BGLs are

from bacterial, plant and mammalian origin with galactosidase activity in addition to

β-glucosidase activity. In Trichoderma reesei, two β-glucosidases (BGL1/Cel3A and

BGLII/Cel1A) were isolated from culture supernatant, but the enzymes were found to

be primarly bound to the cell wall (Kubicek et al., 1981). In brief, endoglucanase

enzymes randomly cleave β-1-4 glucosidic linkages within the backbone of cellulose.

Cellobiohydrolase enzymes cleave from either the reducing or non reducing end of

cellulose chains in a processive manner. Oligosaccharides released as a result of these

activities are converted to glucose by the action of cellodextrinases, whereas the

cellobiose released mainly by the action of cellobiohydrolases is converted to glucose

by β-glucosidases (Fig 1.4).


- 7 -

Fig. 1.4 Mechanism of cellulase action


- 8 -

1.2.2 Synergy among cellulases

Synergy between cellulases have been reviewed and identified between either

different cellobiohydrolases; endo and/or exo-glucanases; endo-glucanases and β-

glucosidases (Walker and Wilson; 1991; Nidetzky et al., 1994; Boisset et al., 2000;

Schwarz, 2001; Zhang and Lynd, 2004).

The degree of synergy or synergism is defined as the ratio of the rate or yield of

product released by enzymes when used together to the sum of the rate or yield of

these products when the enzymes are used separately in the same amount as they were

employed in the mixture (Kumar and Wyman, 2009). Synergy depends on the ratio of

the enzymes involved (Nidetzky et al., 1994), as well as the specific characteristics of

the enzymes and the characteristics of the substrate. The ratios of various cellulases

used in different studies to obtain maximum synergy displayed large variations.

Bothwell et al. (1993) used a ratio of endoglucanase to cellobiohydrolase of 3:1,

while other studies used higher ratios of cellobiohydrolase to endoglucanase. Berger

et al. (2007) used 17:1 cellobiohydrolase to endoglucanase and Jung et al. (2008)

used a ratio of 10:1 cellobiohydrolase to endoglucanase. Hoshino et al. (1997), on

the other hand, found optimal synergy with a combination of 1:1 cellobiohydrolase to

endoglucanase. Boisset et al. (2001) used 98.75% cellobiohydrolase to 1.25%

endoglucanase. It is not clear why such varied ratios have been used, but this may be

as a result of enzyme characteristics, assay conditions or variations in substrate

characteristics, particularly resulting from different pretreatment methods.

1.2.3 Thermostable cellulases

Thermostability is defined as the capacity of an enzyme to retain its active structural

confirmation at a selected temperature for a long period of time. Thermostable

enzymes in the hydrolysis of lignocellulosic materials have several potential

advantages: higher specific activity and higher stability which allow longer life-time,

improved recyclability and increased flexibility for the process configurations. The

two first characteristics would expectedly improve the overall performance of the

enzymatic hydrolysis even at the range of presently used temperatures. Thus, the use

of thermophilic enzymes would ultimately lead to improved performances i.e.

decreased enzyme dosage and reduced hydrolysis time and consequently decreased


- 9 -

hydrolysis costs. Thermostable enzymes would also expectedly allow hydrolysis at

higher consistency due to lower viscosity at elevated temperatures and thus allow more

flexibility in the process configurations.

Several mesophilic or moderately thermophilic fungal strains are known to produce

thermostable enzymes. These enzymes are stable and active at temperatures that can

be clearly higher than the optimum temperatures for the growth of the microorganism.

Filamentous fungi reported to produce cellulases that retain relatively high cellulose

degrading activity at elevated temperatures include particularly those from the species

Talaromyces emersonii, Thermoascus aurantiacus, Chaetomium thermophilum,

Myceliophthora thermophilla and Thielavia terrestris as reviewed by Vikari and

coworkers (Vikari et al., 2007). Thermophilic β-glucosidases have been characterized

from e.g. Aureobasidium pullulans, Clostridium thermphillum, Talaromyces

emersonii, Thermoascus aurantiacus and Thermomyces lanuginose. Although

thermostability is one of the most studied and engineered protein properties, no

generally applicable rules have been established so far. With the availability of

thermostable enzymes a number of new applications in the future are likely. Although,

believed to provide tremendous economical benefits, production of the enzymes to the

required level by the industries has remained a challenge.

1.2.4 Molecular expression and regulation of cellulases

Research on cellulase gene(s) expression is increasing exponentially with a potential to

use the tremendous resources of cellulosic waste for different applications. Owing to

the progress in heterologous expression techniques, recombinant enzyme production

systems are now promising platforms for efficient industrial cellulase production with

many areas where they may enhance the productivity of biomass to biofuel processing

(Alper et al., 2009). Bacterial expression systems for cellulase production have been

modified to efficiently screen heterogenous cellulase genes and organisms like

Zymomonas mobilis (Linger et al., 2010) and Bacillus subtilis (Liu et al., 2012) are

being investigated for roles in consolidated bioprocessing (CBP) (Pottkamper et al.,

2009). Recombinant production of cellulases in bacteria often leads to an increase in

enzyme yield, as compared to the original host, thought to be a result of changes to the

expression systems involving gene copy number, promoters, and inducer types (Afzal

et al., 2010). Yeast expression systems for cellulase production are becoming


- 10 -

increasingly popular even on an individual scale (Mattanovich et al., 2012). Several

yeast expression systems such as Saccharomyces cerevisiae, Pichia pastoris and

Kluyveromyces marxianus have been developed for use in the biofuel industry (Yanase

et al., 2010; Akcapinar et al., 2011). Although important progress has been made

towards industrial, heterologous cellulase production in yeasts, the yield and activity of

enzymes remains substantially lower than Trichoderma reesei that are in currently

industrial use (Elkins et al., 2010). Plant expression systems are potentially an ideal

system for cost effective cellulase production because of their ability to be

economically produced on an industrial scale (Shen et al., 2012; Jung et al., 2012).

Fungal expression systems are the mainstay of cellulase production in current

industrial scale, particularly Trichoderma reesei, Trichoderma longibrachiatum and

Aspergillus niger (Chandel et al., 2012). However, significant efforts have also been

made towards increasing productivity using synthetic biological tools. Although

specific expression strategies in various species of bacteria, yeasts, plants and fungi

show distinct potential, no ideal system has yet been established (Elkins et al., 2010;

Chandel et al., 2012). Thus, an amalgamation of genetically modified plant biomass

and the most promising enzymes, whether identified from natural sources or

synthetically enhanced, incorporated within surface cellulase displaying, ethanol

tolerant yeast or cellulosome displaying bacteria, may be required in combination for

the final crucial breakthrough in this ever expanding field.

Regulation of cellulase production is finely controlled by activation repression

mechanisms (Sukumaran et al., 2008) and genes are found to be coordinately regulated

(Ilmen et al., 1997). Trichoderma reesei cellulases are inducible enzymes and several

inducers were reported in Trichoderma cellulase system where sophorose is one of the

major inducer (Vaheri et al., 1979; Kubicek et al., 1998). Cellobiose, δ-cellobiose-1-5

lactone and other oxidized products of cellulose hydrolysis were also found to act as

inducers. Lactose, another known inducer of cellulase is utilized in commercial

production of enzyme due to its economic reasons. Though mechanism of lactose

induction is not fully understood, it is now known that lack of galactose mutarotase

activity is crucial for cellulase induction in fungus.

Analyses of promoters of cellobiohydrolase I and II has shown binding sites of at least

three transcription activators (ACE I, ACE II and HAP 2/3/5) and one carbon


- 11 -

catabolite repressor (CRE I). Molecular mechanism of gene induction in presence of

cellulose is still unclear. However, it is reported that expression of cellobiohydrolase

and at least two endoglucanases (EG1 and EG2) are believed to be controlled by ACE

II binding to their promoters (Aro et al., 2001; Wurleitner et al., 2003). HAP 2/3/4

binding to cellobiohydrolase I promoter is evidenced by the presence of its binding

sequence in the promoter region (Ilmen et al., 1996a). Glucose repression of cellulase

is supposed to be mediated through carbon catabolite repressor protein CRE I (Ilmen et

al., 1996a) and promoter regions of cbh 1, cbh 2, eg 1 and eg 2 genes have CRE I

binding sites indicating fine control of these genes by carbon catabolite repression

(Kubicek et al., 1998). Glucose repression of cellulase system overrides its induction

(Ilmen et al., 1997) and de-repression is believed to occur by an induction mechanism

mediated by trans-glycosylation of glucose (Sternberg et al., 1979; Fritscher et al.,

1990).

1.2.5 Production of cellulases

1.2.5.1 Microorganisms producing cellulases

Cellulolytic microbes are carbohydrate degraders that primarily utilize cellulose as

energy source for metabolic growth. However, bacteria belonging to Cellulomonas sp.

Cytophaga sp. and fungi utilize a variety of other carbohydrates in addition to cellulose.

Whereas, the anaerobic cellulolytic species have a restricted carbohydrate range,

limited to cellulose and or its hydrolytic products. Most commonly studied cellulolytic

organisms include: Fungal species-Trichoderma, Humicola, Penicillium and

Aspergillus; Bacteria-Bacillus, Psuedomonas, Cellulomonas; and Actinomycetes-

Streptomycetes, Actinomucor and Streptomyces. One of the most extensively studied

fungi is Trichoderma reesei, which converts native cellulose and its derivatives to

glucose by the action of cellulolytic enzymes namely cellulases. Besides Trichoderma

reesei, several fungi like Humicola, Penicillium and Aspergillus can metabolize

cellulose as an energy source, but only few strains are capable of secreting a complex

of cellulase enzymes, which could have potential application in the enzymatic

hydrolysis of cellulose.


- 12 -

Cellulase production is a major research area and with the rejuvenated interest created

due their applications in lignocellulose conversion, several investigations worldwide

are working on these aspects of cellulase (Sukumaran et al., 2005). Production of low

titres of cellulase has always been major concern and researchers are trying to improve

the production titres by adopting multifaceted approaches, which include the use of

better bioprocess technologies and use of cheaper or crude raw materials as substrate

for enzyme production (Sukumaran et al., 2005). Majority of the reports on microbial

production of cellulases utilizes submerged fermentation (SmF) technology. However,

in nature, the growth and cellulose utilization of cellulase producing aerobic

microorganisms probably resembles solid-state fermentation (SSF) than a liquid

culture (Holker et al., 2004; Zhu et al., 2009).

