Hydrolysis Of Pretreated Rice Straw Research Articles

Novel buffalo rumen metagenome derived acidic cellulase Cel-3.1 cloning, characterization, and its application in saccharifying rice straw and corncob biomass

Lignocellulosic biomass (LCB) is a prominent option for second-generation biofuels production. Cellulase hydrolyses cellulose, a component of LCB by attacking the β-1,4-glycosidic bonds, thus liberating mono, di, and oligosaccharides, which subsequently, can be converted to biofuel. In this study, a novel cellulase (Cel-3.1) of 1593 bp which encodes a 530 amino acid protein was identified from buffalo rumen metagenomic fosmid library, and functional expression was achieved through transformation into Escherichia coli. The molecular weight was estimated as 58 kDa on SDS-PAGE. Cel-3.1 belongs to glycosyl hydrolase family-5 (GH-5) and is predicted to have 14 α-helices and 15 β-strands. The optimal temperature and pH for Cel-3.1 were experimentally determined as 5.0 and 50 °C respectively. The synergistic effect of Ca2+ with K+ ions improved Cel-3.1 activity significantly (25%) and 1% Polyethylene Glycol (PEG-400), 1% β-mercaptoethanol enhanced the relative activity Cel-3.1 by 31.68%, 12.03% respectively. Further, the enzymatic (Cel-3.1) hydrolysis of pretreated rice straw and corncob released 13.41 ± 0.26 mg/mL and 15.04 ± 0.08 mg/mL reducing sugars respectively. High Performance Liquid Chromatography (HPLC), Scanning Electron Microscope (SEM), and Fourier Transformation Infrared spectroscopy (FTIR) analysis revealed the capability of Cel-3.1 for the breakdown and hydrolysis of both rice straw and corncob to generate various fermentable sugars.

Expression and characterization of TbCel12A, a thermophilic endoglucanase with potential in biomass hydrolysis

Abstract To increase economic benefit and reduce pollutants, agricultural biomass can be hydrolyzed to monosaccharides, which serve as a feedstock for biorefinery production of fuels and fine chemicals. In a search for new enzymes to apply to this process, Thermobispora bispora TbCel12A endoglucanase was identified by rice straw compost metagenome analysis and expressed in recombinant Escherichia coli. TbCel12A hydrolyzed both soluble and insoluble β-glucan substrates. Its highest hydrolysis rate was observed for cellopentaose compared to other cellooligosaccharides, barley mixed linkage β-glucan, and carboxymethyl cellulose. Low hydrolysis rates were observed for α-cellulose, and avicel. The primary product detected was cellobiose and a smaller amount of glucose. TbCel12A exhibited highest activity at 65 °C and pH 5.0 and retained 86% relative activity after incubation at 60 °C overnight. TbCel12A conserved >80% activity in 10% alcohol and at high salt concentrations. Hydrolysis of pretreated rice straw using commercial cellulase and TbCel12A released slightly more reducing sugars than hydrolysis using only commercial cellulase. Therefore, TbCel12A is a promising enzyme for further development for use in biomass conversion.

High-concentrated substrate enzymatic hydrolysis of pretreated rice straw with glycerol and aluminum chloride at low cellulase loadings

Rice straw was pretreated with glycerol and AlCl3 for enzymatic hydrolysis at low cellulase loadings. Based on a central composite design, 83% delignification, 94% hemicellulose removal, and 92% cellulose recovery (or 76% cellulose in solid residue) were achieved under the optimized pretreatment conditions (0.08 mol/L AlCl3 as catalyst at 146.8 °C for 20 min with 90% glycerol). During glycerol-AlCl3 pretreatment, the lignin-carbohydrate complex was depolymerized, resulting in the complex and recalcitrant construction of straw effectively being destroyed. The enzyme adsorption ability of pretreated straw was 16.5 times that for the original sample. After pretreatment, glucose yield was increased by 2.4 times to 74% for 48 h. Moreover, concentrated solid (15%) with low cellulase loading (3.3 FPU/g dry substrate) achieved 58.6% glucose yield, and further increased by 12% to 65.7% by adding Tween 80. Glycerol-AlCl3 pretreatment was a promising approach to realize high-concentrated solid hydrolysis for sugars at low cellulase loadings.

