Pretreatment of Extruded Corn Stover with Polyethylene Glycol to Enhance Enzymatic Hydrolysis: Optimization, Kinetics, and Mechanism of Action

Due to the high potential of the extrusion technique for pretreatment of lignocellulosic substrates, several attempts have been conducted in previous studies to further increase the subsequent sugar yield from extrusion pretreatment. Examples include application of chemicals along with extrusion, such as alkali-extrusion and ethylene glycol-extrusion, or before extrusion, such as hot water extraction. In this study, a new sequential technique has been developed for pretreatment of corn stover (CS), which utilizes an initial extrusion pretreatment (155?rpm screw speed and temperatures of 90°C, 180°C and 180°C corresponding to feed, barrel and die zones, respectively at a reaction time of 45?C90?s) followed by pretreatment with polyethylene glycol 6,000 (PEG). In order to fully characterize the response for sugar yield over the range of surfactant treatment conditions assessed, response surface methodology was used. Treatment temperature, incubation time and PEG concentration were varied between 45?C55°C, 1?C4?h, 0.15?C0.6?g PEG/g glucan, respectively. Statistical analysis was conducted by fitting the glucose and xylose yields to a quadratic polynomial model. PEG concentration and temperature were found to be the most significant factors in surfactant pretreatment. The optimum condition resulted in 25.4% and 10.3% increase in glucose and xylose yield, respectively. Using the combination of 10.8?FPU/g glucan of Ctec₂ and 0.3?g PEG/g glucan, the glucose yield of extruded CS reached 98%. A yield was 64% resulted from application of similar amounts of Ctec and Htec. Decreased adsorption of enzyme to the lignocellulosic substrate as well as increased enzyme activity and reaction velocity indicated by kinetic parameter evaluation and nitrogen combustion analysis suggested an increased solubilization of cellulase in the presence of PEG. We propose that a non-productive adsorption of enzymes occur during hydrolysis not only due to lignin but also due to crystalline cellulose. Comparison of enzyme adsorptions and increase in sugar yields between Avicel and CS suggests the presence of other potential mechanisms of action for PEG in addition to increase of enzyme solubilization.     

酶水解制备花生粕多肽工艺的优化

目的：优化研究花生粕制备多肽的工艺。方法：采用酶法水解提取多肽、用总蛋白试剂盒（TP）双缩脲比色法在540nm处测定其含量。结果：用木瓜蛋白酶水解花生粕蛋白得到多肽的最佳工艺条件为：加酶量6 300u/g原料、温度45℃、底物浓度10%、酶水解时间5h。     

Novel Single-step Pretreatment of Steam Explosion and Choline Chloride to De-lignify Corn Stover for Enhancing Enzymatic Edibility

It is important to develop efficient and economically feasible pretreatment methods for lignocellulosic biomass, to increase annual biomass production. A number of pretreatment methods were introduced to promote subsequent enzymatic hydrolysis of biomass for green energy processes. Pretreatment with steam explosion removes the only xylan at high severity but increases lignin content. In this study, corn stover soaked in choline chloride solution before the steam explosion is economically feasible as it reduced cost. Enzymatic hydrolysis of de-lignified corn stover is enhanced by combinatorial pretreatments of steam explosion and choline chloride. Corn stover pretreated with choline chloride at the ratio of 1:2.2 (w/w), 1.0 MPa, 184 °C, for 15 min efficiently expelled 84.7% lignin and 78.9% xylan. The residual solid comprised of 74.59% glucan and 7.51% xylan was changed to 84.2% glucose and 78.3% xylose with enzyme stacking of 10FPU/g. This single-step pretreatment had ∼ 4.5 and 6.4 times higher glucose yield than SE-pretreated and untreated corn stover, respectively. Furthermore, SEM, XRD and FTIR indicated the porosity, crystalline changes, methoxy bond-cleavage respectively due to the lignin and hemicellulose expulsion. Thus, the released acetic acid during this process introduced this novel strategy, which significantly builds the viability of biomass in short pretreatment time.     