1.2.5.2 Solid-state fermentation

Solid-state fermentation is defined as the fermentation in absence or near absence of

free water (Pandey, 1994). SSF is rapidly gaining interest as a cost-effective

technology due to high titres of microbial cellulase production, especially from fungal

cultures (Jha et al., 1995; Cen and Xia. 1999). Filamentous fungi such as Trichoderma

reesei, Aspergillus niger, Penicillium sp. etc. have been employed for cellulase

production using SSF where a basal mineral salts medium is used for moistening the

substrate. Solid state cultures are strongly recommended for producing cellulases than

submerged cultures since the product concentration in SSF remains quite high thereby

reducing the downstream processing steps, in turn reducing the cost of operation

(Vintila et al., 2009). Chahal (1985) had reported a higher yield of cellulases from

Trichoderma reesei in SSF cultures compared to liquid cultures. Tengerdy (1996)

compared cellulase production in SmF and SSF systems and had indicated that there is

about a 10-fold reduction in the production cost when SSF is employed. Nigam and

Singh (1996) have reviewed the use of agricultural wastes as substrates for cellulolytic

enzyme production under SSF and strongly believe that with the appropriate

technology, improved bioreactor design, and operation controls, SSF may become a

competitive method for the production of cellulases. Cellulases produced in solid-state

culture shows remarkable stability towards temperature, pH and metal ions, etc.

Optimization of SSF conditions when attempted may still improve the overall

production economics and also make it an attractive technology for cellulase


- 13 -

production. It offers many advantages over submerged fermentation, including high

volumetric productivity, higher concentration of products, less effluent generation and

low catabolic repression which make it a promising technology in the near future

(Pandey et al., 1999; Singhania et al., 2009).

1.2.5.3 Submerged fermentation

Submerged fermentation is defined as fermentation in the presence of excess water.

Almost all the large-scale enzyme producing facilities are using this proven technology

due to better monitoring and ease of handling. Though bacteria and actinomycetes are

also reported for cellulase production, the titers are very low to make the technology

economically feasible. Most of the cellulases that are produced commercially under

SmF are from filamentous fungi-Trichoderma reesei or Aspergillus niger and can

perform at diverse pH range and temperature (Cherry et al., 2003). A large-scale

production of cellulases requires understanding and proper controlling of the growth

and enzyme production capabilities of the microbe. Cellulase production in cultures is

highly influenced by various parameters including the nature of the cellulosic

substrate, pH of the medium, nutrient availability, inducer supplementation,

fermentation temperature, etc. The media formulation for fermentation is of significant

concern since no general composition can give the optimum growth and cellulase

production which is dependent on the microbe. In Trichoderma reesei, a basal medium

after Mandels and Reese (1957) or Mandel and Weber (1969) has been most frequently

used with or without modifications.

Mostly, pure cellulose preparations like Solka-Floc and Avicel have been used in the

liquid cultures of cellulolytic microbes for production of the enzymes and when natural

cellulosic materials are used as the carbon source the enzyme yields are significantly

lower (Tangnu et al., 1981). Cellulases produced by SSF and SmF have been listed in

Table 1.2.


- 14 -

Table 1.2 Types of bioprocesses and substrates involved for cellulase production (Singhania et al., 2010)

Microorganism Substrate Method Magnitude Enzyme activity

Trichoderma reesei RUT C30 Wheat bran SSF Shake flask 3.8 U/gds FPU

Aspergillus niger A 20 Cellulose SmF Shake flask Cellobiase 27.5 U/ml

Neurospora crassa Wheat straw SmF Shake flask FPA 1.33 U/ml, CMCase 19.7 U/ml, BGL 0.58 U/ml

Streptomyces sp. T3-1 Carboxy-methyl cellulose

SmF 50-l fermentor CMCase 148 IU/ml, Avicellase45 IU/ml, BGL 137 IU/ml

Trichoderma reesei RUT C30 Cellulose (Avicel)

SmF Microbubble dispersion Bioreactor

FPA 1.8 U/ml

Trichoderma reesei RUT C30 Corrugated cardboard

SmF 30-l fermentor FPA 2.27 U/ml

Trichoderma reesei ZU-02 Cornstover residue

SmF 30-l fermentor Cellulase 5.48 IU/ml, FPA 0.25 U/ml

Trichoderma viridae Sugarcane bagasse

SmF Shake flask FPA 0.88 U/ml, CMCase 33.8 U/ml, BGL 0.33 U/ml

Aspergillus niger NRRL3 Wheat bran/Corn cob

SSF Flask Cellobiase 215 IU/g, cellulose

Mixed culture: Trichoderma reesei Aspergillus niger

Rice chaff/Wheat bran (9:1)

SSF Flask FPA 5.64 IU/g

Thermoascus auranticus Wheat straw SSF Perforated drum bioreactor

FPA 4.4 U/gds, CBH 2.8 U/gds, Endoglucanase 987 U/gds, BGL 48.8 U/gds

Trichoderma reesei ZU 02 Corncob residue

SSF Tray fermentor FPA 158 U/gds


- 15 -

The carbon sources in majority of the commercial cellulase fermentations are

cellulosic biomass ranging from pure cellulose to straw, spent hulls of cereals and

pulses, rice or wheat bran, bagasse, paper industry waste, dairy manure and various

other lignocellulosic residues (Szabo et al., 1996; Reczey et al., 1996; Romero et al.,

1999; Heck et al., 2002; Wen et al., 2005). Rice straw is reported to be an excellent

cheap carbon source for growth of various microorganisms to produce industrially

important microbial enzymes (Gao et al., 2008). Thus, for developing an

economically feasible technology, use of cheaper raw material as a substrate under

SSF for cellulase production could bring down the production costs (Sukumuran et al.,

2005).

1.3 Purification of cellulases and their characterization

Purification of cellulases to homogeneity is necessary for detailed biochemical and

molecular studies and also for the successful determination of primary amino-acid

sequence and three dimensional structure of the protein (Sa-Pereira et al., 2003).

Ideally the final purification process consists of sample preparation, extraction and

clarification followed by three major purification steps i.e. capture (isolate,

concentrate and stabilize), intermediate purification (remove bulk impurities),

polishing (achieve final high level purity). One of the most important steps in

purification of extracellular cellulase is clarification. This step is mainly used to

remove polyphenols, pigments, nucleic acids etc. Clarification can be done by

precipitation, centrifugation or filtration methods, respectively.

Large number of purification protocols for cellulases includes ammonium sulfate

precipitation as the preliminary step in purification process. As an alternative method

to precipitation, ultrafiltration could also be performed where 5-10 kDa molecular cut-

off membranes are generally used. Further, chromatographic methods such as ion

exchange and gel filtration chromatography are used as a final purification step to

obtain high purity cellulase proteins. Microbial cellulases are mainly purified by

chromatographic methods using two to five purification steps. Apart from ion

exchange and gel filtration chromatography, other techniques such as affinity

chromatography and high performance liquid chromatography (HPLC) etc. have also

been used.


- 16 -

Bara et al. (2003) purified exoglucanase by gel filtration and ion exchange

chromatography and obtained 35.7- fold purification. However, a 69-fold purified

protein was obtained using similar techniques by Noronha et al., 2000. For purifying

β-glucosidase Wei et al. (1996) employed ammonium sulfate precipitation, gel

filtration and ion-exchange chromatography step. Using gel filtration chromatography

a 13-fold and 105-fold purified β-glucosidase were obtained from Monascus

purpureus and Paecilomyces thermophila, respectively (Dariot et al., 2008).

Endoglucanases from Aspergillus was purified to homogeneity using ammonium

sulfate precipitation, acetone fractionation and various types of chromatography

methods (gel filtration, sephadex G-100, ion exchange: DEAE-sephadex A-50,

affinity: sepharose-4B). Endoglucanase from Aspergillus terreus M11 was purified

18-fold with a yield of 14% (Gao et al., 2008), 27-fold with a yield of 10.5% from

Aspergillus terreus DSM 826 (Elshafei et al., 2009), 40-fold with a yield of 1.32%

from Aspergillus terreus AN1(Nazir et al., 2009), 2.09-fold with a yield of 18.4%

from Aspergillus niger VTCC F021 (Thai et al., 2010), 12-fold from Aspergillus

awamori F 18 (Nguyen et al., 2010), 5.8-fold with a yield of 3.6% from Penicillium

pinophilum MS 20 (Dipali et al., 2012), respectively.

Many workers have purified and characterized cellulases from different bacteria viz.

Thermomomonospora sp. (George et al., 2001), Pseudomonas fluorescens (Bakare et

al., 2005), Melanocarpus sp. MTCC 3922 (Kaur et al., 2007), Pyrococcus horikoshi

(Kang et al., 2007), Cellulomonas sp. Y15 (Yin et al., 2010), Bacillus sp. (Singh et

al., 2004; Kim et al., 2005; Bischoff et al., 2006; Bajaj et al., 2009; Acharya and

Chaudhary 2011; Ashabil et al., 2011). Table 1.3 shows the summary of biochemical

properties of purified endoglucanases from different microorganisms.


- 17 -

Table 1.3 Summary of biochemical properties of purified endoglucanases from

different microorganisms (Saha, 2004; Thai et al., 2010; Dipali et al., 2012)

Microorganism No. of

steps

Yield (%)

Purification (fold)

Molecular mass (kDa)

Optimum

temp. (°C)

pH Specific activity (U/mg)

Fusarium

oxysporum 5 29.60 6.44 34 50 6.0 32.20

Streptomyces

lividans 2 10.40 6.47 46 50 5.5 539.00

Rhizopus oryzae

REC 1 6 3.10 103.10 41 55 5-6 257.80

Melanocarpus sp.

MTCC3922 EGI 4 13.40 4.51 40 50 6.0 24.61

Melanocarpus sp.

MTCC3922 EGII 4 8.90 9.00 50 70 5.0 35.39

Mucor

circinelloides 3 3.00 408.00 25 55 5.0 43.00

Aspergillus

terreus M11 3 14.00 18.00 23 60 2.0 67.00

Aspergillus

oryzae cmc-1 5 32.30 12.33 25 55 4.4 92.50

Aspergillus niger

VTCC-F021 2 18.40 2.09 31 55 5.0 14.12

Aspergillus

terreus DSM 826 - 10.50 27.00 - 50 4.8 4.35

Aspergillus

terreus AN1 - 1.30 40.00 - 60 4.0 200


- 18 -

1.4 Applications of cellulases

1.4.1 Biofuel industry

Cellulases were initially investigated several decades back for the bioconversion of

biomass to glucose and other fermentable sugars which gave way to research in the

industrial applications of the enzyme in animal feed, food, textiles and detergents and

in the paper industry. With the shortage of fossil fuels and the rising need to feed

alternative source for renewable energy and fuels, there is a renewal interest in the

bioconversion of lignocellulosic biomass using cellulases. Ethanol production of

lignocellulosic biomass comprises the following main steps: hydrolysis of cellulose

and hemicelluloses, sugar fermentation, separation of lignin residue and finally

recovery and purifying the ethanol to meet the fuel specifications. The task of

hydrolyzing lignocellulosic biomass to fermentable sugars is hindered by physico-

chemical, structural compositional factors. Owing to these structural characteristics,

pretreatment is an essential step for obtaining fermentable sugars (Mosier et al.,

2005).

