Brazilian Journal of Microbiology (2012): 844-856 ISSN 1517-8382 BIOPROSPECTING THERMOPHILES FOR CELLULASE PRODUCTION: A REVIEW Somen Acharya*; Anita Chaudhary Division of Environmental Sciences, Indian Agricultural Research Institute, New Delhi-110012, India. Submitted: June 01, 2010; Returned to authors for corrections: November 25, 2011; Approved: June 07, 2012. ABSTRACT Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production. Key words: Lignocelluloses, Bioethanol, cellulase, Thermophiles INTRODUCTION biotechnology, many chemical processes in different Industries such as textile, leather, pulp and paper, fruits and vegetables Bioprospecting is defined as the exploration of processing and animal feed are being replaced with biocatalysts biodiversity for commercially valuable biochemical and i.e. enzymes. Besides playing a crucial role in our food genetic resources for achieving economic and conservation production and other consumer goods and services, they have goals (28). This holds substantial promise for the development recently become vital in the production of fuel for our of novel compounds for food production and processes, automobiles. Enzymes can be harvested from a variety of consumer goods, public health, and environmental and energy sources i.e., animal tissue, plants and microbes. uses. To serve these purposes, existing diversity of Microorganisms represent an attractive source of enzymes microorganisms can act as a resource reservoir from which because they can be cultured in large quantities in a relatively individual species with special traits can be exploited (16, 26). short time period and as such they can produce an abundant, Due to the environmental related issues and advances in regular supply of desired enzyme products. Moreover, *Corresponding Author. Mailing address: Defence Institute of High Altitude Research, DRDO, C/o 56 APO, Leh-194101, India.; E-mail: [email protected] 844 Acharya, S. et al. Thermophiles for cellulase production microbial proteins are often more stable than enzymes of similar environments as the thermal vents at the bottom of the ocean, in specificity obtained from plant or animal sources and often may be Antarctica's ice, in the thermal or hot springs, in the bellies of stored under less than ideal conditions for weeks without termites, translucent stones in the dry desert valleys of Antarctica significant loss of biological activity (41). Hence most of the etc. (36, 44, 91). The third largest industrial enzyme worldwide is commercial enzymes are derived from the microorganisms (95) cellulases, because of their utility in paper recycling, cotton and are often being used in commercial processes that were processing, detergent industry and food processing industry. previously either mechanical or cellular. Microorganisms have Recently the interest in cellulases has grown across the globe, been used for millennia in the production of beer, wine, vinegar, because of its importance in the production of transportation fuel, yoghurt and cheese, but the number of potential and realized which is a driver of any economy of a country. To produce a wide applications continues to grow in other industries as well viz., variety of cellulases, both fungi and bacteria have been heavily baking industry, leather industry, paper industry, textile industry exploited. Till now, the emphasis has been placed on fungal etc. (Table 1). Many enzymes have very specific requirements of cellulases because of large amount of less complex extracellular pH and temperature before they will function and quite often, cellulases which used to be more readily cloned and produced via these requirements are different from the real situations in the recombination in a rapidly growing bacterial host. However, industrial plant. However, we now know that some recently the shift has been towards the bacterial cellulases, because microorganisms such as extremophiles can produce enzymes of robust bacterial growth, survival in harsh conditions of which can survive and function in extreme conditions, which are bioconversion processes, stability and presence of multi-enzyme generally required for these applications (34, 85). Often the complexes which provides increased function and synergy. extremophiles (14) are found in such diverse and harsh Table 1. Enzymes contributing to sustainable industrial development (41) Industry segment Enzymes