Hydrolysis of Ammonia-pretreated Sugar Cane Bagasse with Cellulase, β-Glucosidase, and Hemicellulase Preparations

Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH₃: 1 biomass, 100 °C, 30 min) or with NH₄OH (0.5 g NH₄OH of a 28% [v/v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.     

Fractionation of Corn Fiber Treated by Soaking in Aqueous Ammonia (SAA) for Isolation of Hemicellulose B and Production of C5 Sugars by Enzyme Hydrolysis

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of β-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.     

Kinetics of Lime Pretreatment of Sugarcane Bagasse to Enhance Enzymatic Hydrolysis

The objective of this work was to determine the optimum conditions of sugarcane bagasse pretreatment with lime to increase the enzymatic hydrolysis of the polysaccharide component and to study the delignification kinetics. The first stage was an evaluation of the influence of temperature, reaction time, and lime concentration in the pretreatment performance measured as glucose release after hydrolysis using a 2³ central composite design and response surface methodology. The maximum glucose yield was 228.45 mg/g raw biomass, corresponding to 409.9 mg/g raw biomass of total reducing sugars, with the pretreatment performed at 90°C, for 90 h, and with a lime loading of 0.4 g/g dry biomass. The enzymes loading was 5.0 FPU/dry pretreated biomass of cellulase and 1.0 CBU/dry pretreated biomass of β-glucosidase. Kinetic data of the pretreatment were evaluated at different temperatures (60°C, 70°C, 80°C, and 90°C), and a kinetic model for bagasse delignification with lime as a function of temperature was determined. Bagasse composition (cellulose, hemicellulose, and lignin) was measured, and the study has shown that 50% of the original material was solubilized, lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (60–90°C). The delignification was highly dependent on temperature and duration of pretreatment.     

Relatively High-Substrate Consistency Hydrolysis of Steam-Pretreated Sweet Sorghum Bagasse at Relatively Low Cellulase Loading

Sweet sorghum bagasse (SSB) was steam pretreated in the conditions of 190 °C for 5 min to assess its amenability to the pretreatment and enzymatic hydrolysis. Results showed that pretreatment conditions were robust enough to pretreat SSB with maximum of 87% glucan and 72% xylan recovery. Subsequent enzymatic hydrolysis showed that the pretreated SSB at 2% substrate consistency resulted in maximum of 70% glucan-glucose conversion. Increasing substrate consistency from 2% to 16% led to a significant reduction in glucan conversion. However, the decrease ratio of glucan-glucose conversion was the minimum when the consistency increased from 2% to 12%. When the pretreated SSB consistency of 12% was applied for hydrolysis, increase in cellulase loading from 7.5 up to 20 filter paper units (FPU)/g glucan resulted only in 14% increase in glucan-glucose conversion compared to 20% increase with cellulase loading varying from 2.5 to 7.5 FPU/g glucan. More than 10 cellobiase units (CBU)/g glucan β-glucosidase supplementation had no noticeable improvement on glucan-glucose conversion. Additionally, supplementation of xylanase was found to significantly increase glucan-glucose conversion from 50% to 80% with the substrate consistency of 12%, when the cellulase and β-glucosidase loadings were at relatively low enzyme loadings (7.5 FPU/g and 10 CBU/g glucan). It appeared that residual xylan played a critical role in hindering the ease of hydrolysis of SSB. A proper xylanase addition was suggested to achieve a high hydrolysis yield at relatively high substrate consistency with relatively low enzyme loadings.     

Evaluation and Characterization of Forage Sorghum as Feedstock for Fermentable Sugar Production

Sorghum is a tropical grass grown primarily in semiarid and drier parts of the world, especially areas too dry for corn. Sorghum production also leaves about 58 million tons of by-products composed mainly of cellulose, hemicellulose, and lignin. The low lignin content of some forage sorghums such as brown midrib makes them more digestible for ethanol production. Successful use of biomass for biofuel production depends on not only pretreatment methods and efficient processing conditions but also physical and chemical properties of the biomass. In this study, four varieties of forage sorghum (stems and leaves) were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy and X-ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and the enzymatic hydrolysis process. Forage sorghums with a low syringyl/guaiacyl ratio in their lignin structure were easy to hydrolyze after pretreatment despite the initial lignin content. Enzymatic hydrolysis was also more effective for forage sorghums with a low crystallinity index and easily transformed crystalline cellulose to amorphous cellulose, despite initial cellulose content. Up to 72% hexose yield and 94% pentose yield were obtained using modified steam explosion with 2% sulfuric acid at 140 °C for 30 min and enzymatic hydrolysis with cellulase (15 filter per unit (FPU)/g cellulose) and β-glucosidase (50 cellobiose units (CBU)/g cellulose).     