1.4.2 Pretreatment of lignocellulosic biomass

Biomass contains a significant percentage of lignin which has been demonstrated to be

the most important preventive factor in biomass hydrolysis by cellulolytic and

hemicellulolytic enzymes (Dijkerman et al., 1997; Varnai et al., 2010). Dijkerman et

al. (1997) established a correlation between the percentage of lignin and release of

sugars from lignocellulose substrates. It has also been suggested that residual lignin

blocks the progress of cellulase down the cellulose chain (Zhang and Lynd, 2004).

Some researchers indicate that it is not just the presence of lignin, but the type and

distribution that has an impact on enzymatic hydrolysis (Zhang and Lynd, 2004).

Regardless of the cause of reduced bioconversion, the removal or disruption of lignin

is essential for efficient bioconversion of lignocellulose to sugars. During the past few years a large number of pretreatment methods have been

developed which include physical, chemical, physico-chemical and biological

pretreatment etc. (Sun and Cheng, 2002). Pretreatment of lignocellulosic biomass is

costly and crucial for achieving effective hydrolysis of substrates since enzymatic

hydrolysis of native lignocellulose produces less than 20% glucose from the cellulose

fraction (Zhang and Lynd, 2004). But the cost of using un-treated biomass for


- 19 -

enzymatic hydrolysis is much higher when compared to the pretreated substrate

(Eggeman and Elander, 2005). Fig 1.5 shows schematic representation on effect of

pretreatment on lignocellulosic material.

Fig. 1.5 Schematic representation on effect of pretreatment on lignocellulosic material

Depending on the specific pretreatment, different effects may be observed on the

substrate that may contribute to improve hydrolysis. These effects includes: removal

of some or all of the lignin which causes increased porosity in the substrate (Mansfield

et al., 1999); disruption of the lignin structure and its linkages with the rest of the

biomass; redistribution of lignin (Zhang and Lynd, 2004); removal of hemicellulose

that hampers access of cellulases to cellulose; disruption of the hemicellulose structure;

reduction in crystallinity of the cellulose; reduction in the degree of polymerisation of

cellulose; reduction in the size of the particles (Chundawat et al., 2007), respectively.

Each pretreatment technology has advantages and disadvantages (Table 1.4).


- 20 -

Table 1.4 Advantages and disadvantages of various pretreatment processes for lignocellulosic materials (Alvira et al., 2010)

Pretreatment process

Advantages Limitations and disadvantages

Mechanical comminution

Reduces cellulose crystalinity

Power consumption usually higher than inherent biomass energy

Steam explosion Causes hemicellulose degradation and lignin transformation, cost-effective

Destruction of aportion of the xylan fraction, incomplete disruption of the lignin- carbohydrate matrix, generation of compounds inhibitory to microorganisms, not effective for biomass with lignin content

AFEX Increases accessible surface area, removes lignin and hemicelluloses to an extent; does not produce inhibitors for down-stream processes, increases accessible surface area, cost effective

Not efficient for biomass with high lignin content

CO2 explosion Increases accessible surface area, cost effective, does not cause formation of inhibitory compounds

Does not modify lignin or hemicelluloses

Ozonolysis Reduces lignin content, does not produce toxic residues

Large amount of ozone required, expensive

Acid hydrolysis Hydrolyses hemicelluloses to xylose and other sugars, alters lignin structure

High cost, equipment corrosion, formation of toxic substances

Alkaline hydrolysis

Removes hemicelluloses and lignin, increases accessible surface area

Long resistance time required, irrecoverable salts formed and incorporated into biomass

Organosolv Hydrolyses lignin and hemicelluloses Solvents need to be drained from the reactor

Pyrolysis Produces gas and liquid products High temperature, ash production

Microwave

Short process time, high uniformity and selectivity, less energy input than the conventional heating

Degrade hemicelluloses

Biological

Simple equipment, degrades lignin and hemicelluloses, low energy requirements

Rate of hydrolysis is very low


- 21 -

Finally, an effective and economical pretreatment should meet the following

requirements: (a) production of reactive cellulose fiber for enzymatic attack (b)

avoiding destruction of hemicelluloses and cellulose (c) avoiding formation of

possible inhibitors for hydrolytic enzymes and fermenting microorganisms (d)

minimizing the energy demand (e) reducing the cost of size reduction of feed stocks

(e) reducing the cost of material for construction of pretreatment reactors and (f)

consumption of little or no chemical and using a cheap chemical.

1.4.3 Enzymatic hydrolysis (Saccharification)

Enzymatic hydrolysis of lignocellulose has long been studied as a method to

depolymerize the biomass into fermentable sugars and conversion to biofuels. It

requires less energy and mild environment conditions (Ferreira et al., 2009). It has

several advantages due to low toxicity and utility cost, less corrosion and no

inhibitory compounds formation as compared to acid hydrolysis (Sun and Cheng,

2002; Taherzadeh et al., 2008; Ferreira et al., 2009). Since, enzymatic hydrolysis is

carried out by cellulase enzymes that are highly substrate specific (Taherzadeh et al.,

2008).

The performance of cellulase mixtures in biomass conversion processes depends on

several properties including enzyme stability, product inhibition, synergism among

the different enzymes, productive binding to the cellulose, physical state as well as the

composition of cellulosic biomass (Heinelman et al., 2009). The choice of the enzyme

preparation for a particular biomass would be more dependent on biomass

characteristics rather than on standard enzyme activities measured (Kabel et al.,

2005), such as filter paper activity (FPU), carboxymethylcellulase (CMCase), β-

glucosidase and xylanase activities. It would not be feasible to predict the efficiency

of cellulases for bioconversion only on the basis of standard cellulase assays, as there

are no clear relationships between cellulase activities on soluble and insoluble

substrates (Zhang et al., 2006). Thus, the soluble substrates should not be used to

predict the efficiency of cellulases for processing relevant solid substrates such as

plant cell walls. Since filter paper is highly crystalline cellulose, preparations having

higher FPUs are desirable for bioconversion and the degradation of which depends on

the combination of activities of EG and CBH (Vincken et al., 1994).


- 22 -

Preparations of cellulase from a single organism may not be very efficient for

hydrolysis of a particular feedstock. Though, the filamentous fungi are the major

source of cellulases and hemicellulases and the mutant strains of Trichoderma

including Trichoderma reesei, Trichoderma viride and Trichoderma longibrachium

are the best known producers, these species have a low level of β-glucosidase activity

(Duff et al., 1996). The key to developing cellulases that are effective towards a

particular biomass feedstock is to artificially construct them either by enzyme

assembly to form cocktails or to engineer the cellulase producers to express desired

combination of cellulase enzymes (Schulein et al., 2000). There are several reports

available where an enzyme cocktail has been employed successfully for biomass

conversion (Zhang et al., 2006; Park et al., 2009; Singh et al., 2009; Sukumuran et

al., 2009). Cellulases for biomass conversion could be a blend or enzyme cocktail

containing endo- and exo-cellulase, xylanase, β-glucosidase, pectinase, etc. which

could vary for different biomass on the basis of their composition. Enzyme cocktails

are also developed by mixing Trichoderma reesei cellulase with other enzymes

including xylanases, pectinases and β-glucosidases and these cocktails have been tried

for hydrolysis of various feedstocks (Xin et al., 1993; Berlin et al., 2007). The

hydrolytic efficiency of a multi enzyme cocktail for lignocellulose saccharification

depends both on biochemical properties of individual enzymes and their ratio

(Gusakov et al., 2007). Some of the following synergies have been reported for

cellulase interaction on complex substrates (degree of synergy shown in brackets

where reported): Endo xylanase and endo-glucanase on sugarcane bagasse (3.59)

(Beukes et al., 2008); Endo xylanase and cellulases on corn cell walls (1.6)

(Murashima et al., 2003); Cellobiohydrolase, endo xylanase, acetyl xylan esterase and

ferulic acid esterase on pretreated corn stover (84% increase) (Selig et al., 2008).

The development of these enzyme cocktails for lignocellulose degradation therefore,

appears to focus on finding the appropriate accessory enzymes to enhance the

degradation of cellulose. There are many factors which influence the enzymatic

hydrolysis of cellulose in lignocellulosic feedstock such as temperature, pH, substrate

load, enzyme load and mixing rate etc. (Olsson et al., 1996), but recently the focus is

on operating at high solid loadings. The process at high solid loading offers many

advantages (≥15% solid, w/w) over conversions performed at low or moderate solid

loads, including increased sugar and ethanol concentrations and decreased capital and


- 23 -

investment costs (Modernbach et al., 2013). The use of high solid operations would

make biofuels more economical and more price-competitive with petroleum.

As solid loading increases, challenges that were negligible in low-solid systems

become more prominent, this has also been noted in high solid pretreatment

(Modernbach et al., 2012). One of the major challenges for enzymatic hydrolysis at

high solid loading is the lack of available water in the reactor. Water is essential for

effective hydrolysis mainly for two reasons: mass transfer and lubricity. Water

increases the effectiveness of the enzymatic and chemical reactions by providing a

medium for solubilizing and aiding in the mass transfer of products. Water also

reduces the viscosity of the slurry by increasing the lubricity of the particles, which

decreases the required shear stress necessary to produce a given shear rate, allowing

lower power input for mixing (Hodge et al., 2009; Kristensen et al., 2009). The

physical and chemical properties of the specific biomass affect the way biomass

absorbs water. As solid loadings approach 20% (w/w), the liquid fraction becomes

fully absorbed into the biomass leaving little free water (Hodge et al., 2009). With

lower amounts of free water, the apparent viscosity of the mixture increases, and

consequently mixing and handling of material become more difficult. Other

challenges specific to high-solid enzymatic hydrolysis include long hydrolysis times.

Enzymatic hydrolysis is typically thought to be the bottleneck of the entire

bioconversion process in terms of both time and money, since the reaction time

needed for most enzymes to convert lignocellulose into sufficient glucose

concentrations for fermentation is on the order of days (usually ≥3 days). Long

hydrolysis times could only be reduced by increasing enzyme loading. Recent studies

have suggested that enzymes can overcrowd accessible cellulose sites. Thus not

reaching the full hydrolytic potential for the given enzyme loading (Xu et al., 2007;

Bommarius et al., 2008). Enzyme is usually added based on per weight of a substrate.

As the solid loading increases the amount of enzyme should also increase

propotionately. The cost of enzyme is still at a level that makes this step in the

conversion process one of the most expensive. Therefore, is important to evaluate the

economics when determining the balance between the loadings applied to the

lignocellulose and the amount of time needed to reach sufficient glucose

concentrations. Researchers are addressing these issues from many angles,

experimenting with different pretreatment methods and various enzyme sources and


- 24 -

cocktails, while modifying operating conditions and slurry properties. Although there

has been some success at the pilot and demonstration scale, many questions must be

resolved before the full potential of high-solids lignocellulose conversion will be

realized. Several studies have been reported on the conversion of cellulosic biomass

to sugars by enzymatic hydrolysis at high solids using a horizontal orientation of the

reactor (Jorgensen et al., 2007; Larsen et al., 2008; Roche et al., 2009). One of the

highest solid loadings in enzymatic hydrolysis reported to date is 40% (w/w)

(Jorgensen et al., 2007 and 2009). A horizontal oriented rotating drum was utilized as

the reactor in these studies in order to effectively mix the solids. The studies found

that cellulose and hemicelulose conversion decreased from 90% to 33% and 70%-

35%, respectively, with the increase in solid loadings from 2% to 40%, but the reactor

was providing adequate mixing as evidenced by the conversion of lignocellulose into

fermentable saccharides (86 g glucose/kg at 40% solids).

