Lignocellulolytic Enzymes: Potential for Biorefinery

It is considered that the enzymes secreted from filamentous fungi grown on ligno- cellulosic material produce enzymes with high activity and specificity (da Silva Delabona et al. 2012). However, due to complex nature of lignocellulosic feedstocks and the regulation of fungal cellulolytic enzyme systems, the type of secreted enzyme, enzyme titers, rate of biosynthesis, etc., varies widely (da Silva Delabona et al. 2013). Currently, most commercial cellulases are produced by Trichoderma and Aspergillus species (Banerjee et al. 2010a, b, c; Navarro et al. 2010). Table 2.4 includes some examples of commercial enzymes. The filamentous fungus Tricho- derma reesei (syn. Hypocrea jecorina) is a well-known producer of enzymes that are used for depolymerization of lignocellulosic biomass (Durand et al. 1988). Their extracellular cellulolytic system is composed of cellobiohydrolases or exoglucanases, 60-80 % (EC3.2.1.74); endoglucanases, 20-36 % (EC3.2.1.4); and P-glucosidases, 1 % (EC3.2.1.21); and all of them act in synergism in the conversion of cellulose into glucose. It has been shown that the production of enzymes

Table 2.4 Cellulolytic enzymes encoded in the T. reesei genome (Martinez et al. 2008)

Cellulase

typea

CBHI

(Cel7A)

CBHII

(Cel6)

EG1

(Cel7B)

EG2

(Cel5)

EG3

(Cel12)

EG4

(Cel61)

EG5

(Cel45)

Sum

T. reesei

1

1

1

2

1

3

1

10

aEnzymes: CBHI exocellobiohydrolase I, GH7; CBHII exocellobiohydrolase II, GH6; EG1 endoglucanase I, GH7; EG2 endoglucanase II, GH5_5; EG3 endoglucanase III, GH12_1; EG4 glycoside hydrolase family, Cel61, GH61; EG5 endoglucanaseV, Cel45

from T. reesei is dependent on carbon source and is transcriptionally regulated (Juhasz et al. 2005; Strieker et al. 2008). Transcription of the major T. reesei cellu- lase genes (CBH I/Cel7a, CBH II/Cel6a, EGI/Cel7b, EGII/Cel5a, EGIII/Cel12a, EGIV/Cel61a, and EGV/Cel45a) is induced by cellulose and wide variety of mono- or disaccharides including cellobiose, xylose, lactose, and sophorose (Nogawa et al. 2001; Olsson et al. 2003). T. reesei is also an excellent producer of hemicellulases, and more than ten hemicellulase genes have been identified from T. reesei genome (Martinez et al. 2008). The major hemicellulolytic enzymes produced by T. reesei are xylanases (Xynl, Xyn2, Xyn3, and Xyn4) and mannanase (Man1), p-xylosidase (Bxll), and three a-galactosidases (Agl1, Agl2, and Agl3) (Margolles-Clark et al. 1996; Foreman et al. 2003). Like cellulase, hemicellulase genes are also dependent on carbon source, for example, xylanase and p-xylosidase production is induced specifically by lactose and xylose, respectively (Kristufek et al. 1995; Xiong et al. 2004). When T. reesei Rut C-30 was grown on cellulose with partially removed hemicellulose, lignin and pectin were used as carbon source resulting in endogluca- nase (0.19 U/mL) and endoxylanase (0.17 U/mL) activity production, whereas a-arabinosidase (0.009 U/mL) after 106 h of fermentation (Olsson et al. 2003). When T. reesei was cultivated on xylose and lactose, FPase activity was found to be 0.62 and 2.07 pmol glucose/min/mg protein and xylanase activity 6.18 and 4.91 pmol xylose/min/mg protein, on xylose and lactose, respectively. There are more p-glucosidase (bgl1) and p-galactosidase (Bga1) in the lactose-grown culture, whereas more xylanases and p-xylosidase were produced in the xylose-grown culture than in the lactose-grown culture (Jun et al. 2013). It was also noticed that xylanase activity was nine times higher (~510 IU/mL) when T. reesei Rut C-30 was grown on L-arabinose-rich plant hydrolysates as carbon sources such as oat husk as compared to those grown on lactose (~60 IU/mL) (Xiong et al. 2005). It has further been shown that T. reesei Rut C-30 cultures on alkali-treated samples result in higher enzyme production than acid-treated samples, 0.72 and 0.44 x 106 U m-3 (cellulase) from alkali-treated and acid-treated sugarcane bagasse, respectively, and 0.77 and 0.54 x 106 U m-3 from alkali-treated and acid-treated corn stover, respectively. Xylanase productions were 2.15 and 1.55 x 106 U m-3 (cellulase) from alkali- treated and acid-treated sugarcane bagasse, respectively, and 2.14 and 1.51 x 106 U m-3 from alkali-treated and acid-treated corn stover, respectively (Zhang et al. 2012). Doppelbauer et al. (1987) produced cellulases from T. reesei using steam- pretreated wheat straw as the carbon source and obtained 1.9 FPU mL-1 of the product.

