Cellulase Hydrolysis Research Articles

A Single Mutation in the Carbohydrate-Binding Module Enhances Cellulase Activity in Bacillus Amyloliquefaciens Mutant

From our earlier work, we modified the carbohydrate-binding module (CBM) of Bacillus amyloliquefaciens to increase cellulase activity using cold plasma technology. The cellulase gene (BglC-M) from the mutant was expressed in Escherichia coli BL21(DE3) under the T7 promoter. The hydrolysis activity of the cellulase mutant (BglC-M) was approximately 2.5-fold higher than the control (BglC-W) over a wide range of pH and temperature conditions. The amino acid sequence of the mutant BglC-M contained 471 residues that were almost identical to the control BglC-W. Only a single amino acid, lysine, was replaced by glutamic acid at position 370 (K370E) within the carbohydrate-binding module (CBM). Structure prediction and substrate docking of BglC-M indicated that the single mutation (K370E) might involve cellulose binding of the β-sandwich facilitated by hydrogen bonding. The docking study of cellopentaose with the model structure of BglC-M indicated that the replacement of lysine-370 led to the formation of a hydrogen bond with 436Y, which has a shorter distance (2.6 Å) compared with the control (5.4 Å). As a result, the structure becomes more compact and stable, resulting in increased catalytic efficiency. Finally, the biomass hydrolysis ability of cellulase was investigated on lignocellulosic wastes such as pineapple peel, corncob, and durian peel. The BglC-M enzyme showed a more significant amount of reducing sugar released from all lignocellulosic wastes than the control. This was the first evidence that altering the base composition of the cellulose binding module enhanced the catalytic activity.
 HIGHLIGHTS
 
 Increasing cellulase activity of Bacillus amyloliquefaciens using plasma technology
 Mutation at cellulose-binding module enhance cellulase hydrolysis activity
 Greater cellulase activity in the hydrolysis on lignocellulosic wastes
 
 GRAPHICAL ABSTRACT

Effect of carbohydrate binding modules alterations on catalytic activity of glycoside hydrolase family 6 exoglucanase from Chaetomium thermophilum to cellulose

Exoglucanase (CBH) is the rate limiting enzyme in the process of cellulose degradation. The carbohydrate binding module (CBM) can improve the accessibility of cellulase to substrate, thereby promoting the enzymatic hydrolysis of cellulase. In this study, the influence of CBM on the properties of GH6 exoglucanase from Chaetomium thermophilum (CtCBH) is systematically explored from three perspectives: the fusion of exogenous CBM, the exogenous CBM replacement of its own CBM, and the deletion of its own CBM. The parental and reconstructed CtCBH presented the same optimum pH (6.0) and temperature (60 °C) for maximum activity. Fusion of exogenous CBM increased the binding capacity of CtCBH to Avicel by 8% and 9%, respectively, but it had no significant effect on its catalytic activity. The exogenous CBM replacement of its own CBM resulted in a 12% reduction in the binding ability of CtCBH to Avicel, and a 26% reduction in the catalytic activity of Avicel. The deletion of its own CBM significantly reduced the binding ability of CtCBH to Avicel by approximately 53%, but its catalytic activity was not obviously reduced. These observations suggest that binding ability of CBM is not necessary for the catalysis of CtCBH.

The influences of acetylation, hydroxypropylation, enzymatic hydrolysis and crosslinking on improved adsorption capacities and in vitro hypoglycemic properties of millet bran dietary fibre

The effects of acetylation, hydroxypropylation, cellulase hydrolysis and crosslinking on adsorption capacities and in vitro hypoglycemic activities of millet bran dietary fibre (MBDF) were studied. The results demonstrated that both acetylation and hydroxypropylation improved water swelling ability of MBDF, and adsorption capacities of cholesterol, cholate and copper ion on MBDF. Acetylation and hydroxypropylation also enhanced α-glucosidase and α-amylase inhibition activities, glucose-binding ability and glucose diffusion retardation index (GDRI) of MBDF. Acetylated MBDF showed the highest cholate (77.31 mg/g) and cholesterol (13.97 mg/g) adsorption capacities. The crosslinking improved adsorption of cholate, cholesterol, copper ion (25.64 mg/g) and nitrite ion (181.59 μg/g) on MBDF; but reduced α-amylase inhibition activity (p < 0.05). Moreover, cellulase hydrolyzed MBDF exhibited the highest GDRI (39.60%) and α-amylase inhibition activity (34.53%), but the lowest oil and cholate adsorption capacities. The results suggest that the modified MBDFs can be used as an ingredient of hypoglycemic foods.

