Treatment of rice straw with selected Cyathus stercoreus strains to improve enzymatic saccharification

Seventeen Cyathus stercoreus isolates were tested for their ability to treat rice straw for improved enzymatic saccharification. These isolates showed a negative correlation between cellulase and xylanase activity and enzymatic saccharification yields. Incubation of rice straw pretreated at 60 °C for 15 min with strain C. stercoreus TY-2 for 25 days resulted in an enzymatic saccharification yield of 57% as compared to a yield of 11% for the same straw in the absence of the fungus. These findings highlight the potential of this isolate for biological pretreatment of rice straw under conditions of low energy input.     

Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The lignin accounted for about 18.8–21.8% of pretreated rice straw, which was less than the 23.1–26.5% of pretreated bagasse and the 21.5–24.1% of pretreated silvergrass. The maximum glucose yield achieved, under an enzyme loading 6.5 FPU g^?1 DM for 72 h, was close to 0.8 g glucose/g glucan from the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A decrease in initial rate of glucose production was observed in all cases when the raw materials underwent enzymatic saccharification with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of β-glucosidase activity. Fourier transform infrared (FTIR) spectroscopy further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic. The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the cellulase and this could become very important for raw materials such as silvergrass that contain more lignin.     

湿氧化爆破稻秆的同步糖化发酵条件研究

探讨了木质纤维素经过湿氧化爆破后在同步糖化发酵过程中酵母产乙醇的基本规律.采用单因素方法对湿氧化爆破条件、酶系组成和添加量以及预酶解时间和温度进行了优化.不同湿氧化爆破预处理条件下的稻秆对同步糖化发酵工艺的影响较大,在预处理温度160 ℃,进氧压力为4×105 Pa,碱用量为6%(w/w),反应时间为20 min的条件...     

Efficient Recovery of Glucose and Fructose via Enzymatic Saccharification of Rice Straw with Soft Carbohydrates

Soft carbohydrates, defined as readily-recoverable carbohydrates via mere extraction from the biomass or brief enzymatic saccharification, were found in significant amounts in rice straw as forms of free glucose, free fructose, sucrose, starch, and β-1,3-1,4-glucan. In this study, we investigated their amounts in rice straw (defined as culm and leaf sheath), and developed an easy method for glucose and fructose recovery from them with heat-pretreatment and subsequent 4-h enzymatic saccharification with an enzyme cocktail of cellulase and amyloglucosidase. The recovery of glucose and fructose exhibited good correlation with the amounts of soft carbohydrates. The maximum yields of glucose and fructose in the rice straw per dry weight at the heading stage and the mature stage were 43.5% in cv. Habataki and 34.1% in cv. Leafstar. Thus, rice straw with soft carbohydrates can be regarded as a novel feedstock for economically feasible production of readily-fermentable glucose and fructose for bioethanol.     

Enhanced enzymatic saccharification of rice straw by microwave pretreatment

In this study, Box-Behnken design and response surface methodology were employed to plan experiments and optimize the microwave pretreatment of rice straw. Experimental results show that microwave intensity (MI), irradiation time (IT) and substrate concentration (SC) were main factors governing the enzymatic saccharification of rice straw. The maximal efficiencies of cellulose, hemicellulose and total saccharification were respectively increased by 30.6%, 43.3% and 30.3% under the optimal conditions of MI 680 W, IT 24 min and SC 75 g/L. The chemical composition analysis of straw further confirmed that microwave pretreatment could disrupt the silicified waxy surface, break down the lignin-hemicellulose complex and partially remove silicon and lignin.     

RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.     

DiSC (direct saccharification of culms) process for bioethanol production from rice straw

A simple process (the direct-saccharification-of-culms (DiSC) process) to produce ethanol from rice straw culms, accumulating significant amounts of soft carbohydrates (SCs: glucose, fructose, sucrose, starch and β-1,3-1,4-glucan) was developed. This study focused on fully mature culms of cv. Leafstar, containing 69.2% (w/w of dried culms) hexoses from SCs and cellulose. Commercially-available wind-separation equipment successfully prepared a culm-rich fraction with a SC recovery of 83.1% (w/w) from rice straw flakes (54.1% of total weight of rice straw). The fraction was suspended in water (20%, w/w) for starch liquefaction, and the suspension was subjected to a simultaneous saccharification and fermentation with yeast, yielding 5.6% (w/v) ethanol (86% of the theoretical yield from whole hexoses in the fraction) after 24 h fermentation. Thus, the DiSC process produced highly-concentrated ethanol from rice straw in a one vat process without any harsh thermo-chemical pretreatments.     

