Enzymatic hydrolysis of sodium-hydroxide-pretreated sallow in an ultrafiltration membrane reactor

An ultrafiltration membrane reactor was used to investigate the recovery of biocatalysts during enzymatic hydrolysis of pretreated sallow. Product inhibition could be eliminated by continuous removal of products through the ultrafiltration membrane, thus retaining the macromolecular substrate and enzymes. In this way, the degree of conversion was improved from 40% in a batch hydrolysis to 95% (within 20 h), and the initial hydrolysis rate was increased up to seven times. The recovery studies were focused on mechanical deactivation and irreversible adsorption on to the nonconvertible fraction of the substrate. Cellulase deactivation during mechanical agitation was not significant, and the loss of activity was attributed mainly to strong adsorption of the enzymes onto undigested material. This process was studied in semicontinuous hydrolyses, where fresh substrate was added intermittently. The amount of reducing sugars produced in this experiment was 25.7 g/g enzyme, compared to 4.7 g/g enzyme in a batch hydrolysis.     

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5 FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively. However, when higher enzyme loadings were employed, the greater availability of the enzyme could overcome the limitations imposed by both the lignin’s restrictions on cellulose accessibility and direct binding of the enzymes, resulting in a near complete hydrolysis of the cellulose.     

Three-stage enzymatic hydrolysis of steam-exploded corn stover at high substrate concentration

The feasibility of three-stage hydrolysis of steam-exploded corn stover at high-substrate concentration was investigated. When substrate concentration was 30% and enzyme loading was 15-30 FPU/g cellulose, three-stage (9+9+12 h) hydrolysis could reach a hydrolysis yield of 59.9-81.4% in 30 h. Compared with one-stage hydrolysis for 72 h, an increase of 34-37% in hydrolysis yield could be achieved. When steam-exploded corn stover was used as the substrate for enzyme synthesis and hydrolysis was conducted at a substrate concentration of 25% with an enzyme loading of 20 FPU/g cellulose, a hydrolysis yield of 85.1% was obtained, 19% higher than that the commercial cellulase could reach under the same conditions. The removal of end products was suggested to improve the adsorption of cellulase on the substrate and enhance the productivity of enzymatic hydrolysis.     

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.     

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.     

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.     

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).     

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％.     

Optimization of enzyme loading and hydrolytic time in the hydrolysis of mixtures of cotton gin waste and recycled paper sludge for the maximum profit rate

The hydrolytic kinetics of mixtures of cotton gin waste (CGW) and recycled paper sludge (RPS) at various initial enzyme concentrations of Spezyme™ AO3117 was investigated. The experiments showed that the concentrations of reducing sugars and the conversions of the mixtures increased with increasing initial enzyme concentration. The reducing sugar concentration and conversion of the mixture of 75% CGW and 25% RPS were higher than those of the mixture of 80% CGW and 20% RPS. The conversion of the former can reach 73.8% after a 72-h hydrolysis at the initial enzyme loading of 17.4 Filter Paper Unit (FPU)/g substrate. A three-parameter kinetic model based on enzyme deactivation and its analytical expression were derived. Using nonlinear regression, the parameters of the model were determined for the experimental data of hydrolytic kinetics of the mixtures. Based on this kinetic model of hydrolysis, two profit rate models, representing two kinds of operating modes with and without feedstock recycling, were developed. Using the profit rate models, the optimal enzyme loading and hydrolytic time can be predicted for the maximum profit rate in ethanol production according to the costs of enzyme and operation, enzyme loading, and ethanol market price. Simulated results from the models based on the experimental data of hydrolysis of the mixture of 75% CGW and 25% RPS showed that use of a high substrate concentration and an operating mode with feedstock recycle can greatly increase the profit rate in ethanol production. The results also demonstrated that the hydrolysis at a low enzyme loading is economically required for systematic optimization of ethanol production.     

Sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust enzymatic saccharification of hardwoods

This study demonstrates sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust bioconversion of hardwoods. With only about 4% sodium bisulfite charge on aspen and 30‐min pretreatment at temperature 180°C, SPORL can achieve near‐complete cellulose conversion to glucose in a wide range of pretreatment liquor of pH 2.0–4.5 in only about 10 h enzymatic hydrolysis. The enzyme loading was about 20 FPU cellulase plus 30 CBU β‐glucosidase per gram of cellulose. The production of fermentation inhibitor furfural was less than 20 mg/g of aspen wood at pH 4.5. With pH 4.5, SPORL avoided reactor corrosion problem and eliminated the need for substrate neutralization prior to enzymatic hydrolysis. Similar results were obtained from maple and eucalyptus. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance

In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme–inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.     

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.     

Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion

The slow down in enzymatic hydrolysis of cellulose with conversion has often been attributed to declining reactivity of the substrate as the more easily reacted material is thought to be consumed preferentially. To better understand the cause of this phenomenon, the enzymatic reaction of the nearly pure cellulose in Avicel was interrupted over the course of nearly complete hydrolysis. Then, the solids were treated with proteinase to degrade the cellulase enzymes remaining on the solid surface, followed by proteinase inhibitors to inactive the proteinase and successive washing with water, 1.0 M NaCl solution, and water. Next, fresh cellulase and buffer were added to the solids to restart hydrolysis. The rate of cellulose hydrolysis, expressed as a percent of substrate remaining at that time, was approximately constant over a wide range of conversions for restart experiments but declined continually with conversion for uninterrupted hydrolysis. Furthermore, the cellulose hydrolysis rate per adsorbed enzyme was approximately constant for the restart procedure but declined with conversion when enzymes were left to react. Thus, the drop off in reaction rate for uninterrupted cellulose digestion by enzymes could not be attributed to changes in substrate reactivity, suggesting that other effects such as enzymes getting "stuck" or otherwise slowing down may be responsible.     