Chemical process replaced Detergents Lipases, proteases, cellulases, amylases Phosphates, silicates, high temperature Textile Amylases, cellulases, catalases Acid, alkali, oxidizing agents, reducing agents, water, pumis, energy, new garment manufacture Starch Amylases, pullulanases Acids, high temperatures Backing Amylases, proteases, xylanases Emulsifying agents, sodium bisulfate Pulp and paper Xylanases, mannanases Chlorine, toxic waste Leather Proteases, lipases Sulfides, high temperature Biocatalyst Isomerases, lipases, reductases, acylases Acids, organic solvents, high temperature Plant Biomass Structure and Enzymatic Degradation oils, waxes, resin and many pigments) and proteins. These Plant biomass viz: agricultural residues have a great components build up plant biomass. Cellulose, the most potential for the production of biofuels because of its abundant polysaccharide on earth, is a highly ordered polymer abundance and inexpensive nature. There are multiple sources of cellobiose (D-glucopyranosyl-β-1,4-D-glucopyranose), of waste from agricultural and industrial processes e.g. corn representing over 50% of the biomass. Chemically cellulose fibre, corn stover, sugarcane bagasse, rice hulls, forest residues, microfibrils is a β-1,4 linked anhydro D-glucose homopolymer industrial waste, municipal solid waste and paper mill sludge. (42) with hydroxyl group in equilateral and H atoms in the Plant cell wall is mainly constituted of cellulose, axial position (Fig. 1). The glycosidic linkages acting as hemicellulose, lignin, water soluble sugars, amino acids and functional groups and three hydroxyl groups together not only aliphatic acids, ether and alcohol-soluble constituents (e.g. fats, determine the chemical properties of cellulose but also act as a 845 Acharya, S. et al. Thermophiles for cellulase production place for significant chemical and enzymatic reactions. enzymes and other helping proteins. In nature, a variety of Hemicelluloses (polyoses) are the linking material enzymes (hydrolytic and oxidative) produced by a variety of between cellulose and lignin: Wood hemicelluloses are short, fungi and bacteria, work in synergy to perform lignocellulose highly branched heteropolymers of the predominant xylose, degradation (76). Cellulose degrading enzymes (cellulases) are along with glucose, mannose, galactose and arabinose, as well widely spread in nature, predominantly produced by as different sorts of uronic acids. Depending on the microorganisms, such as molds, fungi and bacteria (8, 11). predominant sugar type, the hemicelluloses are referred to as Cellulases are the enzyme systems that hydrolyze the β-1,4 mannans, xylans or galactans. The C and C sugars, linked glycosidic bonds in the cellulose polymer to release glucose 5 6 through 1,3, 1,6 and 1,4 glycosidic bonds and often acetylated, units (71). The biochemical transformation of cellulose form a loose, very hydrophilic structure that acts as a glue molecule during biodegradation by microorganisms is between cellulose and lignin (12). catalyzed by extra cellular cellulases enzyme system. In contrast, lignin is a three-dimensional polyphenolic Three components of cellulases are responsible for network built up of dimethoxylated (syringyl), cellulose breakdown: monomethoxylated (guaiacyl) and non-methoxylated (p- 1. β-1,4 glucan glucanohydrolase (an endoglucanase) which hydroxyphenil) phenylpropanoid units, derived from the breaks down long cellulose chain to shorter fragments. corresponding p-hydroxycinnamyl alcohols, which give rise to 2. β-1,4 glucan cellobiohydrolase (an exoglucanase) acting a variety of sub-units including different ether and C-C bonds. from non reducing end of cellulose chain. Lignin is hydrophobic and highly resistant towards chemical 3. β-1,4 glucosidase, the third component breaks down and biological degradation. Lignin is phenolic in nature and is glucosidic bond of cellobiose and cellodextrins to give deposited during lignification of the plant tissue. It gets glucose molecules which can easily permeate into the cell intimately associated within the cell walls with cellulose and (10). hemicellulose and imparts the plant an excellent strength and In general two types of cellulase systems exists: one type rigidity (12, 76). consists of extracellular cellulases in filamentous fungi and in Other non-structural components of plant tissues including aerobic bacteria