BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

The influence of pretreatment and enzyme loading on the effectiveness of batch and fed-batch hydrolysis of corn stover

To try to improve hydrolysis yields at elevated solids loadings, a comparison was made between batch and fed-batch addition of fresh substrate at the initial and later phases of hydrolysis. Both ethanol (EPCS) and steam-pretreated corn stover (SPCS) substrates were tested at low (5 FPU) and high (60 FPU) loadings of cellulase per gram of cellulose. The fed-batch addition of fresh substrate resulted in a slight decrease in hydrolysis yields when compared with the corresponding batch reactions. A 72-h hydrolysis of the SPCS substrate resulted in a hydrolysis yield of 66% compared with 51% for the EPCS substrate. When the enzyme adsorption and substrate characteristics were assessed during batch and fed-batch hydrolysis, it appeared that the irreversible binding of cellulases to the more recalcitrant original substrate limited their access to the freshly added substrate. After 72-h hydrolysis of the SPCS substrate at low enzyme loadings, ～40-50% of the added cellulases were desorbed into solution, whereas only 20% of the added enzyme was released from the EPCS substrate. Both simultaneous and sequential treatments with xylanases and cellulases resulted in an up to a 20% increase in hydrolysis yields for both substrates at low enzyme loading. Simons' stain measurements indicated that xylanase treatment increased cellulose access, thus facilitating cellulose hydrolysis.     

Acetone–butanol–ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment

In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone–butanol–ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.     

Benefits from tween during enzymic hydrolysis of corn stover

Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. (The critical relationship was the Tween loading on the biomass, not the Tween concentration in solution.) The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 degrees C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector. Copyright 1998 John Wiley & Sons, Inc.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.     

Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase

Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition.     

Enzymatic hydrolysis of lime-pretreated corn stover and investigation of the HCH-1 Model: inhibition pattern, degree of inhibition, validity of simplified HCH-1 Model

The inhibition pattern was identified for a reaction system composed of Trichoderma reesei cellulase enzyme complex and lime-pretreated corn stover. Also, the glucose inhibition effect was quantified for the aforementioned reaction system over a range of enzyme loadings and substrate concentrations. Lastly, the range of substrate concentrations and enzyme loadings were identified in which the linear form of the simplified HCH-1 Model is valid. The HCH-1 Model is a modified Michaelis-Menton Model with non-competitive inhibition and the fraction of insoluble substrate available to bind with enzyme. With a high enzyme loading, the HCH-1 Model can be integrated and simplified in such a way that sugar conversion is linearly proportional to the logarithm of enzyme loading. A wide range of enzyme loadings (0.25-50 FPU/g dry biomass) and substrate concentrations (10-100g/L) were investigated. All experiments were conducted with an excess cellobiase loading to ensure the experimental results were not influenced by cellobiose inhibition. A non-competitive inhibition pattern was identified for the corn stover-cellulase reaction system, thereby validating the assumptions of the HCH-1 Model. At a substrate concentration of 10 g/L, glucose inhibition parameters of 0.986 and 0.979 were measured for enzyme loadings of 2 FPU/g dry biomass and 50 FPU/g dry biomass, respectively. At 5 FPU/g dry biomass, glucose inhibition parameters of 0.985 and 0.853 were measured for substrate concentrations of 10 and 100g/L, respectively. The linear form of the HCH-1 Model predicted biomass digestibility for lime-pretreated corn stover over an enzyme loading range of 0.25-50 FPU/g dry biomass and substrate concentration range of 10-100g/L.     

Influence of enzyme loading and physical parameters on the enzymatic hydrolysis of steam-pretreated softwood

