Biotechnological processes for conversion of corn into ethanol

Ethanol has been utilized as a fuel source in the United States since the turn of the century. However, it has repeatedly faced significant commercial viability obstacles relative to petroleum. Renewed interest exists in ethanol as a fuel source today owing to its positive impact on rural America, the environment and United States energy security. Today, most fuel ethanol is produced by either the dry grind or the wet mill process. Current technologies allow for 2.5 gallons (wet mill process) to 2.8 gallons (dry grind process) of ethanol (1 gallon = 3.785 l) per bushel of corn. Valuable co-products, distillers dried grains with solubles (dry grind) and corn gluten meal and feed (wet mill), are also generated in the production of ethanol. While current supplies are generated from both processes, the majority of the growth in the industry is from dry grind plant construction in rural communities across the corn belt. While fuel ethanol production is an energy-efficient process today, additional research is occurring to improve its long-term economic viability. Three of the most significant areas of research are in the production of hybrids with a higher starch content or a higher extractable starch content, in the conversion of the corn kernel fiber fraction to ethanol, and in the identification and development of new and higher-value co-products.     

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.     

Integrating alkaline extraction of proteins with enzymatic hydrolysis of cellulose from wet distiller’s grains and solubles

Fractionation of distiller’s grains into value added products may serve to improve the economic viability of dry grind corn ethanol facilities in the wake of variable corn and ethanol prices. This research is aimed at creating a high protein, high lysine product from the grain using alkaline protein extractions in conjunction with hydrolysis of the remaining fiber to sugars which are then fermented to ethanol. Alkaline extractions improved the lysine content in protein products, although protein solubility did not exceed 45% of the total protein. In addition, oligomeric carbohydrates, starch, and other water solubles were also extracted, leading to a low purity protein product. Resulting sugar yields following ammonia fiber expansion (AFEX) pretreatment were also lower for extracted distiller’s grains. From these experiments, it does not appear likely that alkaline extraction is a useful tool for fractionation of distiller’s grains. However, pretreatment and hydrolysis can be an effective tool for further fractionation of protein.     

不同生态环境与播种期下小麦籽粒品质变异规律的研究

选用 6个不同品质类型小麦品种在 4个生态点进行分期播种试验, 系统分析了不同生态环境下小麦籽粒产量与品质的变异特征及其与主要气候生态因子间的关系。结果表明 :生态点、品种以及地点×品种互作对籽粒产量、千粒重、蛋白质、湿面筋和淀粉含量、沉降值与降落值的影响均达到显著水平 ;不同播种期处理对产量与淀粉含量的影响达到极显著水平, 而播种期×品种互作对千粒重、降落值、淀粉含量及沉降值的效应达到显著水平 ;地点×播种期×品种互作仅对产量、湿面筋、淀粉含量与沉降值有显著的影响。在 4个不同生态点中, 南京点的籽粒蛋白质与湿面筋含量最低, 但淀粉含量最高 ;徐州点的产量和千粒重最大 ;泰安点的蛋白质含量与湿面筋含量最高, 沉降值最小 ;保定点的产量、千粒重最小, 但沉降值最大。不同播种期处理下, 适播与晚播的籽粒蛋白质含量、湿面筋含量、淀粉含量、沉降值与降落值都显著高于早播, 而早播期的产量和千粒重最大。各小麦品种在不同地点与播种期下产量与品质性状的变异中以降落值的变异系数为最大, 淀粉的变异为最小。开花至成熟期的日均温与淀粉含量呈线性负相关, 与产量、蛋白质和湿面筋含量及降落值呈二次曲线相关关系 ;日温差与产量和千粒重呈二次曲线相关关系, 籽粒蛋白质和湿面筋含量、沉降值及降落值则随昼温差的增加线性递增 ;开花至成熟期降雨量与产量、千粒重都呈现先升后降的二次曲线相关关系, 而与籽粒蛋白质含量和降落值呈现线性负相关关系 ;籽粒蛋白质和湿面筋含量与降落值随开花至成熟期的累计日照时数呈现线性正相关关系。     

Genetic diversity of bread wheat genotypes in Iran for some nutritional value and baking quality traits