that act synergistically to degrade cellulose, phenols, tannins, fats and sterols, water soluble compounds while the second type is an enzyme complex called the such as sugars and starch, as well as proteins and ashes, usually "cellulosome," in anaerobic bacteria such as Clostridium represent less than 5% of the wood dry weight (63). Due to thermocellum which consists of a nonenzymatic scaffolding resistant structure of lignocellulosic biomass, efficient protein associated with various enzymatic subunits that act pretreatment technologies are needed prior to enzymatic synergistically to degrade cellulose and hemicellulose (64). hydrolysis. Mainly the enzymes involved in lignocellulosics Non complexed cellulase systems are more common and are hydrolysis are: Cellulases, Hemicellulases, lignin modifying currently most exploited for industrial applications. Figure 1. Cellulose structure 846 Acharya, S. et al. Thermophiles for cellulase production Physiological and Adaptive Aspects of Thermophilic by increased electrostatic, disulphide and hydrophobic Microorganisms interactions in their proteins (53, 74). Certain thermophilic To produce a wide variety of cellulases and enzymes are stabilized by certain conformational changes (29). hemicellulases, both fungi and bacteria have been heavily However, certain metals, inorganic salts and substrate exploited. However, the focus has been more towards the fungi molecules are also reported to impart the thermostability (94). because of their capacity to excrete abundant amount of non Based on the thermal behaviour of these enzymes, the complex cellulases and hemicellulases. Recently, this trend is Equilibrium Model has been described to reveal the effect of shifting towards the bacteria, due to their higher growth rates, temperature on enzyme activity by reversible active-inactive presence of more complex multi-enzymes and their presence in transition states (22). Due to the increasing demand of highly wide variety of environmental niches. Not only can these thermostable industrial enzymes, certain computational bacteria survive the harsh conditions, but they often produce algorithms and bioinformatic tools have been designed, which stable enzymes which may increase rates of bioconversion can predict protein rigidity and stability. Protein stabilization processes. The cell membranes of thermophiles contain can be carried out by site-directed mutagenesis, and gene saturated fatty acids which provide a hydrophobic environment shuffling (39). for the cell and maintain the cell rigidity at elevated The fungus Hypocrea jecorina (anamorph Trichoderma temperatures (43). Further, the hyperthermophilic archae have reesei) produces a complete set of cellulases classified as lipids linked with ether on the cell wall and formation of a cellobiohydrolases (CBH) endoglucanases (EG) and ß- monolayer rather than bi-layer has been suggested for stability glucosidases. Two genes encoding CBHs, cbh1 and cbh2, four of membranes at high temperatures (24). Recently, tetraether encoding EGs, egl1, egl2, egl3, and egl5, and one encoding a membrane lipids were reported in a thermoacidophilic ß-glucosidase have now been reported from this organism. The euryarchaeota Candidatus Aciduliprofundum boonei from deep promoter regions of cbh1, cbh2, egl1 and egl2 genes has CRE1 sea hydrothermal vents (81). In addition to the structural binding sites indicating fine control of these genes by carbon adaptations of cell wall and cell membrane, DNA of catabolic repression (51). ACEII binds to the promoters of cbhI thermophiles contains reverse DNA gyrase, which enhance the in Hypocrea jecorina (anamorph Trichoderma reesei) and is melting point by producing positive super coils in the DNA believed to control expression of cbh1, cbh2, egl1 and egl2 (6). imparting temperature stability (59). In Sulfolobus solfataricus Activator of Cellulase Expression protein I (ACEI) gene has a small DNA binding protein, Sso7d, not only imparts binding sites in cbh1 promoters but it acts as a repressor of thermostability to the DNA but also promotes the annealing of cellulase gene expression (5). Glucose repression of cellulase is complementary strands above the melting point and the supposed to be mediated through carbon catabolic repressor ATPase-dependent rescue of the aggregated proteins (18). protein CRE1 in Hypocrea jecorina (anamorph Trichoderma Thermophiles are