Softwood is an interesting raw material for the production of fuel ethanol as a result of its high content of hexoses, and it has attracted attention especially in the Northern hemisphere. However, the enzymatic hydrolysis of softwood is not sufficiently efficient for the complete conversion of cellulose to glucose. Since an improvement in the glucose yield is of great importance for the overall economy of the process, the influence of various parameters on the cellulose conversion of steam-pretreated spruce has been investigated. The addition of beta-glucosidase up to 50 IU g(-)(1) cellulose to the enzymatic hydrolysis process resulted in increased cellulose conversion at a cellulase loading up to 48 FPU g(-)(1) cellulose. Despite very high enzyme loading (120 FPU g(-)(1) cellulose) only about 50% of the cellulose in steam-pretreated spruce was converted to glucose when all of the material following pretreatment was used in the hydrolysis step. The influence of temperature, residence time, and pH were investigated for washed pretreated spruce at a dry matter (DM) content of 5% and a cellulase activity of 18.5 FPU g(-)(1) cellulose. The optimal temperature was found to be dependent on both residence time and pH, and the maximum degree of cellulose conversion, 69.2%, was obtained at 38 degrees C and pH 4.9 for a residence time of 144 h. However, when the substrate concentration was changed from 5% to 2% DM, the cellulose conversion increased to 79.7%. An increase from 5% to 10% DM resulted, however, in a similar degree of cellulose conversion, despite a significant increase in the glucose concentration from 23 g L(-)(1) to 45 g L(-)(1). The deactivation of beta-glucosidase increased with increasing residence time and was more pronounced with vigorous agitation.     

响应面法优化玉米秸秆同步酶解发酵产乙醇条件

对汽爆玉米秸秆同步酶解发酵生产乙醇的条件进行优化。首先利用Fractional Factorial设计法对影响乙醇产量的7个因素进行评价,筛选出具有显著效应的3个因素,即反应温度、酶添加量、总反应时间,再以Box—Behnken设计法及响应面分析法确定主要因素的最佳水平,即反应温度37℃,每g纤维素添加纤维素酶32u,反应时间87h,此时乙醇体积分数达到3．69％。新工艺条件实验结果表明,乙醇体积分数在87h可达到3．76％,和原工艺相比,反应时间缩短了9h,乙醇体积分数提高了13％。     

Hydrolysis of cellulose derived from steam exploded bagasse by Penicillium cellulases: Comparison with commercial cellulase

A complete cellulase from Penicillium pinophilum was evaluated for the hydrolysis of α-cellulose derived from steam exploded sugarcane bagasse and other cellulosic substrates. α-Cellulose at 1% substrate concentration was completely hydrolyzed by Penicillium cellulase within 3 h wherein at 10% the hydrolysis was 100% within 24 h with an enzyme loading of 10 FPU/g. The hydrolysate yielded glucose as major end product as analyzed by HPLC. Under similar conditions, hydrolysis of Sigmacell (microcrystalline cellulose), CP-123 (pulverized cellulose powder) and ball milled Solka Floc were 42%, 56% and 52%, respectively. Further the hydrolysis performance of Penicillium sp. cellulase is compared with Trichoderma reesei cellulase (Accellerase^TM 1000) from Genencore. The kinetics of hydrolysis with respect to enzyme and substrate concentration will be presented.     

Recycle of cellulases and the use of lignocellulosic residue for enzyme production after hydrolysis of steam-pretreated aspenwood

Summary Various modes of substrate and enzyme addition were used to hydrolyze a 10% concentration (w/v) of steam-exploded, water-and-alkali extracted aspenwood withTrichoderma harzianum E58 cellulases. Although cellulose conversion was high (94–100%), enzyme recovery was low in all cases. Low enzyme recovery was due to a combination of thermal inactivation and adsorption of the cellulases onto the lignocellulosic residue. Enzyme recycle was not feasible as the activity of the recovered cellulases towards crystalline cellulose was low. However, the residual material from enzyme hydrolysis was a suitable carbon source for cellulase enzyme production byT. harzianum based on enzyme yield and hydrolytic potential. These residues could only be used up to a 1% substrate concentration, since at higher substrate loadings cellulase production was reduced, likely because of lignin inhibitors.     

