Autohydrolysis extraction process as a pretreatment of lignocelluloses for their enzymatic hydrolysis

Autohydrolysis was studied as a pretreatment to enhance sugar yields from enzymatic hydrolysis of wheat and rape straw, beech, birch and poplar sawdust. Reaction temperatures were 185°C to 212°C and the reaction time 20 min. The pretreated slurries were hydrolyzed with “Novo” cellulase and Fusarium sp. 27 cellulase at 45°C and pH 4.8 for 24 h with addition of Fusarium sp. 27 cellbound cellobiase. From 85% to 90% sugar content of substrates were converted to reducing sugars after 24 h enzymatic hydrolysis, with exception of poplar wood. 10.8 g biomass was obtained after cultivation of Fusarium sp. 27 with water solution hemicellulose fraction from 100 g beech sawdust autohydrolyzed at 200°C during 20 min.     

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H₂SO₄ with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2³ central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.     

Column cellulose hydrolysis reactor: the effect of retention time,temperature, cellulase concentration and exogenously added cellobiase on the overall process

Summary A novel column cellulose hydrolysis reactor with constant enzyme recycling was operated under various conditions to determine the effects of retention time, temperature, cellulase concentration and exogenously added cellobiase on the concentration of the product stream and the productivities of the reactor. Short term (7 days) hydrolysis was best at 42°C while longer term (14 days) hydrolysis was better at 37°C. A retention time of 11 h and reactor cellulase concentration of 30 filter paper units per gram of cellulose gave the best compromise for efficient operation by minimizing product inhibition, maximizing product concentration and minimizing enzyme consumption. The addition of cellobiase to the reactor increased cellulose hydrolysis and raised the proportion of monomeric sugars in the hydrolysate. Continuous cellulose hydrolyses were maintained for 7 and 14 days at 42°C and 37°C, respectively, resulting in volumetric productivities of 6.82 and 4.84 g/l/h and average sugar concentrations of 7.3% and 6.0% (w/v), respectively. Greater than 95% (w/w) of the sugars produced were in the monomeric state. Average cellulase used for the two runs were 8.4 and 5.3 filter paper units per gram of sugar produced, respectively.     

Statistical optimization of enzymatic degradation process for oil palm empty fruit bunch (OPEFB) in rotary drum bioreactor using crude cellulase produced from Aspergillus niger EFB1

Oil palm empty fruit bunch (OPEFB) was pretreated with 2% (v/v) HNO₃ and degraded by Aspergillus niger EFB1 crude cellulase. Through 2 Level Factorial Design (2LFD), it was found that OPEFB concentration, temperature, incubation time, concentration of Tween 80 and agitation speed have significant effect in reducing sugar production. A standard Response Surface Methodology (RSM) design known as Central Composite Design (CCD) was used to optimize the enzymatic degradation condition of OPEFB in rotary drum bioreactor. Reducing sugar level of 1.183 g/L was obtained with the following optimized degradation conditions: 1.95% (w/v) OPEFB, 0.5% (v/v) Tween 80, 55 °C, 87.5 rpm in the incubation period of 3 days and 16 h. The optimal degradation condition improved reducing sugar production by 1.07 fold compared to that before optimization in shake flasks culture. The optimization strategy of enzymatic degradation of OPEFB inside rotary drum bioreactor led to increase in glucose, xylose, arabinose, galactose and mannose production by 3, 2.5, 1.64, 19.37 and 22.52 fold, respectively. The improvement in reducing sugar and polyoses production were comparable with the reduction in OPEFB cellulose and hemicellulose content by 89.32% and 48.17% respectively after enzymatic degradation in optimized condition.     

Cellulases as an aid to enhance saccharification of cassava root

Summary The conventional saccharification of cassava root by enzymatic hydrolysis is improved by using a little amount of cellulase and cellobiase in addition to conventional enzyme, glucoamylase. With new saccharification[glucoamylase 0.45SGU/g cassava, cellulase 4.5NCU/g cassava, cellobiase 0.09U/g cassava, pH 4.3, temperature 60°C, total volume 465ml : 100g of cassava/400ml of water], the reaction time was reduced by about 5 hours, the concentration of reducing sugar was increased by 40%, glucose production was enhanced by 10% , and the viscosity was reduced by 30%.     