鲜鹿茸酶降解过程的研究

研究了胰蛋白酶、Alcalase 碱性蛋白酶、木瓜蛋白酶对鲜鹿茸的降解过程,确定了优化降解工艺条件,具有一定的理论意义和实践价值。确定了Alcalase 碱性蛋白酶的降解效率最高,通过单因素实验确定了降解过程中底物浓度、酶解温度、pH值和酶解时间为影响鲜鹿茸降解率的主要因素。正交试验确定最佳的酶解条件为:底物浓度0.08 g/ml、酶解温度65 ℃、pH 9.0、酶解时间6.0 h。在此条件下,鲜鹿茸降解率高达92.6%,氨基酸产品收率达12.1%。     

Understanding the Physicochemical Characteristics and the Improved Enzymatic Saccharification of Corn Stover Pretreated with Aqueous and Gaseous Ammonia

Physicochemical characteristics of corn stover pretreated by soaking in aqueous ammonia (SAA) and low-moisture anhydrous ammonia (LMAA) were compared and investigated. The glucan digestibility of the treated biomass reached 90 % (SAA) and 84 % (LMAA). The LMAA pretreatment enhanced the digestibility by cleaving cross-linkages between cell wall components, whereas the SAA pretreatment additionally improved the digestibility by efficiently removing a major portion of the lignin under mild reaction conditions without significant loss of carbohydrates. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC) revealed the structural and chemical transformations of lignin during the pretreatments. Both pretreatments effectively cleaved ferulate cell wall cross-linking that is associated with the recalcitrance of grass lignocellulosics toward enzymatic saccharification. Extracted lignin from SAA pretreatment was extensively depolymerized but retained “native” character, as evidenced by the retention of β-ether linkages.     

植物纤维的水解与SSF过程的数学模型

采用一种植物纤维木糖渣为原料,对纤维素酶水解及同时糖化和发酵（SSF）过程进行了研究,研究结果表明,在纤维素含量为42g.L^-1,纤维素酶的加入量为280FPIU.L^-1,乳酸菌用量为0．5g.L^-1时,乳酸对纤维素的转化率可达80％,并从反应机理及动力学角度出发,建立了纤维素酸水解与SSF过程的数学模型。     

Aerobic and Anaerobic Storage of Single-pass,Chopped Corn Stover

Corn stover has great potential as a biomass feedstock due its widespread availability. However, storage characteristics of moist corn stover harvested from single-pass harvesters have not been well quantified. In 2007, whole-plant corn stover at 19.1–40.3% (w.b.) moisture content was stored for 237 days in aerobic piles, one covered and one uncovered, as well an anaerobic silo bag. In 2008, two stover materials—whole plant and cob/husk from 31.7% to 58.1% (w.b.) moisture—were stored for 183 or 204 days in covered and uncovered anaerobic piles, ventilated bags, or anaerobic silo bags. Stover stored in uncovered piles was rehydrated by precipitation, which increased biological activity resulting in dry matter (DM) losses from 8.2% to 39.1% with an average of 21.5%. Stover in covered piles was successfully conserved when the average moisture was less than 25% (w.b.) with DM losses of 3.3%. Stover above 36% (w.b.) moisture and piled under a plastic cover had DM losses from 6.4% to 20.2% with an average of 11.9%. Localized heating occurred in the aerobic piles when moisture was above 45% (w.b.) which lead to temperatures where spontaneous combustion might be a concern (i.e., >70°C). Ambient air blown through a center tube in the ventilated storage bag dried stover near the tube to an average of 24.2% (w.b.), but the remainder of the bag averaged 46.8% (w.b.) at removal. Loss of DM ranged from 7.4% to 22.0% with an average of 11.8% with this storage method. Stover was most successfully conserved in the bags where anaerobic conditions were maintained. Under anaerobic conditions, DM losses ranged from 0.2% to 0.9%. When anaerobic conditions were not maintained in the silo bag, DM losses averaged 6.1% of DM. Anaerobic storage is the best solution for conserving the value of moist corn stover.     