Biomimetic catalysis for hemicellulose hydrolysis in corn stover

Efficient and economical hydrolysis of plant cell wall polysaccharides into monomeric sugars is a significant technical hurdle in biomass processing for renewable fuels and chemicals. One possible approach to overcoming this hurdle is a biomimetic approach with dicarboxylic acid catalyst mimicking the catalytic core microenvironment in natural enzymes. This paper reports developments in the use of a dicarboxylic acid catalyst, maleic acid, for hemicellulose hydrolysis in corn stover. Hemicellulose hydrolysis and xylose degradation kinetics in the presence of maleic acid was compared to sulfuric acid. At optimized reaction conditions for each acid, maleic acid hydrolysis results in minimal xylose degradation, whereas sulfuric acid causes 3-10 times more xylose degradation. These results formed the basis for optimizing the hydrolysis of hemicellulose from corn stover using maleic acid. At 40 g/L dry corn stover solid-loading, both acid catalysts can achieve near-quantitative monomeric xylose yield. At higher solids loadings (150-200 g dry stover per liter), sulfuric acid catalyzed hydrolysis results in more than 30% degradation of the xylose, even under the previously reported optimal condition. However, as a result of minimized xylose degradation, optimized biomimetic hydrolysis of hemicellulose by maleic acid can reach approximately 95% monomeric xylose yields with trace amounts of furfural. Fermentation of the resulting unconditioned hydrolysate by recombinant S. cerevisiae results in 87% of theoretical ethanol yield. Enzyme digestibility experiments on the residual corn stover solids show that >90% yields of glucose can be produced in 160 h from the remaining cellulose with cellulases (15 FPU/g-glucan).     

Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature

Background

Simultaneous saccharification and fermentation (SSF) is a promising process for bioconversion of lignocellulosic biomass. High glucan loading for hydrolysis and fermentation is an efficient approach to reduce the capital costs for bio-based products production. The SSF of steam-exploded corn stover (SECS) for ethanol production at high glucan loading and high temperature was investigated in this study.

Results

Glucan conversion of corn stover biomass pretreated by steam explosion was maintained at approximately 71 to 79% at an enzyme loading of 30 filter paper units (FPU)/g glucan, and 74 to 82% at an enzyme loading of 60 FPU/g glucan, with glucan loading varying from 3 to 12%. Glucan conversion decreased obviously with glucan loading beyond 15%. The results indicated that the mixture was most efficient in enzymatic hydrolysis of SECS at 3 to 12% glucan loading. The optimal SSF conditions of SECS using a novel Saccharomyces cerevisiae were inoculation optical density (OD)₆₀₀?=?4.0, initial pH 4.8, 50% nutrients added, 36 hours pre-hydrolysis time, 39°C, and 12% glucan loading (20% solid loading). With the addition of 2% Tween 20, glucan conversion, ethanol yield, final ethanol concentration reached 78.6%, 77.2%, and 59.8 g/L, respectively, under the optimal conditions. The results suggested that the solid and degradation products’ inhibitory effect on the hydrolysis and fermentation of SECS were also not obvious at high glucan loading. Additionally, glucan conversion and final ethanol concentration in SSF of SECS increased by 13.6% and 18.7%, respectively, compared with separate hydrolysis and fermentation (SHF).

Conclusions

Our research suggested that high glucan loading (6 to 12% glucan loading) and high temperature (39°C) significantly improved the SSF performance of SECS using a thermal- and ethanol-tolerant strain of S. cerevisiae due to the removal of degradation products, sugar feedback, and solid’s inhibitory effects. Furthermore, the surfactant addition obviously increased ethanol yield in SSF process of SECS.

     

Fractal kinetic analysis of the enzymatic saccharification of CO2 laser pretreated corn stover

The enzymatic hydrolyses of laser pretreated corn stover as a novel pretreatment method were examined to establish a simplified kinetic model for the complicated hydrolysis process. The time dependence of the total reducing sugars amount was closely related to the amounts of cellulosic materials and amounts of cellulase. The evaluated model fitted very well with the experimental data of enzymatic hydrolysis of laser pretreated corn stover under different conditions, including cellulase loading, nature of substrate, substrate loading in the reaction medium. The results indicated that the complex kinetics of cellulase enzymatic saccharification could be assessed with the fractal kinetic model. The cellulase enzymatic reaction process was effectively predicted and controlled with the kinetic model. The result showed that the model could effectively reflect dynamic process of enzyme hydrolysis.     

Enzyme regeneration during hydrolysis of steam-pretreated willow and requirement for cellulase complex composition

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willow Salix caprea by mixed cellulase of Trichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and permanent mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5:1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5-10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identified T. reesei cellobiohydrolase II as the predominant component, whereas clear domination of T. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to hydrolysis residual solids.     