Efficient production of xylooligosaccharides and fermentable sugars from corncob by propionic acid and enzymatic hydrolysis

Xylooligosaccharides (XOS) are usually produced by xylan isolation from lignocellulose by alkaline followed by enzymatic hydrolysis, but the process is complicated. Recently acid hydrolysis for XOS preparation has become popular as it is faster and easier. This study investigated a novel strategy for producing XOS from corncob using propionic acid (PA) hydrolysis, then producing monosaccharides from solid residues by cellulase hydrolysis. The effect of alkaline post-treatment on enzymatic hydrolysis was studied. The maximum XOS yield of 68.5% was achieved using 5% PA at 170 °C for 50 min. About 84% of lignin in PA-hydrolyzed corncob was removed using alkaline post-treatment. The yields of glucose and xylose reached 89.8% and 80.1%, respectively, using 5 FPU cellulase/g dry matter. The results indicated that alkaline post-treatment reduced 50% cellulase loading and improved the saccharification of PA-hydrolyzed corncob. This study presents an innovative option for efficient production of XOS and monosaccharides from corncob.

Experimental study on optimization of initial pH for photo-fermentation bio-hydrogen under different enzymatic hydrolysis of chlorella vulgaris

In the paper, Use Chlorella as raw material, HAU-M1 Photosynthetic bacteria (PSB) as hydrogen-producing bacteria, the influence of initial pH on bio-hydrogen by photosynthetic organisms from Chlorella vulgaris with diverse enzyme addition was studied. The results showed that when using cellulase as hydrolase, the optimum initial pH was 7.0 and highest bio-hydrogen was 25.99 mL/g dry cell weight. Using neutral protease as hydrolase, the optimum initial pH was 8.0 and highest bio-hydrogen was 16.47 mL/g dry cell weight. Using mixed enzyme of cellulase and protease as hydrolase, the optimal initial pH was 7.0 and highest bio-hydrogen was 27.43 mL/g dry cell weight. The bio-hydrogen from Chlorella after mixed enzymatic hydrolysis is better than that of single enzymatic hydrolysis, we think the mixed enzymatic hydrolysis of cellulase and protease was superior to the single enzymatic hydrolysis of the two enzymes, which provides a scientific reference and low-cost bio-hydrogen technology by microalgae.

Effect of four modification methods on adsorption capacities and in vitro hypoglycemic properties of millet bran dietary fibre

To improve the adsorption capacities and hypoglycemic properties of millet bran dietary fibre (MBDF), four methods including acrylate-grafting, carboxymethylation, heat assisted with cellulase hydrolysis, and enzymatic hydrolysis combined with acrylate-grafting were used. The results demonstrated that all carboxymethylation, acrylate-grafting, and enzymatic hydrolysis combined with acrylate-grafting improved soluble dietary fibre content, water swelling ability and α-amylase-inhibition activity of MBDF. They also increased oil, cholesterol, sodium cholate, copper ion and nitrite ion adsorption capacities of MBDF. But carboxymethylation, acrylate-grafting and enzymatic hydrolysis combined with acrylate-grafting decreased polyphenol content, glucose-binding ability and glucose dialysis retardation index of MBDF (p < 0.05). The heat assisted with cellulase hydrolysis increased soluble dietary fibre content, polyphenol content, sodium cholate-adsorption capacity, and hypoglycemic properties of MBDF including glucose-binding ability, glucose dialysis retardation index and α-amylase-inhibition activity; but reduced adsorption capacity of MBDF on cholesterol and copper ion (p < 0.05). Changes in structure of MBDF caused by these modification methods were proved by the results of scanning electron microscopy and Fourier-transformed infrared spectroscopy analysis. These results highlight potential applications of these modified MBDFs as ingredients of hypolipidemic and hypoglycemic foods, or scavenger of nitrite and copper ion.

