Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover

In this study, a newly isolated Trametes hirsuta yj9 was used to pretreat corn stover in order to enhance enzymatic digestibility. T. hirsuta yj9 preferentially degraded lignin to be as high as 71.49% after 42-day pretreatment. Laccase and xylanase was the major ligninolytic and hydrolytic enzyme, respectively and filter paper activity (FPA) increased gradually with prolonged pretreatment time. Sugar yields increased significantly after pretreatment with T. hirsuta yj9, reaching an enzymatic digestibility of 73.99% after 42 days of pretreatment. Scanning electron microscopy (SEM) showed significant structural changes in pretreated corn stover, the surface of pretreated corn stover became increasingly coarse, the gaps between cellulose fibers were visible, and many pores were developed. Correlation analysis showed that sugar yields were inversely proportional to the lignin contents, less related to cellulose and hemicellulose contents.     

Enhanced enzymatic hydrolysis of rapeseed straw by popping pretreatment for bioethanol production

The objective of this study was to find a pretreatment process that enhances enzymatic conversion of biomass to sugars. Rapeseed straw was pretreated by two processes: a wet process involving wet milling plus a popping treatment, and a dry process involving popping plus dry milling. The effects of the pretreatments were studied both in terms of structural and compositional changes and change in susceptibility to enzymatic hydrolysis. After application of the wet and dry processes, the amounts of cellulose and xylose in the straw were 37-38% and 14-15%, respectively, compared to 31% and 12% in untreated counterparts. In enzymatic hydrolysis performance, the wet process presented the best glucose yield, with a 93.1% conversion, while the dry process yielded 69.6%, and the un-pretreated process yielded <20%. Electron microscopic studies of the straw also showed a relative increase in susceptibility to enzymatic hydrolysis with pretreatment.     

Fractal kinetic analysis of the enzymatic saccharification of CO2 laser pretreated corn stover

The enzymatic hydrolyses of laser pretreated corn stover as a novel pretreatment method were examined to establish a simplified kinetic model for the complicated hydrolysis process. The time dependence of the total reducing sugars amount was closely related to the amounts of cellulosic materials and amounts of cellulase. The evaluated model fitted very well with the experimental data of enzymatic hydrolysis of laser pretreated corn stover under different conditions, including cellulase loading, nature of substrate, substrate loading in the reaction medium. The results indicated that the complex kinetics of cellulase enzymatic saccharification could be assessed with the fractal kinetic model. The cellulase enzymatic reaction process was effectively predicted and controlled with the kinetic model. The result showed that the model could effectively reflect dynamic process of enzyme hydrolysis.     

玉米秸秆酶水解正交试验的研究

经蒸汽爆破预处理的玉米秸杆用里氏木霉（Ｔｒｉｃｈｏｄｅｒｍａ ｒｅｅｓｅｉ Ｒｕｔ Ｃ３０）制备的纤维素酶进行水解,其影响因素主要为ｐＨ值、温度、微量元素,考虑了上述三因素对酶解的影响,以酶解得率为指标来评价酶水解效果,设计了三因素三水平正交实验。研究表明,酶最佳工艺条件为：ｐＨ＝４．８,温度４５℃,微量元素０．５ｍｌ。     

Corn stover pretreatment by inorganic salts and its effects on hemicellulose and cellulose degradation

Inorganic salts, NaCl, KCl, CaCl₂, MgCl₂, FeCl₂, FeSO₄, FeCl₃, and Fe₂(SO₄)₃, were studied as catalysts for the degradation of hemicellulose in corn stover. FeCl₃ significantly increased the hemicellulose degradation in aqueous solutions heated between 140 and 200 °C with high xylose recovery and low cellulose removal, amounting to 90% and <10%, respectively. Hemicellulose removal increased 11-fold when the corn stover was pretreated with 0.1 M FeCl₃ compared to pretreatment with hot water under otherwise the same conditions, which was also 6-fold greater than pretreatment with dilute sulfuric acid at the same pH. Optimum pretreatment conditions were found where the corn stover was pretreated with 0.1 M FeCl₃ at 140 °C for 20 min. Under such conditions, 91% of hemicellulose was removed, and the recovery of monomeric and oligomeric xylose in liquid fraction achieved 89%, meanwhile, only 9% of cellulose was removed.     