Many attempts have been made to reduce the enzymatic hydrolysis process cost and improve the overall process yield with the emphasis on enzyme production and enzyme activity (Dashtban et al. 2009).

Cellulase production from T. reesei-T. reesei is the main industrial source of cel- lulases and hemicellulases (Tables 2.4 and 2.5) used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels. T. reesei is an extraordinarily efficient producer of extracellular enzymes, with certain industrial strains producing 100 g of extracellular protein per liter (Cherry and Fidantsef 2003). Figure 2.5 demonstrates simple flow diagram of cellulase production from

Table 2.5 Hemicellulose-degrading enzymes encoded in T. reesei genome, arranged by GH family (Martinez et al. 2008)

Family3

GH43

GH10

GH11

GH51

GH74

GH62

GH53

GH54

GH67

GH29

GH26

GH95

Sum

T. reesei

2

1

4

0

1

1

0

2

1

0

0

4

16

aEnzymes abbreviated based on CAZyme classification20

Simplified flow diagram of the enzymatic hydrolysis using on-site enzyme from T. reesei

Fig. 2.5 Simplified flow diagram of the enzymatic hydrolysis using on-site enzyme from T. reesei

T. reesei. Cellulase production from T. reesei depends on three major factors: (1) carbon source, (2) operating factors such as agitation and pH, and (3) type of fermentation. Other factors effecting cellulase production in cultures include the nature of the cellulosic substrate, pH of the medium, nutrient availability, inducer supplementation, and fermentation temperature.

Carbon Source Cellulase preparations have been based mainly on expensive high- purity cellulose as the carbon source. Other carbon sources, such as lactose (Chaudhuri and Sahai 1993) and xylose (Schafner and Toledo 1992), have also been proposed as carbon source for the cellulase production. Moreover, lignocellulosic materials (pretreated wheat straw, corn stover, bagasse, and wood) have been showed promising results when used as carbon source in cellulase production (Juhasz et al. 2005; Jun et al. 2013). Growth of T. reesei is slow on lignocellulose compared to glucose and fructose; it can grow on ammonium phosphate, ammonium sulfate, and ammonia, but it is not able to use nitrate (Ryu and Mandels 1980). Previously it has been reported that expression of hemicellulase genes in T. reesei is also carbon source-dependent. For example, xylanase and p-xylosidase production by T. reesei is induced specifically by lactose (Kristufek et al. 1995), xylose (Kristufek et al. 1995; Xiong et al. 2004), xylan, and xylobiose (Purkarthofer and Steiner 1995).

Operating Factors Due to the nature of their filamentous growth form, mycelial suspensions are generally viscous and behave in a non-Newtonian manner because of interactions between the suspended filaments (Metz et al. 1979; Olsvik and Kristiansen 1994). One of the possible strategies to increase enzyme yields relies on enhancing biomass concentration before production. However, when biomass concentration is increased, the culture broth becomes highly shear-thinning, affecting both mixing and oxygen transfer (Gabelle et al. 2012). Rheology influences transport properties and results in poor oxygen mass transfer. This makes it difficult to achieve well-mixed and aerated suspensions.

Fermentation (Solid State and Submerged) Cellulases can be produced by both bacterial and fungal fermentation in submerged or solid-state systems (Persson et al. 1991). Solid-state fermentation (SSF) is defined as the growth of microorganisms on moist solid substrates without free flowing aqueous phase (Krishna 2005). The objective of SSF is to bring cultivated fungi or bacteria in close contact with the insoluble substrate in order to obtain the maximum nutrient concentration from the substrate for the fermentation. Previous studies have indicated that solid-state fermentation (SSF) is an attractive process for cellulases production, lower capital investment, and lower operating expenses, and high volumetric productivity (Panagiotou et al. 2003; Yang et al. 2004) makes it economical. Its disadvantages include lower overall productivity, high labor intensity, nonuniform state in the culture substrate (gradient formations of temperature, pH, water content, and concentration during the culture), difficulty in heat removal due to low thermal conductivity of substrate, and intricate operational control due to limited water availability.