Enzymatic hydrolysis of the gelatinous layer in tension wood of Salix varieties as a measure of accessible cellulose for biofuels

BackgroundSalix (willow) species represent an important source of bioenergy and offer great potential for producing biofuels. Salix spp. like many hardwoods, produce tension wood (TW) characterized by special fibres (G-fibres) that produce a cellulose-rich lignin-free gelatinous (G) layer on the inner fibre cell wall. Presence of increased amounts of TW and G-fibres represents an increased source of cellulose. In the present study, the presence of TW in whole stems of different Salix varieties was characterized (i.e., physical measurements, histochemistry, image analysis, and microscopy) as a possible marker for the availability of freely available cellulose and potential for releasing d-glucose. Stem cross sections from different Salix varieties (Tora, Björn) were characterized for TW, and subjected to cellulase hydrolysis with the free d-glucose produced determined using a glucose oxidase/peroxidase (GOPOD) assay. Effect of cellulase on the cross sections and progressive hydrolysis of the G-layer was followed using light microscopy after staining and scanning electron microscopy (SEM).ResultsTension wood fibres with G-layers were developed multilaterally in all stems studied. Salix TW from varieties Tora and Björn showed fibre G-layers were non-lignified with variable thickness. Results showed: (i) Differences in total % TW at different stem heights; (ii) that using a 3-day incubation period at 50 °C, the G-layers could be hydrolyzed with no apparent ultrastructural effects on lignified secondary cell wall layers and middle lamellae of other cell elements; and (iii) that by correlating the amount of d-glucose produced from cross sections at different stem heights together with total % TW and density, an estimate of the total free d-glucose in stems can be derived and compared between varieties. These values were used together with a literature value (45%) for estimating the contribution played by G-layer cellulose to the total cellulose content.ConclusionsThe stem section-enzyme method developed provides a viable approach to compare different Salix varieties ability to produce TW and thus freely available d-glucose for fermentation and biofuel production. The use of Salix stem cross sections rather than comminuted biomass allows direct correlation between tissue- and cell types with d-glucose release. Results allowed correlation between % TW in cross sections and entire Salix stems with d-glucose production from digested G-layers. Results further emphasize the importance of TW and G-fibre cellulose as an important marker for enhanced d-glucose release in Salix varieties.

Co-production of functional xylo-oligosaccharides and fermentable sugars from corn stover through fast and facile ball mill-assisted alkaline peroxide pretreatment

The aim of this work was to develop a feasible ball mill-assisted alkaline peroxide pretreatment followed by stepwise hydrolysis to improve the yield of xylo-oligosaccharides (XOS) and fermentable sugars. The hydrogen peroxide charge, ball-milling time, and solid-to-liquid ratio affected the compositions, particle sizes, morphology, and crystallinity of the corn stover, directly improving the following hydrolytic efficiency. The optimal pretreatment was with 0.45 g/g (H2O2: substrate) and 1:3 solid-to-liquid ratio (w/v) for 1.0 h ball-milling, resulting in 84.29% delignification. Physicochemical properties of the pretreated samples were characterized and their correlations to the enzymatic hydrolysis were revealed. Compared with one-step cellulase hydrolysis, the two-step xylanase-cellulase hydrolysis of the pretreated corn stover showed significant advance in preparing XOS, producing 69.65% (on the base of xylan content in pretreated sample) of XOS, along with 20.55% xylose, 68.94% glucose, and 21.15% gluco-oligosaccharides. The yield of XOS was 2–7 times higher than those in previous studies.

Naganishia cerealis IN1S2.5 oil production from the hydrolysate of NaOH-impregnated and catalyst steam explosion pretreated oil palm empty fruit bunch

NaOH-impregnation with catalyst steam explosion was found to be an efficient pretreatment method for oil palm empty fruit bunch (OPEFB) as a substrate for oil production by Naganishia cerealis IN1S2.5. Cellulase hydrolysis of the pretreated OPEFB yielded glucose at 0.364 g/g. Investigation of N. cerealis IN1S2.5 oil production in the OPEFB hydrolysate revealed a maximum oil yield (2.46 g/L) when the C/P molar ratio of the OPEFB hydrolysate was adjusted to 25.71, supplemented with Ca2+ and Zn2+, and set to pH 4. The N. cerealis IN1S2.5 oil was comprised of oleic (37.6%), palmitic (36.2%), and steric (17.9%) acids, all (w/w), as the major fatty acids. Predicted properties of the produced biodiesel indicated the potential of N. cerealis IN1S2.5 oil as a biodiesel feedstock.

