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.     

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.     

玉米秸秆生物炭制备及结构特性分析

为了高效、经济、环保地解决华北平原地区玉米秸秆处置问题并寻求有效途径,该研究以玉米秸秆为原料,采用限氧裂解法在不同温度(200℃、300℃、400℃、500℃)下制备生物炭,并对生物炭的热解动力学、结构形貌、元素组成、比表面积、孔径分布、官能团等理化特征进行了分析表征。结果表明:不同裂解温度制备的生物炭具有不同的差热曲线,其官能团的组成也存在差异,这表明了样品中不同生物质的热解反应过程。随着热解温度的升高,生物炭产率、氢和氧含量下降,同时H/C和(O+N)/C比值也降低,而碳和氮含量却升高,说明生物炭芳香性增强,亲水性和极性减弱,性质趋于稳定。生物炭热重曲线和差热曲线分为三个过程,热解温度高时失重比例低,曲线趋向平缓。生物炭的比表面积、微孔比表面积、中孔体积和微孔体积随着热解温度的升高而增大,但最可几孔径却减小,吸附能力增强。综上所述,400℃的温度制备生物炭,其产率相对较高、结构最稳定、吸附性能最佳,有助于最大程序的利用农业废弃物资源、降低耗能,提高农产品附加值。     

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.     

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.     

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.     

Effect of using a nitrogen atmosphere on enzyme hydrolysis at high corn stover loadings in an agitated reactor

A comprehensive review of the literature shows that enzyme hydrolysis efficiency decreases with increased solids loadings at constant enzyme:cellulose ratios for pretreated lignocellulosic substrates. In seeking a mechanistic explanation for this phenomenon, we found that a nitrogen atmosphere enhances enzyme hydrolysis and minimizes the decrease in glucose yields as solids loadings are increased in an agitated bioreactor. For liquid hot water pretreated corn stover, at solids loadings of both 100 and 200 g/L and hydrolyzed for 72 hr in a 1 L bioreactor at pH 5.0 with 3.6 mg protein per g biomass, glucose yields were 55% in a nitrogen atmosphere versus 45% in air with agitation and about 34% without agitation. While mixing promotes biomass/enzyme contact and disperses sugars released during hydrolysis that would otherwise cause product inhibition, nitrogen gas displaces air, avoiding deactivation of cellulases by oxygen. The nitrogen effect points to a facile approach of enhancing hydrolysis at high solids loadings.     

Shock treatment of corn stover

Corn stover digestibility was enhanced via shock treatment. A slurry of lime‐treated corn stover was placed in a partially filled closed vessel. From the ullage space, either a shotgun shell was fired into the slurry, or a gas mixture was detonated. Various conditions were tested (i.e., pressures, depth, solids concentrations, gas mixtures). A high pressurization rate (108,000 MPa/s shotgun shells; 4,160,000 MPa/s hydrogen/oxygen detonation) was the only parameter that improved enzymatic digestibility. Stoichiometric propane/air deflagration had a low pressurization rate (37.2 MPa/s) and did not enhance enzymatic digestibility. Without shock, enzymatic conversion of lime‐treated corn stover was 0.80 g glucan digested/g glucan fed with an enzyme loading of 46.7 mg protein/g glucan. With shock, the enzyme loading was reduced by ～2× while maintaining the same conversion. Detonations are extraordinarily fast; rapidly cycling three small vessels (0.575 m³ each) every 7.5 s enables commercially relevant shock treatment (2,000 tone/day). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:815–823, 2017     

Acidogenic fermentation of corn stover

Corn stover was fermented by anaerobic acidogenic bacteria to produce volatile (C₂–C₆) organic acids. Mild pretreatment with dilute alkali solutions produced a two-fold increase in fermentability. A mixture of lime and sodium carbonate was found to be a better pretreatment agent than sodium hydroxide. Methane generation was inhibited by low temperature (? 25°C) and high solids [≥ 2.5% (w/v)] fermentation. Volatile acid yields of 0.5–0.55 g acetic acid equiv/g dry ash-free (DAF) stover could be obtained in batch fermentations. Several extractants and extraction solvents for organic acids were found to be nontoxic to acidogenic fermentation. The data show that acidogenic fermentation can produce useful volatile fatty acids in high yields from a complex lignocellulosic feedstock. These fermentations are nonsterile, need no stirring, and are easy to run. Moreover, cellulose, pentosans, and other carbohydrates are directly utilized by acidogenic bacteria. Hence, acidogenic fermentation could be useful in converting biomass to chemical feedstocks and fuel.     