1.4.4 Recovery of cellulases

It is necessary to develop efficient methods to produce renewable fuels from

lignocellulosic biomass. Recent commercial cellulase preparations have been shown

to be effective at hydrolyzing cellulose under industrially relevant conditions;

however, the high cost of enzymes remains a significant barrier to the economical

production of ethanol from lignocellulosic biomass (Weiss et al., 2013). It is

therefore necessary to reduce the amount of enzymes required for the enzymatic

hydrolysis step. A variety of methods have been suggested to achieve increased

hydrolysis yields, including surfactant addition (Borjesson et al., 2007), gradual

substrate loading (Rosgaard et al., 2007) or advance reactor configurations coupled

with product removal to avoid inhibition (Andric et al., 2010; Yang et al., 2010).

However, these methods have yet to be shown to be cost effective. One method that

may reduce the amount of enzyme used and increase enzymatic productivity is to

recover/recycle the enzymes (Ramos et al., 1993; Tjerneld et al., 1994). The majority

of cellulase recycling methods that have been reported involved either separating the

enzymes from the solids or liquid phases or recycling of the solid and/or liquid phase

directly. Approaches have been demonstrated where free enzymes were recovered

from the liquid fraction by membrane filtration (Tjerneld et al., 1994). Similarly, the

enzymes associated with the solids have been recovered by washing with excess


- 25 -

volumes of buffer, sometimes with surfactants to desorb the enzymes, which were

then concentrated and added to the fresh substrate (Tu et al., 2009). These methods

have shown different levels of success under controlled laboratory conditions and

with specially prepared feedstocks.

1.4.5 Bioethanol production

Each year a large portion of agricultural residues is disposed of as “waste” can

potentially be used to produce various value added products like biofuels, animal

feeds, chemicals, enzymes etc. Rice straw is one of major crop produced adundantly

all over the world. It is annually produced about 731 million tons which is distributed

in Africa (20.9 million tons), Asia (667.6 million tons), Europe (3.9 million tons),

America (37.2 million tons) and Oceania (1.7 million tons) (Balat et al., 2008). The

options for the disposal of rice straw are limited by the bulk of material, slow

degradation in the soil, harboring of rice stem diseases and high mineral content. Only

a small portion of globally produced rice straw is used as animal feed, the rest is

removed from the field by burning, a common practice all over the world, thus

increasing air pollution and affecting human health (Ogawa et al., 2005; Chen et al.,

2007; Wati et al., 2007). This huge amount of rice straw can potentially produce 205

billion liters bioethanol per year, which is the largest amount from a single biomass

feedstock.

Even though the fermentative process for ethanol production is well known, the

production costs are still the key impediment. Intense research has been carried out

for obtaining efficient fermentative microorganisms, low-cost fermentation substrates

and optimal environmental conditions for fermentation. The ideal strain for ethanol

production of lignocellulosic biomass should possess the following characteristics:

stable conversion of C5 and C6 sugars, resistance against inhibitory compounds,

tolerance concerning temperature, ethanol as well as sugar and industrial stability.

Regardless of the feedstock, the degradation of celluloses or hemicelluloses yields

hexoses and pentoses that need to be fermented to ethanol using yeast strains.

Current approaches are inefficient, since no native microorganisms can convert all

sugars into ethanol at a high yield (Chen, 2011). Therefore, microorganisms that can

efficiently convert both hexoses and pentoses to ethanol would be more useful.


- 26 -

Saccharomyces cerevisiae is the most favoured organism for ethanol production from

hexoses and does not have genes encoded for xylose reductase (XR) and xylitol

dehydrogenase (XDH) and thus cannot utilize xylose. A way to overcome this

obstacle of pentose utilization is through recombinant DNA technology (genetic

engineering). Other approach is to use pentose fermenting microorganisms. Yeasts

such as Pichia stipitis, Candida shehatae and Pachysolen tannophilus are capable of

fermenting both hexose and pentose sugars to ethanol (Chandel et al., 2007; Chen,

2011). Thermophilic bacteria have also been considered for fermentation of pentose

containing lignocellulosic hydrolysates. Thermophilic and saccharolytic Clostridia

are an important group of ethanol producing microorganisms and include species of

Clostridium thermohydrosulfuricum, Thermoanaerobacter (formerly Clostridium)

Thermosaccharolyticum and Clostridium thermocellum. These bacteria may translate

pentoses and aminoacids into ethanol and can synthesize upto 2 mol EtOH/mol

hexose. Having saccharolytic properties, these microorganisms have the ability to

grow on a wide variety of non treated wastes. Clostridium thermocellum can even

directly convert lignocellulosic materials into ethanol (McMillan, 1997). On the other

hand, there are a few cases where the immobilization of these yeasts increases the

ethanol productivity (Chandrakant and Bisaria, 1998), unlike the case of hexose

fermenting yeasts or Zymomonas mobilis.

The processes usually employed in the fermentation of lignocellulosic hydrolysate are

simultaneous saccharification and fermentation (SSF) and separate hydrolysis and

fermentation (SHF). SSF is a single step process whereas SHF is a two step process.

During SSF process the solid fraction of pretreated lignocellulosic material

simultaneously undergoes hydrolysis (saccharification) and once hydrolysis is

completed, the resulting cellulose hydrolysate is fermented and converted into

ethanol. However, one of the main features of the SHF process is that each step can

be performed at its optimal operating conditions. The most important factors to be

taken into account for saccharification step are reaction time, temperature, pH,

enzyme dosage and substrate load. The SHF process has been employed either

conventionally or traditionally but SSF is superior for ethanol production since it can

improve ethanol yields by removing end product inhibition and simultaneously

eliminate the need for separate reactors. In SSF, the cellulases and microorganisms

are added to the same process allowing that the glucose formed during the enzymatic


- 27 -

hydrolysis of cellulose be immediately consumed by the microbial cells converting it

into ethanol. Thus, the inhibition causes by the sugars over the cellulases is

neutralized. Since this process operates at non-optimal conditions for hydrolysis and

requires high enzyme dosage, which positively influence on substrate conversion, but

negatively on process costs. Considering that enzymes account for an important part

of production costs, it is necessary to find methods reducing the cellulase doses to be

utilized (Bjerre et al., 1996; Hamelinck et al., 2005). Apart from SSF or SHF, CBP is

also performed for ethanol production (Cardona et al., 2008). In CBP, mono-or co-

culture of microorganisms is generally used to ferment cellulose directly to ethanol.

Bacteria such as Clostridium thermocellum and some fungi including Neurospora

crassa, Fusarium oxysporum and Paecilomyces sp. have been used in CBP.

However, this process is not efficient because of poor ethanol yield and long

fermentation periods (3-12 days). Sequential fermentation with two different

microorganisms in different time periods of the fermentation process for better

utilization of sugars has also been employed using Saccharomyces cerevisiae in the

first phase for hexose utilization and Candida shehatae in the second phase for

pentose utilization but ethanol yields achieved are not high (Cardona et al., 2008).

Some studies on separate fermentation of individual hydrolysates using C6 specific

yeast strains have been reported (Gupta et al., 2009; Laser et al., 2009). The ethanol

yield obtained from various cellulosic hydrolysate using Saccharomyces cerevisiae

are: Sweet sorghum (0.40 g g-1) (Mamma et al., 1995), Corn cob (0.48 g g-1) (Chen et

al., 2007), Prosopis juliflora (0.49 g g-1) (Gupta et al., 2009) and Lanata camara

(0.33 g g-1) (Kuhad et al., 2010). Moniruzza et al. (1997) reported sequential

fermentation of glucose and xylose from rice straw by using Saccharomyces

cerevisiae ATCC 26603 and Pichia stiptis NRRL Y-7124 and yielded 4 g l-1 and 6 g l-

1 ethanol, respectively. Other microorganisms capable of fermenting a great variety

of sugars including hexoses and pentoses is the fungus Mucor indicus reaching

ethanol yields of 0.46 g g-1 glucose when cultivated under anaerobic conditions (Sues

et al., 2005).


- 28 -

1.4.6 Anaerobic digestion of liquid waste generated during ethanol production

process

While developing an integrated process technology for transport fuel production from

lignocellulosic material, a large amount of solid and liquid wastes are generated,

therefore, it is important to extract energy potential to maximum extent from the

unutilized side streams, which will help us to have a zero waste sustainable process.

Anaerobic digestion (AD) is a process that occurs only under strict anaerobic

conditions where several populations of bacteria react in concert to decompose

complex organic material to methane and carbon dioxide without an external electron

acceptor such as oxygen or nitrate. Anaerobic digestion can be considered as one of

the oldest technologies for waste and wastewater treatment. It is also probably the

major biological process involved in landfill wastes decomposition. There are several

advantages recognized to the process when used for wastewater treatment plants such

as high capacity to treat slowly degradable substrates at high concentrations, very low

sludge production (5-10 times less than in aerobic processes), potentiality for valuable

intermediate metabolites production, low energy requirements (no aeration is

required), reduction of odors in a closed system, pathogens reduction and possibility

for energy recovery through methane combustion or even hydrogen production

(Steyer et al., 2002).

1.4.6.1 Anaerobic treatment systems

All modern high rate biomethanation processes are based on the concept of retaining

highly concentrated viable biomass by some mode of bacterial sludge immobilization.

The processes are attractive alternative to anaerobic lagoon and conventional

digesters. A wide variety of process configurations have been made available which

hold the concept of immobilized cells. The various configurations developed so far

include Upflow anaerobic filter by Young and McCarty (1969), Anaerobic attached

film expanded bed by Switzen Baum and Jewell (1980); Upflow anaerobic sludge

blanket by Lettinga et al., (1980); Anaerobic rotating biological contactor by Tait and

Friedman (1980); Anaerobic fluidized bed by Jeris (1982); and Downflow stationary

fixed film by van den Berg et al., (1985). Switzen Baum and Jewell (1980) developed

the concept of fixed film reactors from the initial idea of anaerobic filters by Young


- 29 -

and McCarty (1969). Anaerobic hybrid reactor which combines advantages of both

anaerobic filter and up flow anaerobic sludge blanket designs, operates with a sludge

blanket in the lower zone and packing media forming a filter in the upper zone, was

first developed in 1981 (Maxham and Wakamiya, 1981). This type of reactor has been

also successfully and widely applied for the treatment of low to medium strength

wastewater (Kumar et al., 2007). The most important aspects in the AHR design are

the selection of appropriate support material. A variety of natural materials such as

smooth quartzite pebbles, shells, granite stones, wooden blocks, brick bats and

synthetic materials like polyvinyl chloride sheets, rasching rings, tyre rubber and

other materials have been used for the attachment and growth of anaerobic biomass

(Gourari and Achkari-Begdouri, 1997; Reyes et al., 1999; Show and Tay, 1999;

Michaud et al., 2002; Melidis et al., 2003; Rovirosa et al., 2004; Yang et al., 2004). It

was reported that the reactors packed with high media porosity obtained better organic

removal efficiency compared to that using non-porous support. Thus, a hybrid

treatment system will reduce investment costs through the use of cheaper local media

and also its flexibility to deal with almost all kinds of wastewater.

