Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400

The two main sugars in the agricultural by-product corn stover are glucose and xylose. Co-fermentation of glucose and xylose at high content of water-insoluble solids (WIS) without detoxification is a prerequisite to obtain high ethanol concentration and to reduce production costs. A recombinant strain of Saccharomyces cerevisiae, TMB3400, was used in simultaneous saccharification and fermentation (SSF) of whole pretreated slurry of corn stover at high WIS. TMB3400 co-fermented glucose and xylose with relatively high ethanol yields giving high final ethanol concentration. The ethanol productivity increased with increasing concentration of pretreatment hydrolysate in the yeast production medium and when SSF was performed in a fed-batch mode.     

Evaluation of ion exchange resins for removal of inhibitory compounds from corn stover hydrolyzate for xylitol fermentation

The use of dilute acids to catalyze the hydrolysis of hemicellulose to its sugar constituents is well-known and effective. However, a major problem associated with this pretreatment is the poor fermentability of the produced hydrolyzate as a result of the presence of the microorganism's inhibitory compounds. In the present work, seven ion-exchange resins were tested in order to detoxify corn stover hydrolyzate. Regarding xylose recovery, it was observed that more than 92% recovery was feasible. Furfural removal varied from 53.% to 99.%, and hydroxymethylfurfural (HMF) removal was effective between 37% and 100%. Acetic acid was totally removed by Purolite A 103 S resin. Corn stover hydrolyzate (CSH) treated with Purolite A 103 S, and Finex CS 14 GC resins, was tested as substrate for xylitol production using a yeast, Candida mogii. Product yields, Yp/s, of 0.41 and 0.37 g/g and cellular yields, Yx/s, of 0.24 and 0.13 g/g, respectively, were obtained using the two types of resin-treated hydrolyzates.     

酵母发酵蔗渣半纤维素水解物生产木糖酶

采用二次正交旋转组合设计研究了蔗渣半纤维素水解过程中硫酸浓度与液 固比对木糖收率的影响。回归分析表明 ,这两个因素与木糖的收率之间存在显著的回归关系。通过回归方程优化水解条件 ,当硫酸浓度 2 .4g L ,液 固 =6 .2 ,在蒸汽压力 2 .5× 10 4Pa的条件下水解 2 .5h ,10 0g蔗渣可水解生成木糖约 2 4g。大孔树脂吸附层析处理蔗渣半纤维素水解物 ,能有效地减少其中的酵母生长抑制物含量 ,显著改善水解物的发酵性能。用大孔树脂在pH 2条件下处理过的蔗渣半纤维素水解物作基质 ,含木糖 2 0 0g L ,产木糖醇酵母菌株CandidatropicalisAS2 .1776发酵 110h耗完基质中的木糖 ,生成木糖醇 12 7g L ,产物转化率 0 .6 4(木糖醇g 木糖g) ,产物生成速率 1.15g L·h .     

Development of a yeast strain for xylitol production without hydrolysate detoxification as part of the integration of co-product generation within the lignocellulosic ethanol process

The present study verified an applicable technology of xylitol bioconversion as part of the integration of co-product generation within second-generation bioethanol processes. A newly isolated yeast strain, Candida tropicalis JH030, was shown to have a capacity for xylitol production from hemicellulosic hydrolysate without detoxification. The yeast gives a promising xylitol yield of 0.71 g(p) g(s)(-1) from non-detoxified rice straw hydrolysate that had been prepared by the dilute acid pretreatment under severe conditions. The yeast's capacity was also found to be practicable with various other raw materials, such as sugarcane bagasse, silvergrass, napiergrass and pineapple peel. The lack of a need to hydrolysate detoxification enhances the potential of this newly isolated yeast for xylitol production and this, in turn, has the capacity to improve economics of lignocellulosic ethanol production.     

Rheology of corn stover slurries at high solids concentrations--effects of saccharification and particle size

The rheological characteristics of untreated and dilute acid pretreated corn stover (CS) slurries at high solids concentrations were studied under continuous shear using plate-plate type measurements. Slurry rheological behavior was examined as a function of insoluble solids concentration (10-40%), extent of pretreatment (0-75% removal of xylan) and particle size (-20 and -80 mesh). Results show that CS slurries exhibit shear-thinning behavior describable using a Casson model. Further, results demonstrate that the apparent viscosity and yield stress increase with increasing solids concentration (which corresponds to a decrease in free water). Dilute acid pretreatment leads to lower viscosity and yield stresses at equivalent solids concentrations, as does smaller particle size. Taken together, these findings are consistent with the hypothesis that the availability of free water in the slurry plays a significant role in determining its rheological behavior. In particular, as the free water content of the slurry decreases, e.g., with increasing solids concentration, the greater interaction among particles likely increases the apparent viscosity and yield stress properties of the slurry. The results also suggest that the availability of free water, and thereby slurry rheological properties, depend on the chemical composition of the corn stover as well as its physical characteristics such as particle size and porosity. Hydrophilic polymers within the cell wall, such as xylan or pectin, or larger pores within bigger particles, facilitate sequestration of water in the solid phase resulting in decreased availability of free water. Thus, dilute acid pretreated slurries, which contain smaller size particles having significantly lower xylan content than slurries of untreated milled stover, exhibit much lower viscosities and yield stresses than untreated slurries containing large particles at similar solid concentrations.     