reported to have a zigzag structure of surface reesei) (84). The promoter region of cellulases harbour binding layer proteins which are thermostable and resist denaturation sites for the CREI catabolic repressor protein as well as sites and proteolysis (52). Certain specialized proteins, known as for the transcriptional activators including Activator of ‘chaperons’, are produced by these organisms, which help to Cellulase Expression protein II (ACEII) besides CCAAT refold the proteins to their native form and restore their sequence, which binds general transcriptional activator functions (54, 83). Besides the above strategies, thermophilic complexes designated as ‘HAP’ proteins (69). Suto and Tomita bacteria, actinomycetes and archae tolerate high temperatures (87) has given a detailed review on cellulase expression, in 847 Acharya, S. et al. Thermophiles for cellulase production which a sensor enzyme is constitutively expressed which Moreover, not only bacteria grow rapidly compared to fungi, hydrolyzes cellulose into oligosaccharides that enter the allowing for higher recombinant production of enzymes. The bacterium and activate the expression of the cellulase genes. capacity of bacteria inhabiting wide variety of environmental niches such as high temperature, alkaline pH, and acidic pH help Cellulase Producing Thermophiles these strains to produce biocatalysts which are stable under harsh Cellulolytic fungi: The cellulolytic fungus Trichoderma conditions found in the bioconversion processes leading to sp. was considered as the best source of cellulases (79), increase rates of hydrolysis and finally efficient product recovery. however, the major bottleneck with Trichoderma cellulases is The bacterial cellulases have very high activities against that they produce very low β-glucosidase activity in culture crystalline celluloses like cotton or avicel and are also more supernatants and the enzyme is subject to product inhibition thermostable and are able to perform in an alkaline pH in (50). Mandels (62) observed that some species of thermophilic comparison to fungal cellulases (13, 61, 97). The cellulolytic fungi degraded cellulose rapidly but that their culture filtrates property has been reported in Bacillus strains (65) and in had low cellulase activity. This was contradicted by reports that thermophilic anaerobic bacterium Clostridium thermocellum the thermophilic fungi Sporotrichum thermophile (20) and (66). Fangdong (27) isolated thermophilic cellulolytic bacteria Talaromyces emersonii (30) produced cellulase activity nearly by using three different kinds of medium and determined its comparable to that of the mesophilic fungus Hypocrea jecorina enzyme activity. Acharya and Chaudhary (2) also isolated (anamorph Trichoderma reesei). Characterization of Hypocrea thermophilic cellulolytic Bacillus strains from hot spring, India jecorina (anamorph Trichoderma reesei) cellulase and and reported 600C for their optimal activity. Thermostable xylanase promoters has recently been evaluated by Rahman et cellulases of archaeal origin showing optimal activity at 102- al. (77). Cellulolytic rates of some thermophilic fungi 1050C have been isolated from Pyrococcus furiosus (48) and Chaetomium thermophile, Sporotrichum thermophile and Pyrococcus horikoshii (3). Sulfolobus solfataricus MT4, Thermoascus aurantiacus has been observed to be two or three Sulfolobus acidocaldarius and Sulfolobus shibatae have also times greater than that of Trichoderma viridae (89). been shown to produce significant amount of β-glucosidases (35). Chellapandi and Jani (17) studied on production of Highly thermostable cellulases acting at 950C, pH 6.0 and 7.0 has endoglucanase by the native strains of Streptomyces isolates in been reported from Thermotoga maritima MSB8 (15). The other submerged fermentation in Brazil. species of the same organism i.e. Thermotoga sp. FjSS3-B1 also In general, crystalline cellulose has been found to be a produced highly thermostable cellobiose which was active at superior carbon source for cellulase production in thermophilic 1150C at pH 6.8-7.8 (80). Endocellulase, with the ability to fungi than its amorphous or impure forms (31, 78) except hydrolyze microcrystalline cellulose, was isolated from the Thermoascus aurantiacus (47), Humicola insolens (40) and H. extremely thermophilic bacterium Anaerocellum thermophilum grisea var. thermoidea which showed