1.4.7 Textile industry

Cellulases have become the third largest group of enzymes used in the industry since

their introduction only since a decade. They are used in biostoning of the denim

garments for producing softness and the fade look replacing the use of pumice stones

which were traditionally employed. They act on the cellulose fiber to release the

indigo dye used for coloring the fabric, producing the faded look of denim. Though

use of acidic cellulase from Trichoderma along with the proteases is found to be

equally good, Humicola insolens cellulase is commonly employed in the biostoning

process. Cellulases are utilized for digesting off the small fiber ends protruding from

the fabric resulting in a better finish. Cellulases have also been used in softening,

defibrillation and in processes for providing localized variation in the color density of

fibers.


- 30 -

1.4.8 Laundry and detergents Cellulases, in particular EG III and CBH I, are commonly used in detergents for

cleaning textiles. Several reports disclose that EG III variants, in particular from

Trichoderma reesei and Trichoderma harzianum and Aspergillus niger are

industrially utilized natural sources of cellulases. Cellulase preparations, mainly from

species of Humicola that are active under mild alkaline conditions and at elevated

temperature, are commonly added in washing powders and in detergents.

1.4.9 Food and animal feed In food industry, cellulases are used in extraction and clarification of fruit and

vegetable juices, production of fruit nectars and purees and in the olive oil. Cellulases

are also used in carotenoid extraction in the production of food coloring agents.

Enzyme preparations containing hemicellulases and pectinase addition to cellulases

are used to improve the nutritive quality of forages. Improvements in feed

digestibility and animal performance are reported with the use of cellulases in feed

processing.

1.4.10 Pulp and paper industry In the pulp and paper industry, cellulases and hemicellulases have been employed for

biochemical pulping for modification of coarse mechanical pulp and hand sheet

strength properties, de-inking of recycled fibers and for improving drainage and run

ability of paper mills. Cellulases are employed in the removal of inks, coating and

toners from paper. Bio-characterization of pulp fibers is another application where

microbial cellulases are employed. They are also used in preparation of easily

biodegradable cardboard and in the manufacture of soft paper including paper towels

and sanitary paper and preparations containing cellulases are used to remove adhered

paper.


- 31 -

1.5.0 Cellulase market scenario

Globally, there are two major companies known for cellulase production for biomass

conversion-“Genencor” and “Novozyme”. Both the companies has played a

significant role in bringing down the cost of cellulase several folds by their active

research and are continuing to bring down the cost by adopting novel technologies.

Recently, Genencor has launched Accelerase® 1500, a cellulase complex intended

specifically for lignocellulosic biomass processing industries

(http://www.genecor.com/ wps/wcm/coonect/genecor/products). It is claimed to be

more cost effective and efficient for bioethanol industries than its predecessor-

Accelerase® 1000. Accelerase® 1500 is produced with a genetically modified strain of

Trichoderma reesei. This enzyme preparation is claimed to contain higher levels of β-

glucosidase activity than all other commercial cellulases available today, so as to

ensure almost complete conversion of cellobiose to glucose (http://www.genecor.com/

wps/wcm/coonect/genecor/products).

Genencor has also launched Accelerase® XY accessory xylanase enzyme complex

that enhances both xylan (C5) and glucan (C6) conversion when blended with other

Accelerase® enzyme products. Similarly, Accelerase® XC is an accessory

xylanase/cellulase enzyme complex that contains a broad profile of hemicellulase and

cellulase activities and enhances both xylan (C5) and glucan (C6) conversion when

blended with other Accelerase® enzyme products. Also, Accelerase® BG is an

accessory β-glucosidase enzyme that enhances glucan (C6) conversion when blended

with cellulase products. There are several potential cellulases which may prove to be

effective for biomass hydrolysis when supplemented with β-glucosidase, indicating

the importance of Accelerase® BG.

Novozymes also have a diverse range of cellulase preparations available based on

application as Cellusoft® AP and Cellusoft® CR for bioblasting in textile mills,

Carezyme® and Celluclean for laundry in detergent, Denimax® 601l for stonewash

industry at low temperature as well as many others specific for particular application

(http://www.bioenergy.novozymes.com). Amano Enzyme Inc. in Japan and MAP’s

India in India are another enzyme industry actively involved in cellulase production

(Table 1.5).


- 32 -

Table 1.5 Commercial cellulases produced by companies and their sources (Singhania et al., 2010)

Enzyme samples Supplier Source Cellubrix (Celluclast) Novozymes,

Denmark Trichoderma longibrachiatum and

Aspergillus niger Novozymes 188 Novozymes Aspergillus niger Viscostar 150L Dyadic (Jupiter, USA) Trichoderma longibrachiatum/

Trichoderma reesei Multifect CL Genencor

Intl. (S.San Francisco, CA) Trichoderma reesei

Energex L Novozymes Trichoderma longibrachiatum/

Trichoderma reesei Ultraflo L Novozymes Trichoderma longibrachiatum/

Trichoderma reesei Viscozyme L Novozymes Trichoderma longibrachiatum/

Trichoderma reesei GC 440

Genencor-Danisco (Rochester, USA)

Trichoderma longibrachiatum/

Trichoderma reesei

GC 880 Genencor Trichoderma longibrachiatum/

Trichoderma reesei Spezyme CP Genencor Trichoderma longibrachiatum/

Trichoderma reesei Accelerase® 1500 Genencor Trichoderma reesei Cellulase AP30K Amano Enzyme Aspeergillus niger Cellulase TRL

Solvay Enzymes (Elkhart, IN)


Trichoderma reesei

Econase CE Alko-EDC (New York, NY)


Trichoderma reesei Cellulase TAP106 Amano Enzyme (Troy,VA) Trichoderma viride


- 33 -

Problem delineated

Energy crisis and environmental pollution from the use of fossil fuels has become a

serious global threat. Thus, we need to move towards a sustainable development path

and explore the use of biofuel as cheaper and cleaner alternate to fossil fuels.

Bioethanol obtained from lignocellulosic materials has received major attention due to

their abundance and immense potential for conversion into sugars and fuels. Typical

bioprocesses for conversion of lignocellulose into biofuels involve production of

enzymes, pretreatment, hydrolysis of biomass and fermentation of sugars released.

Although bioethanol production has been greatly improved by new technologies,

there are still challenges that need further investigation. The major research

challenges are significant reduction in cost of enzyme production, improvement of

enzymatic hydrolysis with effective enzymes, development of novel technologies for

high solid handling during enzymatic hydrolysis and development of robust

fermentating organisms which are more tolerant to inhibitors and ferment all sugars

present in concentrated hydrolysate with high ethanol yields. Sincere efforts are also

required to adapt concepts of integration of the processes, reuse of enzymes and

appropriate technologies for the treatments of the solids and liquid wastes generated.

These efforts will significantly reduce energy and thereby cost of the fuel ethanol.

Thus, the present study was undertaken with the following research objectives:

1. Screening of potential cellulolytic microorganisms from various sources

2. Production of cellulases by solid state fermentation

3. Purification of endoglucanase and its biochemical characterization

4. Pretreatment and enzymatic hydrolysis of rice straw

5. Bioethanol production from enzymatic hydrolysate of rice straw

6. Biomethanation of waste generated during bioconversion of lignocellulosic

material


- 34 -

References Acharya, S., and Chaudhury, A., 2011. Effect of nutritional and environmental

factors on cellulase activity by thermophillic bacteria isolated from hot spring. Journal of Scientific and Industrial Research, 70, 142-148.

Afzal, A., Bano, A., Fatima, M., 2010. Higher soybean yield by inoculation with N-

fixing and P-solubilizing bacteria. Agronomy Sustainable Development, 30, 487-495.

Akcapinar, G.B., Gul, O., Sezerman, U., 2011. Effect of codon optimization on the

expression of Trichoderma reesei endoglucanase 1 in Pichia pastoris. Biotechnology Progress, 27, 1257-1263.

Alper, H., and Stephanopoulos, G., 2009. Engineering for biofuels: exploiting innate

microbial capacity or importing biosynthetic potential? Nature Review in Microbiology, 7, 715-723.

Alvira, P., Tomas-Pejo, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment

technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresource Technology, 101, 4851-4861.

Andric, P., Meyer, A. S., Jensen, P. A., Johansen, K. D., 2010. Reactor design for

minimizing product inhibition during enzymatic lignocellulose hydrolysis I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes. Biotechnology in Advances, 28, 308-324.

Aro, N., Saloheimo, A., Ilmen, M., Penttila, M., 2001. ACEII, a novel transcriptional

activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei. Journal of Biological Chemistry, 276, 24309-24314.

Ashabil, A., Lutfiye, K., Burhan, A., 2011. Alkaline thermostable and halophilic

endoglucanase from Bacillus licheniformis C108. African Journal of Biotechnology, 10, 789-796.

Bajaj, B.K., Pangotra, H., Wani, A.M., Sharma, P., Sharma, A., 2009. Partial

purification and characterization of a highly thermostable and pH stable endoglucanase from a newly isolated Bacillus strain M-9. Indian Journal of Chemical Technology, 16, 382-387.

Bakare, M.K., Adewale, I.O., Ajayi, A., Shonukan, O.O., 2005. Purification and

characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens. African Journal of Biotechnology, 4, 898-904.

Balat, M., Balat, H., Oz, C., 2008. Progress in bioethanol processing. Progress in

Energy and Combustion Science, 4, 551-573.


- 35 -

Bara, M.T.F., Lima, A.L., Ulhoa, C.J., 2003. Purification and characterization of an exo- β-1, 3-glucanase produced by Trichoderma asperellum. FEMS Microbiology Letters, 219, 81-85.

Bayer, E.A., Shimon, L.J.W., Shoham, Y., Lamed, R., 1998. Journal of Structural

Biology. 124, 221-234. Beg, Q.K., Kapoor, M., Mahajan, L., Hoondal, G.S., 2001. Microbial xylanases and

their industrial application: a review. Applied Microbiology and Biotechnology, 56, 326-338.