Neural network prediction of biomass digestibility based on structural features

Plots of biomass digestibility are linear with the natural logarithm of enzyme loading; the slope and intercept characterize biomass reactivity. The feed-forward back-propagation neural networks were performed to predict biomass digestibility by simulating the 1-, 6-, and 72-h slopes and intercepts of glucan, xylan, and total sugar hydrolyses of 147 poplar wood model samples with a variety of lignin contents, acetyl contents, and crystallinity indices. Regression analysis of the neural network models indicates that they performed satisfactorily. Increasing the dimensionality of the neural network input matrix allowed investigation of the influence glucan and xylan enzymatic hydrolyses have on each other. Glucan hydrolysis affected the last stage of xylan digestion, and xylan hydrolysis had no influence on glucan digestibility. This study has demonstrated that neural networks have good potential for predicting biomass digestibility over a wide range of enzyme loadings, thus providing the potential to design cost-effective pretreatment and saccharification processes.     

木质素降解菌株的分离及其降解玉米秸秆过程中产酶特点

【目的】筛选高效降解木质素的菌株,并研究其以玉米秸秆为底物时木素降解酶活性。【方法】本研究以愈创木酚培养基和苯胺蓝培养基从吉林省不同经纬度的自然朽木及腐朽玉米秸秆土壤样品中分离、筛选得到高效降解木质素的菌株,并对其形态学鉴定,通过ITS序列分析构建系统发育树,分析菌株的分类地位。通过秸秆固体发酵过程产生的胞外木质素酶的活性分析,选出高效秸秆降解菌。【结果】筛选出1株高效降解秸秆的真菌,对其进行形态学特征和ITS序列分析,命名为白囊耙齿菌W2(Irpex lacteus W2)。该菌株在4–8 d内产生的锰过氧化物酶(Manganese peroxidase)呈上升趋势,并且在8 d达到峰值86.31 U/mL,与黄孢原毛平革菌(Phanerochaete chrysosporium)的最高酶活力45.86 U/mL相比,高出了88.20%(P0.01);该菌株的漆酶(Laccase)活力8 d时达到20.60 U/mL,比对照高40.76%(P0.05)。【结论】本研究分离到一株具有较强降解秸秆能力的真菌,初步鉴定为Irpex lacteus W2,具有较强的降解秸秆能力,其降解秸秆过程中产生较高的锰过氧化物酶与漆酶活力。     

秸秆颗粒还田对黑土土壤酶活性及细菌群落的影响

为探讨不同玉米秸秆颗粒还田量对黑土生物学特性及细菌群落的影响,在内蒙古兴安盟扎赉特旗农业科技示范园试验地设置秸秆0%还田,还田量0kg/hm^2(CK)、秸秆60%还田,还田量4500kg/hm^2(JG1)、秸秆70%还田,5250kg/hm^2(JG2)、秸秆80%还田,6000kg/hm^2(JG3)、秸秆90%还田,6750kg/hm^2(JG4)和秸秆100%还田,7500kg/hm^2(JG5)6个处理,通过连续2年大田试验,研究土壤蔗糖酶、脲酶、过氧化氢酶、碱性磷酸酶活性、微生物生物量碳氮以及细菌群落的变化。结果表明:秸秆还田能够增加土壤蔗糖酶(3.29%—32.12%),脲酶(5.32%—52.66%),过氧化氢酶(0.60%—27.11%),碱性磷酸酶的活性(10.89%—64.20%),土壤微生物生物量碳(1.32%—7.07%)、氮(16.35%—80.46%)含量;秸秆施入土壤也提高了黑土变形菌门和厚壁菌门相对丰度,提高了土壤固氮、分解养分及抵御病害能力,并降低了放线菌门相对丰度,降低了土壤病害发生概率,还出现了具有固氮、吸磷、改良土壤特性的新细菌,可见玉米秸秆还田具有重要的生态学意义,可在一定程度上增加细菌数量和种类多样性,进而使土壤系统向稳定健康的方向发展。综合研究结果在本试验条件下,以6750kg/hm^2为较适宜的玉米秸秆颗粒还田量。     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Partial flow of compressed-hot water through corn stover to enhance hemicellulose sugar recovery and enzymatic digestibility of cellulose