Submerged fermentation can be defined as fermentation in the presence of excess water. Large-scale enzyme-producing facilities are using the technology of SmF due to better monitoring and ease of handling. Most of the commercial cellulases are produced by the filamentous fungi such as T. reesei and A. niger under SmF (Cherry and Fidantsef 2003). Submerged cultures (SmF) of filamentous fungi are widely used for the cellulase production, but SSF is preferred over SmF due to a large number of extracellular enzyme productions (Holker et al. 2004; Krishna 2005).

Some of the attempts described below focus on alternate process design and genetic engineering.

Progress in Improving Enzyme Specific Activity There are three main ways to achieve robust fungal strains, namely, cloning, mutagenesis, and co-culturing. A wide variety of thermostable cellulolytic enzymes have been cloned and successfully expressed in mesophilic microorganisms, such as Escherichia coli, T. reesei (Ramchuran et al. 2002), which increases the operating temperature of enzymes and eventually decreases the protein dosage of thermophilic enzymes (Volynets and Dahman 2011). However, the differences in codon use or improper folding of the proteins can reduce enzyme activity or lower the level of expression (Ciaramella et al. 1995; Duffner et al. 2000). Moreover, expression of heterooligomers, complex enzymes, or those requiring covalently bound cofactors can be very difficult in mesophilic hosts. Recombinant cellulase technology has been used widely. Yeast has been widely used as a host for T. reesei gene expression, whereas filamentous fungus A. oryzae has been extensively studied as a host for high expression of genome and cDNA of fungal cellulase genes of humicola species (Dalb0ge and Heldt-Hansen 1994; Takashima et al. 1996). In the previous study, higher cellulase production was studied when T. reesei genes were expressed in A. oryzae and maltose was used as carbon source and endoglucanase I showed broad substrate specificities and high activity toward several substrates compared to cellobiohydrolases I and II (Takashima et al. 1998). A new Trichoderma strain, T. harzianum, was recently studied for evaluating its potential for producing lignocellulolytic enzymes. T. harzianum was grown on delignified steam-exploded bagasse resulted into FPase

  • 0. 78 FPU/mL, xylanase 96.09 IU/mL, and p-glucosidase 19.61 IU/mL using submerged fermentation, at an initial concentration of 1 % (w/v) of substrate (da Silva Delabona et al. 2013). In order to enhance cellobiohydrolase (CBH II) production in Trichoderma reesei as CBH II component has higher specific activity than CBH
  • 1, the cbh1 strong promoter was transformed into T. reesei using Agrobacterium- mediated transformation to overexpress the cbh2 gene. The recombinant transformation resulted in T. reesei transformants with filter paper activity 28.92 ± 2.45 IU/ mL, 4.3-fold higher than that of parent strain T. reesei ZU-02, 6.71 ± 0.79 IU/mL and cellobiohydrolase activity 122.44 ± 7.42 U/mL, 5.4 times higher than that of parent strain T. reesei ZU-02, 22.49 ± 2.27 U/mL (Fang and Xia 2013).

Recently research is focusing more on the cell immobilization to facilitate the reuse of fungal spores and increase the reactor’s productivity due to increased cell density and further to simplify the product recovery. The drawback of cell immobilization is entrapment in porous matrices that imposes mass transfer limitation and thus reduces the reactor’s productivity. Recently, a rotating fibrous bed bioreactor (RFBB) was developed to immobilize mutant strain, T. viride mycelia. Cell immobilization using RFBB resulted into more FPase (0.31 IU/mL) and xylanase (0.76 IU/mL) but slightly lower cellobiase (0.22 IU/mL) and CMCase (0.61 IU/mL) activities compared to the free-cell fermentation (Lan et al. 2013). Major enzyme complex of T. viride includes the endoglucanases Cel7B (EG I), Cel12A (EG III), and Cel61A (EG IV); the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), and Cel6B (CBH IIb); and the p-glucosidase. In contrast to other Trichoderma strains, T. viride exhibits high p-glucosidase activity (5.6 ^mol/min/mg protein) and prevents end-product inhibition due to cellobiose accumulation by converting them to glucose (Zhou et al. 2008). The p-glucosidase activity of strain T. viride is similar to the activity of Penicillium verruculosum (7.0 U) (Solov’eva et al. 2005). Another study reported FPase (2.19 IU/mL), CMCase (16.46 IU/mL), p-glucosidase (4.04 IU/mL), xylanase (42.37 IU/mL), and p-xylosidase (0.12 IU/mL) from T. viride produced by mutant EU2-77 (Jiang et al. 2011). Another potential candidate for lignocellulolytic enzyme production in biorefinery can be Trichoderma sp. strain