Mechanochemical-assisted hydrolysis of pretreated rice straw into glucose and xylose in water by weakly acidic solid catalyst

In this work, carbonaceous materials bearing only weakly acidic COOH and phenolic OH groups were directly prepared by the pyrolysis of lignin and KOH in black liquor generating from rice straw that was pretreated with KOH aqueous solution. The synthesized carbon materials were used for the hydrolysis of cellulose or the alkali pretreated rice straw in water, after mixed ball-milling pretreatment, and provided a high glucose yield of 76.3% for cellulose, high yields of 52.1% glucose and 66.5% xylose for alkali pretreated rice straw, respectively, in 0.015 wt% HCl aqueous solution at 200 °C for 60 min. The weakly acidic catalyst showed good stability and recyclability in the aqueous reaction system. This work provides an efficient process for the hydrolysis of lignocellulose by biomass-derived weakly acidic catalysts in water and should have wide applications in biomass utilization.

Production of β-xylosidase from Trichoderma asperellum KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase.

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.

Black liquor-derived carbonaceous solid acid catalyst for the hydrolysis of pretreated rice straw in ionic liquid

Lignin-containing black liquor from pretreatment of rice straw by KOH aqueous solution was applied to prepare a carbonaceous solid acid catalyst, in which KOH played dual roles of extracting lignin from rice straw and developing porosity of the carbon material as an activation agent. The synthesized black liquor-derived carbon material was applied in catalytic hydrolysis of the residue solid from the pretreatment of rice straw, which was mainly composed of cellulose and hemicellulose, and showed excellent activity for the production of total reducing sugars (TRS) in ionic liquid, 1-butyl-3-methyl imidazolium chloride. The highest TRS yield of 63.4% was achieved at 140°C for 120min, which was much higher than that obtained from crude rice straw under the same reaction conditions (36.6% TRS yield). Overall, this study provides a renewable strategy for the utilization of all components of lignocellulosic biomass.

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 106 spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.

Evaluation of Amberlyst15 for hydrolysis of alkali pretreated rice straw and fermentation to ethanol

Solid acid catalysts are potent alternatives to enzymes or mineral acids currently used in the hydrolysis of lignocellulosic biomass to generate sugars for further applications in biofuel or chemicals manufacturing. They are considered green, environmentally safe, and are reusable. In the present work, Amberlyst 15 was evaluated as a solid acid catalyst for the hydrolysis of alkali pretreated biomass. A response surface methodology was used to optimize the hydrolysis of pretreated rice straw. Optimized conditions of 7.0% biomass and catalyst loading resulted in a sugar yield of 255mg/g of pretreated biomass in 4.0h. The catalytic hydrolysate generated lesser amount of fermentation inhibitors compared to similar treatment by mineral acids. Recovery and reusability of the catalyst was also studied. The catalytic hydrolysate could be used for fermentative production of bioethanol. Results indicate the possibility to use solid acid catalysts as an alternative strategy for biomass hydrolysis.

Influence of alkaline catalyst addition on compressed liquid hot water pretreatment of rice straw

Liquid hot water (LHW) pretreatment of cellulosic feedstock improves its enzymatic digestibility by removing hemicellulose/lignin, thus making the remaining cellulose more accessible to cellulases. In this study, alkaline-catalyzed (NaOH) LHW pretreatment of rice straw was explored. Various NaOH concentrations (0.25–1.0%) were tested for removal of hemicellulose and lignin as well as enzymatic hydrolysis of the pretreated material at 140–160°C for 5–20min. It was found that glucose recovery from enzymatic hydrolysis was markedly higher for rice straw pretreated by LHW in the presence of NaOH compared with LHW pretreatment in the absence of NaOH. Alkaline catalysis of LHW pretreatment also increased removal of hemicellulose and lignin from solid residue and increased the pentose yield in the liquid phase. Under optimum LHW condition of 140°C for 10min in the presence of 0.25% NaOH, a maximum glucose yield of 71.8% was obtained from the enzymatic hydrolysis of pretreated rice straw; combined with hydrolyzed glucose in the liquid, this represents a maximum glucose recovery of 74.6% from rice straw. The results thus demonstrate the usefulness of NaOH catalyst for LHW pretreatment of rice straw for increasing sugar recovery and reducing energy requirement.