Genetic variation among 78 irrigated bread wheat genotypes was studied for their nutritional value and baking quality traits as well as some agronomic traits. The experiment was conducted in a randomized complete block design with three replicates under normal and terminal drought stress conditions in Kermanshah, Iran during 2012–2013 cropping season. The results of combined ANOVA indicated highly significant genotypic differences for all traits. All studied traits except grain yield, hectoliter weight and grain fiber content were significantly affected by genotype × environment interaction. Drought stress reduced grain yield, thousand kernel weight, gluten index, grain starch content and hectoliter weight and slightly promoted grain protein and fiber contents, falling number, total gluten and ratio of wet gluten to grain protein content. Grain yield by 31.66% and falling number by 9.20% attained the highest decrease and increase due to drought stress. There were negative and significant correlations among grain yield with grain protein and fiber contents under both conditions. Results of cluster analysis showed that newer genotypes had more grain yield and gluten index than older ones, but instead, they had the lower grain protein and fiber contents. It is thought that wheat breeders have bred cultivars with high grain yield, low protein content, and improved bread-making attributes during last seven decades. While older genotypes indicated significantly higher protein contents, and some of them had higher gluten index. We concluded from this study that it is imperative for breeders to pay more attention to improve qualitative traits coordinated to grain yield.     

Novel carbohydrate binding modules in the surface anchored α‐amylase of Eubacterium rectale provide a molecular rationale for the range of starches used by this organism in the human gut

Gut bacteria recognize accessible glycan substrates within a complex environment. Carbohydrate binding modules (CBMs) of cell surface glycoside hydrolases often drive binding to the target substrate. Eubacterium rectale, an important butyrate‐producing organism in the gut, consumes a limited range of substrates, including starch. Host consumption of resistant starch increases the abundance of E. rectale in the intestine, likely because it successfully captures the products of resistant starch degradation by other bacteria. Here, we demonstrate that the cell wall anchored starch‐degrading α‐amylase, Amy13K of E. rectale harbors five CBMs that all target starch with differing specificities. Intriguingly these CBMs efficiently bind to both regular and high amylose corn starch (a type of resistant starch), but have almost no affinity for potato starch (another type of resistant starch). Removal of these CBMs from Amy13K reduces the activity level of the enzyme toward corn starches by ～40‐fold, down to the level of activity toward potato starch, suggesting that the CBMs facilitate activity on corn starch and allow its utilization in vivo. The specificity of the Amy13K CBMs provides a molecular rationale for why E. rectale is able to only use certain starch types without the aid of other organisms.     

Structural basis for carbohydrate-binding specificity--a comparative assessment of two engineered carbohydrate-binding modules

Detection, immobilization and purification of carbohydrates can be done using molecular probes that specifically bind to targeted carbohydrate epitopes. Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that can be engineered to bind and detect specifically a number of carbohydrates. Design and engineering of CBMs have benefited greatly from structural studies that have helped us to decipher the basis for specificity in carbohydrate-protein interactions. However, more studies are needed to predict which modifications in a CBM would generate probes with predetermined binding properties. In this report, we present the crystal structures of two highly related engineered CBMs with different binding specificity profiles: X-2, which is specific for xylans and the L110F mutant of X-2, which binds xyloglucans and β-glucans in addition to xylans. The structures of the modules were solved both in the apo form and complexed with oligomers of xylose, as well as with an oligomer of glucose in the case of X-2 L110F. The mutation, leucine to phenylalanine, converting the specific module into a cross-reactive one, introduces a crucial hydrogen-π interaction that allows the mutant to retain glucan-based ligands. The cross-reactivity of X-2 L110F is furthermore made possible by the plasticity of the protein, in particular, of residue R142, which permits accommodation of an extra hydroxymethyl group present in cellopentaose and not xylopentaose. Altogether, this study shows, in structural detail, altered protein-carbohydrate interactions that have high impact on the binding properties of a carbohydrate probe but are introduced through simple mutagenesis.     

Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan

Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1-->4)-beta-mannan/galacto-(1-->4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.     

Lipids-free waxy corn starch as a substrate for distillery yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Utilization of lipids-free waxy starch by distillery yeasts in fuel ethanol production can contribute to better management of renewable resources, like cereals, especially maize Zea mays L. But the efficient conversion of starch into glucose-rich fermentable substrate, and subsequently to ethanol, needs more research on hydrolysis and fermentation conditions. The aim of this study was to evaluate the lack of natural corn grain lipids on the process of simultaneous saccharification and fermentation using chemometric techniques of designed experiments, commercial enzymatic preparations and distillery yeasts Saccharomyces cerevisiae CCY-11-3. Based on the results and statistical software support we can conclude that extraction of lipids from corn grains did not lead to statistically significant increase or decrease of glucose concentration in starch hydrolysis. The ethanol concentration in fermentation mash according to analysis was not statistically significantly affected by lipids extraction. The separated lipids could serve as a source of very valuable corn oil.     