Kinetic modeling of rapid enzymatic hydrolysis of crystalline cellulose after pretreatment by NMMO

Pretreatment of cellulose with an industrial cellulosic solvent, N-methylmorpholine-N-oxide, showed promising results in increasing the rate of subsequent enzymatic hydrolysis. Cotton linter was used as high crystalline cellulose. After the pretreatment, the cellulose was almost completely hydrolyzed in less than 12 h, using low enzyme loading (15 FPU/g cellulose). The pretreatment significantly decreased the total crystallinity of cellulose from 7.1 to 3.3, and drastically increased the enzyme adsorption capacity of cellulose by approximately 42 times. A semi-mechanistic model was used to describe the relationship between the cellulose concentration and the enzyme loading. In this model, two reactions for heterogeneous reaction of cellulose to glucose and cellobiose, and a homogenous reaction for cellobiose conversion to glucose was incorporated. The Langmuir model was applied to model the adsorption of cellulase onto the treated cellulose. The competitive inhibition was also considered for the effects of sugar inhibition on the rate of enzymatic hydrolysis. The kinetic parameters of the model were estimated by experimental results and evaluated.     

On the mode of action of fungal cellulases

Summary The mode of action of the cellulolytic enzymes of two strong cellulose decomposing fungi, Penicillium oxalicum Curie et Thom and Helminthosporium cyclops Drechsler, was studied. The culture filtrates and enzyme preparations obtained from them showed high cellulase activity and very weak cellobiase activity. The cellulolytic system of both experimental organisms seems to be multicomponent. The cellulase component showed its activity mainly extracellulary and the cellobiase component, mainly intracellulary. It seems, therefore, that during growth of both fungi on a cellulose medium, the extracellular cellulase acts hydrolytically on the cellulose substrate forming cellobiose which is further acted upon by intracellular cellobiase to form glucose. Paper chromatographic assay of the products of the enzymatic reaction sub-stantiated this conclusion.     

Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase

Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. In addition, the difficulty in the recovery of cellulase also leads to high cost for the enzymatic hydrolysis of cellulose. In this study, enzymatic degradation of cellulose was carried out in pH–pH recyclable aqueous two-phase systems (ATPS) composed by copolymers poly (AA-co-DMAEMA-co-BMA) (abbreviated P_ADB3.8) and poly (MAA-co-DMAEMA-co-BMA) (abbreviated P_MDB). In the systems, cellulase was immobilized on pH-response copolymer P_MDB by using 1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in the systems was 2.45, in the presence of 40 mM (NH₄)₂SO₄. Insoluble substrate and immobilized enzyme were biased to bottom phase, while the product was partitioned to top phase. Microcrystalline cellulose was hydrolyzed into reducing sugar, and the product entered into top phase. The yield of saccharification in ATPS could reach 70.57% at the initial substrate concentration of 0.5% (w/v), and the value was 9.3% higher than that in the single aqueous phase. Saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS.     

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K_m (15.6260 g/L) and the rate constant of deactivation, K_de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V_max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.     

固定化纤维二糖酶的研究

黑曲霉 (AspergillusnigerLORRE 0 12 )的孢子中富含纤维二糖酶 ,将这些孢子用海藻酸钙凝胶包埋后 ,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定 ,半衰期为 38d ,耐热性和适宜的pH范围均比固定化前有所增加 ,其Km 和Vmax值分别为 6 .0 1mmol L和 7.0 6mmol (min·L)。利用固定化纤维二糖酶重复分批酶解10g L的纤维二糖 ,连续 10批的酶解得率均可保持在 97%以上 ;采用连续酶解工艺 ,当稀释率为 0 .4h- 1 ,酶解得率可达 98.5 %。玉米芯经稀酸预处理后 ,其纤维残渣用里氏木霉 (Trichodermareesei)纤维素酶降解 ,酶解得率为6 9.5 % ;通过固定化纤维二糖酶的进一步作用 ,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除 ,酶解得率提高到 84.2 % ,还原糖中葡萄糖的比例由 5 3 .6 %升至 89.5 % ,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。     