青贮饲料的研究、发展及现状

我国从上世纪50年代开始对青贮饲料技术进行研究和推广,但受当时生产水平和经济条件等因素制约,工作多属于生产性试探,对青贮饲料发酵机制,二次发酵问题及防腐措施等方面的研究甚少。在我国,主要以利用黄秸秆为饲料,而鲜青贮饲料的利用和推广尚有一定困难。     

响应面法优化米糠淀粉酶法水解工艺

以米糠为原料,对米糠淀粉酶法水解生产葡萄糖的液化工艺进行研究和优化,来提高葡萄糖收率。在单因素试验的基础上,用响应面法对液化工艺进行优化。结果表明,液化工艺的最佳条件为酶用量0.11%、醪浓度25%、pH=6.0、温度88℃,在此条件下得到的液化葡萄糖值(即DX值)平均值为6.54%。然后对此液化液进行糖化,最终得到的糖化液DX值为97.07%。     

Development of Sustainable Corn Stover Harvest Strategies for Cellulosic Ethanol Production

To prepare for a 2014 launch of commercial scale cellulosic ethanol production from corn/maize (Zea mays L.) stover, POET-DSM near Emmetsburg, IA has been working with farmers, researchers, and equipment dealers through “Project Liberty” on harvest, transportation, and storage logistics of corn stover for the past several years. Our objective was to evaluate seven stover harvest strategies within a 50-ha (125 acres) site on very deep, moderately well to poorly drained Mollisols, developed in calcareous glacial till. The treatments included the following: conventional grain harvest (no stover harvest), grain plus a second-pass rake and bale stover harvest, and single-pass grain plus cob-only biomass, grain plus vegetative material other than grain [(MOG) consisting of cobs, husks, and upper plant parts], grain plus all vegetative material from the ear shank upward (high cut), and all vegetative material above a 10 cm stubble height (low cut), with a John Deere 9750 STS combine, and grain plus direct baling of MOG with an AgCo harvesting system. Average grain yields were 11.4, 10.1, 9.7, and 9.5 Mg ha^?1 for 2008, 2009, 2010, and 2011, respectively. Average stover harvest ranged from 0 to 5.6 Mg ha^?1 and increased N, P, and K removal by an average of 11, 1.6, and 15 kg Mg^?1, respectively. Grain yield in 2009 showed a significant positive response to higher 2008 stover removal rates, but grain yield was not increased in 2010 or 2011 due to prior-year stover harvest. High field losses caused the direct-bale treatment to have significantly lower grain yield in 2011 because the AgCo system could not pick up the severely lodged crop. We conclude that decreases in grain yield across the 4 years were due more to seasonal weather patterns, spatial variability, and not rotating crops than to stover harvest.     

Low Temperature and Long Residence Time AFEX Pretreatment of Corn Stover

Low temperature and long residence time pretreatments have been proposed as an alternative to conventional pretreatments within a centralized biorefinery, allowing for a decentralized pretreatment without high energy costs. Ammonia fiber expansion (AFEX?) pretreatment may be uniquely suitable for decentralized pretreatment, and this study considers the possibility of decreasing the temperature in AFEX pretreatment of corn stover. AFEX pretreatment at 40°C and 8?h produced comparable sugar and ethanol yields as conventional AFEX pretreatment at high temperatures and short residence time during subsequent hydrolysis and fermentation. Increasing the ammonia loading at these temperatures tends to increase digestibility, although the moisture content of the reaction has little effect. This study suggests a greater flexibility in AFEX pretreatment conditions than previously thought, allowing for an alternative approach for decentralized facilities if the economic conditions are appropriate.     