Use of hemicellulose hydrolysate for β-glucosidase fermentation

Hydrolysis of cellulose byTrichoderma cellulases often results in a mixture of glucose, cellobiose, and low-mol-wt cellodextrins. Cellobiose is nonfermentable for most yeasts, and therefore it has to be hydrolyzed to glucose by β-glucosidase prior to ethanol fermentation. In the present study, the β-glucosidase production of onePenicillium and threeAspergillus strains, which were previously selected out of 24 strains, was investigated on steam pretreated willow. Both steam-pretreated willow and hemicellulose hydrolysate, released during steam explosion of willow, were used as carbon sources. Reference cultivation runs were performed using prehydrolyzed Solka Floc and glucose: The four strains were compared withTrichoderma reesei regarding sugar consumption and β-glucosidase production.Aspergillus niger andAspergillus phoenicis proved to be the best enzyme producers on hemicellulose hydrolysate. The maximum β-glucosidase activity, 4.60 IU/mL, was obtained whenA. phoenicis was cultivated on the mixture of hemicellulose hydrolysate and steam-pretreated willow. The maximum yield of enzyme activity, 502 IU/g total carbohydrate, was obtained whenAspergillus foetidus was cultivated on the hemicellulose hydrolysate.     

Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates

A quantitative approach was taken to determine the inhibition effects of glucose and other sugar monomers during cellulase and β-Glucosidase hydrolysis of two types of cellulosic material: Avicel and acetic acid-pretreated softwood. The increased glucose content in the hydrolysate resulted in a dramatic increase in the degrees of inhibition on both β-Glucosidase and cellulase activities. Supplementation of mannose, xylose, and galactose during cellobiose hydrolysis did not show any inhibitory effects on β-Glucosidase activity. However, these sugars were shown to have significant inhibitory effects on cellulase activity during cellulose hydrolysis. Our study suggests that high-substrate consistency hydrolysis with supplementation of hemicellulose is likely to be a practical solution to minimizing end-product inhibition effects while producing hydrolysate with high glucose concentration.     

Cloning and Characterization of Two β-Glucosidase/Xylosidase Enzymes from Yak Rumen Metagenome

Two β-glucosidase/xylosidase genes, Rubg3A and Rubg3B, were cloned from yak rumen uncultured microorganisms by metagenome method and function-based screening. Recombinant RuBG3A and RuBG3B purified from Escherichia coli were characterized for enzymatic properties, and they exhibited activity against 4-nitrophenyl-β-d-glucopyranoside and 4-nitrophenyl-β-d-xylopyranoside, suggesting bifunctional β-glucosidase/xylosidase activity. Chromatography analysis showed that they could effectively hydrolyze cellooligosaccharide substrates, indicating the facilitation in saccharification of cellulose. RuBG3A and RuBG3B can also increase the reducing sugar released in xylan hydrolysis to 218% and 169%, respectively, through synergism with xylanase, suggesting their application in hemicellulose saccharification. Molecular modeling and substrate docking showed that there should be one active center responsible for the bifunctional activity in each enzyme, since the active site pocket is substantially wide to allow the entry of both β-glucosidic or β-xylosidic substrates, which elucidated the structure–function relationship in substrate specificities. Therefore, the enzymatic properties, the participation in hydrolysis of cellooligosaccharides, and the synergism with xylanase make RuBG3A and RuBG3B very interesting candidates for saccharification of both cellulose and hemicellulose.     

Maximum Saccharification of Cellulose Complex by an Enzyme Cocktail Supplemented with Cellulase from Newly Isolated <Emphasis Type="BoldItalic">Aspergillus fumigatus</Emphasis> ECU0811

Either the natural biodegradation process or the industrial hydrolytic process requires synergistic interactions between various cellulases. However, it is sometimes impeded by low hydrolytic rate of existing cellulases and the lack of accessory enzymes. Herein, the ability of a commercial cellulase (Spezyme CP, from Genencor) to degrade steam explosion-pretreated corn stover was significantly improved. Firstly, a fungal cellulase producer, Aspergillus fumigatus ECU0811, was isolated from hundreds of soil samples. A 96-deep-well microscale-based platform was developed here to reduce the labor-intensive screening work and proved to be consistent with macroscale screening work. After optimization of fermentation, 3% corn cob could induce A. fumigatus ECU0811 to yield the highest cellulase production. Based on the high activities of β-glucosidase and xylanase by A. fumigatus ECU0811, 0.91 and 125 U/mg protein, respectively, an enzyme cocktail was composed with a fixed dosage of Spezyme CP (CPCel) at 14.2 filter paper units (FPU)/g glucan and varied dosages of A. fumigatus cellulase (AFCel). Consequently, the glucan-to-glucose conversion of corn stover was increased from 25.6% in the presence of CPCel at a dosage of 14.2 FPU/g glucan to 99.5% in the presence of the enzyme cocktail (14.2 FPU CPCel plus 1.21 FPU AFCel per gram of glucan). On the other side, it reduced the total protein amount of CPCel by as much as tenfold, which extremely improved the hydrolytic rate of Spezyme CP and reduced its dosage.     