Enhanced enzymatic saccharification of kenaf powder after ultrasonic pretreatment in ionic liquids at room temperature

This study demonstrates for the first time that the enzymatic hydrolysis of cellulose is drastically enhanced following ultrasonic pretreatment of lignocellulosic material in ionic liquids (ILs) when compared to conventional thermal pretreatment. Five types of ILs, 1-buthyl-3-methylimidazolium chloride (BmimCl), 1-allyl-3-methylimidazolium chloride (AmimCl), 1-ethyl-3-methylimidazolium chloride (EmimCl), 1-ethyl-3-methylimidazolium diethyl phosphate (EmimDep), and 1-ethyl-3-methylimidazolium acetate (EmimOAc) were tested. Cellulose saccharification ratio was about 20% for kenaf powders pretreated in BmimCl, AmimCl, EmimCl, and EmimDep by conventional heating at 110 °C for 120 min. Conversely, 60-95% of cellulose was hydrolyzed to glucose, subsequent to ultrasonic pretreatment in the same ILs for 120 min at 25 °C. The cellulose saccharification ratio of kenaf powder in EmimOAc was 86% after only 15 min of the ultrasonic pretreatment at 25 °C, compared to only 47% in that case of thermal pretreatment in the IL.     

Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis

Production of ethanol by bioconversion of lignocellulosic biomass has attracted much interest in recent years. However, the pretreatment process for increasing the enzymatic digestibility of cellulose has become a key step in commercialized production of cellulosic ethanol. During the last decades, many pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. From the point of view for integrated utilization of lignocellulosic biomass, organosolv pretreatment provides a pathway for biorefining of biomass. This review presents the progress of organosolv pretreatment of lignocellulosic biomass in recent decades, especially on alcohol, organic acid, organic peracid and acetone pretreatments, and corresponding action mechanisms. Evaluation and prospect of organosolv pretreatment were performed. Finally, some recommendations for future investigation of this pretreatment method were given.     

玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化

木质纤维素高浓度还原糖水解液的获得是纤维乙醇产业化发展的方向。在发酵工业领域,分批补料法是实现这一目标的重要研究途径。本研究采用分批补料法对获得高浓度玉米秸秆酶解还原糖的条件进行了优化。以稀硫酸预处理的玉米秸秆为原料,考察了液固比、补加量与补加时间对分批补料糖化的影响。结果表明,秸秆高浓度酶解液条件的初始物料为20％ (重量/体积),木聚糖酶220 U/g (底物),纤维素酶6 FPU/g (底物),果胶酶50 U/g (底物),在24 h、48 h后分批补加8％预处理后的物料,同时添加与补料量相应的木聚糖酶20 U/g (底物),纤维素酶2 FPU/g (底物),72 h后,最终糖化结果与非补料法相比,还原糖浓度从48.5 g/L提高到138.5 g/L,原料的酶解率最终达到理论值的62.5％。试验结果表明补料法可以显著提高秸秆水解液还原糖浓度。     

The study of factors affecting the enzymatic hydrolysis of cellulose after ionic liquid pretreatment

Cellulose resource has got much attention as a promising replacement of fossil fuel. The hydrolysis of cellulose is the key step to chemical product and liquid transportation fuel. In this paper a serials of chloride, acetate, and formate based ionic liquids were used as solvents to dissolve cellulose. The cellulose regenerated from ILs was characterized by FTIR and X-ray powder diffraction. From the characterization and analysis, it was found that the original close and compact structure has changed a lot. After enzymatic hydrolysis, different kinds of ionic liquids (ILs) have different yields of the reducing sugar (TRS). They are 100%, 90.72%, and 88.92% from 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), 1-butyl-3-methylimidazolium formate ([BMIM][HCOO]) respectively after enzymatic hydrolysis at 50 °C for 5 h. The results indicated that the yields and the hydrolysis rates were improved apparently after ILs pretreatment comparing with the untreated substrates.     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.     

The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods

Selective white-rot fungi have shown potential for lignocellulose pretreatment. In the study, a new fungal isolate, Echinodontium taxodii 2538, was used in biological pretreatment to enhance the enzymatic hydrolysis of two native woods: Chinese willow (hardwood) and China-fir (softwood). E. taxodii preferentially degraded the lignin during the pretreatment, and the pretreated woods showed significant increases in enzymatic hydrolysis ratios (4.7-fold for hardwood and 6.3-fold for softwood). To better understand effects of biological pretreatment on enzymatic hydrolysis, enzyme–substrate interactions were investigated. It was observed that E. taxodii enhanced initial adsorption of cellulase but which did not always translate to high initial hydrolysis rate. However, the rate of change in hydrolysis rate declined dramatically with decreasing irreversible adsorption of cellulase. Thus, the enhancement of enzymatic hydrolysis was attributed to the decline of irreversible adsorption which may result from partial lignin degradation and alteration in lignin structure after biological pretreatment.     

Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid

Enzymatic hydrolysis of pretreated lignocellulosic substrates has emerged as an interesting option to produce sugars that can be converted to liquid biofuels and other commodities using microbial biocatalysts. Lignocellulosic substrates are pretreated to make them more accessible to cellulolytic enzymes, but the pretreatment liquid partially inhibits subsequent enzymatic hydrolysis. The presence of pretreatment liquid from Norway spruce resulted in a 63% decrease in the enzymatic saccharification of Avicel compared to when the reaction was performed in a buffered aqueous solution. The addition of 15 mM of a reducing agent (hydrogen sulfite, dithionite, or dithiothreitol) to reaction mixtures with the pretreatment liquid resulted in up to 54% improvement of the saccharification efficiency. When the reducing agents were added to reaction mixtures without pretreatment liquid, there was a 13-39% decrease in saccharification efficiency. In the presence of pretreatment liquid, the addition of 15 mM dithionite to Avicel, α-cellulose or filter cake of pretreated spruce wood resulted in improvements between 25 and 33%. Positive effects (6-17%) of reducing agents were also observed in experiments with carboxymethyl cellulose and 2-hydroxyethyl cellulose. The approach to add reducing agents appears useful for facilitating the utilization of enzymes to convert cellulosic substrates in industrial processes.     

Microscopic examination of changes of plant cell structure in corn stover due to hot water pretreatment and enzymatic hydrolysis

Particle size associated with accessible surface area has a significant impact on the saccharification of plant cell walls by cellulolytic enzymes. Small particle sizes of untreated cellulosic substrate are more readily hydrolyzed than large ones because of higher specific surface area. Pretreatment enlarges accessible and susceptible surface area leading to enhanced cellulose hydrolysis. These hypotheses were tested using ground corn stover in the size ranges of 425-710 and 53-75 microm. Ultrastructural changes in these particles were imaged after treatment with cellulolytic enzymes before and after liquid hot water pretreatment. The smaller 53-75 microm corn stover particles are 1.5x more susceptible to hydrolysis than 425-710 microm corn stover particles. This difference between the two particle size ranges is eliminated when the stover is pretreated with liquid hot water pretreatment at 190 degrees C for 15 min, at pH between 4.3 and 6.2. This pretreatment causes ultrastructural changes and formation of micron-sized pores that make the cellulose more accessible to hydrolytic enzymes.     

Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw

In order to defray the cost of biodiesel production, the ensuing work was to further investigate utilization of the crude glycerol (CG) from oleochemicals industry in the atmospheric autocatalytic organosolv pretreatment (AAOP) to enhance enzymatic hydrolysis.

The AAOP–CG enabled wheat straw to achieve with reasonable enzymatic hydrolysis yields, reaching 75% for the wet substrate and 63% for the dried. Lipophilic compounds from the CG formed pitch deposition on the fiber, which was responsible for low delignification (30%) and also troublesome in practical operation. Pitch deposits itself had no significant role on enzymatic hydrolysis. A striking finding of the lignin recondensation and/or lignin–carbohydrate complex helped explain why dried pretreated wheat straw had a low enzymatic hydrolysis yield. The CG was suitable for the AAOP to enhance enzymatic hydrolysis of lignocellulosic biomass. But it was advisable to remove lipophilic compounds from crude glycerol before utilization.     

白腐菌协同热水预处理对毛白杨化学组分和酶水解效果的影响

【目的】增强真菌预处理的效率和降低热水预处理对反应条件的要求。【方法】综合利用白腐菌和热水预处理毛白杨,分析此方法对毛白杨化学组分和酶水解效果的影响。【结果】白腐菌Lenzites betulinus C5617协同热水处理,损失率最高达70.70%。纤维素在2个预处理阶段都有损失,其中L.betulinus C5617达到29.62%。木质素的降解主要集中在白腐菌预处理阶段,其中L.betulinus C5617降解的酸不溶木素较多,达到了16.98%。综合预处理显著改善了毛白杨的酶水解效果。与只经热水预处理的样品相比较,L.betulinus C5617和P.sanguineus D9497协同热水处理分别引起还原糖得率上升了20.60%和12.23%。【结论】综合预处理降低了热水解对反应条件的要求,节约了预处理成本。     

Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose

Compared with batch systems, flowthrough and countercurrent reactors have important potential advantages for pretreating cellulosic biomass, including higher hemicellulose sugar yields, enhanced cellulose digestibility, and reduced chemical additions. Unfortunately, they suffer from high water and energy use. To better understand these trade-offs, comparative data are reported on xylan and lignin removal and enzymatic digestibility of cellulose for corn stover pretreated in batch and flowthrough reactors over a range of flow rates between 160 degrees and 220 degrees C, with water only and also with 0.1 wt% sulfuric acid. Increasing flow with just water enhanced the xylan dissolution rate, more than doubled total lignin removal, and increased cellulose digestibility. Furthermore, adding dilute sulfuric acid increased the rate of xylan removal for both batch and flowthrough systems. Interestingly, adding acid also increased the lignin removal rate with flow, but less lignin was left in solution when acid was added in batch. Although the enzymatic hydrolysis of pretreated cellulose was related to xylan removal, as others have shown, the digestibility was much better for flowthrough compared with batch systems, for the same degree of xylan removal. Cellulose digestibility for flowthrough reactors was related to lignin removal as well. These results suggest that altering lignin also affects the enzymatic digestibility of corn stover.     