Rapid Biomass Quality Determination of Corn Stover Using Near-Infrared Reflectance Spectroscopy

Near-infrared reflectance spectroscopy (NIRS) has been used extensively in the forage industry for rapid measurement of forage constituents and could be useful for determining quality of biomass feedstocks at the point of delivery. In previous work, we developed an assay that partitions feedstock carbohydrates based on their availability to be converted to fermentable sugars, including non-structural carbohydrates (C _N), biochemically available carbohydrates (C _B) with an associated first-order availability rate constant (k _B), and unavailable carbohydrates (C _U ). Additional quality parameters measured included neutral detergent lignin (NDL), total available carbohydrates (C _A), and total carbohydrates (C _T). We evaluated the variability of biomass quality parameters in a set of corn stover samples and developed calibration equations for determining parameter values using NIRS. Fifty-two corn stover samples harvested in Iowa and Wisconsin in 2005 and 2006 were analyzed using a high-throughput assay for determining feedstock quality for biochemical conversion. Non-structural carbohydrates ranged from 84 to 155?g?kg^?1 dry matter (DM); C _B ranged from 354 to 557?g?kg^?1 DM; k _B ranged from 0.199 to 0.330?h^?1; C _A ranged from 463 to 699?g?kg^?1 DM, and NDL ranged from 32 to 74?g?kg^?1 DM. Significant differences (P?<?0.0001) among samples were observed for all parameters, except k _B. Near-infrared reflectance spectroscopy calibration equations were developed for C _N, C _B, C _A, C _U , C _T, and NDL. It was not possible to generate a meaningful calibration equation for k _B. There is significant variability within the corn stover population for several key quality-related carbohydrate and lignin constituents which can be predicted reliably using NIRS.     

Structural Base for Enzymatic Cyclodextrin Hydrolysis

Cyclodextrins resist hydrolysis by burying all bridge oxygens at their interior. Still, the rings can be opened by a small group of specialized enzymes, the cyclomaltodextrinases. Among them, the enzyme from Flavobacterium sp. no. 92 was mutated, crystallized and soaked with cyclodextrins, giving rise to four complex structures. One of them showed an α-cyclodextrin at the outer rim of the active center pocket. In the other complexes, α-, β-and γ-cyclodextrins were bound in a competent mode in the active center. The structures suggest that Arg464 functions as a chaperone guiding the substrates from the solvent into the active center. Over the last part of this pathway, the cyclodextrins bump on Phe274, which rotates the glucosyl group at subsite (+1) by about 120° and fixes it in the new conformation. This induced fit was observed with all three major cyclodextrins. It makes the bridging oxygen between subsites (+1) and (−1) available for protonation by Glu340, which starts the hydrolysis. The mechanism resembles a spring-lock. The structural data were supplemented by activity measurements, quantifying the initial ring opening reaction for the major cyclodextrins and the transglucosylation activity for maltotetraose. Further activity data were collected for mutants splitting the tetrameric enzyme into dimers and for active center mutants.     

An Eco-Friendly and Sustainable Process for Enzymatic Hydrolysis of Penicillin G in Cloud Point System

Enzymatic hydrolysis of penicillin G by immobilized penicillin acylase in a nonionic surfactant mediated cloud point system was presented. The effect of the operation parameters on equilibrium pH of this enzymatic hydrolysis process without pH control was examined. A relatively high equilibrium pH in cloud point system without pH control can be obtained. The feasibility of recycling utilization of the nonionic surfactant, a novel green solvent, was also investigated experimentally. Enzymatic hydrolysis of penicillin G in a discrete semi-batch mode, which simulates a semi-continuous process, envisages a completely eco-friendly, sustainable and efficient process for production of 6-aminopenicillanic acid.     

Enhancement of Enzymatic Adipyl-7-ADCA Hydrolysis

We studied enzymatic adipyl-7-ADCA hydrolysis as a new process for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the building blocks for cephalosporin antibiotics like cephalexin and cefadroxil. Adipyl-7-ADCA hydrolysis carried out with immobilised glutaryl acylase was considerably enhanced by addition of phenylglycine amide, the side-chain donor used for cephalexin synthesis; unlike reactions carried out with free enzyme. The rate enhancing effect was not specifically related to phenylglycine amide; we found a linear relationship between the reaction rate and the buffering capacity of the added substance. These observations can be explained by a pH-gradient in the immobilised enzyme, the pH inside the particle being lower (corresponding to low enzyme activity) than outside. It was concluded that the buffer reduced the pH-gradient inside the biocatalyst, and therewith, caused the reaction rate enhancing effects. Further, chloride ions decreased the reaction rate strongly, while sodium, magnesium, sulphate, and potassium did not influence the reaction rate much. For an actual process, it is important to use a buffer that is appropriate for the reaction-pH. In that way the amount of enzyme required in a process can be reduced considerably, in our case a factor of three was found.     

