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。     

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.     

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。     

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.     

玉米秸秆不同部位的组成及其对预处理的影响

不同玉米秸秆部位的成分组成及分布对预处理和酶解影响显著。研究表明:韧皮部与髓芯的成分相近,但叶子的差异较大,其木聚糖和总糖的质量分数最高,分别为29.48%和66.15%,而木质素的质量分数最低,因而叶子更容易预处理。玉米秸秆在稀酸预处理过程中可回收96.9%葡聚糖和50.0%～70.0%木聚糖,其中50.0%～60.0%木聚糖水解成木糖溶出;不同部位的木聚糖损失率与初始的木聚糖含量正相关;经稀酸预处理后,叶子中葡聚糖的质量分数最高,达72.40%,叶子和髓芯易于被纤维素酶水解生成葡萄糖,而韧皮部困难。不同部位的酶解得率与自身的葡聚糖含量正相关,与酸不溶木质素含量负相关,同时受原料的物理结构、葡聚糖和木质素大分子的化学组成等影响。     

Fermentation of corn stover to carboxylic acids

This article describes countercurrent fermentation to anaerobically convert corn stover and pig manure to mixed carboxylic acids using a mixed culture of mesophilic microorganisms. Corn stover was pretreated with lime to increase digestibility. The Continuum Particle Distribution Model (CPDM) was used to simulate continuous fermentors based on data collected from batch experiments. This model saves considerable time in determining optimum operating conditions. For 80% corn stover/20% pig manure, the highest total carboxylic acid productivity was 1.81 g/(L of liquid. d) at a concentration of 21.4 g total acid/L. The highest total acid selectivity, yield, and conversion were 0.714 g total acid/g volatile solids (VS) digested, 0.550 g total acid/g VS fed, and 0.770 g VS digested/g VS fed, respectively, at a concentration of 16.0 g total acid/L. CPDM predicted the acid concentration and conversion within 13.4 and 11.6%, respectively.     

萃取耦合技术对玉米秸秆水解液发酵产丁醇的影响

本研究以玉米秸秆水解液为原料,通过萃取发酵技术生产燃料丁醇,以提高丁醇产量,降低生产成本。通过对萃取剂的筛选与条件优化,确定纤维丁醇发酵的萃取剂为油醇,添加时间为发酵0 h,添加比例为1:1 (V/V)。该条件下发酵32 g/L糖浓度的玉米秸秆水解液,丁醇和总溶剂产量分别为3.28 g/L和4.72 g/L,比对照分别提高958.1%和742.9%。以D301树脂脱毒后5%总糖浓度的玉米秸秆水解液进行丁醇萃取发酵,丁醇和总溶剂产量分别达到10.34 g/L和14.72 g/L,发酵得率为0.31 g/g,与混合糖发酵结果相当。研究结果表明萃取发酵技术能够显著提高原料的利用率和丁醇产量,为纤维丁醇工业化生产提供了技术支撑。     

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.     

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).     

Effect of additives on the digestibility of corn stover solids following pretreatment by leading technologies

Bovine serum albumin (BSA), Tween‐20, and polyethylene glycol (PEG6000) were added to washed corn stover solids produced by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute sulfuric acid (DA), lime, controlled pH, and sulfur dioxide (SO₂) pretreatments and to untreated corn stover (UT) and pure Avicel glucan prior to adding cellulase supplemented with β‐glucosidase at an activity ratio of 1:2/g and a moderate enzyme loading of 16.1 mg/g glucan in the raw corn stover. The additives were applied individually at 150, 300, and 600 mg/g glucan in the pretreated solids and in combinations of equal amounts of each that totaled 600 mg/g. The greatest increase in total sugar release was by Tween‐20 with SO₂ pretreated solids followed by PEG6000 with ARP solids and Tween‐20 with lime solids. The effectiveness of the additives was observed to depend on the type of sugars left in the solids, suggesting that it may be more beneficial to use the mixture of these additives to realize a high total sugar yield. In addition, little enhancement in sugar release was possible beyond a loading of 150 mg additives/g glucan for most pretreatments, and combinations did not improve sugar release much over use of additives alone for all except SO₂. Additives were also found to significantly increase concentrations of cellobiose and cellooligomers after 72 h of Avicel hydrolysis. Biotechnol. Bioeng. 2009;102: 1544–1557. © 2008 Wiley Periodicals, Inc.     