稀碱预处理棕榈残渣制备纤维乙醇

以棕榈残渣(Empty fruit bunch,EFB)为原料,通过预处理、酶解、发酵等过程制备纤维乙醇.首先对比了碱、碱/过氧化氢等预处理条件对棕榈残渣组成及酶解的影响,结果表明稀碱预处理效果较好.适宜的稀碱预处理条件为:NaOH浓度为1％,固液比为1∶10,在40℃浸泡24 h后于121℃下保温30 min,在该条件下,EFB的固体回收率为74.09％,纤维素、半纤维素和木质素的含量分别为44.08％、25.74％和13.89％.对该条件下预处理后的固体样品,以底物浓度5％、酶载量30 FPU/g底物酶解72 h,纤维素和半纤维素的酶解率分别达到84.44％和89.28％.进一步考察了酶载量和底物浓度对酶解的影响以及乙醇批式同步糖化发酵,当酶载量为30 FPU/g底物,底物浓度由5％增加至25％时,利用酿酒酵母Saccharomyces cerevisiae(接种量为5％,VIV)发酵72 h后乙醇的浓度分别为9.76 g/L和35.25 g/L,可分别达到理论得率的79.09％和56.96％.     

Single cell oil production in solid-state fermentation by Microsphaeropsis sp. from steam-exploded wheat straw mixed with wheat bran

Microsphaeropsis sp. was used to produce SCO in solid-state fermentation (SSF) from a substrate consisting of steam-exploded wheat straw (SEWS) and wheat bran (WB). The yield of SCO was 42 mg/g dry substrate (gds) without adding cellulase. To achieve a higher SCO yield, cellulase was added to the solid-state medium, resulting in an increase of SCO from 42 to 74 mg/gds with a cellulase loading of 10 FPU/gds. Other SSF parameters such as ratio of SEWS to WB of the dry substrate, initial moisture content, and incubation temperature were optimized under the condition of cellulase loading of 10 FPU/gds. So optimized, the SCO yield was 80 mg/gds, and the SCO content of the dry fermented mass was 10.2%. This research explored a novel method to produce SCO from the abundant and cheap agricultural residues - wheat straw and wheat bran.     

Optimization of pH controlled liquid hot water pretreatment of corn stover

Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 degrees C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.     

Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery

A cycle spray flow-through reactor was designed and used to pretreat corn stover in dilute sulfuric acid medium. The dilute sulfuric acid cycle spray flow-through (DCF) process enhanced xylose sugar yields and cellulose digestibility while increasing the removal of lignin. Within the DCF system, the xylose sugar yields of 90–93% could be achieved for corn stover pretreated with 2% (w/v) dilute sulfuric acid at 95 °C during the optimal reaction time (90 min). The remaining solid residue exhibited enzymatic digestibility of 90–95% with cellulase loading of 60 FPU/g glucan that was due to the effective lignin removal (70–75%) in this process. Compared with flow-through and compress-hot water pretreatment process, the DCF method produces a higher sugar concentration and higher xylose monomer yield. The novel DCF process provides a feasible approach for lignocellulosic material pretreatment.     

High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue

Cellulase production using corn cob residue from xylose manufacture as substrate was carried out by Trichoderma reesei ZU-02. It was found that on the same cellulose basis, the cellulase activity and yield produced on corn cob residue were comparable with that on purified cellulose. Under batch process, the optimum concentration of substrate was 40 g/l and the optimum C/N ratio was 8.0. In 500 ml flasks, cellulase activity reached 5.25 IU/ml (213.4 IU/g cellulose) after seven days' cultivation. In a 30 m(3) stirred fermenter for large scale production, cellulase and cellobiase activity were 5.48 IU/ml (222.8 IU/g cellulase) and 0.25 IU/ml (10.2 IU/g cellulose), respectively, after four days' submerged fermentation. The produced cellulase could effectively hydrolyze the corn cob residue, and the yield of enzymatic hydrolysis reached 90.4% on 10% corn cob residue (w/v) when the cellulase dosage was 20 IU/g substrate.