Directly convert lignocellulosic biomass to H2 without pretreatment and added cellulase by two-stage fermentation in semi-continuous modes

Cost-competitive production of lignocellulosic H2 using biological methods is limited by energy-intensive pretreatments and expensive cellulase hydrolysis. Here we report stable two-stage fermentation to convert unpretreated energy sorghum without added cellulase to H2, where Clostridium thermocellum was fed semi-continuously with 20 g/L sorghum in the first stage and then Rhodobacter sphaeroides consumed the effluent wastes in the second stage to achieve residence times (RTs) of 60, 48, and 24 h. Glutamic acid was the preferred nitrogen source for coordinating the two-stage, two-organism fermentation. 25.0 ± 1.1 mL-H2/day in consolidated bioprocessing stage and 133.0 ± 1.5 mL-H2/day in photofermentation stage were obtained for 200 mL cultures at RT 48 h. Propionate is the main product accumulated, with cellobiose, xylose, arabinose, formate, acetate and ethanol also observed in the effluent wastes, which were degraded effectively by Rhodobacter sphaeroides with COD removal of 72.6%. These results provide references for developing low-cost H2 production from lignocellulose.

Characteristics of a recombinant Fusarium verticillioides cutinase and its effects on enzymatic hydrolysis of rice straw

The current study heterologously expressed a cutinase from Fusarium verticillioides by Pichia pastoris and investigated its properties and effects on the hydrolysis of rice straw. The optimal pH and temperature for F. verticillioides cutinase were 8.0 and 50 °C, respectively. F. verticillioides cutinase had poor thermal stability and could be inhibited by some metal ions, inhibitors, and detergents (5 mM), including Ni2+, Zn2+, Cu2+, Ca2+, Mn2+, sodium dodecyl sulfate, EDTA, and Tween-20. F. verticillioides cutinase could tolerate 15% methanol and dimethyl sulfoxide but was significantly repressed by 15% ethanol and acetone with 48% and 63% residual activity, respectively. F. verticillioides cutinase could degrade the cuticle of rice straw with palmitic acid and stearic acid as the main products. However, the dissolving sugars released from the rice straw treated with F. verticillioides cutinase were significantly reduced by 29.2 μg/mL compared with the control (107.9 μg/mL). Similarly, the reducing sugars produced from the cellulase hydrolysis of rice straw pretreated with F. verticillioides cutinase were reduced by 63.5 μg/mL relative to the control (253.6 μg/mL). Scanning electron microscopy results showed that numerous tuberculate or warty protrusions were present nearly everywhere on the surface of rice straw treated with F. verticillioides cutinase, and some protrusions even covered and blocked the stomata of the rice straw surface. Current limited data indicate that F. verticillioides cutinase might not be an appropriate choice for improving the utilization of agricultural straws.

Production of xylo-oligosaccharides from poplar by acetic acid pretreatment and its impact on inhibitory effect of poplar lignin

Recently, efficient production of xylo-oligosaccharides (XOS) from poplar by acetic acid (AA) pretreatment was developed; but the effect of residual lignin on subsequent cellulase hydrolysis was unclear. Herein, XOS was produced from poplar by AA pretreatment and the effect of AA pretreatment on lignin inhibition to cellulase hydrolysis was investigated. The results indicated that a high XOS yield of 55.8% was obtained, and the inhibition degree of lignin in poplar increased from 1.0% to 6.8% after AA pretreatment. Lignin was acetylated and its molecular weight decreased from 12,211 to 2871 g/mol after AA pretreatment. The increase of S/G ratio, phenolic hydroxyl, and condensed units of lignin after AA pretreatment might be reasons for this intensified inhibition. The results advanced our understanding of the structural and inhibitory properties of lignin after production of XOS from poplar with AA pretreatment, and provided references for efficient cellulase hydrolysis of poplar after AA pretreatment.

Adsorption Study of Lignin Removal from Recycled Alkali Black Liquor by Adsorption Resins for Improved Cellulase Hydrolysis of Corn Straw

Previous studies showed that aromatic compounds such as lignin, phenols, and furans were main inhibitors of cellulase hydrolysis in recycled alkali black liquor (RBL), which should be removed to improve alkali utilization. In this study, three polymeric resins, XAD-4, XAD-16N, and XAD-7HP, were evaluated for their abilities to remove lignin from alkali black liquor recycled at the third time. Adsorption conditions of adsorbent dose and equilibrium time, isotherms, and kinetics were investigated. Of three tested adsorbents, XAD-16N was the most efficient, which can remove 89.84% of lignin after adsorption at an adsorbent-to-solution ratio of 1:4 for 2.5 h. Pseudo-second-order model was efficient to represent XAD-16N and XAD-7HP adsorption kinetics. Adsorption behavior of XAD-4 on RBL was fitted better to Langmuir model, while XAD-16N and XAD-7HP adsorption were more consistent with Freundlich model. The cellulase hydrolysis rate of corn straw treated with RBL after XAD-16N adsorption combined with ozone was 86.89%, which was only 0.89% lower than that of sodium hydroxide combined with ozone treatment. Structure characterization proved that the damage of XAD-16N adsorbed RBL to corn straw was similar to that of sodium hydroxide. It indicated that adsorption was effective in inhibitor removal from RBL to improve alkali utilization.