414. It was shown that when Trichoderma sp. strain 414 was cultivated in Tanaka media based on corn fiber pulp, cellulase produced showed activity of 34 U/cm3 and xylanase activity, 37 U/cm3 (Vlaev et al. 1997). T. reesei QM 9414 was shown to produce cellulase of activity 0.09 IU/mL from alkaline-treated sugarcane bagasse (Aiello et al. 1996). Trichoderma reesei QM 9414 when grown on wheat straw as a carbon source showed very high activity of p-xylanase, p-xylosidase, and arylxylo- side (Estrada et al. 1990).

The microbial strains have undergone several screenings and mutation to increase the cellulase production and efficiency (Kubicek et al. 2009). However, the current protein level of cellulolytic enzymes still cannot meet the need of industrial application (Banerjee et al. 2010a, b, c; Navarro et al. 2010). Therefore, some other methods to improve the total activity of cellulase are highly desired. Recently, extensive efforts in cellulase engineering have been made by improving individual or non- complexed cellulase mixture using rational design or directed evolution (Percival Zhang et al. 2006). However, this method ignores the synergism between all three enzyme components. Another strategy is to reduce the enzyme cost by reconstitution of defined cellulase cocktail. It has been demonstrated that a careful combination of cellulase mixtures from fungi and bacteria show improved biomass can be biomass hydrolysis (Banerjee et al. 2010a, b, c; Liao et al. 2011). Nevertheless, the reconstitution cellulase cocktail requires several organisms (each producing individual enzyme), which again increases the cost of fermentation. Alternative strategy for cost reduction is co-expression of a whole cellulase system in one organism which means cellulase production from single organism and can focus on rational improvement of a whole cellulase system. Moreover, co-expression would help in assembling different cellulolytic characteristics such as stability at elevated temperature, pH tolerance, extreme conditions, etc., from different microorganisms into one organism, which can result into a cellulase system with higher catalytic efficiency, increased thermostability and stability at range of pH, and higher tolerance to end-product inhibition (Percival Zhang et al. 2006). To date, E. coli is most commonly used as a host for recombinant protein expression due to its rapid growth, low production cost, high yields, and ease of genome modifications. Typically, coexpression can be conducted by using either single or multiple plasmids in E. coli. In the single plasmid case, it can be either polycistronic (having a single promoter for multiple genes that are transcribed in the same mRNA) or the plasmid that can contain multiple genes, each controlled by a separate promoter (transcribed each in a distinct mRNA) (Busso et al. 2011). There are a few reports describing the coexpression of biomass-degrading enzymes in E. coli (Nakazawa et al. 2008; Grange et al. 2010). Liu and Yu (2012) developed bicistronic and dual-promoter constructs based on pET30a for co-expressing an endo-p-xylanase gene (xyn) and a P-glucosidase gene (bgl) from Trichoderma reesei QM 9414 in E. coli.

One of the ways to enhance enzymatic activity is mutagenesis. Mutagenesis represents an excellent tool to analyze the importance of specific residues in biocatalysis (Knowles 1987; Plapp 1995). In late 1969, Mandels and Weber (1969) isolated T. reesei (formerly called T. viride QM6a) as the best cellulolytic strain after screening more than 100 wild-type strains of Trichoderma species. Many mutant strains were produced using different mutagenesis techniques: UV radiation and chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) which showed higher cellu- lase activity (FPA) such as NG14 (Montenecourt and Eveleigh 1977) and RUT-C30 (Montenecourt and Eveleigh 1979). Site-directed mutagenesis methods such as saturation mutagenesis, error-prone PCR, and DNA shuffling have been used to improve specific properties of enzymes such as catalytic efficiency, thermostability (engineering a disulfide bridge at N-terminal region), and high pH tolerance (above 6) (Dashtban et al. 2009). T. reesei RUT-C30 was one of the best known mutants. T. reesei mutants isolated by mutagenesis have showed significantly higher cellu- lase activity (4-5 times higher) compared to wild strains (Montenecourt and Eveleigh 1979; Le Crom et al. 2009). Kovacs et al. (2008) have reported that wild- type Trichoderma atroviride (F-1505) produces the maximum cellulase among 150 wild-type Trichoderma. Wild strain T. atroviride was mutated using N-methyl-M- nitro-Wnitrosoguanidine (NTG) and UV light to produce T. atroviride mutants (e.g., T. atroviride TUB F-1724) which produced high levels of extracellular cellu- lases and p-glucosidase on pretreated willow as carbon source (Kovacs et al. 2008). Cellulase and hemicellulase (xylanase) activities in Penicillium verruculosum 28K mutants were improved about threefold using four cycles of UV mutagenesis. The two-stage fermentation process further provided two- to threefold increase in the production of carbohydrate-degrading enzymes (Solov’eva et al. 2005).