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute‐acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose–xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute‐acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical‐based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently. Biotechnol. Bioeng. 2010;105: 992–996. © 2009 Wiley Periodicals, Inc.     

Assessing solid digestate from anaerobic digestion as feedstock for ethanol production

Ethanol production using solid digestate (AD fiber) from a completely stirred tank reactor (CSTR) anaerobic digester was assessed comparing to an energy crop of switchgrass, and an agricultural residue of corn stover. A complete random design was fulfilled to optimize the reaction conditions of dilute alkali pretreatment. The most effective dilute alkali pretreatment conditions for raw CSTR AD fiber were 2% sodium hydroxide, 130 °C, and 3 h. Under these pretreatment conditions, the cellulose concentration of the AD fiber was increased from 34% to 48%. Enzymatic hydrolysis of 10% (dry basis) pretreated AD fiber produced 49.8 g/L glucose, while utilizing 62.6% of the raw cellulose in the AD fiber. The ethanol fermentation on the hydrolysate had an 80.3% ethanol yield. The cellulose utilization efficiencies determined that the CSTR AD fiber was a suitable biorefining feedstock compared to switchgrass and corn stover.     

Effects of sulfuric acid loading and residence time on the composition of sugarcane bagasse hydrolysate and its use as a source of xylose for xylitol bioproduction

A 2(2) full factorial design was employed to evaluate the effects of sulfuric acid loading and residence time on the composition of sugarcane bagasse hydrolysate obtained in a 250-L reactor. The acid loading and the residence time were varied from 70 to 130 mg acid per gram of dry bagasse and from 10 to 30 min, respectively, while the temperature (121 degrees C) and the bagasse loading (10%) were kept constant. Both the sulfuric acid loading and the residence time influenced the concentrations of xylose and inhibitors in the hydrolysate. The highest xylose concentration (22.71 g/L) was achieved when using an acid loading of 130 mg/g and a residence time of 30 min. These conditions also led to increased concentrations of inhibiting byproducts in the hydrolysate. All of the hydrolysates were vacuum-concentrated to increase the xylose concentration, detoxified by pH alteration and adsorption into activated charcoal, and used for xylitol bioproduction in a stirred tank reactor. Neither the least (70 mg/g, 10 min) nor the most severe (130 mg/g, 30 min) hydrolysis conditions led to the best xylitol production (37.5 g/L), productivity (0.85 g/L h), and yield (0.78 g/g).     

Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery

A cycle spray flow-through reactor was designed and used to pretreat corn stover in dilute sulfuric acid medium. The dilute sulfuric acid cycle spray flow-through (DCF) process enhanced xylose sugar yields and cellulose digestibility while increasing the removal of lignin. Within the DCF system, the xylose sugar yields of 90–93% could be achieved for corn stover pretreated with 2% (w/v) dilute sulfuric acid at 95 °C during the optimal reaction time (90 min). The remaining solid residue exhibited enzymatic digestibility of 90–95% with cellulase loading of 60 FPU/g glucan that was due to the effective lignin removal (70–75%) in this process. Compared with flow-through and compress-hot water pretreatment process, the DCF method produces a higher sugar concentration and higher xylose monomer yield. The novel DCF process provides a feasible approach for lignocellulosic material pretreatment.     

Optimization of pH controlled liquid hot water pretreatment of corn stover

Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 degrees C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.     

Chemical and enzymic pretreatment of corn stover to produce soluble fermentation substrates

Corn stover was pretreated with various chemical agents, including sodium hydroxide, sulfuric acid, ethylenediamine, n-butylamine (either alone or in solution with methanol), and acetonitrile or ethanol containing hydrochloric acid. Of these chemicals, n-butylamine was the best reagent for pretreatment of corn stover, considering the degree of loss of total carbohydrate, delignification, cumulative weight loss, cumulative yield of reducing sugars per original total carbohydrate, and the potential ease of recovery and reuse of reagent. In comparison to the other reagents tested, n-butylamine (n-BA) selectively delignified corn stover. The best conditions were as follows: a 12-h presoak of about a 155 g dry wt/L slurry (1 mm average particle size) in 100% n-BA at room temperature, followed by 30 min of refluxing (86.5 degrees C) with 40% (w/w) n-BA-distilled water solution. The cumulative yield of reducing sugars after enzymic hydrolysis was 44.5% of the original total carbohydrate and the cumulative total weight loss (dry basis) was 59%. Degradative loss of total carbohydrate during pretreatment was not detected.     