Cloning of AA9 Polysaccharide Monooxygenase gene AN3860 into pEX2B for expression in Aspergillus oryzae

Polysaccharide monooxygenases (PMOs) catalyze oxidative degradation of recalcitrant carbohydrate chains in cellulose, starch, and chitin. In biofuel industry, the conversion of rich lignocellulose source to fermentable sugars by hydrolytic cellulases can be synergistically boosted by cellulose-active PMOs (AA9 PMOs) found in a vast number of fungi that grow on biomass. Aspergillus nidulans, a filamentous fungus, possess a dozen of PMO-encoding genes, but only the AN3860 is expressed at a high level when cultured with wheat straw as the sole carbon source. Bioinformatic analysis indicates that AN3860 belongs to type 3 AA9 PMO subfamily that is capable of hydroxylating both C1 and C4 of the glycosidic linkages. Therefore, AN3860 may be a potential enzyme to improve cellulose hydrolysis efficiency, which has not been characterized. In this study, we describe the AN3860 cloning into Aspergillus oryzae AUT1-PlD. To facilitate the purification of AN3860, we added a CBM20 tag to its C-terminal. The recombinant vector was designed and constructed successfully. Simultaneously, we have obtained the clone of A. oryzae carrying the target gene by the ATMT method. Further expression optimization and characterization of AN3860 by both activity assays and spectroscopic techniques are underway.

Comprehensively Understanding Enzymatic Hydrolysis of Lignocellulose and Cellulase–Lignocellulose Adsorption by Analyzing Substrates’ Physicochemical Properties

In order to understand the relationship among enzymatic hydrolysis of lignocellulose, cellulase adsorption characteristics, and physicochemical properties of lignocellulose, a series of investigations was conducted, including composition, surface morphology, chemical bonds, functional groups, pore characteristics, cellulase accessibility, productive cellulase adsorption, cellulase adsorption isotherm, and kinetics of sodium hydroxide (NaOH) and ammonia-treated sugarcane bagasse (SCB) substrates. The results showed that the hydrolysis efficiency of NaOH-treated SCB (87.85%) was higher than that of ammonia-treated SCB (33.65%) and NaOH-treated SCB had less lignin, looser surface, and larger specific surface area and pore volume. This suggested that better accessibility of NaOH-treated SCB to cellulase resulted in higher enzymatic hydrolysis efficiency. The data on cellulase adsorption showed that NaOH-treated SCB had more productive adsorption, less non-productive adsorption, better adsorption capacity, and smaller binding strength with cellulase, which was also beneficial to the enhancement of enzymatic hydrolysis of NaOH-treated SCB. In addition, it was found that once cellulase molecules were adsorbed onto these two SCB substrates, two types of bonds as follows might be formed: one was C-OR (R means organic groups) and the other was C-OH··NH that was a type of hydrogen bond formed between the hydrogen of amino group in cellulase and the hydroxyl group in lignocellulose. This study would help further learn the link between lignocellulosic substrate characteristics and enzymatic hydrolysis or cellulase adsorption properties of lignocellulose.

Physico-chemical properties of lignin fractions from acid pretreated corn stover and their effects on enzymatic hydrolysis of microcrystalline cellulose

Lignin has been shown to be a recalcitrance factor in many biomass conversion studies. To better understand the effects of lignin on cellulose conversion, different lignin fractions were extracted from the same dilute acid pretreated corn stover by three sequential isolation methods, namely ethanol extraction, dioxane extraction, and enzyme purification. The physicochemical properties of each lignin fraction including molecular weight distribution, surface area, surface charge, and other structural features varied, depending on the isolation methods. All three lignin fractions had negative surface charges, and ethanol-extracted lignin carried the highest surface charges, followed by dioxane-extracted lignin and cellulase-purified residual lignin. These physicochemical properties of lignin fractions also resulted in different extent of inhibitory effects on enzymatic hydrolysis of microcrystalline cellulose (MCC). Dioxane-extracted lignin exhibited the highest inhibitory effect on glucose release from MCC, followed by the cellulase-purified residual lignin fraction and ethanol-extracted lignin. Furthermore, lignin fractions with higher contents of syringyl (S) substructure and β-O-4 aryl ether interunit linkages showed a stronger negative effect on cellulase hydrolysis of MCC.