Optimal operating policy of the ultrafiltration membrane bioreactor for enzymatic hydrolysis of cellulose

The dilution rate of an ultrafiltration membrane bioreactor in the enzymatic hydrolysis of cellulose was optimized using the kinetic model developed by Fan and Lee.(4) The sequence of optimal dilution rates was found to generally consist of an initial period of a minimal value (batch period), a subsequent period of maximum dilution rate, a period of a second batch, and a final period of a singular dilution rate. The effects of operating conditions, such as beta-glucosidase activity, operating time, maximum dilution rate, substrate feeding rate, and enzyme-to-substrate ratio on both the conversion yield and the sequence of optimal dilution rates were investigated. To evaluate the validity of kinetic model employed in this work, enzymatic hydrolysis was carried out using alpha-cellulose as a substrate in the ultrafiltration membrane bioreactor. The experimental data were well consistent with the simulation results. (c) 1993 John Wiley & Sons, Inc.     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

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.     

Cellulolytic and physiological properties of Clostridium thermocellum

Three strains of Clostridium thermocellum obtained from various sources were found to have nearly identical deoxyribonucleic acid guanosine plus cytosine contents that ranged from 38.1–39.5 mole-%. All strain examined fermented only cellulose and cellulose derivatives, but not glucose, or xylose or other sugars. The principal cellulose fermentation products were ethanol, lactate, acetate, hydrogen and carbon dioxide. Growth of C. thermocellum on cellulose resulted in the production of extracellular cellulase that was non-oxygen labile, was thermally stable at 70° C for 45 min and adsorbed strongly on cellulose. Production of cellulase during fermentation correlated linearly with growth and cellulose degradation. Both the yield and specific activity of crude cellulase varied considerably with the specific growth substrates. Highest cellulase yield was obtained when grown on native cellulose, -cellulose and low degree of polymerization cellulose but not carboxymethylcellulose or other carbohydrate sources. Cellulase activity was not detected when cells were grown on cellobiose. Crude extracellular protein preparations lacked proteolytic and cellobiase activity. The pH and temperafure optima for endoglucanase activity were 5.2 and 65° C, respectively, while that of the exoglucanase activity were 5.4 and 64° C, respectively. The specific activity at 60° c for exoglucanase and endoglucanase of crude cellulase obtained from cells grown on cellulose (MN 300) was 3.6 moles reducing sugar equivalents released per h (unit)/mg of protein and 1.5 mole reducing sugar equivalent released per min (unit)/mg of protein, respectively. The yield of endoglucanase was 125 units per g of cellulose MN 300 degraded and that of exoglucanase was 300 units per g of cellulose MN 300 degraded. Glucose and cellobiose were the hydrolytic end products of crude cellulase action on cellulose, cellotraose and cellotriose in vitro.     

Saccharification, fermentation, and protein recovery from low-temperature AFEX-treated coastal bermudagrass

Coastal bermudagrass was pretreated by a low-temperature ammonia fiber explosion (AFEX) process, which soaked the grass in liquid ammonia and then explosively released the pressure. Saccharifying enzymes were systematically applied to the AFEX-treated grass corresponding to low, medium, and high loadings of cellulase/hemicellulase (from Trichoderma reesei), cellobiase, glucoamylase, and pectinase. Three-day sugar yields linearly correlated with the logarithm of the cellulase loading. Supplemental enzymes (cellobiase, pectinase) caused upward shifts in the lines. The linearity and upward shifts are consistent with the HCH-1 model of cellulose hydrolysis. The hydrolysis sugars were converted to ethanol using yeast (Saccharomyces cerevisiae). The solid residues were treated with proteases to attempt recovery of valuable proteins. The low-temperature AFEX pretreatment was able o nearly double sugar yields. At the highest cellulase loadings (30 IU/g), the best reducing sugar and ethanol yields were 53% and 44% of the maximum potential, respectively. Protein recovery was, at most, 59% (c) 1994 John Wiley & Sons, Inc.     