采用玉米秸秆水解糖和玉米浆发酵生产丁二酸

研究了以玉米秸秆水解糖为碳源,不同氮源条件下琥珀酸放线杆菌Actinobacillus succinogenesSF-9的丁二酸发酵产酸能力。结果表明玉米浆可以替代酵母膏作为丁二酸发酵的廉价氮源。厌氧摇瓶丁二酸发酵单因素试验,得到在初糖浓度50 g/L时,玉米浆的较佳用量为20 g/L。在5 L搅拌罐上,考察了不同初始玉米秸秆水解糖浓度对A.succinogenes SF-9发酵生产丁二酸的影响,结果显示高初始秸秆糖浓度对琥珀酸放线杆菌的生长有抑制作用。采用补料分批发酵,发酵60 h丁二酸的产量达到42.7g/L,丁二酸产率82.7%,生产强度0.81 g/(L·h)。丁二酸的产量和生产强度较分批发酵有明显提高。     

Kinetic behavior of Candida guilliermondii yeast during xylitol production from Brewer's spent grain hemicellulosic hydrolysate

Brewer's spent grain, the main byproduct of breweries, was hydrolyzed with dilute sulfuric acid to produce a hemicellulosic hydrolysate (containing xylose as the main sugar). The obtained hydrolysate was used as cultivation medium by Candidaguilliermondii yeast in the raw form (containing 20 g/L xylose) and after concentration (85 g/L xylose), and the kinetic behavior of the yeast during xylitol production was evaluated in both media. Assays in semisynthetic media were also performed to compare the yeast performance in media without toxic compounds. According to the results, the kinetic behavior of the yeast cultivated in raw hydrolysate was as effective as in semisynthetic medium containing 20 g/L xylose. However, in concentrated hydrolysate medium, the xylitol production efficiency was 30.6% and 42.6% lower than in raw hydrolysate and semisynthetic medium containing 85 g/L xylose, respectively. In other words, the xylose-to-xylitol bioconversion from hydrolysate medium was strongly affected when the initial xylose concentration was increased; however, similar behavior did not occur from semisynthetic media. The lowest efficiency of xylitol production from concentrated hydrolysate can be attributed to the high concentration of toxic compounds present in this medium, resulting from the hydrolysate concentration process.     

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。     

Reducing acid in dilute acid pretreatment and the impact on enzymatic saccharification

Dilute acid pretreatment is a leading pretreatment technology for biomass to ethanol conversion due to the comparatively low chemical cost and effective hemicellulose solubilization. The conventional dilute acid pretreatment processes use relatively large quantities of sulfuric acid and require alkali for pH adjustment afterwards. Significant amounts of sulfate salts are generated as by-products, which have to be properly treated before disposal. Wastewater treatment is an expensive, yet indispensable part of commercial level biomass-to-ethanol plants. Therefore, reducing acid use to the lowest level possible would be of great interest to the emerging biomass-to-ethanol industry. In this study, a dilute acid pretreatment process was developed for the pretreatment of corn stover. The pretreatment was conducted at lower acid levels than the conventional process reported in the literature while using longer residence times. The study indicates that a 50% reduction in acid consumption can be achieved without compromising pretreatment efficiency when the pretreatment time was extended from 1–5 min to 15–20 min. To avoid undesirable sugar degradation and inhibitor generation, temperatures should be controlled below 170°C. When the sulfuric acid-to-lignocellulosic biomass ratio was kept at 0.025 g acid/g dry biomass, a cellulose-to-glucose conversion of 72.7% can be achieved at an enzyme loading of 0.016 g/g corn stover. It was also found that acid loading based on total solids (g acid/g dry biomass) governs the pretreatment efficiency rather than the acid concentration (g acid/g pretreatment liquid). While the acid loading on lignocellulosic biomass may be achieved through various combinations of solids loading and acid concentration in the pretreatment step, this work shows that it is unlikely to reduce acid use without undermining pretreatment efficiency simply by increasing the solid content in pretreatment reactors, therefore acid loading on biomass is indicated to be the key factor in effective dilute acid pretreatment.     