Enhancement of Enzymatic Adipyl-7-ADCA Hydrolysis

We studied enzymatic adipyl-7-ADCA hydrolysis as a new process for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the building blocks for cephalosporin antibiotics like cephalexin and cefadroxil. Adipyl-7-ADCA hydrolysis carried out with immobilised glutaryl acylase was considerably enhanced by addition of phenylglycine amide, the side-chain donor used for cephalexin synthesis; unlike reactions carried out with free enzyme. The rate enhancing effect was not specifically related to phenylglycine amide; we found a linear relationship between the reaction rate and the buffering capacity of the added substance. These observations can be explained by a pH-gradient in the immobilised enzyme, the pH inside the particle being lower (corresponding to low enzyme activity) than outside. It was concluded that the buffer reduced the pH-gradient inside the biocatalyst, and therewith, caused the reaction rate enhancing effects. Further, chloride ions decreased the reaction rate strongly, while sodium, magnesium, sulphate, and potassium did not influence the reaction rate much. For an actual process, it is important to use a buffer that is appropriate for the reaction-pH. In that way the amount of enzyme required in a process can be reduced considerably, in our case a factor of three was found.     

蛋白质的酶水解过程研究

进行了蛋白质酶水解过程的研究。结果表明木瓜蛋白酶对混合蛋白质的亲和力最强 ,而 1398蛋白酶的亲和力最弱。也表明作用位点和亲和力之间有一定的对应关系 ,Km值和作用位点氨基酸含量比例的相关系数为 0 .90 9。温度影响结果表明温度较低时温度升高加速水解反应过程处主要地位 ;当温度较高时 ,酶失活过程处主导地位。在一定水解时间内的讨论最适温度条件具有更明确的针对性 ,从本研究的采用胰酶 (胰蛋白酶和胰凝乳蛋白酶 )水解 4h的条件下 ,反应温度控制在45～ 5 0℃之间最适     

Vertical Distribution of Corn Stover Dry Mass Grown at Several US Locations

Corn (Zea mays L.) stover was identified as a renewable non-food agricultural feedstock for production of liquid fuels, biopower, and other bioproducts, but it is also needed for erosion control, carbon sequestration, and nutrient cycling. To help balance these multiple demands, our objectives were to (1) determine height distribution of corn stover biomass, (2) quantify the percentage of stover that is corn cob, and (3) develop a general relationship between plant harvest height and stover remaining in the field for a broad range of growing conditions, soil types, and hybrids in different regions. Plant height, dry grain, stover, and cob yield data were collected at eight US locations. Overall, stover yield increased about 0.85 Mg ha^-1 and cob yield increased about 0.10 Mg ha^-1 for each 1.0 Mg ha^-1 increase in dry grain yield. At grain harvest, the stover-to-grain ratio ranged from 0.64 to 0.96 and cob-to-grain ratio ranged from 0.11 to 0.19. A strong nearly 1:1 linear (r ²?=?0.93) relationship between the relative cutting height and relative biomass remaining in the field was observed across all sites. These data were requested by the US Department of Agriculture-Natural Resource Conservation Service to help improve version 2 of the Revised Universal Soil Loss Equation (RUSLE2) and Wind Erosion Prediction System and better estimate corn stover harvest rates based on cutting height or selective organ harvest (e.g., grain and cob only). This information will improve the capacity of RUSLE2 and similar models to predict the erosion risk associated with harvesting corn residues.