Berger, E., Zhang, D., Zverlow, V.V., Schwarz, W.H., 2007. Two noncellulosomal

cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically, FEMS Microbiology Letters, 268, 194-201.

Berlin, A., Maximenko, V., Gilkes, N., Saddler, J., 2007. Optimization of enzyme

complexes for lignocellulose hydrolysis. Biotechnology and Bioengineering, 97, 287-296.

Beukes, N., Chan, H., Doi, R. H., Pletschke, B.L., 2008. Synergistic associations

between Clostridium cellulovorans enzymes XynA, ManA and EngE against sugarcane bagasse. Enzyme and Microbial Technology, 42, 492-498.

Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshimi, S.,

Kurien, N., Sukumaran, R.K., Pandey, A., 2010. Bioethanol production from rice straw: an overview, Bioresource Technology, 101, 4767-4774.

Bischoff, K.M., Rooney, A.P., Li, X.L., Liu, S., Hughes, S.R., 2006. Purification and

characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnology Letters, 28, 1761-1765.

Bjerre, A.B., Olesen, A.B., Fernqvist, T., 1996. Pre-treatment of wheat straw using

combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and hemicellulose. Biotechnology and Bioengineering, 49, 568-577.

Boisset, C., Fraschini, C., Schulein, M., Henrissat, B., Chanzy, H., 2000. Imaging the

enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A, Applied Environmental Microbiology, 66, 1444-1452.

Bommarius, A.S., Katona, A., Cheben, S.E., Patel, A.S., Ragauskas, A.J., Knudson,

K., 2008. Cellulase kinetics as a function of cellulose pretreatment. Metabolic Engineering, 10, 370-381.

Borjesson, J., Peterson, R., Tjerneld, F., 2007. Enhanced enzymatic conversion of

softwood lignocellulose by polyethylene glycol addition. Enzyme and Microbial Technology, 40, 54-762.


- 36 -

Cadoche, L., and Loopez, G.D., 1989. Assessment of size reduction as a preliminary step in the production of ethanol from lignocellulosic wastes. Biological Wastes, 30, 153-157.

Cardona, C.A., and Sanchez, O.J., 2008. Trends in biotechnological production of

fuel ethanol from different feedstocks. Bioresource Technology, 99, 5270-5295.

Cen, P.L., and Xia, L.M., 1999. Production of cellulase by solid state fermentation.

Advances in Biochemical Engineering and Biotechnology, 65, 69-92. Chahal, D.S., 1985. Solid-state fermentation with Trichoderma reesei for cellulase

production. Applied Environmental Microbiology, 49, 205-210. Chandel, A.K., Chandrasekhar, G., Silva, M.B., Silverio, D., Silva, S., 2012. The

realm of cellulases in biorefinery development. Critical Reviews in Biotechnology, 32, 187-202.

Chandel, A.K., Kapoor, R.K., Singh, A., Kuhad, R.C., 2007. Detoxification of

sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501. Bioresource Technology, 98, 1947-1950.

Chandrakant, P., and Bisaria, V.S., 1998. Simultaneous bioconversion of cellulose

and hemicellulose to ethanol. Critical Review of Biotechnology, 18, 295-331. Chen, G., Li, S., Jiao, F., Yuan, Q., 2007. Catalytic dehydration of bioethanol to

ethylene over TiO2/γ-Al2O3 catalysts in micro channel reactors. Catalyst, 125, 111-119.

Chen, Y., 2011. Development and application of co-culture for ethanol production by

co-fermentation of glucose and xylose: a systematic review. Journal of Indian Microbiology and Biotechnology, 38, 581-597.

Cherry, J.R., Fidantsef, A.L., Cherry, Fidantsef, 2003. Directed evolution of industrial

enzymes: an update. Current Opinion in Biotechnology, 14, 438-443. Chundawat, S.P., Venkatesh, B., Dale, B.E., 2007. Effect of particle size based

separation of milled corn stover on AFEX pretreatment and enzymatic digestibility. Biotechnology and Bioengineering, 96, 219-31.

Dariot, D.J., Simonetti, A., Plinho, F., Brandeli, A., 2008. Purification and

characterization of extracellular β-glucosidase from Monascus purpureus. Journal of Microbial Biotechnology, 18, 933-941.

Dijkerman, R., Op den Camp, H.J., Van der Drift, C., Vogels, G.D., 1997. The role of

the cellulolytic high molecular mass (HMM) complex of the anaerobic fungus Piromyces sp. strain E2 in the hydrolysis of microcrystalline cellulose. Archives of Microbiology, 167, 137-142.


- 37 -

Dipali, P., Seeta, L. R., Mala, R., 2012. Purification and biochemical characterization of endoglucanase from Penicillium pinophilum MS 20. Indian Journal of Biochemistry and Biophysics, 49, 189-194.

Divne, C., Stahlberg, J., Teeri, T.T., Jones, T.A., 1998. High-resolution crystal

structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei. Journal of Molecular Biology, 275, 309-325.

Duff, S.J.B., and Murray, W.D., 1996. Bioconversion of forest products industry

waste cellulosic to fuel ethanol: a review. Bioresource Technology, 55, 1-33. Eggeman, T., and Elander, R.T., 2005. Process and economic analysis of pretreatment

technologies. Bioresource Technology, 96, 2019-2025. Elkins, J.G., Raman, B., Keller, M., 2010. Engineered microbial systems for enhanced

conversion of lignocellulosic biomass. Current Opinion in Biotechnology, 21, 657-662.

Elshafei, A.M., Mohamed, M.H., Bakry, M.H., Osama, M.A., Housam, M.A.,

Abdelmageed, M.O. 2009. Purification and properties of an endoglucanase of Aspergillus terreus DSM 826. Journal of Basic Microbiology, 426-432.

Eriksson, K.E.L., and Bermek, H., 2009. Lignin, lignocellulose, ligninase. Applied

Microbiology, 373-384. Ferreira, S.M.P., Duarte, A.P., Queiroz, J.A., Domingues, F.C., 2009. Influence of

buffer systems on Trichoderma reesei Rut C-30 morphology and cellulase production. Electronic Journal of Biotechnology, 12, 1-9.

Fritscher, C., Messner, R., Kubicek, C.P., 1990. Cellobiose metabolism and

cellobiohydrolase I biosynthesis by Trichoderma reesei. Experimental Mycology, 14, 405-415.

Gao, J., Weng, H., Zhu, D., Yuan, M., Guan, F., Y. Xi, 2008. Production and

characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover. Bioresource Technology, 99, 7623-7629.

George, P.S., Ahmad, A., Rao, M.B., 2001. Studies on carboxy-methyl cellulase

produced by an alkalothermophilic actinomycete. Bioresource Technology, 77, 171-175.

Gourari, S., and Achkari-Begdouri, A., 1997. Use of baked clay media as biomass

supports for anaerobic filters. Applied Clay Science, 12, 365-375. Gupta, R., Sharma, K.K., Kuhad, R.C., 2009. Separate hydrolysis and fermentation

(SHF) of Prosopis juliflora, a woody substrate for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498.


- 38 -

Gusakov, A.V., Salanovich, T.N., Antonov, A.I., Ustinov, B.B., Okunev, O.N., Burlingame, R., 2007. Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose. Biotechnology and Bioenergy, 97, 1028-1038.

Hamelinck, C.N., Hooijdonk, G.V., Faaij. A.P., 2005. Ethanol from lignocellulosic

biomass: techno-economic performance in short-middle-and long-term. Biomass and Bioenergy, 28,384-410.

Heck, J.X., Hertz, P.F., Ayub, M.A.Z., 2002. Cellulase and xylanase production by

isolated Amazon Bacillus strains using soya been industrial residue based solid-state cultivation. Brazilian Journal of Microbiology, 33, 213-218.

Heinelman, P., Snow, C.D., Wu, I., Nguyen, C., Villalobos, A., 2009. A family of

thermostable fungal cellulases created by structure-guided recombination. PNAS, 106, 5451-5452. DOI:10.1073/pnas.0901417106.

Hendriks, A.T.W.M., and Zeeman, G., 2009. Pretreatments to enhance the

digestibility of lignocellulosic biomass. Bioresource Technology, 100, 10-18. Henrissat, B., 1991. A classification of glycosyl hydrolases based on amino acid

sequence similarities. Biochemical Journal, 280, 309-316.1991. Hodge, D.B., Karim, M.N., Schell, D.J., McMillan, J.D., 2009. Model-based fed-

batch for high-solid enzymatic cellulose hydrolysis. Applied Biochemistry and Biotechnology, 152, 88-107.

Holker, U., Hofer, M., Lenz, J., 2004. Biotechnological advantages of laboratory-

scale solid-state fermentation with fungi. Applied Microbiology and Biotechnology, 64, 175-186.

Hoshino, E., Shiroishi, M., Amano, Y., Nomura, M., Kanda, T., 1997. Synergistic

action of exo-type cellulases in the hydrolysis of cellulose with different crystallinities. Journal of Fermentation and Bioengineering, 84, 300-306.

Ilmen, M., Thrane, C., Penttila, M., 1996a. The glucose repressor gene cre 1 of

Trichoderma; isolation and expression of a full-length and a truncated mutant form. Molecular and General Genetics, 251, 451-460.

Ilmen, Onnela, M.L., Klemsadals, Keranans, Pentilla, M., 1996b. Functional analysis

of the cellobiohydrolase I promoter of the filamentous fungus. Trichoderma reesei. Molecular and General Genetics, 253, 303-314.

Ilmen, M., Saloheimo, A., Onnela, M.L., Penttila, M.E., 1997. Regulation of

cellulase gene expression in the filamentous fungus Trichoderma reesei. Applied Environmental Microbiology, 63, 1298-1306.

Jeris, J.S., 1982. Industrial wastewater treatment using anaerobic fluidised bed

reactors. Proceedings of IAWPRC Seminar on Anaerobic Treatment, Copenhagen, Denmark.


- 39 -

Jha, P.K., Nair, S., Gopinathan, M.C, Babu. C.R., 1995. Suitability of rhizobia inoculated wild legumes Argurolobium flaccidum, Astragalus graveolens, Indigofera gangetica and Lespedeza stenocarpa in providing a vegetational cover in unreclaimed limestone quarry. Plant Soil, 177, 139-149.

Jorgensen, H., Kristensen, J.B., Felby, C., 2007. Enzymatic conversion of

lignocellulose into fermentable sugars: Challenges and opportunities, Biofuels, Bioproducts and Biorefining 1, 119-134.

Jorgensen, H., Vibe-Pedersen, J., Larsen, J., Felby, C., 2009. Liquefaction of

lignocellulose at high-solid concentrations. Biotechnology and Bioengineering, 96, 862-870.

Jung, H., Yoon, H.G., Park, W., Choi, C., Wilson, D.B., Shin, D.H., 2008. Effect of

sodium hydroxide treatment of bacterial cellulose on cellulase activity. Cellulose, 15, 465-471.