蒸汽爆破处理沙柳原料酶解条件优化

研究蒸汽爆破预处理对沙柳原料酶解效果的影响,通过响应曲面实验设计法优化蒸汽爆破处理沙柳原料的酶解工艺。结果表明,蒸汽爆破预处理沙柳原料的最佳蒸汽爆破处理条件：压力3.5 MPa、维压时间300 s; 蒸汽爆破最佳酶解条件：pH 4.8、温度53.5 ℃、 每克底物酶加量29.8 FPU。在最优条件下,蒸汽爆破处理沙柳原料的酶解率可以达到最大值87.92%,并验证了数学模型的有效性,试验结果表明蒸汽爆破预处理可以有效提高沙柳原料的水解率。     

High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids: a comparison between SPORL and dilute acid pretreatments

Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA pretreatment, the SPORL pretreatment reduced the energy required for wood chip size-reduction, and reduced mixing energy of the resultant substrate for solid liquefaction. Approximately 60% more ethanol was produced from the solid SPORL substrate (211 L/ton wood at 59 g/L with SSF efficiency of 76%) than from the solid DA substrate (133 L/ton wood at 35 g/L with SSF efficiency 47%) at a cellulase loading of 10 FPU/g glucan after 120 h. When the cellulase loading was increased to 15 FPU/g glucan on the DA substrate, the ethanol yield still remained lower than the SPORL substrate at 10 FPU/g glucan.     

Kinetics of the extracellular cellulases of Clostridium thermocellum acting on pretreated mixed hardwood and Avicel

Initial hydrolysis rates were examined for mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220 °C (PTW220) and Avicel. Linear rates were observed for fractional conversion relative to the theoretical up to 0.2 for PTW220 and 0.4 for Avicel. Initial rates were essentially unaffected by the presence of growth medium components over a range of pH values. Avicel-hydrolyzing activity was inhibited linearly by ethanol, with a 50% rate reduction at 8 wt.% ethanol. Rate saturation with either substrate or enzyme was observed in a manner qualitatively consistent with previously reported adsorption data. Although somewhat less reactive than Avicel at very low enzyme loadings, much higher reaction rates were observed for PTW220 at moderate and high enzyme loading because of its higher capacity to bind cellulase. At equal subtrate concentrations (as potential glucose) and fractional substrate coverage of 0.09, the initial rate of pretreated wood hydrolysis exceeded that of Avicel by 15-fold. For fractional substrate coverage values up to 0.09 (the maximum value achieved for PTW220), the initial rate was proportional to adsorbed enzyme for PTW220. However, the rate per adsorbed enzyme declined sharply with increasing fractional coverage for Avicel hydrolysis.     

Enzyme adsorption on SO2 catalyzed steam-pretreated wheat and spruce material

Lignocellulose is widely recognized as a sustainable substrate for biofuels production, and the enzymatic hydrolysis is regarded as a critical step for the development of an effective process for the conversion of cellulose into ethanol. One key factor affecting the overall conversion rate is the adsorption capacity of the cellulase enzymes to the surface of the insoluble substrate. Pretreatment has a strong impact on hydrolysis, which could be related to both chemical changes and morphological changes of the material. In the current work, the accessibility of four differently pretreated wheat straw substrates, two differently pretreated spruce materials, and Avicel cellulose was investigated. Adsorption isotherms (at 4 °C and 30 °C) for a cellulase preparation were obtained, and the rates of hydrolysis were determined for the different materials. Furthermore, the surface area and pore size distribution of the various materials were measured and compared to adsorption and hydrolysis properties, and the structures of the pretreated materials were examined using scanning electron microscopy (SEM).The results demonstrated a positive correlation between enzyme adsorption and the substrate specific surface area within each feedstock. Overall, the amount of enzyme adsorbed was higher for pretreated spruce than for the pretreated wheat straw, but this was not accompanied by a higher initial rate of hydrolysis for spruce. Also, the difference in the measured endoglucanase adsorption and overall FPU adsorption suggests that a larger fraction of the enzyme adsorbed on spruce was unproductive binding. The SEM analysis of the material illustrated the structural effects of pretreatment harshness on the materials, and suggested that increased porosity explains the higher rate of hydrolysis of more severely pretreated biomass.     

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.

     

Influence of steam pretreatment severity on post‐treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings

It is recognized that some form of post‐treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post‐treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post‐treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post‐treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post‐treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post‐treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post‐treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post‐treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used. Biotechnol. Bioeng. 2011;108: 2300–2311. © 2011 Wiley Periodicals, Inc.     

Enzymatic liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a horizontal rotating bioreactor

A self-designed horizontal rotating bioreactor (HRR) was applied for enzymatic hydrolysis of pretreated corn stover to improve the process economics of ethanol production. The mixing principle was based on gravity and free fall employed with tank-rotating. The liquefaction performances using the HRR and the vertical stirred-tank reactor (VSTR) with a helical impeller were compared and analyzed by measuring rheological properties of the slurry. During the enzymatic hydrolysis, viscosity decreased dramatically in the initial phase for both bioreactors and more pronouncedly for the HRR. Rheological parameters fitted to the power law showed that shear thinning properties of the slurry weakened during the reaction. The glucose concentration was used to define the efficiency of the saccharification reaction. The HRR also proved to be more efficient for glucose release with both the constant and fed-batch substrate addition modes. Liquefaction and saccharification at 25 % w/w dry matter (DM) and enzyme loading of 7 FPU/g DM resulted in the optimal glucose concentration of 86 g/kg. Results revealed a decrease in cellulose conversion at increasing initial DM, which was slighter in the HRR compared with that in the VSTR.