Production of xylanase under solid-state fermentation by <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> JP-1 and its application

The production of extracellular xylanase by a locally isolated strain of Aspergillus tubingensis JP-1 was studied under solid-state fermentation. Among the various agro residues used wheat straw was found to be the best for high yield of xylanase with poor cellulase production. The influence of various parameters such as initial pH, moisture, moistening agents, nitrogen sources, additives, surfactants and pretreatment of substrates were investigated. The production of the xylanase reached a peak in 8 days using untreated wheat straw with modified MS medium, pH 6.0 at 1:5 moisture level at 30 °C. Under optimized conditions yield as high as 6,887 ± 16 U/g of untreated wheat straw was achieved. Crude xylanase was used for enzymatic saccharification of agro-residues like wheat straw, rice bran, wheat bran, sugarcane bagasse and industrial paper pulp. Dilute alkali (1 N NaOH) and acid (1 N H₂SO₄) pretreatment were found to be beneficial for the efficient enzymatic hydrolysis of wheat straw. Dilute alkali and acid-pretreated wheat straw yielded 688 and 543 mg/g reducing sugar, respectively. Yield of 726 mg/g reducing sugar was obtained from paper pulp after 48 h of incubation.     

Utilization of pretreated coir pith for the optimized bioproduction of cellulase by Aspergillus nidulans

The present study is aimed at simultaneous cellulase synthesis and coir pith degradation by Aspergillus nidulans using coir pith as chief substrate. The lignocellulosic biomass, coir pith is known to be an excellent carbon source for microbial cellulase production under solid state fermentation. The alkali pretreatment with sodium hydroxide was seen to enhance enzymatic hydrolysis. The effect of coir pith weight, moisture content, initial pH and growth temperature on cellulase activity and yield were investigated by response surface methodology (RSM) employing a four-factor-five-level central composite design (CCD). The results of Fourier transform infrared spectroscopy (FTIR), X-Ray diffraction (XRD) and Scanning electron microscopy (SEM) of coir pith showed structural changes through pretreatment, in favor of enzymatic hydrolysis. Maximum carboxy methyl cellulase activity (CMCase) of 28.64 U/g and cellulase yield of 66.32% were achieved with 8 g coir pith at 70% moisture content and 40 °C temperature with pH 5 as evident from run numbers 25 and 30. Filter paper (FPase) and cellobiase (CBase) activities of 10.23 U/g and 4.31 U/g respectively were observed on the 11th day after the inoculation.     

Cellulases: Biosynthesis and applications

Strains of Trichoderma, particularly T. reesei and its mutants, are good sources of extracellular cellulase suitable for practical saccharification. They secrete a complete cellulase complex containing endo- and exo-glucanases plus β-glucosidase (cellobiase) which act syngergistically to degrade totally even highly resistant crystalline cellulose to soluble sugars. All strains investigated to date are inducible by cellulose, lactose, or sophorose, and all are repressible by glucose. Induction, synthesis and secretion of the β-glucanase enzymes appear to be closely associated. The composition and properties of the enzyme complex are similar regardless of the strain or inducing substrate although quantities of the enzyme secreted by the mutants are higher. Enzyme yields are proportional to initial cellulose concentration. Up to 15 filter paper cellulase units (20 mg of cellulase protein) per ml and productivities up to 80 cellulase units (130 mg cellulase protein) per litre per hour have been attained on 6% cellulose. The economics of glucose production are not yet competitive due to the low specific activity of cellulase (0.6 filter paper cellulase units/mg protein) and poor efficiency (about 15% of predicted sugar levels in 24 h hydrolyses of 10–25% substrate). As hydrolysis proceeds, the enzyme reaction slows due to increasing resistance of the residue, product inhibition, and enzyme inactivation. These problems are being attacked by use of pretreatments to increase the quantity of amorphous cellulose, addition of β-glucosidase to reduce cellobiose inhibition, and studies of means to overcome instability and increase efficiency of the cellulases. Indications are that carbon compounds derived from enzymatic hydrolysis of cellulose will be used as fermentation and chemical feedstocks as soon as the process economics are favourable for such application.