One of the well-known cellulase producers as a result of mutagenesis is T. reesei RUT-C30. T. reesei RUT-C30 produces insignificant amount of p-glucosidase (~1 %), which is responsible for cellobiose conversion to glucose. Some approaches have been developed to enrich cellulase mixture produced by T. reesei RUT-C30 by P-glucosidase: (1) external addition of p-glucosidase from other fungi to T. reesei cellulase preparation and (2) co-culturing of T. reesei with fungi that produce high P-glucosidase activity such as Aspergillus niger (Gutierrez et al. 1994), Aspergillus phoenicis (Duff et al. 1987; Wen et al. 2005), and A. awamori (Friedrich et al. 1987). The difference in growth conditions and other factors increases the complexity in mixed culture fermentation and (Lynd et al. 2002) (3) heterologous gene transforma- tion—where gene from one microorganism is transformed to another. For example, a p-glucosidase I coding sequence from Penicillium decumbens was transformed to T. reesei RUT-C30 into the genome of T. reesei strain RUT-C30 by Agrobacterium- mediated transformation and the transformant displayed six- to eightfold increase in P-glucosidase activity and 30 % increase in filter paper activity compared to the parent strain (Chen et al. 2013). Another example, a p-glucosidase gene, was ligated from thermophilic fungus, Talaromyces emersonii, and transformed into T. reesei RUT-C30. The transformants displayed p-glucosidase activity while no P-glucosidase activity was observed in the parent strain (Murray et al. 2004). Overexpression of bgl1 under either the egl3 or the xyn3 gene promoter increased the p-glucosidase activity 4- to 7.5-fold in transgenic strain of T. reesei PC-3-7 (Rahman et al. 2009).

Other fungal species in Penicillium genus such as P. decumbens, P. echinulatum, and P. purpurogenum have been reported to produce higher cellulase and P-glucosidase activities than T. reesei (J0rgensen et al. 2005; Martins et al. 2008;

Jeya et al. 2010; Joo et al. 2010; Chen et al. 2013). Some Penicillium strains are thermostable at varying temperature and, for larger duration such as cellulolytic preparation from Penicillium funiculosum, showed stability at both 37 and 50 °C for at least 300 h and cellulase activity 1.14 (FPase) and p-glucosidase, 2.26 U/mL, in the crude extract from sugarcane bagasse (Maeda et al. 2013). Penicillium funiculosum have shown 16 and 0.4 U mL-1 of D-xylanase (1,4-p-D-xylan xylano- hydrolase, EC 3.2.1.8) and p-D-xylosidase (1,4-p-D-xylan xylohydrolase, EC 3.2.1.37), respectively (Mishra et al. 1985). Penicillium echinulatum is among the microorganisms that show excellent potential for enzyme complex secretion to enable the efficient enzymatic conversion of biomass into ethanol. Mutants of P echinulatum are able to secrete high cellulase titers in both submerged fermentation (Dillon et al. 2006, 2011) and solid-state fermentation (Camassola and Dillon 2007). Moreover, the FPase and p-glucosidases of P echinulatum have relatively good thermal stability at 50 °C (Camassola et al. 2004), and the cellulase complex presents a ratio of FPase and p- glucosidase that favors more efficient hydrolysis of cellulose, when compared to the cellulases of T. reesei (Martins et al. 2008). Jorgensen and Olsson (J0rgensen and Olsson 2006) used steam-pretreated spruce as the carbon source to produce cellulase from Penicillium brasilianum and reported low filter paper activity, i.e., 0.59 FPU mL-1.