high cellulase and (100) and maximal activity of this enzyme was observed at pH xylanase activities even on hemicellulosic substrates without 5.0-6.0 and 85-950C. cellulose. Like the mesophilic fungi, the thermophilic fungi produced multiple forms of the cellulase components. Fermentative Processes for Microbial Cellulase Production Cellulolytic Bacteria: In nature, fungi tend to produce more Currently, industrial demand for microbial production of cellulases than bacteria, however, cellulases produced by bacteria cellulases is being met by production methods using are better catalyst as they encounter less feedback inhibition. submerged fermentation (SmF) processes and widely studied 848 Acharya, S. et al. Thermophiles for cellulase production organism used generally genetically modified strains of (73) also describes the tremendous potential of SSF for the Hypocrea jecorina (anamorph Trichoderma reesei). Although production of various enzymes of industrial importance and the high cost of production in SmF systems due to increased their direct agro-biotechnological applications as silage or feed fermentation time with low productivity, has resulted in a shift additive, ligno-cellulosic hydrolysis and natural fibre (e.g. jute) towards the solid state fermentation (SSF) systems but the processing. Da-Silva et al. (23) also produced xylanase and advantage of better monitoring and handling are still associated CMCase on SSF in different residues by Theroascus with the submerged cultures (86). Though there are reports on aurantiacus. There has been attempts to produce cellulase cellulases production by SSF, the large scale commercial through fed batch instead of batch processes which helps to processes are still using the proven technology of SmF because overrides the repression caused by accumulation of reducing the SSF is still non competitive. The appropriate technology, sugars. operation controls and improved bioreactor design may make it viable e.g. the enzyme in SSF crude product after concentration Industrial Uses of Thermophilic Cellulases can be directly use in agro-biotechnological applications viz. Thermophilic enzymes are ideal biocatalysts for modern silage or feed additive, ligno-cellulosic hydrolysis, and for biotechnology because of their thermostability (38) and better processing of natural fiber. Solid state fermentation (SSF) may yields under extreme operational conditions (4). The wide become a competitive method for the production of cellulases range of applications of thermophilic cellulases is being listed as it offers numerous advantages such as high productivity, in Table 2. Potential applications are in food, animal feed, relatively high concentrations of the products and less effluent textile, fuel, chemical industries, paper and pulp industry, generation. Tengerdy (92) compared cellulase production in waste management, medical/pharmaceutical industry, SmF and SSF systems and indicated that there was a 10 fold protoplast production, genetic engineering and pollution reduction in the production cost in SSF than SmF. Pandey et al. treatment (90). Table 2. Bioconversion reactions and applications of thermostable enzymes (38) Enzyme Temperature range Bioconversions Applications (ºC) α-amylase (bacterial) 90-100 Starch to dextrose syrups Starch hydrolysis, brewing, baking, detergents α-amylase (fungal) 50-60 Starch to dextrose syrups Production of maltose Pullulanase 50-60 Starch to dextrose syrups Production of glucose syrups Xylanase 45–65, 105a Craft pulp to xylan+lignin Pulp and paper industry Chitinase 65–75b Chitin to chitobiose Food, cosmetics, pharmaceuticals, Chitin to N-acetyl glucosamine agrochemicals (chitibiase) N-acetyl glucosamine to glucosamine (deacetylation) Chitin to chitosan (deacetylase) Cellulase 45–55, 95c Cellulose to glucose Cellulose hydrolysis, polymer degradation in detergents Protease 65–85 Protein to amino acids and Baking, brewing, detergents, leather peptides industry Lipase 30–70 Fat removal, hydrolysis, Dairy, oleo chemical, detergent, interesterification, alcholysis, pulp, pharmaceuticals, cosmetics aminolysis and leather industry DNA polymerase 90–95 DNA amplification Genetic engineering/PCR a Xylanase from Thermotoga sp. b Within this range enzyme activity was high. c Cellulases from Thermotoga sp. 849 Acharya, S. et al. Thermophiles for cellulase production Food Processing: Thermophilic cellulases