Orthogonal experimental study on preparation of chitosan oligosaccharide based on cellulase hydrolysis

The conditions of enzymatic hydrolysis of chitosan by cellulase were studied by orthogonal experiments. The effects of temperature, pH, reaction time and enzyme dosage on the decomposition efficiency of chitosan were investigated and the following conclusions were obtained by experiments: the major and minor relationship between the factors affecting the conversion of chitosan oligosaccharide prepared by cellulase hydrolysis is temperature > pH > time. The optimum conditions are as follows: at the reaction temperature of 50 °C, the pH of 4.8 and the hydrolysis time of 3.5 h. Under these reaction conditions, the content of reducing sugar is the highest, which is 1.59 mg⋅mL-1, and the economic benefit is the best.

Study on preparation of chito-oligosaccharide by cellulase hydrolysis

By using cellulase enzymatic hydrolysis preparation to obtain low molecular weight chito-oligosaccharide. The conditions for the hydrolysis of chitosan by cellulase were studied by single factor experiment. The effects of temperature, pH value, reaction time and enzyme dosage on the decomposition efficiency of chitosan were investigated.The conclusions from the experiment are as follows: the optimum conditions for the preparation of chito-oligosaccharide by cellulase hydrolysis are reaction temperature of 50 °C, pH of 4.8, enzymatic hydrolysis time of 3.5 h, the enzyme dosage of 50 U/mg, in this case its economic benefits is greatest.

Recombinant Lentinula edodes xylanase improved the hydrolysis and in vitro ruminal fermentation of soybean straw by changing its fiber structure

Soybean straw cannot be efficiently degraded and utilized by ruminants due to the complex cross-linked structure among cellulose, hemicellulose, and lignin in its cell wall. Xylanase can degrade the xylan component of hemicellulose, destroy the xylan-lignin matrix and, consequently, would theoretically improve the hydrolysis effectiveness of cellulose. Therefore, this study was performed to investigate the effects of recombinant Lentinula edodes xylanase (rLeXyn11A) on fiber structure, hydrolysis, and in vitro ruminal fermentation of soybean straw. Treatment with rLeXyn11A enhanced the hydrolysis of soybean straw with an evident increase in productions of ribose, rhamnose, and xylose. Soybean straw treated by rLeXyn11A had lower hemicellulose content and greater cellulose and lignin contents. The rLeXyn11A could remove xylan, loosen unordered fibrous networks, enhance substrate porosity, and rearrange lignin, consequently increasing the exposure of cellulose and improving the cellulase hydrolysis of soybean straw. Supplemental rLeXyn11A stimulated the dry matter digestion, volatile fatty acids production, and microbial protein synthesis during in vitro ruminal incubation. This paper demonstrated that rLeXyn11A could strengthen the cellulase hydrolysis and in vitro ruminal fermentation of soybean straw by degrading xylan and changing fiber structure, showing its potential for improving the utilization of soybean straw in ruminants.

Enzymatic hydrolysis of cassava stems for butanol production of isolated Clostridium sp.

This research focused on the hydrolysis of cassava stems (CS) and subsequent utilization as a carbon source for the cultivation of isolated Clostridium sp. To yield the highest amount of reducing sugars (RS), the studies on the pretreatment with sodium hydroxide (NaOH) and the hydrolysis with cellulases, amylases, and mixed enzymes were carried out. Afterwards, the hydrolysate was utilized for the cultivation of isolated Clostridium sp. Experimental results revealed that CS after 1.0 M NaOH pretreatment at 121 °C for 15 min and cellulase hydrolysis (Accellerase® 1500, 2500 CMC U/g CS) obtained 10.94 ± 0.29 g/L RS concentration. Hydrolysis of CS with amylases (Termamyl® 120, 1.2 U/g CS and AMG 300L™ 3.5 U/g CS) provided 34.85 ± 0.75 g/L RS and the maximum RS amount of 47.90 ± 0.39 g/L was obtained from the hydrolysis with mixed enzymes (Termamyl® 120, 1.2 U/g CS, AMG 300L™ 3.5 U/g CS followed by Accellerase® 1500, 2500 CMC U/g CS). From the cultivation of Clostridium sp. G10 using CS hydrolysate, the highest dry cell weight concentration of 1.28 ± 0.07 g/L was obtained with 11.68 ± 0.31 g/L butanol. It could be concluded that CS hydrolysate was comparable with glucose for utilization as a carbon source for butanol production.