Improve the Recovery of Fermentable Sugar from Rice Straw by Sonication and Its Mathematical Modeling

Rice straw is waste renewable agricultural biomass, which contains sufficient amount of fermentable sugars like glucose, galactose fructose, xylose etc. These sugars can be treated with fermentation pathway to produce ethanol. Hydrolysis of pretreated rice straw in dilute sulfuric acid was investigated at different acid concentrations (0.25–0.75 % w/v), and sonication was carried out to improve the extent of sugar extraction. The current work examines the effect of sonication on extraction of total reducing sugar (TRS) and an empirical mathematical model has been established to predict it. Effects of various operating variables of sonication, including amplitude (60–100 %), cycle (0.6–1.0), treatment time (0–15 min) have been analyzed for each acid concentration. Observation shows that on optimization of the sonication conditions (100 % amplitude, 0.8 cycle and 10 min) around 90 % improvement of TRS extraction occurs at 0.5 % (w/v) acid concentration.

Kinetic Studies on the Product Inhibition of Enzymatic Lignocellulose Hydrolysis

In order to understand the product inhibition of enzymatic lignocellulose hydrolysis, the enzymatic hydrolysis of pretreated rice straw was carried out over an enzyme loading range of 2 to 30 FPU/g substrate, and the inhibition of enzymatic hydrolysis was analyzed kinetically based on the reducing sugars produced. It was shown that glucose, xylose, and arabinose were the main reducing sugar components contained in the hydrolysate. The mass ratio of glucose, xylose, and arabinose to the total reducing sugars was almost constant at 52.0%, 29.7% and 18.8%, respectively, in the enzyme loading range. The reducing sugars exerted competitive inhibitory interferences to the enzymatic hydrolysis. Glucose, xylose, and arabinose had a dissociation constant of 1.24, 0.54 and 0.33 g/l, respectively. The inhibitory interferences by reducing sugars were superimposed on the enzymatic hydrolysis. The enzymatic hydrolysis could be improved by the removal of the produced reducing sugars from hydrolysate.

Novel renewable ionic liquids as highly effective solvents for pretreatment of rice straw biomass by selective removal of lignin

Cholinium amino acids ionic liquids ([Ch][AA] ILs), a novel type of bio-ILs that can easily be prepared from renewable biomaterials, were investigated for pretreatment of rice straw by selective extraction of lignin from this abundant lignocellulosic biomass material. Of the eight ILs examined, most were demonstrated to be excellent pretreatment solvents. Upon pretreatment using these ILs, the initial saccharification rates of rice straw residues were substantially improved as well as the extent to which polysaccharides could be digested (>90% for cellulose and >60% for xylan). Enzymatic hydrolysis of pretreated rice straw by Trichoderma reesei cellulase/xylanase furnished glucose and xylose with the yields in excess of 80% and 30%, respectively. Detailed spectroscopic characterization showed that the enhancement of polysaccharides degestibility derived mainly from delignification rather than changes in cellulose crystallinity. The yields of fermentable reducing sugars were significantly improved after individual optimization of pretreatment temperature and duration. With [Ch][Lys] as the solvent, the sugar yields of 84.0% for glucose and 42.1% for xylose were achieved after pretreatment at 90°C for 5 h. The IL [Ch][Lys] showed excellent reusability across five successive batches in pretreatment of rice straw. These bio-ILs performed as well as or better than previously investigated non-renewable ILs, and thus present a new and environmentally friendly way to pretreat lignocellulose for production of fermentable sugars and total utilization of the biomass.

Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production

Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification. Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase, 0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g(p)/g(s) by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO(2) released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail was good, and enzymatic hydrolyzate could be fermented to produce ethanol.