Horticultural Waste as the Substrate for Cellulase and Hemicellulase Production by Trichoderma reesei Under Solid-State Fermentation

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), β-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and β-xylosidase (10.4 U/g SDM). The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than the commercial enzyme blend.     

Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field

The use of the intensive mass transfer reactor (IMTR) for enzymatic saccharification of cellulose, where the reaction mixture is intensively stirred by ferromagnetic particles (FMP), enhances the process rate and productivity drastically. The most significant enhancement of the process was observed when microcrystalline cellulose was used as a substrate. A concentration of sugars up to 5% was obtained after 1 h of cellulose hydrolysis using a cellulase activity level of 2 filter paper units (FPU)/mL (20 FPU/g substrate). In the hydrolysis of two types of industrial cellulosic wastes, the enhancement effects were less pronounced. Parameters related to the IMTR design, such as the shape, dimensions, and mass of FMP, as well as the magnetic field strength, strongly affected the process of hydrolysis. Among various kinds of FMP tested, the most efficient were found to be cylindrical particles (0.25 x 4 mm). In general, the hydrolysis rate enhanced when the magnetic field strength increased from 26,000 to 64,000 A/m. An optimal FMP loading existed at each level of the field strength. Hydrolyzates obtained in the IMTR under the action ofTrichoderma reesei andPenicillium verruculosum cellulases contained glucose and cellobiose as soluble products, cellobiose being predominant (> 50%). Only when a high level of extra Β-glucosidase was added to the IMTR (10 CBU/mL), did glucose made up more than 90% of the products. Owing to extreme shear conditions in the IMTR, significant enzyme inactivation took place.     

Efficient cellulose solubilization by a combined cellulosome-β-glucosidase system

The cellulosome, the multienzyme complex of the cellulase system ofClostridium thermocellum, that mediates the solubilization of insoluble cellulose, is strongly inhibited by the major end product, cellobiose. By combining a purified β-glucosidase fromAspergillus niger with the cellulosome, accumulated cellobiose was hydrolyzed thereby resulting in a dramatic enhancement (up to 10-fold) of cellulose degradation. The observed enhancement was expressed both in the rate and degree of solubilization of microcrystalline cellulose, compared with that observed for the unsupplemented cellulosome. Near-complete conversion of cellulose to glucose could be obtained from dense substrate suspensions (up to at least 200 g/L).     

The effect of agitation speed,enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 °C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2³ full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

Determination by the enzyme thermistor of cellobiose formed on degradation of cellulose

A calorimetric assay procedure for the determination of cellobiose has been developed. The cellobiose is hydrolyzed by β-glucosidase and the glucose formed is measured calorimetrically by an enzyme thermistor containing co-immobilized glucose oxidase and catalase. The system was optimized with regard to the arrangement of the enzymes, the pH-dependence of the separate enzymic steps, and of the total system. By placing the β-glucosidase in a precolumn that could be switched in and out of the flow through the enzyme thermistor, both cellobiose and glucose present in the sample could be determined. The performance with standard solutions and with crude samples from cellulose degradation experiments was investigated.     

A β-glucosidase from Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β-d-glucopyranoside hydrolysis, apparent K _m and V _max values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly inhibited by Fe²⁺ and activated about 35% by Ca²⁺. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and ¹³C nuclear magnetic resonance spectroscopy.     

Profiling Differential Expression of Cellulases and Metabolite Footprints in Aspergillus terreus

This study reports differential expression of endoglucanase (EG) and β-glucosidase (βG) isoforms of Aspergillus terreus. Expression of multiple isoforms was observed, in presence of different carbon sources and culture conditions, by activity staining of poly acrylamide gel electrophoresis gels. Maximal expression of four EG isoforms was observed in presence of rice straw (28 U/g DW substrate) and corn cobs (1.147 U/ml) under solid substrate and shake flask culture, respectively. Furthermore, the sequential induction of EG isoforms was found to be associated with the presence of distinct metabolites (monosaccharides/oligosaccharides) i.e., xylose (X), G₁, G₃ and G₄ as well as putative positional isomers (G₁/G₂, G₂/G₃) in the culture extracts sampled at different time intervals, indicating specific role of these metabolites in the sequential expression of multiple EGs. Addition of fructose and cellobiose to corn cobs containing medium during shake flask culture resulted in up-regulation of EG activity, whereas addition of mannitol, ethanol and glycerol selectively repressed the expression of three EG isoforms (Ia, Ic and Id). The observed regulation profile of βG isoforms was distinct when compared to EG isoforms, and addition of glucose, fructose, sucrose, cellobiose, mannitol and glycerol resulted in down-regulation of one or more of the four βG isoforms.     

