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 and structural features of corn stover by FeCl3 pretreatment

Corn stover was pretreated with FeCl₃ to remove almost all of the hemicellulose present and then hydrolyzed with cellulase and β-glucosidase to produce glucose. Enzymatic hydrolysis of corn stover that had been pretreated with FeCl₃ at 160 °C for 20 min resulted in an optimum yield of 98.0%. This yield was significantly higher than that of untreated corn stover (22.8%). FeCl₃ pretreatment apparently damaged the surface of corn stover and significantly increased the enzymatic digestibility, as evidenced by SEM and XRD analysis data. FTIR analysis indicated that FeCl₃ pretreatment could disrupt almost all the ether linkages and some ester linkages between lignin and carbohydrates but had no effect on delignification. The FeCl₃ pretreatment technique, as a novel pretreatment method, enhances enzymatic hydrolysis of lignocellulosic biomass by destructing chemical composition and altering structural features.     

A novel mechanism and kinetic model to explain enhanced xylose yields from dilute sulfuric acid compared to hydrothermal pretreatment of corn stover

Pretreatment of corn stover in 0.5% sulfuric acid at 160 °C for 40 min realized a maximum monomeric plus oligomeric xylose yield of 93.1% compared to a maximum of only 71.5% for hydrothermal (no added mineral acid) pretreatment at 180 °C for 30 min. To explain differences in dilute acid and hydrothermal yields, a fast reacting xylan fraction (0.0889) was assumed to be able to directly form monomeric xylose while a slow reacting portion (0.9111) must first form oligomers during hydrothermal pretreatment. Two reactions to oligomers were proposed: reversible from fast reacting xylan and irreversible from slow reacting xylan. A kinetic model and its analytical solution simulated xylan removal data well for dilute acid and hydrothermal pretreatment of corn stover. These results suggested that autocatalytic reactions from xylan to furfural in hydrothermal pretreatment were controlled by oligomeric xylose decomposition, while acid-catalytic reactions in dilute acid pretreatment were controlled by monomeric xylose decomposition.     

The effect of high intensity mixing on the enzymatic hydrolysis of concentrated cellulose fiber suspensions

Enzymatic hydrolysis of lignocellulosic biomass in a high shear environment was examined. The conversion of cellulose to glucose in samples mixed in a torque rheometer producing shear flows similar to those found in twin screw extruders was greater than that of unmixed samples. In addition, there is a synergistic effect of mixing and enzymatic hydrolysis; mixing increases the rate of cellulose conversion while the increased conversion facilitates mixing. The synergy appears to result in part from particle size reduction, which is more significant when hydrolysis occurs during intense mixing.     

中和剂对稀酸蒸爆玉米秸秆酶解效果的影响

以稀酸蒸爆的玉米秸秆为研究对象,考察直接水洗、Ca(OH)2、NaOH、氨水中和物料至pH 5,在固液比1∶10、酶添加量为每克纤维素14 U(滤纸酶活)的酶解条件下对纤维素转化率的影响。结果表明:水洗、Ca(OH)2、NaOH、氨水中和物料酶解72 h后,纤维素转化率分别为91.7%、80.7%、83.1%及81.7%。同时对影响纤维素酶解效率的各种因素进行了探讨。从综合成本及后续发酵过程考虑,用氨水中和稀酸蒸爆物料更适合于工业化生产。     

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。     

Biogas production and saccharification of Salix pretreated at different steam explosion conditions

Different steam explosion conditions were applied to Salix chips and the effect of this pretreatment was evaluated by running both enzymatic hydrolysis and biogas tests. Total enzymatic release of glucose and xylose increased with pretreatment harshness, with maximum values being obtained after pretreatment for 10 min at 210 °C. Harsher pretreatment conditions did not increase glucose release, led to degradation of xylose and to formation of furfurals. Samples pretreated at 220 and 230 °C initially showed low production of biogas, probably because of inhibitors produced during the pretreatment, but the microbial community was able to adapt and showed high final biogas production. Interestingly, final biogas yields correlated well with sugar yields after enzymatic hydrolysis, suggesting that at least in some cases a 24 h enzymatic assay may be developed as a quick method to predict the effects of pretreatment of lignocellulosic biomass on biogas yields.     