Acremonium cellulolyticus is another fungal strain used for cellulases production and was first isolated in 1982 from soil in Japan (Yamanobe et al. 1987). A. cellulo- lyticus produces several cellulolytic enzymes and displays high activity of P-glucosidase. Fujii et al. (2009) reported that cellulases derived from A. cellulolyti- cus are more efficient and has higher activity for cellulose conversion to glucose than T. reesei. Some study included mutagenesis to further increase the cellulase activity from A. cellulolyticus. The mutant strain A. cellulolyticus CF-2612 was recently isolated from the mutant strain C1 by random mutagenesis and found to be the most efficient producer of cellulolytic enzymes within this species (Fang et al. 2009). Hideno et al. (2011) studied cellulase production from mutant strain A. cellulolyticus CF-2612 using rice straw as carbon source and reported cellulase activity (FPase) 0.58 Umg-1, p-glucosidase 1.25 Umg-1, xylanase activity ф-xylanase) 32.13 Umg-1, p-xylosidase 0.06 Umg-1, and a-arabinofuranosidase 0.06 Umg-1.

Bacteria are another group of microorganisms that are capable of depolymeriz- ing lignocellulose. There are three categories of cellulolytic bacteria: (1) aerobes such as Pseudomonas and Actinomyces, (2) strict anaerobes such as Clostridium, and (3) facultative anaerobes such as Bacillus and Cellulomonas. Bacillus species produce a wide range of extracellular cellulolytic enzymes (Priest 1977), including strains of B. cereus (Thayer and David 1978), B. subtilis (Robson and Chambliss 1984), B. licheniformis (Dhillon et al. 1985), Bacillus sp. KSM-330 (Ozaki and Ito 1991), and alkaliphilic Bacillus (Horikoshi 1997). Previously, it has been reported that bacilli lack the complete cellulase system and that endoglucanase is the major enzyme component which are not able to hydrolyze crystalline cellulose (Robson and Chambliss 1984; Ozaki and Ito 1991; Aa et al. 1994). However, there are a few findings on some Bacillus that have shown activity on microcrystalline cellulose over detection limit (Aa et al. 1994; Kim 1995).

Co-culturing refers to cultivation of two or more microorganisms which complement each other to obtain enzyme cocktail with essentially all enzymes required for lignocellulose degradation. It is a method to improve hydrolysis of lignocellulosic biomass by use of efficient enzyme cocktail. For example, T. reesei RUT-C30 the best mutant so far produces EG and CBH in high quantity, but very low amount of p-glucosidase, an essential component of enzyme complex required for cellobiose conversion to glucose (Stockton et al. 1991). Another fungal sp. Aspergillus, especially A. niger, produces high amount of p-glucosidase, but EG and CBH production is limited (Kumar et al. 2008). Co-culturing of T. reesei and A. niger was studied intensively, and results showed enzyme cocktail with higher catalytic activity, better productivity, and substrate utilization (Maheshwari et al. 1994; Ahamed and Vermette 2008). Other fungi used for co-culture with T. reesei RUT-C30 include A. phoenicis (Duff et al. 1985) or A. niger and A. oryzae (Hu et al. 2011). Co-culturing can also be used for producing enzyme cocktails with high cellulolytic and hemicellulolytic activities as some fungal strains are good producers of cellulase and some hemicellulase (Johnson 1990; Howard et al. 2003). Examples of co-culture of cellulase- and hemicellulase-producing strains include T. reesei D1-6 and A. wentii Pt 2804 grown in a mixed submerged culture (Panda et al. 1983) and co-culture of T. reesei LM-UC4 and A. phoenicis QM329 using ammonia-treated bagasse (Duenas et al. 1995). In both cases, enzyme activities of cellulases and hemicellulases were significantly increased. Growing different fungi with different growth conditions in the same culture makes the process complex and difficult to optimize (Lynd et al. 2002).

Another strategy to increase the enzyme efficiency is heterologous gene expression. In posttranslational protein processing, fungi are used as an expression host for proteins requiring posttranslational modification, notably, protein glycosylation, proteolytic cleavage, or multiple disulfide bond formation. Typically, a fungal used for the gene expression is a high protein-secreting mutant made by random mutagenesis, and their characteristics are further modified by genetic engineering. Since T. reesei and Aspergillus are excellent producers of cellulase expressing their enzyme genes in host, filamentous fungi are a highly attractive option. Filamentous fungi Neurospora crassa and Aspergillus nidulans are genetically tractable and are among the viable options for expressing heterologous genes (Su et al. 2012). Neurospora crassa has been commonly used as a host for production of proteins used in vaccines (Allgaier et al. 2009) and other valuable enzymes (Allgaier et al.