Jung, J.H., Fouad, W.M., Vermerris, W., Gallo, M., Altpeter, F., 2012. RNAi

suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass. Plant Biotechnology Journal, 10, 1067-1076.

Kabel, M.A., Van der Maarel, M.J.E.C., Klip, G., Voragen, A.G.J., Schols, H.A.,

2005. Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials. Biotechnology and Bioengineering, 93, 56-63.

Kang, H.J., Uegaki, K., Fukada, H., Ishikawa, K., 2007. Improvement of the

enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshi. Extremophiles, 11, 251- 256.

Kaur, J., Chadha, B.S., Kumar, B.A., Saini, H.S., 2007. Purification and

characterization of two endoglucanases from Melanocarpus sp. MTCC 3922. Bioresource Technology, 98, 74-81.

Kim, J.Y., Hur, S.H., Hong, J.H., 2005. Purification and characterization of an

alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810. Biotechnology Letters, 27,313- 316.

Koukiekolo, R., Kosugi, A., Inui, M., Yukawa, H., Doi, R.H., 2005. Degradation of

corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA. Applied Environmental Microbiology, 71, 3504-3511.

Kristensen, J.B., Felby, C., Jorgensen, H., 2009. Yield-determining factors in high-

solids enzymatic hydrolysis of lignocellulose, Biotechnology and Biofuels, 2. Kubicek, C.P., 1981. Release of carboxy-methyl cellulase and β-glucosidase from the

cell walls of Trichoderma reesei. European Journal of Applied Microbiology and Biotechnology, 13, 226-231.


- 40 -

Kubicek, C.P., and Penttila, M., 1998. Regulation of production of plant polysaccharide degrading enzymes by Trichoderma reesei in Trichoderma and Gliocladium, Volume 2, edition by GE Harman and CP Kubicek (Taylor and Francis, London), 49-72.

Kuhad, R.C., Gupta, R., Khasa, Y.P., Singh, A., 2010. Bioethanol production from

Lantanacamara (red sage): pretreatment, saccharification and fermentation. Bioresource Technology, 101, 8348-8354.

Kumar, G. S, Gupta, S. K., Singh G, 2007. Anaerobic hybrid reactor - a promising

technology for the treatment of distillery spent wash, Journal of Indian School of Mines, 11, 1 25-138.

Kumar, R., and Wyman, C.E., 2009. A Cellulase adsorption and relationship to

features of corn stover solids produced by leading pretreatments. Biotechnology and Bioengineering.103, 252-67.

Larsen, J., Petersen, M.O., Thirup, L., Li, H.W., Iversen, F.K., 2008. The IBUS

process -Lignocellulosic bioethanol close to a commercial reality. Chemical Engineering Technology, 31, 765-772.

Laser, M., Larson, E., Dale, B., Wang, M., Greene, N., Lynd, L.R., 2009.

Comparative analysis of efficiency, environmental impact and process economics for mature biomass refining scenarios Biofuels, Bioproducts and Biorefining, 3, 247-270.

Lettinga, G., van Velsen, A.F.M., Hobma, S.W., de Zeeuw, W.J., Klapwijk, A., 1980.

Use of the upflow sludge blanket (USAB) reactor. Biotechnology and Bioengineering, 22, 699-734.

Linger, J.G., Adney, W.S., Darzins, A., 2010. Heterologous expression and

extracellular secretion of cellulolytic enzymes by Zymomonas mobilis. Applied Environmental Microbiology, 76, 6360-6369.

Liu, D., Zhang, R., Yang, X., Zhang, Z., Song, S., Miao, Y., 2012. Characterization of

a thermo-stable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33. Microbial Cell, 11:25.

Maxham, J.V., and Wakamiya, W., 1981. Innovative biological wastewater treatment

technologies applied to the treatment of biomass gasification wastewater. In. Proceedings of the 35th Ind. Waste Conference, Purdue University, 80-94.

Mamma, D., Christakopoulos, P., Koullas, D., Kekos, D., MaMammacris, B.J.,

Koukios, E., 1995. An alternative approach to the bioconversion of sweet sorghum carbohydrates to ethanol. Biomass and Bioenergy, 8, 99-103.

Mandels, M., and Weber, J., 1969. Production of cellulases. Advances in Chemistry

Series, 95, 391-414.


- 41 -

Mandels, M., and Reese, E.T., 1957. Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. Journal of Bacteriology, 73, 269-278.

Mansfield, S.D., Mooney, C., Saddler, J.N., 1999. Substrate and enzyme

characteristics that limit cellulose hydrolysis. Biotechnology Programmes, 15, 804-816.

Mattanovich, D., Branduardi, P., Dato, L., Gasser, B., Sauer, M., Porro, D., 2012.

Recombinant protein production in yeasts recombinant gene expression. Methods in Molecular Biology, 824, 329-358.

McMillan, J.D., 1997. Biethanol production: Status and prospects. Renewable

Energy, 10, 295,-302. Melidis, P., Georgiou, D., Aivasidis, A., 2003. Scale-up and design optimization of

anaerobic immobilized cell reactors for wastewater treatment. Chemical Engineering and Processing, 42, 897-908.

Michaud, S., Bernet, N., Buffiere, P., Roustan, M., and Moletta, R., 2002. Methane

yield as a monitoring parameter for the start-up of anaerobic fixed film reactors. Water Research, 36, 1385 -1391.

Modenbach, A. A., and Nokes, S.E., 2012. The use of high solids loadings in biomass

pretreatment- a review. Biotechnology and Bioengineering, 109, 1430. Modenbach, A. A., and Nokes, S.E., 2013. Enzymatic hydrolysis of biomass at high

solids loadings- a review. Biomass and Bioenergy, 56, 526-544. Moniruzza, A.M., Dien, B.S., Skory, C.D., Chen, Z.D., Hespell, R.B., Ho, N.W.Y.,

Dale, B.E., Bothast R.J., 1997. Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain. World Journal of Microbiology and Biotechnology, 341-346.

Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M., Ladisch, M.,

2005. Features of promosing technologies for pretreatment of lignocellulosic biomass. Bioresource Technology, 96, 673-686.

Murashima, K., Kosugi, A., Doi, R., 2003. Synergistic effects of cellulosomal

xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation. Journal of Bacteriology, 185, 1518-1524.

Nazir, A., Soni, H., Saini, H.S., Manhas, R.K., Chadha, B.S., 2009. Purification and

characterization of an endoglucanase from Aspergillus terreus highly active against barley β-glucan and xyloglucan, World Journal of Microbial Biotechnology, 25, 1189-1197.

Nguyen, V.T., and Quyen, D.T., 2010. Purification and properties of a novel

thermoactive endoglucanase from Aspergillus awamori VTCC-F099. Australian Journal of Basic Applied Sciences, 4, 6211-6216.


- 42 -

Nidetzky, B., Steiner, W., Hayn, M., Claeyssens, M., 1994. Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction. Biochemical Journal, 3, 705-710.

Nigam, P., and Singh, D., 1996. Processing of agriculture wastes in solid state

fermentation for cellulolytic enzyme production. Journal of Scientific and Industrial Research, 55, 457-463.

Noronha, E.F., Kipnis, A., Kipinis, A.P.J., Ulhoa, C.J., 2000. Regulation of 36 kDa 1-

3 glucanase synthesis in Trichoderma harzianum. FEMS Microbiology Letters, 188, 19-22.

Ogawa, S., Lozach, J., Benner, C., Pascual, G., Tangirala, R.K., Westin, S.,

Hoffmann, Subramaniam, A., David, M., Rosenfeld, M.G., 2005. Molecular determinants of crosstalk between nuclear receptors and toll-like receptors. Cell, 122, 707-721.

Olsson, P.A., Baath, E., Jakobsen, I., Soderstrom, B., 1996. Soil bacteria respond to

presence of roots but not to mycelium of Arbuscular mycorrhizal fungi. Soil Biology and Biochemistry, 28, 463-470.

Pandey, A., 1994. Solid state fermentation-an overview. In: Pandey, A. (Ed), Solid

state fermentation. Wiley Eastern Limited, New Delhi, 3-10. Pandey, A., Benjamin, S., Soccol, C.R., Nigam, P., Krieger, N., Soccol, U.T., 1999.

The realm of microbial lipases in biotechnology. Biotechnology and Applied Biochemistry, 29, 119-131.

Park, Y.H., Kim, J., Kim, S.S., Park, Y.K., 2009. Pyrolysis characteristics and kinetics

of oak trees using thermo gravimetric analyzer and micro-tubing reactor. Bioresource Technology, 100, 400-405.

Pottkamper, J., Barthen, P., Ilmberger, N., Schwaneberg, U., Schenk, A., Schulte, M.,

Ignatiev, N., Streit. W. R., 2009. Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids. Green Chemistry, 11, 957-965.

Ramos, L.P., Nazhad, M.M., Saddler, J.N., 1993. Effect of enzymatic hydrolysis on

the morphology and fine structure of pretreated cellulosic residues. Enzyme and Microbial Technology, 15, 821-831.

Raven, P.H., Evert, R.F., Eichhorn, S.E., 1999. Biology of plants. New York; W.H.

Freeman and company. Reczey, K., Szengyel, Z.S., Eklund, R., Zacchi, G., 1996. Cellulase production by

Trichoderma reesei. Bioresource Technology, 57, 25-30. Reyes, O., Sanchez, E., Rovirosa, N., Borja, R., Cruz, M., Colmenarejo, M.F.,

Escobedo, R., Ruiz, M., Rodriguez, X., Cprrea, G., 1999. Low strength wastewater treatment by a multistage anaerobic filter packed with waste tyre rubber. Bioresource Technology, 70, 55-60.


- 43 -

Romero, M.D., Aguado, J., González, L., Ladero, M., 1999. Cellulase production by Neurospora crassa on wheat straw. Enzyme and Microbial Technology, 25, 244-250.

Rosgaard, L., Pedersen, S., Meyer, A.S., 2007. Comparison of different pretreatment

strategies or enzymatic hydrolysis of wheat and barley straw. Applied Biochemistry and Biotechnology, 143, 284-296.

Rovirosa, N., Sanchez, E., Cruz, M., Veiga, M.C., Borja, R., 2004. Coliform

concentration reduction and related performance evaluation of a down-flow anaerobic fixed bed reactor treating low-strength saline wastewater. Bioresource Technology, 94, 119-127.

Saha, B.C., 2004. Production, purification and properties of endoglucanase from a

newly isolated strain of Mucor circinelloides. Process Biochemistry, 39, 1871- 1876.

Saha, B.C., 2003. Hemicellulose bioconversion, Journal of Industrial Biotechnology,

30, 279- 291. Sandgren, M., Stahlberg, J., Mitchinson, C., 2005. Structural and biochemical studies

of GH family 12 cellulases: improved thermal stability and ligand complexes. Progress in Biophysics and Molecular Biology, 89, 246-291.