play a primary Paper Processing: In the Pulp and Paper mill, mechanical role in food biotechnology. Macerating enzymes complex pulping process of the woody materials lead to bulky and stiffed (cellulases, xylanases and pectinases) is being used for extraction pulp, however the pulp prepared with thermophilic cellulases not and clarification of fruit and vegetable juices (9, 70). Their use only leads to energy saving but also improves the mechanical improves cloud stability and texture and decrease viscosity of the strength of the pulp (75). Besides this cellulases have also been nectars and purees from tropical fruits. By infusion of enzymes shown to enhance the bleachability of the pulp (25) and deinking such as pectinases and β-glucosidases, texture, flavour and aroma of different types of paper wastes. The main benefit which is being properties of fruits and vegetables can be improved. The mixture drawn from the biobleaching is that of less alkali usage, reduction of pectinases, cellulases and hemicellulases can also be used for in fine particles in pulp and improved fiber brightness (25, improved extraction of olive oil. 68). Thus the use of cellulases alone or in combination with Textile Processes: The textile industry uses thermophilic xylanases has not only improved the overall performance of the cellulases for creating the stone washed look in jeans, biopolishing paper mills but they can also be used in preparation of easily of cotton and other cellulosic fabrics. Commonly the thermophilic biodegradable cardboard, manufacturing of paper towels and cellulases are used in the stone washing of jeans to make them sanitary paper. appear faded (93, 94). During the stone washing process, Ethanol Fuel Production: Two main approaches have been cellulases act on the cotton fabric and break off the small fiber developed in parallel for conversion of lignocellulose to ethanol - ends on the yarn surface, thereby loosening the dye, which is “acid based” and “enzyme based” (32, 37, 57). Biomass easily removed in the wash cycle. These enzymes are also being hydrolysis, i.e. the depolymerization of the biomass added in the detergents for decreasing the discolouration and polysaccharides to fermentable sugars, must be performed via fuzzing effects caused by numerous washes (21, 99). During environmentally friendly and economically feasible technologies repeated washing most cotton blended garments, tend to become (60). The enzyme based ethanol production (Fig. 2) has an fluffy and dull, due to the presence of partially detached advantage over chemical procedure, because of its higher microfibrils on the surface. The cellulases present in these conversion efficiency, the absence of substrate loss due to detergents can remove these microfibrils and restore a smooth chemical modifications and the use of more moderate and non- surface and original colour to the garments. Svetlana et al. (88) corrosive physical-chemical operating conditions. Atsushi et al. used the cellulases for treatment of raw cotton fibers i.e. non (7) directly produced ethanol from barley β-glucan by shake yeast woven fabrics. using Aspergillus oryzae β-glucosidase and endoglucanase. Figure 2. Enzyme production component within the ethanol production 850 Acharya, S. et al. Thermophiles for cellulase production Cellulase Production Costs and Challenges sources are desired. Recently agricultural and other waste The search for greener fuels, as well as more residues are being used for cellulase production, however, due environmental friendly technologies in different industries has to the high cost of the utilization process, the renewed the interests in cellulases. However, the enzyme cost commercialization of this technology has been hampered (56). is considered to be a major impediment in its extensive The biomass needs to be pre-treated to expose the cellulose commercialization. Production of enzymes by microorganisms fibre to the enzymes and high concentrations of enzymes are offers enormous advantages over the conventional chemical required which accounts for more than 50% of the ethanol techniques, as they can be produced in copious amounts by production cost (96). Three different approaches have been established fermentation techniques. Cellulase yields depend proposed to reduce the cost of cellulase: (i) improve on-site on a complex interrelationship of variety of factors such as