Cellulase Hydrolysis of Rice Straw and Inactivation of Endoglucanase in Urea Solution

In order to optimize the cellulase (from Aspergillus glaucus) hydrolysis of pretreated rice straw, the effects of varying enzyme concentration, temperature, and pH were studied. The best experimental conditions found to degrade the pretreated rice straws were 24 h of incubation at 55 °C and pH 5.0, with an enzyme concentration of 48 mg/L. Urea is one of the important nitrogen sources used in fungi culture, but it is also a denaturant. The model of denaturation of endoglucanase (EG) in urea solutions was established. The denaturation was a slow, reversible reaction. Determination of microscopic rate constants showed k(+0) > k'(+0), indicating that EG was protected by the substrate to a certain extent during denaturation. Comparison with the results from fluorescence emission spectroscopy revealed that the inactivation of EG occurred before the marked conformational changes could be detected.

Production and characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carbon source

Cellulases and hemicellulases are key enzymes in the production of alternative fuels and chemicals from lignocellulosic biomass—an abundant renewable resource. Carbon source selection is an important factor in the production of cellulases and hemicellulases. Rice straw – a potential ethanol source – has recently gained considerable interest in Asian countries. Here, we investigated the production of cellulases by using rice straw subjected to various pretreatments as substrates in order to produce cellulases at low costs; we also identified the enzymes’ characteristics. Rice straw cutter milled to <3 mm was pretreated by wet disk milling, dry ball milling, or hot-compressed water treatment (HCWT). Pretreated rice straw and commercial cellulose, Solka Floc (SF), were used as carbon sources for cellulase production by the fungus Acremonium cellulolyticus. Filter paper cellulase, β-xylanase, and β-xylosidase production from ball- and disk-milled samples were higher than those from SF. Enzymatic activity was absent in cultures where HCWT rice straw was used as carbon source. Wet disk-milled rice straw cultures were more suitable for enzymatic hydrolysis of pretreated rice straw than SF cultures. Thus, wet disk milling may be a suitable pretreatment for producing substrates for enzymatic hydrolysis and generating inexpensive carbon sources for cellulase production.

Pretreatment of Rice Straw by a Hot-Compressed Water Process for Enzymatic Hydrolysis

Hot-compressed water (HCW) is among several cost-effective pretreatment processes of lignocellulosic biomass for enzymatic hydrolysis. The present work investigated the characteristics of HCW pretreatment of rice straw including sugar production and inhibitor formation in the liquid fraction and enzymatic hydrolysis of pretreated material. Pretreatment was carried out at a temperature ranging from 140 to 240 degrees C for 10 or 30 min. Soluble oligosaccharides were found to constitute almost all the components of total sugars in the liquid fraction. The maximal production of total glucose at 180 degrees C and below accounted for 4.4-4.9% of glucan in raw material. Total xylose production peaked at 180 degrees C, accounting for 43.3% of xylan in raw material for 10-min pretreatment and 29.8% for 30-min pretreatment. The production of acetic acid increased at higher temperatures and longer treatment time, indicating more significant disruption of lignocellulosic structure, and furfural production achieved the maximum (2.8 mg/ml) at 200 degrees C for both 10-min and 30-min processes. The glucose yield by enzymatic hydrolysis of pretreated rice straw was no less than 85% at 180 degrees C and above for 30-min pretreatment and at 200 degrees C and above for 10-min pretreatment. Considering sugar recovery, inhibitor formation, and process severity, it is recommended that a temperature of 180 degrees C for a time of 30 min can be the most efficient process for HCW pretreatment of rice straw.

Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis

Combination pretreatment of rice straw using microwave and alkali and its enzymic hydrolysis were investigated and compared with the alkali-alone pretreated process. First, the effect of microwave power and pretreatment time on the weight loss and composition of rice straw was examined. The results show that higher microwave power with shorter pretreatment time and the lower microwave power with longer pretreatment time had almost the same effect on the weight loss and composition at the same energy consumption. The comparison was then made between the effect of the microwave/alkali pretreatment and the alkali-alone one on the weight loss and composition of rice straw. The rice straw had a weight loss of 44.6% and composition cellulose 69.2%, lignin 4.9% and hemicellulose 10.2% after 30-min microwave/alkali pretreatment at 700 W while it only had a weight loss of 41.5% and composition cellulose 65.4%, lignin 6.0% and hemicellulose 14.3% after 70-min alkali-alone pretreatment. It may be that microwave/alkali pretreatment could remove more lignin and hemicellulose from rice straw with shorter pretreatment time compared with the alkali-alone one. Finally, the enzymic hydrolysis of pretreated rice straw (substrate concentration 50 g l −1, enzyme loading 20 mg g −1 substrate) was also investigated and the results indicate that rice straw pretreated by microwave/alkali had a higher hydrolysis rate and glucose content in the hydrolysate in comparison with the one by alkali alone.

97/04742 Enzymatic hydrolysis of pretreated rice straw: Vlasenko, E. Y. et al. Bioresource Technology, 1997, 59, (2/3), 109–119

97/04742 Enzymatic hydrolysis of pretreated rice straw: Vlasenko, E. Y. et al. Bioresource Technology, 1997, 59, (2/3), 109–119

Enzymatic hydrolysis of pretreated rice straw

California rice straw is being evaluated as a feedstock for production of power and fuel. This paper examines the initial steps in the process: pretreatment of rice straw and enzymatic hydrolysis of the polysaccharides in the pretreated material to soluble sugars. Rice straw was subjected to three distinct pretreatment procedures: acid-catalyzed steam explosion (Swan Biomass Company), acid hydrolysis (U.S. DOE National Renewable Energy Laboratory), and ammonia fiber explosion or AFEX (Texas A & M University). Standard conditions for each pretreatment were used, but none was optimized for rice straw specifically. Six commercial cellulases, products of Genencor International (USA), Novo (Denmark), Iogen (Canada) and Fermtech (Russia) were used for hydrolysis. The Swan- and the acid-pretreatments effectively removed hemicellulose from rice straw, providing high yields of fermentable sugars. The AFEX-pretreatment was distinctly different from other pretreatments in that it did not significantly solubilize hemicellulose. All three pretreatment procedures substantially increased enzymatic digestibility of rice straw. Three commercial Trichoderma-reesei-derived enzyme preparations: Cellulase 100L (Iogen), Spezyme CP (Genencor), and A1 (Fermtech), were more active on pretreated rice straw compared than others tested. Conditions for hydrolysis of rice straw using Cellulase 100L were evaluated. The supplementation of this enzyme preparation with cellobiase (Novozyme 188) significantly improved the parameters of hydrolysis for the Swan- and the acid-pretreated materials, but did not affect the hydrolysis of the AFEX-pretreated rice straw. The three pretreatment techniques were compared on a basis of a total yield and distribution of fermentable carbohydrates released by enzymatic hydrolysis (the highest possible substrate concentrations were used, 150 g/l for the Swan- and the acid- and 100 g/l for the AFEX-pretreated straw; enzyme loading of 6.7 filter paper units (FPU) and 6.7 cellobiase units (CBU) per gram of dry straw was the same for all pretreated materials). A combined yield of monosaccharides produced by a pretreatment step and by enzymatic hydrolysis was found to be 46, 42 and 37 g/l for the Swan-, the acid- and the AFEX-pretreated rice straw, respectively.

Comparison of the rates of enzymatic hydrolysis of pretreated rice straw and bagasse with celluloses

The rates of enzymatic hydrolysis of pretreated rice straw and bagasse have been studied and compared with the hydrolysis rates of microcrystalline cellulose powder (MCCP) and Solka Floc. The effects of particle size reduction and enzyme loading on the rates of hydrolysis of rice straw and bagasse were also studied. It was found that the rates of hydrolysis of pretreated rice straw and bagasse are much higher than that of MCCP and Solka Floc. For both rice straw and bagasse, particle size reduction had very little effect in enhancing the rate of hydrolysis. Lignin present at <10% did not seem to hinder the accessibility of the enzyme to the cellulose surface. An enzyme loading > 40 U g −1 had no effect on the hydrolysis rate of rice straw or bagasse.