2010). There have been some reports of filamentous fungal systems in the production of industrial proteins such as Chrysosporium lucknowense which is used for the homologous and heterologous production of industrial enzymes and other proteins; these showed high transformation frequencies, protein production at neutral pH, and low viscosity in the fermentation broth due to the use of nonfilamentous fungal strain and short fermentation duration (Punt et al. 2001, 2002). Some studies have reported Fusarium graminearum as a host for heterologous proteins. This fungus has been commonly used for the production of therapeutic food proteins (“Quorn”) (Royer et al. 1995). Some patents have reported Aspergillus strain as expression host such as Aspergillus sojae and Aspergillus japonicus (Berka et al. 1995).

Little information is available in literature as most of them are commercial proprietary information.

Although genetic engineering methods have overcome the limitation of T. reesei (product inhibition of cellulase activity and lower p-glucosidase activities) to a significant extent, they involve high capital cost which is preventing large-scale industrial use.

Purification of Individual Enzymes from Microorganisms Specific cellulases and hemicellulases are purified from fungal strains to enhance the hydrolysis of ligno- cellulosic biomass, but the process is expensive.

Addition of Isolated Accessory Enzymes Previous studies have confirmed addition of accessory enzymes such as xylanase, p-glucosidase, and cellulase cofactors such as GH61 enhances the cellulase hydrolysis of pretreated lignocellulosic biomass (Hu et al. 2011). Synergy of enzyme mixtures can be improved by developing cloning techniques for cellulases and accessory enzymes (Viikari et al. 2007). Most widely used cellulase system is T. reesei as production strain with external addition of isolated p-glucosidase. As discussed before, T. reesei is a rich source of endoglu- canase and cellobiohydrolase but produces p-glucosidase in insignificant amounts; therefore adding isolated p-glucosidase will produce enzyme mixture with all three essential enzymes required for cellulose degradation. Addition of isolated P-glucosidase is less intricate and more efficient compared to co-culturing of microorganism to obtain complete cellulase enzyme system.

Another important parameter in addition to improve enzyme efficiency is cost reduction as enzymes are expensive and significant reduction in cost is essential for commercial application of biofuels. Cost reduction is essential to support economical and robust biofuel industry and that requires continuing efforts in optimizing performances and amount of enzymes. A new strategy for achieving this objective is presented as consolidated biomass processing (CBP) that combines saccharification and fermentation together in one step including enzyme production mediated by a single microorganism or a microbial consortium (Panagiotou et al. 2005; Xu et al. 2009). Recent advances in enhancing enzyme performance have led to decrease in enzyme use, but extra cost in making tailored enzyme solutions. Previous studies have estimated the contribution of enzyme cost for the production of lignocellulosic ethanol in the range of $0.10-0.40gal, $0.10/gal (Aden and Foust 2009), $0.30/gal (Lynd et al. 2008), $0.35/gal (Klein-Marcuschamer et al. 2010), and $0.40/gal (Kazi et al. 2010). The enzymatic hydrolysis cost can also be significantly decreased by producing enzymes on-site using biorefinery resources. Improving the cellulase- producing strains, using inexpensive carbon source such as biomass fractions from the biorefinery instead of pure glucose, lactose, sophorose, or wheat bran, would certainly make the enzymatic hydrolysis process economical and sustainable. One of the advantages of using the same lignocellulosic material for enzyme production and hydrolysis is that it could reduce the production costs of second-generation ethanol, since both processes could be co-located and share infrastructure and utilities (da Silva Delabona et al. 2013). Another advantage of cultivating the microorganisms using the same lignocellulosic material that is going to be hydrolyzed could be means of producing enzymes that are optimal for the hydrolysis of that specific material (Jprgensen and Olsson 2006; Sprensen et al. 2011). The feasibility of overall biofuel conversion process can only be improved when individual processing steps will be optimized. The concerted efforts are needed to advance toward a more consolidated process, making biofuel production more economically attractive that includes raw feedstock processing, pretreatment, enzymatic hydrolysis, fermentation, product recovery, and product marketing.

 
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