Synthetic xylan-binding modules for mapping of pulp fibres and wood sections

Background  

The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.     

根部喷施FeSO_4对春小麦籽粒组分和面粉品质的影响

以4个春小麦品种为材料,在温室条件下研究了根部喷施不同量FeSO4对小麦籽粒硫含量、铁含量、蛋白质含量、淀粉组成、SDS沉淀值、面筋品质和面团流变学特性的影响.结果表明:喷施FeSO4后4个品种的硫含量、铁含量、蛋白含量、SDS沉淀值、湿面筋含量和面筋指数均显著高于对照,但各处理间干面筋含量无显著变化,且不同品种、不同品质参数达到最高值的FeSO4喷施量并不一致;2个品种的直链淀粉含量、直支比和总淀粉含量比对照显著降低,另2个品种直链淀粉含量和直支比却无显著变化;各品种的面团形成时间、稳定时间和断裂时间随着FeSO4喷施量的增加呈现出先升高后降低的趋势,并均在15 g/m2或22.5 g/m2 FeSO4喷施量水平达到最佳,但均与FeSO4喷施量无显著相关性.研究发现,根部喷施适宜浓度的FeSO4可在一定程度上改善春小麦籽粒组分和面粉品质,但改善的效果因品种和品质参数而异.     

Allocation procedure in ethanol production system from corn grain i. system expansion

We investigated the system expansion approach to net energy analysis for ethanol production from domestic corn grain. Production systems included in this study are ethanol production from corn dry milling and corn wet milling, corn grain production (the agricultural system), soybean products from soybean milling (i.e. soybean oil and soybean meal) and urea production to determine the net energy associated with ethanol derived from corn grain. These five product systems are mutually interdependent. That is, all these systems generate products which compete with or displace all other comparable products in the market place. The displacement ratios between products compare the equivalence of their marketplace functions. The net energy, including transportation to consumers, is 0.56 MJ_net/MJ of ethanol from corn grain regardless of the ethanol production technology employed. Using ethanol as a liquid transportation fuel could reduce domestic use of fossil fuels, particularly petroleum. Sensitivity analyses show that the choice of allocation procedures has the greatest impact on fuel ethanol net energy. Process energy associated with wet milling, dry milling and the corn agricultural process also significantly influences the net energy due to the wide ranges of available process energy values. The system expansion approach can completely eliminate allocation procedures in the foreground system of ethanol production from corn grain.     

Farm-scale production of fuel ethanol and wet grain from corn in a batch process

The batch production of fuel grade ethanol and distillers' wet grain (wet solids) in a farm-scale process (1240-15,580 L/batch) is described. The employs yeast fermentation of amylase-treated corn mash and a two-stage distillation. Primary emphasis in this study was on the cooking, fermentation, and centrifugation steps. Without recycling, fermentation of the mash yield beers with 10.0-10.5% ethanol. Recycling of stillage supernatant at full, 75, or 50% strengths produced enriched mashes that after 48-h fermentation yielded beers with 5-;14% more ethanol. Recycling twice with full-strength supernatant at pH 7.0 increased the ethanol yield in the final beer 16.5%; however, the time to complete the final fermentation was extended form 48 to 72 h and salt buildup occurred. By recycling at pH 5.4, it was possible to avoid rapids salt buildup and obtain beers with 10.3-10.5% ethanol. Recycling resulted in increased levels of glucose, starch, crude protein, and fat in the beer and a reduced moisture content while the wet solids showed an increased starch content. Centrifugation after cooking or fermentation yield in the subsequently produced beer. Fermentation of a volume-resorted mash supernatant gave a beer with only 9.25% ethanol. Mash wet solids varied somewhat chemically from beer and stillage solids. An economic and energy balance analysis of various modes of plant operation are provided and plant considerations are suggested.     