Effects of a non-ionic surfactant,Tween 20, on adsorption/desorption of saccharification enzymes onto/from lignocelluloses and saccharification rate

In this work, we examined the role of a non-ionic surfactant, Tween 20, on enzymatic hydrolysis of lignocelluloses. Delignified lignocelluloses (pine wood chip) were used as model substrates. Effects of Tween 20 on adsorption/desorption onto/from lignocelluloses with and without hydrolysis were evaluated respectively. Tween 20 lowered the non-biospecific adsorption of β-glucosidase and enhanced the bio-specific adsorption of cellulase. Tween 20 did not affect the liquid phase reaction (cellobiose hydrolysis). However, for the solid surface reaction (cellulose hydrolysis), cellulose conversion for 72 hrs was increased 9–21% and 1–8.5% for samples with high lignin contents (PI) and low lignin contents (PIII) by injection of Tween 20 (0.024–0.24 mM), respectively. Moreover, Tween 20 increased the cellulose conversion rate substantially. It is suggested that the increase of cellulase amount adsorbed due to the increase of effective cellulose surface by Tween 20 contribute to the enhancement of cellulose conversion.     

Sulfolane pretreatment of shrub willow to improve enzymatic saccharification

Pretreatment has been regarded as the most efficient strategy for conversion of lignocellulosic biomass to fermentable sugars. In this work, sulfolane pretreatment was performed to break the intricate structure of shrub willow for inhabitation of the enzymatic accessibility to holocellulose. The effects of varying pretreatment parameters on enzymatic hydrolysis of shrub willow were investigated. It was found that sulfolane was more compatible with lignin instead of carbohydrate, and the loss of carbohydrate could be attributed to water and acid generated from sulfolane. The optimum conditions leading to maximal sugar recovery from enzymatic saccharification were confirmed. After pretreatment of shrub willow powder in sulfolane at 170 °C for 1.5 h with mass ratio of sulfolane to substrate of 5, the sugar release could reach 555 mg/g raw materials (352 mg glucose, 203 mg xylose) when combining 20 FPU cellulase, 20 CBU β-glucosidase, and 1.5 FXU xylanase, representing 78.2 % of glucose and 56.6 % of xylose in shrub willow. This enhanced enzymatic saccharification was due to delignification and removal of a proportion of hemicelluloses, as confirmed by X-ray diffraction analysis, scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis, gas chromatography, and ionic chromatography. Thus, these studies prove sulfolane pretreatment to be an effective and promising approach for biomass to biofuel processing.     

Xylanase and cellulase production byAspergillus fumigatus

When grown on a purified cellulose such as CF11 cellulose,Aspergillus fumigatus produces mainly exoglucanase (Avicelase) and endoglucanase (CMCase) with small amounts of Β-glucosidase and xylanase. In such cultures, the pH drops to 3–3.5 after 2 d incubation, which may account for the low levels of Β-glucosidase. The amounts of extracellular enzymes produced are larger when the organism is grown on hay or straw than when grown on CF11 cellulose. In particular, CMCase levels increase approximately seven times and xylanase levels increase 40–50 times. In such cultures the pH remains fairly constant at 6–7 over the 10-d incubation period used and so Β-glucosidase levels are also increased. Extraction of the hay or straw substrate with ethanol had little effect on enzyme production and so there appears to be no soluble material present that influences enzyme production. The organism produced elevated levels of CMCase and xylanase on barley straw, oat straw, and wheat straw, there being little difference between the varieties of each tested. However, grasses dried at elevated temperatures (260–500‡C) gave enzyme levels similar to CF11 cellulose. Similarly, chemical delignification of hay or straw gave enzyme levels similar to CF11 cellulose. Thus, both these treatments must lead to degradation of the hemicellulose present in the substrate. A. fumigatus was able to grow on a number of laboratory prepared and commercially available xylans (hay, barley straw, oat straw, and larch) as a pelletted mycelium. In all cases xylanase levels were increased 10–30 times over CF11 cellulose as substrate, but CMCase levels were similar to those with CF11 cellulose as substrate. Β-Glucosidase in most cases was not detectable, probably because the pH fell to 3–3.5 during incubation. Thus it appears that cellulase and xylanase can be independently induced in this organism. The optimum incubation time at 37‡C for xylanase production was 4–7 d and the optimum concentration of hay as substrate was 4–5%, even though this produces a very thick slurry that does not shake well.