Sa-Pereira, P., Paveia, H., Costa-Ferreira, M., Aires-Barros, M.R., 2003. A new look

at xylanases: An overview of purification strategies. Molecular Biotechnology, 24, 257-281.

Schulein, M., 2000. Protein engineering of cellulases. Biochemica et Biophysica Acta,

1543, 239-252. Schwarz, W.H., 2001. The cellulosome and cellulose degradation by anaerobic

bacteria. Applied Microbiology and Biotechnology, 56, 634-649. Selig, M., Weiss, N., Ji, Y., 2008. NREL laboratory analytical procedure: Enzymatic

saccharification of lignocellulosic biomass, National Renewable Energy Laboratory Retrieved from http://www.nrel.gov/biomass/pdfs/42629.pdf.

Shen, B., Sun, X., Zuo, X., Shilling, T., Apgar, J., Ross, M., 2012. Engineering a

thermo regulated inter-modified xylanase into maize for consolidated lignocellulosic biomass processing. Nature Biotechnology, 30 1131-1136.

Show, K.Y., and Tay, J.H., 1999. Influence of support media on biomass growth and

retention in anaerobic filters, Water Research, 33, 1471-1481. Singh, J., Batra, N., Sobti, R.C., 2004. Purification and characterization of alkaline

cellulose produced by a novel isolate Bacillus sphaericus JS1. Journal of Industrial Microbiology and Biotechnology, 31, 51-56.


- 44 -

Singh, S., Simmons, B.A., Vogel, K.P., 2009. Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switch grass, Biotechnology and Bioengineering, 104, 68-75.

Singhania, R.R., Patel, A.K., Soccol, C.R., and Pandey, A., 2009. Recent advances in

solid state fermentation, Biochemical Engineering Journal, 44, 13-18. Singhania, R.R., Sukamaran, R.K., Pandey, A., 2007. Improved cellulose production

by Trichoderma reesei RUT C30 under SSF through process optimization. Applied Biochemistry and Biotechnology, 142, 60-70.

Singhania, R.R., Sukumaran, R.K., Patel, A.K., Larroche, C., Pandey, A., 2010.

Advancement and comparative profiles in the production technologies using solid state and submerged fermentation for microbial cellulases, Enzyme and Microbial Technology, 46, 541-549.

Sternberg, D., and Mandels, G.R., 1979. Induction of cellulolytic enzymes in

Trichoderma reesei by sophorose. Journal of Bacteriology, 139, 761-769. Steyer, J.P., Bouvier J.C., Conte, T., Gras, P., Sousbie, P., 2002. Evaluation of a four

year experience with a fully instrumented anaerobic digestion process, Water Science Technology, 45, 495-502.

Sues, A., Millati, R., Edebo, L., Taherzadeh, M. J., 2005. Ethanol production from

hexoses, pentoses, and dilute-acid hydrolyzate by Mucor indicus. FEMS Yeast Research, 5, 669-676.

Sukumaran, R.K., Singhania, R.R., Mathew, G.M., Pandey, A., 2009. Cellulase

production using biomass feedstock and its application in lignocellulose saccharification of bio-ethanol production, Renewable Energy, 34, 421-424.

Sukumaran, R.K., Singhania, R.R., Pandey, A., 2005. Microbial cellulases production,

applications and challenges. Journal of Scientific and Industrial Research 64, 832 - 844.

Sun, Y., and Cheng, J.Y., 2002. Hydrolysis of lignocellulosic materials for ethanol

production: a review. Bioresource Technology, 83, 1-11. Switzen Baum, M.S., and Jewel, W.J., 1980. Anaerobic attached film expanded bed

reactor treatment. Journal of Water Pollution Control Federation, 52. Szabo, I.J., Johansson, G., Pettersson, G., 1996. Optimized cellulase production by

Phanerochaete chrysosporium: Control of catabolite repression by fed-batch cultivation. Journal of Biotechnology, 48, 221-230.

Tait, S.L., and Friedman, A.A., 1980. Anaerobic rotating biological contractor for

carbonaceous wastewater. Journal of Water Pollution Control Federation, 52, 2257-2269.


- 45 -

Taherzadeh, M.J., and Kamiri, K., 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production. A review. International Journal of Molecular Science, 9, 1621-1651.

Tangnu, S.K., Blanch, H.W., Wilke, C.R., 1981. Enhanced production of cellulase,

hemicellulase, and β-glucosidase by Trichoderma reesei (RUT C-30), Biotechnology and Bioengineering, 23, 1837-1849.

Tengerdy, R.P., 1996. Cellulase production by solid state fermentation. Journal of

Scientific and Industrial Research, 55, 313-316. Thai, H.P., Dinh, T.Q., Ngoc, M.N., 2010. Optimization of endoglucanase production

by Aspergillus niger VTCC-F021. Australian Journal of Basic and Applied Sciences, 4, 4151-4157.

Tjerneld, F., 1994. Enzyme-catalyzed hydrolysis and recycling in cellulose

bioconversion. Methods in Enzymology. G. J. Harry Walter, Academic Press, 228, 549-558.

Tu, M., Zhang, X., Paice, M., MacFarlane, P., Saddler, J.N., 2009. The potential of

enzyme recycling during the hydrolysis of a mixed softwood feedstock. Bioresource Technology, 100, 6407-6415.

Vaheri, M.P., Vaheri, M.E.O., Kauppinen, V.S., 1979. Formation and release of

cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol, European Journal of Applied Microbiology and Biotechnology, 8, 73-80.

van den Berg, L., Kennedy, K.J., Samson, R., 1985. Anaerobic down flow stationary

fixed film reactors: Performance under steady-state and non-steady state conditions. Water Science and Technology, 17, 89-102.

Van Dyk, J.S., Pletschke, B.I., 2012. A review of lignocellulose bioconversion using

enzymatic hydrolysis and synergistic cooperation between enzymes-factors affecting enzymes, conversion and synergy. Biotechnology Advances, 30, 1458-1480.

Varnai, A., Siika-aho, M., Viikari, L., 2010. Restriction of the enzymatic hydrolysis

of steam pretreated spruce by lignin and hemicellulose. Enzyme and Microbial Technology, 46, 185-193.

Vikari, L., Alapuranen, M., Puranen, T., Vehmannpera, J., Silka-Aho, M., 2007.

Thermostable enzymes in lignocellulosic hydrolysis. Advances in Biochemical Engineering and Biotechnology, 108, 121-145.

Vincken, J.P., Beldman, G., Voragen, A.G.J., 1994. The effect of xylo glucans on the

degradation of cell-wall embedded cellulose by the combined action of cellobiohydrolase and glucanases from Trichoderma viride. Plant Physiology, 104, 99-107.


- 46 -

Vintila, T., Dragomirescu, M., Jurcoane, S., Caprita, R., Maiu, M., 2009. Production of cellulase by submerged and solid-state cultures and yeast selection for conversion of lignocellulose to ethanol. Romanian Biotechnological Letters, 14, 4275-4281.

Walker, L.P., and Wilson, D.B., 1991. Enzymatic hydrolysis of cellulose: an

overview. Bioresource Technology, 36, 3-14. Wati, L., Kumari, S., Kundu, B.S., 2007. Paddy straw as a substrate for ethanol

production. Indian Journal of Microbiology, 47, 26-29. Wei, D.L., Kohtaro, K., hoji, U.S., Hui, L.T., 1996. Purification and characterization

of an extracellular β-glucosidase from the wood-grown fungus Xylaria regalis. Current Microbiology, 33, 297-301.

Weiss, N., Borjesson, J., Pedersen, L.S., and Meyer, A. S., 2013. Enzymatic

lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling. Biotechnology and Biofuels, 6, 5.

Wen, Z., Liao, W., Chen, S., 2005. Production of cellulase/β-glucosidase by the

mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure. Applied Biochemistry and Biotechnology, 121, 93-104.

Wurleitner, E., L. Pera, C., Wacenovsky, A., Cziferszky, S., Zeilinger, C. P., Kubicek,

R. L., 2003. Transcriptional regulation of xyn2 in Hypocrea jecorina. Eukaryotic Cell, 2,150-158.

Xin, Z., Yinbo, Q., Peiji, G., 1993. Acceleration of ethanol production from paper

mill waste fiber by supplementation with b-glucosidase. Enzyme and Microbial Technology. 15, 62-65.

Xu, Q., Adney, W.S., Ding, S.Y., Himmel, M.E., 2007. Cellulase for biomass

conversion. Industrial enzymes, 1st.ed. National Bioenergy Center, National Renewable Energy Laboratory, USA, 35-50.

Yanase, S., Hasunuma, T., Yamada, R., Tanaka, T., Ogino, C., Fukuda, H., Kondo,

A., 2010. Direct ethanol production from cellulosic materials at high temperature using the thermo tolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes. Applied Microbiology and Biotechnology, 88, 381-388.

Yang, B., and Wyman C.E., 2008. Characterization of the degree of polymerization of

xylooligomers produced by flow through hydrolysis of pure xylan and corn stover with water. Bioresource Technology, 99, 5756-5762.

Yang, J., Zhang, X., Yong, Q., Yu, S., 2010. Three-stage hydrolysis to enhance

enzymatic saccharification of steam-exploded corn stover. Bioresource Technology, 101, 4930-4935.


- 47 -

Yang, S., Jiang, Z., Yan, Q., Zhu, H., 2008. Characterization of a thermostable extracellular a β- glucosdiase with activities of exoglucanse and trans glycosylation from Paecilomyces thermophila. Journal of Agricultural Food Chemistry, 56, 602-608.

Yang, S.F., Tay, J.H., Liu., 2004. Inhibition of free ammonia to the formation of

aerobic granules. Biochemical Engineering Journal, 17, 41-48. Yin, L.J., Huang, P.S., Lin, H.H., 2010. Isolation of cellulase-producing bacteria and

characterization of the cellulase from the isolated bacterium Cellulomonas sp.YJ5. Journal of Agricultural Food Chemistry, 58, 9833-9837.

Young, J.C., and McCarty, P.L., 1969. The anaerobic filter for waste treatment.

Journal of Water Pollution Control Federation, 41, 160-173. Zhang, H.Y., and Lynd, L.R., 2004. Toward an aggregated understanding of

enzymatic hydrolysis of cellulose: Non-complexed cellulase systems. Biotechnology and Bioengineering, 88, 797-824.

Zhang, S., and Lv, X., 2006. Ionic liquids-from basic research to application in

industry. Science Press, ISBN 7-03-016882-8, Beijing. Zhu, Z., Sathitsuksanoh, N., Vinzant, T., Schell, D.J., McMillan, J.D., Zhang, Y.H.P.,

2009. Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: enzymatic hydrolysis, supra-molecular structure and substrate accessibility. Biotechnology and Bioengineering, 103, 715-724.

1.1 Introduction - Shodhganga : a reservoir of Indian...

Documents

Transcript of 1.1 Introduction - Shodhganga : a reservoir of Indian...