Pretreatment of corn stover and hybrid poplar by sodium hydroxide and hydrogen peroxide

Sodium hydroxide and its derivatives are used as pulping reagents, wherein the spent NaOH is recovered in salt form and reused. In this study, use of low concentration NaOH (1–5%) in pretreatment of corn stover and hybrid poplar was investigated. It was done with the understanding that NaOH can be recovered. One of the main objectives in this study is to explore the potential of H₂O₂ with NaOH for pretreatment of high lignin substrate such as hybrid poplar. Pretreatment time has not been optimized in this study but held constant at 24 h. Corn stover, after treatment with NaOH under moderate conditions, attains near quantitative glucan digestibility. On the other hand, hybrid poplar requires treatment at higher temperature and NaOH concentration to attain acceptable level of digestibility. Supplementation of hydrogen peroxide in the pretreatment significantly raises delignification and digestibility of hybrid poplar. It was also helpful in retaining the carbohydrates in the treated solids. Retention of hemicellulose after pretreatment provides a significant economic benefit as it eliminates the need for detoxifying hemicellulose sugars. As the residual xylan remaining after pretreatment is an impediment to enzymatic digestion of glucan, supplementation of xylanase has significantly increased the digestibility of glucan as well as xylan of the treated hybrid poplar. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Yield stress of pretreated corn stover suspensions using magnetic resonance imaging

Cellulose fibers in water form networks that give rise to an apparent yield stress, especially at high solids contents. Measuring the yield stress and correlating it with fiber concentration is important for the biomass and pulp industries. Understanding how the yield stress behaves at high solids concentrations is critical to optimize enzymatic hydrolysis of biomass in the production of biofuels. Rheological studies on pretreated corn stover and various pulp fibers have shown that yield stress values correlate with fiber mass concentration through a power‐law relationship. We use magnetic resonance imaging (MRI) as an in‐line rheometer to measure velocity profiles during pipe flow. If coupled with pressure drop measurements, these allow yield stress values to be determined. We compare our results with literature values and discuss the accuracy and precision of the rheo‐MRI measurement, along with the effects of fiber characteristics on the power‐law coefficients. Biotechnol. Bioeng. 2011;108: 2312–2319. © 2011 Wiley Periodicals, Inc.     

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.     

Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048. © 2010 Wiley Periodicals, Inc.     

Degradation of raw corn stover powder (RCSP) by an enriched microbial consortium and its community structure

A microbial consortium with a high cellulolytic activity was enriched to degrade raw corn stover powder (RCSP). This consortium degraded more than 51% of non-sterilized RCSP or 81% of non-sterilized filter paper within 8 days at 40 °C under facultative anoxic conditions. Cellulosome-like structures were observed in scanning electron micrographs (SEM) of RCSP degradation residue. The high cellulolytic activity was maintained during 40 subcultures in a medium containing cellulosic substrate. Small ribosomal gene sequence analyses showed the consortium contains uncultured and cultured bacteria with or without cellulolytic activities. Among these bacteria, some are anaerobic others aerobic. Analyses of the culture filtrate showed a typical anoxic polysaccharide fermentation during the culturing process. Reducing sugar concentration increased at early stage followed by various fermentation products that were consumed at the late stage.     

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.     

Detecting cellulase penetration into corn stover cell walls by immuno‐electron microscopy

In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild‐, moderate‐ and high‐severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme‐ and polymer‐specific antibodies. Low severity dilute‐acid pretreatment (20 min at 100°C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120°C) allowed the enzymes to penetrate ～20% of the cell wall, and the high severity (20 min pretreatment at 150°C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute‐acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high‐resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes. Biotechnol. Bioeng. 2009;103: 480–489. © 2009 Wiley Periodicals, Inc.     

稀碱预处理棕榈残渣制备纤维乙醇

以棕榈残渣(Empty fruit bunch,EFB)为原料,通过预处理、酶解、发酵等过程制备纤维乙醇.首先对比了碱、碱/过氧化氢等预处理条件对棕榈残渣组成及酶解的影响,结果表明稀碱预处理效果较好.适宜的稀碱预处理条件为:NaOH浓度为1％,固液比为1∶10,在40℃浸泡24 h后于121℃下保温30 min,在该条件下,EFB的固体回收率为74.09％,纤维素、半纤维素和木质素的含量分别为44.08％、25.74％和13.89％.对该条件下预处理后的固体样品,以底物浓度5％、酶载量30 FPU/g底物酶解72 h,纤维素和半纤维素的酶解率分别达到84.44％和89.28％.进一步考察了酶载量和底物浓度对酶解的影响以及乙醇批式同步糖化发酵,当酶载量为30 FPU/g底物,底物浓度由5％增加至25％时,利用酿酒酵母Saccharomyces cerevisiae(接种量为5％,VIV)发酵72 h后乙醇的浓度分别为9.76 g/L和35.25 g/L,可分别达到理论得率的79.09％和56.96％.     

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 ℃。