Corn fiber as a raw material for hemicellulose and ethanol production

Corn fiber (CF) is a potential raw material for the production of various products because it is widely available in corn-producing countries. Corn fiber is a byproduct of the corn wet-milling industry and a very large amount of it (approximately 130 t/day) is produced in Hungary. The major component of corn fiber is the pericarp that consists of 35% hemicellulose, 18% cellulose and 20% remaining starch (protein, fiber oil and lignin are also present in this material). Corn fiber is presently used as animal feed. However, with continuous growth in corn processing to ethanol, there might be problems with the utilization of the surplus fibrous byproducts. In this paper the conversion of corn fiber to ethanol or other products was examined. Destarched corn fiber was pretreated by using different alkaline solutions and dissolved hemicellulose was precipitated with ethanol for the recovery of a valuable coproduct. The residual material consisting mostly of cellulose was hydrolyzed with cellulolytic enzymes and fermented into ethanol by using Saccharomyces cerevisiae.     

Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose

The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.     

A structural and functional analysis of alpha-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a conserved mode of starch recognition

Starch-hydrolyzing enzymes lacking alpha-glucan-specific carbohydrate-binding modules (CBMs) typically have lowered activity on granular starch relative to their counterparts with CBMs. Thus, consideration of starch recognition by CBMs is a key factor in understanding granular starch hydrolysis. To this end, we have dissected the modular structure of the maltohexaose-forming amylase from Bacillus halodurans (C-125). This five-module protein comprises an N-terminal family 13 catalytic module followed in order by two modules of unknown function, a family 26 CBM (BhCBM26), and a family 25 CBM (BhCBM25). Here we present a comprehensive structure-function analysis of starch and alpha-glucooligosaccharide recognition by BhCBM25 and BhCBM26 using UV methods, isothermal titration calorimetry, and x-ray crystallography. The results reveal that the two CBMs bind alpha-glucooligosaccharides, particularly those containing alpha-1,6 linkages, with different affinities but have similar abilities to bind granular starch. Notably, these CBMs appear to recognize the same binding sites in granular starch. The enhanced affinity of the tandem CBMs for granular starch is suggested to be the main biological advantage for this enzyme to contain two CBMs. Structural studies of the native and ligand-bound forms of BhCBM25 and BhCBM26 show a structurally conserved mode of ligand recognition but through non-sequence-conserved residues. Comparison of these CBM structures with other starch-specific CBM structures reveals a generally conserved mode of starch recognition.     

Production and purification of self-assembling peptides in Ralstonia eutropha

Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (N-RADARADARADARADAE-C). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.     

Herbicidal activity of hydrolyzed corn gluten meal on three grass species under controlled environments

US Patent No. 5,030,268 discloses that corn gluten meal, the protein fraction of corn (Zea mays L.) grain, can be used as a natural preemergence herbicide. However, corn gluten meal is insoluble in water, and this characteristic renders it difficult to apply as a herbicide. To seek a watersoluble material with more potent herbicidal activity, the phytotoxicity of various samples derived from corn gluten meal and other related crop materials were evaluated by using three different grass species under controlled environments. Greenhouse and growth chamber bioassays showed that the sample of enzymatically hydrolyzed corn gluten meal was more herbicidally active than the corn gluten itself and was highly water soluble. Gluten hydrolysate prepared with bacterial source proteinase had the greatest inhibitory activity to the root growth of germinating seeds. This water-soluble material derived from corn gluten meal had a growth-regulating effect on the root system and can be used as a natural herbicide.Journal Paper No. J-15609 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project No. 3149.     

Composition of corn and distillers dried grains with solubles from dry grind ethanol processing

Increase in the demand for ethanol has resulted in growth in the dry grind (DG) ethanol industry. In DG processing, the whole corn kernel is fermented, resulting in two main coproducts, ethanol and distillers dried grains with solubles (DDGS). Marketing of DDGS is critical to the economic stability of DG plants. The composition of DDGS can vary considerably; this reduces market value. Factors that cause variation in composition need to be evaluated. The objective was to determine the relationship between composition of corn and composition of DDGS. Samples of corn and DDGS were obtained from a DG ethanol plant and analyzed for protein, fat, starch and other nutrients. Concentrations of protein, fiber and starch were similar to published data for corn but were higher for DDGS. Coefficients of variation for protein fat and fiber concentrations were similar for corn and DDGS. There were no significant correlations between concentrations of components in corn and those in DDGS. Variation in the composition of DDGS was not related to variation in corn composition and probably was due to variation in processing streams or processing techniques. This implies that reducing the variation in composition of DDG will require modification of processing strategies.