Sn-S and Ru-Ru bonds cleavage reactions between [Ph3SnS(CH2)3SSnPh3] and Ru3(CO)12: X-ray crystal structures of [Ph3SnS(CH2)3SSnPh3] and trans-[Ru(CO)4(SnPh3)2]

The organotin complex [Ph₃SnS(CH₂)₃SSnPh₃] (1) was synthesized by PdCl₂ catalyzed reaction between Ph₃SnCl and disodium-1,3-propanedithiolate which in turn was prepared from 1,2-propanedithiol and sodium in refluxing THF. Reaction of 1 with Ru₃(CO)₁₂ in refluxing THF affords the mononuclear complex trans-[Ru(CO)₄(SnPh₃)₂] (2) and the dinuclear complex [Ru₂(CO)₆(μ-κ²-SCH₂CH₂CH₂S)] (3) in 20 and 11% yields, respectively, formed by cleavage of Sn-S bond of the ligand and Ru-Ru bonds of the cluster. Treatment of pymSSnPPh₃ (pymS = pyrimidine-2-thiolate) with Ru₃(CO)₁₂ at 55-60 °C also gives 2 in 38% yield. Both 1 and 2 have been characterized by a combination of spectroscopic data and single crystal X-ray diffraction analysis.     

Effect of particle size based separation of milled corn stover on AFEX pretreatment and enzymatic digestibility

Particle size and compositional variance are found to have a substantial influence on ammonia fiber explosion (AFEX) pretreatment and enzymatic hydrolysis of lignocellulosic biomass. Corn stover was milled and fractionated into particle sizes of varying composition. The larger particle size fractions (rich in corn cob and stalk portions) were found to be more recalcitrant to hydrolysis compared to the smaller size fractions (rich in leaves and husk portion). Electron spectroscopy for chemical analysis (ESCA) and Fourier transform infrared spectroscopy (FTIR) were used for biomass surface and bulk compositional analysis, respectively. The ESCA results showed a 15-30% decrease in the O/C (oxygen to carbon) ratio after the pretreatment indicating an increase in the hydrophobic nature of biomass surface. FTIR results confirmed cleavage of the lignin-carbohydrate complex (LCC) for the AFEX-treated fractions. The spectroscopic results indicate the extraction of cleaved lignin phenolic fragments and other cell wall extractives to the biomass surface upon AFEX. Water washing of AFEX-treated fractions removed some of the hydrophobic extractives resulting in a 13% weight loss (dry weight basis). Phenolic content of wash stream was evaluated by the modified Prussian blue (MPB) method. Removal of ligno-phenolic extractives from the AFEX-treated biomass by water washing vastly improved the glucan conversion as compared to the unwashed samples. Reduction in substrate particle size was found to affect the AFEX process and rate of hydrolysis as well. Implications of the stover particle size, composition, and inhibitory role of the phenolic fragments on an integrated biorefinery are discussed.     

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and β-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase–xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.     

Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature

Alkaline pretreatment of spruce at low temperature in both presence and absence of urea was studied. It was found that the enzymatic hydrolysis rate and efficiency can be significantly improved by the pretreatment. At low temperature, the pretreatment chemicals, either NaOH alone or NaOH-urea mixture solution, can slightly remove lignin, hemicelluloses, and cellulose in the lignocellulosic materials, disrupt the connections between hemicelluloses, cellulose, and lignin, and alter the structure of treated biomass to make cellulose more accessible to hydrolysis enzymes. Moreover, the wood fiber bundles could be broken down to small and loose lignocellulosic particles by the chemical treatment. Therefore, the enzymatic hydrolysis efficiency of untreated mechanical fibers can also be remarkably enhanced by NaOH or NaOH/urea solution treatment. The results indicated that, for spruce, up to 70% glucose yield could be obtained for the cold temperature pretreatment (-15 degrees C) using 7% NaOH/12% urea solution, but only 20% and 24% glucose yields were obtained at temperatures of 23 degrees C and 60 degrees C, respectively, when other conditions remained the same. The best condition for the chemical pretreatment regarding this study was 3% NaOH/12% urea, and -15 degrees C. Over 60% glucose conversion was achieved upon this condition.