Influence of xylan on the enzymatic hydrolysis of steam‐pretreated corn stover and hybrid poplar

The focus of this study was to alter the xylan content of corn stover and poplar using SO₂‐catalyzed steam pretreatment to determine the effect on subsequent hydrolysis by commercial cellulase preparations supplemented with or without xylanases. Steam pretreated solids with xylan contents ranging from ～1 to 19% (w/w) were produced. Higher xylan contents and improved hemicellulose recoveries were obtained with solids pretreated at lower severities or without SO₂‐addition prior to pretreatment. The pretreated solids with low xylan content (<4% (w/w)) were characterized by fast and complete cellulose to glucose conversion when utilizing cellulases. Commercial cellulases required xylanase supplementation for effective hydrolysis of pretreated substrates containing higher amounts of xylan. It was apparent that the xylan content influenced both the enzyme requirements for hydrolysis and the recovery of sugars during the pretreatment process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: SEM imaging (part 2)

In the first part of our work, we combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis experiments coupled with chemical analysis of the different fractions of corn stover showed significant differences in cell wall structure before and after liquid hot water pretreatment. The extent of enzyme hydrolysis followed the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Since similar lignin contents remained after liquid hot water pretreatment of leaves, rind, and pith, our results indicated that the amount of lignin alone is not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. While the role of structural characteristics on enzyme hydrolysis of cell walls is measured as described in part I, the SEM images presented in this part II of our work show that sugar yields from enzymatic hydrolysis of corn fractions correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.     

蒸汽爆破玉米秸秆酶解动力学

为了掌握蒸汽爆破玉米秸秆的酶解特性,研究了不同底物浓度、酶浓度、温度对反应速率的影响。运用米氏方程对酶解动力学过程进行拟合,结果表明,纤维素酶对该玉米秸秆的水解反应在反应前3 h符合一级反应,可用米氏方程对其进行拟合。在转速为120 r/min、酶浓度为1.2 FPU/mL、pH 5.0、温度为45 ℃时米氏常数Km为11.71 g/L,最大反应速率Vm为1.5 g/(L·h)。确立了包括底物浓度、酶浓度、温度在内的酶解动力学模型,该模型适合温度为30 ℃~50 ℃。     

Kinetic modeling analysis of maleic acid-catalyzed hemicellulose hydrolysis in corn stover

Maleic acid-catalyzed hemicellulose hydrolysis reaction in corn stover was analyzed by kinetic modeling. Kinetic constants for Saeman and biphasic hydrolysis models were analyzed by an Arrhenius-type expansion which include activation energy and catalyst concentration factors. The activation energy for hemicellulose hydrolysis by maleic acid was determined to be 83.3 +/- 10.3 kJ/mol, which is significantly lower than the reported E(a) values for sulfuric acid catalyzed hemicellulose hydrolysis reaction. Model analysis suggest that increasing maleic acid concentrations from 0.05 to 0.2 M facilitate improvement in xylose yields from 40% to 85%, while the extent of improvement flattens to near-quantitative by increasing catalyst loading from 0.2 to 1 M. The model was confirmed for the hydrolysis of corn stover at 1 M maleic acid concentrations at 150 degrees C, resulting in a xylose yield of 96% of theoretical. The refined Saeman model was used to evaluate the optimal condition for monomeric xylose yield in the maleic acid-catalyzed reaction: low temperature reaction conditions were suggested, however, experimental results indicated that bi-phasic behavior dominated at low temperatures, which may be due to the insufficient removal of acetyl groups. A combination of experimental data and model analysis suggests that around 80-90% xylose yields can be achieved at reaction temperatures between 100 and 150 degrees C with 0.2 M maleic acid.     

Effect of anatomical fractionation on the enzymatic hydrolysis of acid and alkaline pretreated corn stover

Due to concerns with biomass collection systems and soil sustainability there are opportunities to investigate the optimal plant fractions to collect for conversion. An ideal feedstock would require a low severity pretreatment to release a maximum amount of sugar during enzymatic hydrolysis. Corn stover fractions were separated manually and analyzed for glucan, xylan, acid soluble lignin, acid insoluble lignin, and ash composition. The stover fractions were also pretreated with either 0%, 0.4%, or 0.8% NaOH for 2 h at room temperature, washed, autoclaved and saccharified. In addition, dilute sulfuric acid pretreated samples underwent simultaneous saccharification and fermentation (SSF) to ethanol. In general, the two pretreatments produced similar trends with cobs, husks, and leaves responding best to the pretreatments, the tops of stalks responding slightly less, and the bottom of the stalks responding the least. For example, corn husks pretreated with 0.8% NaOH released over 90% (standard error of 3.8%) of the available glucan, while only 45% (standard error of 1.1%) of the glucan was produced from identically treated stalk bottoms. Estimates of the theoretical ethanol yield using acid pretreatment followed by SSF were 65% (standard error of 15.9%) for husks and 29% (standard error of 1.8%) for stalk bottoms. This suggests that integration of biomass collection systems to remove sustainable feedstocks could be integrated with the processes within a biorefinery to minimize overall ethanol production costs.     