Flowthrough pretreatment with compressed-hot water can significantly increase the removal of xylan and lignin and enhance xylose sugar yields and cellulose digestibility, especially at high flow rates. However, continuous flowthrough operations that realize these benefits suffer from a large amount of water consumption that leads to high energy requirements for pretreatment and downstream processing. Because high flow rates are particularly effective early in hemicellulose hydrolysis and less effective later, flow with compressed-hot water was applied at selected intervals, and performance was compared with that of batch and flowthrough operations for corn stover pretreated with compressed-hot water at 200 degrees C. Partial flow reduced water consumption by 60% compared with continuous flowthrough operation but still achieved higher xylose sugar yields (84-89%) compared to batch pretreatment (46.6%). In addition, corn stover cellulose pretreated by partial flow had higher enzymatic digestibility (88-90%) than batch operations (approximately 85%) at otherwise identical conditions, apparently due to much higher lignin removal for the former (40-45% vs 10-12%). Partial flow also reduced degradation, with recovery of xylose and glucose in the solids and hydrolyzate increased to 90-92% vs only about 76% for batch operation. The partial flow approach could be further improved by optimizing the operating strategy and reaction conditions, suggesting that this novel pretreatment could lead to advanced biomass pretreatment technology.     

Invertase covalent grafting onto corn stover

The covalent coupling of an invertase from baker's yeast onto an agricultural by-product, corn grits, has been developed. The optimal conditions for each step of the chemical modification of the support have been determined: oxidation with sodium metaperiodate, amination with ethylenediamine, reduction with sodium cyanoborohydride, and activation with glutaraldehyde. Activities up to 7.2 x 10(4) mumol reducing sugars produced/min g support could thus be achieved. Invertase coupling onto corn grits yields a derivative with a 25 times higher activity than when coupling this enzyme onto porous silica. The operational stability of invertase immobilized onto corn stover was found to be very high, with a half-life of up to 365 days at 40 degrees C when using a 2M sucrose solution as substrate. This immobilization method could be easily scaled up to the preparation of 10 kg of invertase derivative.     

Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover

Ethanol can be produced from lignocellulosic biomass using steam pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields, from both hemicellulose and cellulose are critical parameters for an economically-feasible ethanol production process. This study shows that a near-theoretical glucose yield (96-104%) from acid-catalysed steam pretreated corn stover can be obtained if xylanases are used to supplement cellulases during hydrolysis. Xylanases hydrolyse residual hemicellulose, thereby improving the access of enzymes to cellulose. Under these conditions, xylose yields reached 70-74%. When pre-treatment severity was reduced by using autocatalysis instead of acid-catalysed steam pretreatment, xylose yields were increased to 80-86%. Partial delignification of pretreated material was also evaluated as a way to increase the overall sugar yield. The overall glucose yield increased slightly due to delignification but the overall xylose yield decreased due to hemicellulose loss in the delignification step. The data also demonstrate that steam pretreatment is a robust process: corn stover from Europe and North America showed only minor differences in behaviour.     

Pretreatment of corn stover by aqueous ammonia

Corn stover was pretreated with aqueous ammonia in a flow-through column reactor, a process termed ammonia recycled percolation (ARP). This method was highly effective in delignifying of the biomass, reducing the lignin content by 70-85%. Most lignin removal occurred within the first 20 min of the process. Lignin removal by ARP was further confirmed by FTIR analysis and lignin staining. The ARP process solubilized 40-60% of the hemicellulose but left the cellulose intact. The solubilized carbohydrate existed in oligomeric form. Carbohydrate decomposition during the pretreatment was insignificant. Corn stover treated for 90 min exhibited enzymatic digestibility of 99% with 60 FPU/g of glucan enzyme loading, and 92.5% with 10 FPU/g of glucan. The digestibility of ARP treated corn stover was substantially higher than that of alpha-cellulose. The enzymatic digestibility was related with the removal of lignin and hemicellulose, perhaps due to increased surface area and porosity. The SEM pictures indicated that the biomass structure was deformed and its fibers exposed by the pretreatment. The crystallinity index increased with pretreatment reflecting removal of the amorphous portion of biomass. The crystalline structure of the cellulose in the biomass, however, was not changed by the ARP treatment.     

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).     

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.