production of cellulase; (ii) optimized reconstitution of inoculums size (carbon source and cellulose quality), pH value, cellulase components from different sources into a more temperature, presence of inducers, medium additives, aeration effective artificial cellulase system and (iii) development of and incubation period etc. (46). Ghosh and Ghosh (33) studied improved ethanologenic biocatalysts which supply a portion of the relationship between growth conditions and cellulase the cellulase needed for the direct microbial conversion of productions. For enzyme to be commercially successful, yield cellulose into ethanol. These improved enzyme preparations of 1100 FPUL-1h-1(FPU= Filter paper unit) is needed which can must present different characteristics, such as higher catalytic be obtained from the culture with a growth rate of 70g L-1h-1 efficiency, increased stability at elevated temperatures and (12). This high growth rate can be achieved if the certain pH and higher tolerance to end-product inhibition (98). microorganisms show any one or both of the following For lowering the cost of cellulase production different properties: strategies such as development of recombinant organisms, i) a high enhanced capacity for cellulase production; metabolic engineering and native engineering etc. can be ii) An ability to produce enzymes with a high adopted (55) (Fig. 3). It is well recognized that the economic specific activity viability of biomass ethanol depends on the enzyme cost Moreover, the amount, nature and composition of contribution. Novozymes the largest enzyme supplier has lignocellulosic substrates (Table 3) and sugars present in already obtained a 40% reduction in cellulase enzyme costs medium also affect the cellulase biosynthesis (67). Acharya et and now they can supply enzymes at a cost of US$0.5 per al. (1) studied the optimization for cellulase production by gallon of ethanol produced. At this price the conversion Aspergillus niger using saw dust as substrate and different processes has started looking attractive. However, to support fungal species producing cellulases were used by Khan et al. an economical and robust biorefinery industry, a further (49) for bioconversion of rice straw. Ojumu et al. (72) reported decrease is still necessary to US$ 0.10/gallon (or 0.026/L). on cellulase production by Aspergillus flavus by using saw Data from the industry indicate that the present industrial dust, bagasse and corn cobs as substrates. The other substrates enzymes cost US$ 2.24/gallon (US$ 0.59/L). The capital costs which also can be used are corn cobs, wheat straw, sugarcane can be lowered by different routes of improving the enzyme bagasse, aspen wood and waste from newspaper industry (58). efficiencies which involves the development of enzymes with Cellulase is an inducible enzyme and Sophorose, is an more heat tolerance and higher specific activities, and better indispensable inducer of cellulase activity. In order to lower the corresponding enzymes for different plant cell-wall polymers. production costs of cellulases, cheaper carbon and nitrogen 851 Acharya, S. et al. Thermophiles for cellulase production Table 3. Percentage composition of different lignocellulosic substances Lignocellulosic substances Cellulose Hemicellulose Lignin Coniferous wood 40 – 50 20 -30 25 – 35 Deciduous wood 40 – 50 30 – 40 15- 20 Bagasse 37 28 21 Nut shells 25 – 30 25 – 30 30 – 40 Corn cobs 45 35 15 Corn stalks 35 25 35 Wheat straw 30 50 15 Rice straw 35 35 10 Figure 3. Organism development strategies and related fundamentals (55) Cellulase Market represent 75% of the industrial enzymes and carbohydrases are Most of the industrial enzymes (60%) are produced in the second largest group of industrial enzymes. Cellulases Europe, whereas the remaining 40% come from the United contribute to 8% of the worldwide industrial enzyme demands. States and Japan. Presently, the world enzyme market is During the period 2004-2014 an increase of approximately estimated to be worth US$ 4 billion, whereof approximately 100% in use of cellulases as a special enzyme has been 60% are attributed to industrial enzymes, with a rising projected (19). Countries such as China, India, South Korea tendency of 5.7% per year (19). In this context, hydrolases and Taiwan, which have recently emerged as industrialized 852 Acharya, S. et al. 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