Saccharification of gamma-ray and alkali pretreated lignocellulosics

Enzymic saccharification of gamma ray and alkali pretreated sawdust, rice straw, and sugar cane bagasse showed higher release of reducing sugar from pretreated substrates. By gamma ray treatment alone (500 kGy) reducing sugar release of 2.8, 9.2, and 10 g/l was obtained from 7.5% (w/v) sawdust, rice straw, and bagasse and the same substrates showed reducing sugar release of 4.2, 30, and 20 g/l respectively when treated with alkali (0.1 g/g). Combination of gamma ray with alkali treatment further increased the reducing sugar release to 10.2, 33, and 36 g/l from sawdust, rice straw, and bagasse respectively. The effects of gamma ray and alkali treatment on saccharification varied with the nature of the substrate.     

Direct ethanol production from hemicellulosic materials of rice straw by use of an engineered yeast strain codisplaying three types of hemicellulolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells

The cost of the lignocellulose-hydrolyzing enzymes used in the saccharification process of ethanol production from biomass accounts for a relatively high proportion of total processing costs. Cell surface engineering technology has facilitated a reduction in these costs by integrating saccharification and fermentation processes into a recombinant microbe strain expressing heterologous enzymes on the cell surface. We constructed a recombinant Saccharomyces cerevisiae that not only hydrolyzed hemicelluloses by codisplaying endoxylanase from Trichoderma reesei, β-xylosidase from Aspergillus oryzae, and β-glucosidase from Aspergillus aculeatus but that also assimilated xylose through the expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. The recombinant strain successfully produced ethanol from rice straw hydrolysate consisting of hemicellulosic material containing xylan, xylooligosaccharides, and cellooligosaccharides without requiring the addition of sugar-hydrolyzing enzymes or detoxication. The ethanol titer of the strain was 8.2g/l after 72h fermentation, which was approximately 2.5-fold higher than that of the control strain. The yield (grams of ethanol per gram of total sugars in rice straw hydrolysate consumed) was 0.41g/g, which corresponded to 82% of the theoretical yield. The cell surface-engineered strain was thus highly effective for consolidating the process of ethanol production from hemicellulosic materials.     

Sugar production from agricultural woody wastes by saccharification with Trichoderma viride cellulase.

The saccharification of agricultural woody wastes was studied using a commercial enzyme preparation, Cellulase onozuka, derived from Trichoderma viride or the solid culture extracts of the fungus. With the intention of producing sugar at low cost, a simple procedure of enzymatic saccharification of rice straw, bagasse, and sawdust was studied. Delignifying methods of these wastes were investigated using dilute sodium hydroxide solution and dilute peracetic acid. Rice straw and bagasse were effectively delignified by boiling in a 1% sodium hydroxide solution for 3 hr or by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a broad leaved tree (Machilus thunbergii) was delignified by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr and by subsequent boiling in diluted 1/5 peracetic acid for 1 hr. This type of sawdust was also delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a coniferous tree (Cryptomeria japonica) was delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr; however, the successive treatment by autoclaving with alkali solution and subsequent boiling with diluted peracetic acid failed to bring about the desired effect. The saccharification of delignified rice straw, bagasse, and sawdust was examined using Cellulase onozuka, wheat bran or rice straw solid culture at various substrate concentrations, resulting in the formation of 5 to 10% sugar solutions after incubation at pH 5.0, 45 degrees C for 48 hr. The optimum substrate concentration existed at around 10%. Reuse of cellulase solution and resaccharification of residual sawdust were considered to be inadequate.     

Analysis of the saccharification capability of high-functional cellulase JN11 for various pretreated biomasses through a comparison with commercially available counterparts

Although the capabilities of Trichoderma reesei cellulases have been greatly improved, these enzymes are still too costly for commercial use. The aim of this research was to assess the biomass saccharification capability of JN11, a recombinant cellulase, compared with that of the commercially available cellulases Accellerase 1500 and Cellic CTec. The activities of JN11, Accellerase 1500, and Cellic CTec were compared by using various types of cellulosic biomass, including rice straw, Erianthus, eucalyptus, and Japanese cedar. JN11 had higher saccharification capability for rice straw, Erianthus, eucalyptus, and Japanese cedar compared with the commercial cellulases. The JN11 saccharification of cellulosic biomasses, including hemicellulose (NaOH-pretreated biomasses), resulted in high glucose and xylose yields because of the high xylanase/xylosidase activity of JN11. Moreover, even JN11 saccharification of hemicellulose-free biomasses (sulfuric acid-, hydrothermally, and steam exploded-pretreated biomasses) resulted in high glucose yields. The cellulase activity of JN11, however, was comparable to that of its commercial counterparts. These findings indicate that the saccharification ability of cellulase is unrelated to its cellulase activity when measured against Avicel, CMC, pNP-lactoside, and other substrates. JN11 showed high activity for all types of pretreated cellulosic biomasses, indicating its usefulness for saccharification of various cellulosic biomasses.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.     

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. Biotechnol. Bioeng. 2012; 109: 2484–2493. © 2012 Wiley Periodicals, Inc.     