假丝酵母发酵玉米芯半纤维素水解液生产木糖醇

采用一株驯化过的假丝酵母(Candida sp.)直接发酵经过简单脱毒处理的玉米芯半纤维素水解液生产木糖醇。确定了水解液的最适浓缩倍数在3.0～3.72的范围内。利用正交实验,确定了摇瓶分批发酵工艺条件的最适组合为：摇床转速180r/min,起始C/N为50,起始pH 5.5,接种量5% (体积比)。在此基础上,重点研究了在发酵罐中通气量对酵母发酵玉米芯水解液生产木糖醇的影响。结果表明采用先高后低的分段通气发酵在木糖醇得率方面明显优于恒定通气发酵;其中,在0～24h,3.75 L/min;24～108h,1.25 L/min的分段通气条件下(装液量为2.5L),木糖醇得率(木糖醇/木糖,g/g) 达到0.75 g/g。该结果将有助于建立一种高效的、大规模的利用玉米芯半纤维素水解液发酵生产木糖醇的工艺。     

Characteristics of Corn Stover Pretreated with Liquid Hot Water and Fed-Batch Semi-Simultaneous Saccharification and Fermentation for Bioethanol Production

Corn stover is a promising feedstock for bioethanol production because of its abundant availability in China. To obtain higher ethanol concentration and higher ethanol yield, liquid hot water (LHW) pretreatment and fed-batch semi-simultaneous saccharification and fermentation (S-SSF) were used to enhance the enzymatic digestibility of corn stover and improve bioconversion of cellulose to ethanol. The results show that solid residues from LHW pretreatment of corn stover can be effectively converted into ethanol at severity factors ranging from 3.95 to 4.54, and the highest amount of xylan removed was approximately 89%. The ethanol concentrations of 38.4 g/L and 39.4 g/L as well as ethanol yields of 78.6% and 79.7% at severity factors of 3.95 and 4.54, respectively, were obtained by fed-batch S-SSF in an optimum conditions (initial substrate consistency of 10%, and 6.1% solid residues added into system at the prehydrolysis time of 6 h). The changes in surface morphological structure, specific surface area, pore volume and diameter of corn stover subjected to LHW process were also analyzed for interpreting the possible improvement mechanism.     

Xylitol production from corn fiber and sugarcane bagasse hydrolysates by Candida tropicalis

A natural isolate, Candida tropicalis was tested for xylitol production from corn fiber and sugarcane bagasse hydrolysates. Fermentation of corn fiber and sugarcane bagasse hydrolysate showed xylose uptake and xylitol production, though these were very low, even after hydrolysate neutralization and treatments with activated charcoal and ion exchange resins. Initial xylitol production was found to be 0.43 g/g and 0.45 g/g of xylose utilised with corn fiber and sugarcane bagasse hydrolysate respectively. One of the critical factors for low xylitol production was the presence of inhibitors in these hydrolysates. To simulate influence of hemicellulosic sugar composition on xylitol yield, three different combinations of mixed sugar control experiments, without the presence of any inhibitors, have been performed and the strain produced 0.63 g/g, 0.68 g/g and 0.72 g/g of xylose respectively. To improve yeast growth and xylitol production with these hydrolysates, which contain inhibitors, the cells were adapted by sub culturing in the hydrolysate containing medium for 25 cycles. After adaptation the organism produced more xylitol 0.58 g/g and 0.65 g/g of xylose with corn fiber hydrolysate and sugarcane bagasse hydrolysate respectively.     

Biomimetic catalysis for hemicellulose hydrolysis in corn stover

Efficient and economical hydrolysis of plant cell wall polysaccharides into monomeric sugars is a significant technical hurdle in biomass processing for renewable fuels and chemicals. One possible approach to overcoming this hurdle is a biomimetic approach with dicarboxylic acid catalyst mimicking the catalytic core microenvironment in natural enzymes. This paper reports developments in the use of a dicarboxylic acid catalyst, maleic acid, for hemicellulose hydrolysis in corn stover. Hemicellulose hydrolysis and xylose degradation kinetics in the presence of maleic acid was compared to sulfuric acid. At optimized reaction conditions for each acid, maleic acid hydrolysis results in minimal xylose degradation, whereas sulfuric acid causes 3-10 times more xylose degradation. These results formed the basis for optimizing the hydrolysis of hemicellulose from corn stover using maleic acid. At 40 g/L dry corn stover solid-loading, both acid catalysts can achieve near-quantitative monomeric xylose yield. At higher solids loadings (150-200 g dry stover per liter), sulfuric acid catalyzed hydrolysis results in more than 30% degradation of the xylose, even under the previously reported optimal condition. However, as a result of minimized xylose degradation, optimized biomimetic hydrolysis of hemicellulose by maleic acid can reach approximately 95% monomeric xylose yields with trace amounts of furfural. Fermentation of the resulting unconditioned hydrolysate by recombinant S. cerevisiae results in 87% of theoretical ethanol yield. Enzyme digestibility experiments on the residual corn stover solids show that >90% yields of glucose can be produced in 160 h from the remaining cellulose with cellulases (15 FPU/g-glucan).