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.     

Pretreatment of corn stover by combining ionic liquid dissolution with alkali extraction

Pretreatment plays an important role in the efficient enzymatic hydrolysis of biomass into fermentable sugars for biofuels. A highly effective pretreatment method is reported for corn stover which combines mild alkali-extraction followed by ionic liquid (IL) dissolution of the polysaccharides and regeneration (recovery of the polysaccharides as solids). Air-dried, knife-milled corn stover was soaked in 1% NaOH at a moderate condition (90°C, 1 h) and then thoroughly washed with hot deionized (DI) water. The alkali extraction solublized 75% of the lignin and 37% of the hemicellulose. The corn stover fibers became softer and smoother after the alkali extraction. Unextracted and extracted corn stover samples were separately dissolved in an IL, 1-butyl-3-methylimidazolium chloride (C(4) mimCl), at 130°C for 2 h and then regenerated with DI water. The IL dissolution process did not significantly change the chemical composition of the materials, but did alter their structural features. Untreated and treated corn stover samples were hydrolyzed with commercial enzyme preparations including cellulases and hemicellulases at 50°C. The glucose yield from the corn stover sample that was both alkali-extracted and IL-dissolved was 96% in 5 h of hydrolysis. This is a highly effective methodology for minimizing the enzymatic loading for biomass hydrolysis and/or maximizing the conversion of biomass polysaccharides into sugars.     

Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover

Ionic liquid (IL) and ammonia fiber expansion (AFEX) pretreatments were studied to develop the first direct side-by-side comparative assessment on their respective impacts on biomass structure, composition, process mass balance, and enzymatic saccharification efficiency. AFEX pretreatment completely preserves plant carbohydrates, whereas IL pretreatment extracts 76% of hemicellulose. In contrast to AFEX, the native crystal structure of the recovered corn stover from IL pretreatment was significantly disrupted. For both techniques, more than 70% of the theoretical sugar yield was attained after 48 h of hydrolysis using commercial enzyme cocktails. IL pretreatment requires less enzyme loading and a shorter hydrolysis time to reach 90% yields. Hemicellulase addition led to significant improvements in the yields of glucose and xylose for AFEX pretreated corn stover, but not for IL pretreated stover. These results provide new insights into the mechanisms of IL and AFEX pretreatment, as well as the advantages and disadvantages of each.     

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.     

Economic impact of total solids loading on enzymatic hydrolysis of dilute acid pretreated corn stover

In process integration studies of the biomass-to-ethanol conversion process, it is necessary to understand how cellulose conversion yields vary as a function of solids and enzyme loading and other key operating variables. The impact of solids loading on enzymatic cellulose hydrolysis of dilute acid pretreated corn stover slurry was determined using an experimental response surface design methodology. From the experimental work, an empirical correlation was obtained that expresses monomeric glucose yield from enzymatic cellulose hydrolysis as a function of solids loading, enzyme loading, and temperature. This correlation was used in a technoeconomic model to study the impact of solids loading on ethanol production economics. The empirical correlation was used to provide a more realistic assessment of process cost by accounting for changes in cellulose conversion yields at different solids and enzyme loadings as well as enzyme cost. As long as enzymatic cellulose conversion drops off at higher total solids loading (due to end-product inhibition or other factors), there is an optimum value for the total solids loading that minimizes the ethanol production cost. The optimum total solids loading shifts to higher values as enzyme cost decreases.     

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: enzymatic hydrolysis (part 1)

Lignin content, composition, distribution as well as cell wall thickness, structures, and type of tissue have a measurable effect on enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. The first part of our work combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. The extent of enzyme hydrolysis follows the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Physical fractionation of corn stalks or other C(4) grasses into soft and hard tissue types could reduce cost of cellulose conversion by enabling reduced enzyme loadings to hydrolyze soft tissue, and directing the hard tissue to other uses such as thermal processing, combustion, or recycle to the land from which the corn was harvested.     

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.