Importance of cellulase cocktails favoring hydrolysis of cellulose

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.     

Ethanol production by <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> CHZ2501 from industrial starch feedstocks

The optimal conditions of ethanol fermentation process by Zymomonas mobilis CHZ2501 were investigated. Brown rice, naked barley, and cassava were selected as representatives of the starch-based raw material commercially available for ethanol production. Considering enzyme used for saccharification of starch, the ethanol productivity with complex enzyme was higher than glucoamylase. With regards to the conditions of saccharification, the final ethanol productions of simultaneous saccharification and pre-saccharified process for 1 h were not significantly different. The result suggested that it is possible for simultaneous saccharification and fermentation as a cost-effective process for ethanol production by eliminating the separate saccharification. Additionally, the fermentation rate in early fermentation stage was generally increased with increase of inoculum volume. As the result, optimal condition for ethanol production was simultaneous saccharification and fermentation with complex enzyme and 5% inoculation. Under the same condition, the volumetric productivities and ethanol yields were attained to 3.26 g/L·h and 93.5% for brown rice, 2.62 g/L·h and 90.4% for naked barley, and 3.28 g/L·h and 93.7% for cassava, respectively.     

Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw

Wheat straw used in this study contained 44.24 +/- 0.28% cellulose and 25.23 +/- 0.11% hemicellulose. Alkaline H(2)O(2) pretreatment and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from wheat straw (8.6%, w/v) by alkaline peroxide pretreatment (2.15% H(2)O(2), v/v; pH 11.5; 35 degrees C; 24 h) and enzymatic saccharification (45 degrees C, pH 5.0, 120 h) by three commercial enzyme preparations (cellulase, beta-glucosidase, and xylanase) using 0.16 mL of each enzyme preparation per g of straw was 672 +/- 4 mg/g (96.7% yield). During the pretreatment, no measurable quantities of furfural and hydroxymethyl furfural were produced. The concentration of ethanol (per L) from alkaline peroxide pretreated enzyme saccharified wheat straw (66.0 g) hydrolyzate by recombinant Escherichia coli strain FBR5 at pH 6.5 and 37 degrees C in 48 h was 18.9 +/- 0.9 g with a yield of 0.46 g per g of available sugars (0.29 g/g straw). The ethanol concentration (per L) was 15.1 +/- 0.1 g with a yield of 0.23 g/g of straw in the case of simultaneous saccharification and fermentation by the E. coli strain at pH 6.0 and 37 degrees C in 48 h.     

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L^?1 for rice straw, wheat bran, peptone, and CaCO₃, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL^?1 were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.     

The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol

Autohydrolysis and ethanol-alkali pulping were used as pretreatment methods of wheat straw for its subsequent saccharification by Trichoderma reesei cellulase. The basic hydrolysis parameters, i.e., reaction time, pH, temperature, and enzyme and substrate concentration, were optimized to maximize sugar yields from ethanol-alkali modified straw. Thus, a 93% conversion of 2.5% straw material to sugar syrup containing 73% glucose was reached in 48 h using 40 filter paper units/g hydrolyzed substrate. The pretreated wheat straw was then fermented to ethanol at 43 degrees C in the simultaneous saccharification and fermentation (SSF) process using T. reesei cellulase and Kluyveromyces fragilis cells. From 10% (w/v) of chemically treated straw (dry matter), 2.4% (w/v) ethanol was obtained after 48 h. When the T. reesei cellulase system was supplemented with beta-glucosidase from Aspergillus niger, the ethanol yield in the SSF process increased to 3% (w/v) and the reaction time was shortened to 24 h.     

Enhanced enzymatic hydrolysis of mild alkali pre-treated rice straw at high-solid loadings using in-house cellulases in a bench scale system

In the present study, scale-up systems for cellulase production and enzymatic hydrolysis of pre-treated rice straw at high-solid loadings were designed, fabricated and tested in the laboratory. Cellulase production was carried out using tray fermentation at 45 °C by Aspergillus terreus in a temperature-controlled humidity chamber. Enzymatic hydrolysis studies were performed in a horizontal rotary drum reactor at 50 °C with 25 % (w/v) solid loading and 9 FPU g^?1 substrate enzyme load using in-house as well commercial cellulases. Highly concentrated fermentable sugars up to 20 % were obtained at 40 h with an increased saccharification efficiency of 76 % compared to laboratory findings (69.2 %). These findings demonstrate that we developed a simple and less energy intensive bench scale system for efficient high-solid saccharification. External supplementation of commercial β-glucosidase and hemicellulase ensured better hydrolysis and further increased the saccharification efficiency by 14.5 and 20 %, respectively. An attempt was also made to recover cellulolytic enzymes using ultrafiltration module and nearly 79–84 % of the cellulases and more than 90 % of the sugars were recovered from the saccharification mixture.