Acetic acid removal from corn stover hydrolysate using ethyl acetate and the impact on Saccharomyces cerevisiae bioethanol fermentation

Acetic acid is introduced into cellulose conversion processes as a consequence of composition of lignocellulose feedstocks, causing significant inhibition of adapted, genetically modified and wild‐type S. cerevisiae in bioethanol fermentation. While adaptation or modification of yeast may reduce inhibition, the most effective approach is to remove the acetic acid prior to fermentation. This work addresses liquid–liquid extraction of acetic acid from biomass hydrolysate through a pathway that mitigates acetic acid inhibition while avoiding the negative effects of the extractant, which itself may exhibit inhibition. Candidate solvents were selected using simulation results from Aspen Plus?, based on their ability to extract acetic acid which was confirmed by experimentation. All solvents showed varying degrees of toxicity toward yeast, but the relative volatility of ethyl acetate enabled its use as simple vacuum evaporation could reduce small concentrations of aqueous ethyl acetate to minimally inhibitory levels. The toxicity threshold of ethyl acetate, in the presence of acetic acid, was found to be 10 g L^?1. The fermentation was enhanced by extracting 90% of the acetic acid using ethyl acetate, followed by vacuum evaporation to remove 88% removal of residual ethyl acetate along with 10% of the broth. NRRL Y‐1546 yeast was used to demonstrate a 13% increase in concentration, 14% in ethanol specific production rate, and 11% ethanol yield. This study demonstrated that extraction of acetic acid with ethyl acetate followed by evaporative removal of ethyl acetate from the raffinate phase has potential to significantly enhance ethanol fermentation in a corn stover bioethanol facility. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:929–937, 2016     

Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase

Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition.     

Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH‐ST)

High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process—continuous hydrolysis and fermentation—has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co‐fermentation) and SSCF (simultaneous saccharification and co‐fermentation) of AFEX? (Ammonia Fiber Expansion) pretreated corn stover (AFEX?‐CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX?‐CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose‐to‐ethanol conversion and 47% xylose‐to‐ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3‐ and 1.8‐fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity. Biotechnol. Bioeng. 2013; 110: 1302–1311. © 2012 Wiley Periodicals, Inc.     

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.     

Simultaneous saccharification and fermentation of ground corn stover for the production of fuel ethanol using Phanerochaete chrysosporium, Gloeophyllum trabeum, Saccharomyces cerevisiae, and Escherichia coli K011

Enzymatic saccharification of corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum and subsequent fermentation of the saccharification products to ethanol by Saccharomyces cerevisiae and Escherichia coli K011 were achieved. Prior to simultaneous saccharification and fermentation (SSF) for ethanol production, solid-state fermentation was performed for four days on ground corn stover using either P. chrysosporium or G. trabeum to induce in situ cellulase production. During SSF with S. cerevisiae or E. coli, ethanol production was the highest on day 4 for all samples. For corn stover treated with P. chrysosporium, the conversion to ethanol was 2.29 g/100 g corn stover with S. cerevisiae as the fermenting organism, whereas for the sample inoculated with E. coli K011, the ethanol production was 4.14 g/100 g corn stover. Corn stover treated with G. trabeum showed a conversion 1.90 and 4.79 g/100 g corn stover with S. cerevisiae and E. coli K011 as the fermenting organisms, respectively. Other fermentation co-products, such as acetic acid and lactic acid, were also monitored. Acetic acid production ranged between 0.45 and 0.78 g/100 g corn stover, while no lactic acid production was detected throughout the 5 days of SSF. The results of our experiment suggest that it is possible to perform SSF of corn stover using P. chrysosporium, G. trabeum, S. cerevisiae and E. coli K011 for the production of fuel ethanol.     

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.     

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose‐metabolizing pathways. In this study, a novel two‐stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non‐Saccharomyces microorganism and coupled to a glucose‐utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two‐stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae‐based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014     

Maximizing the liquid fuel yield in a biorefining process

Biorefining strives to recover the maximum value from each fraction, at minimum energy cost. In order to seek an unbiased and thorough assessment of the alleged opportunity offered by biomass fuels, the direct conversion of various lignocellulosic biomass was studied: aspen pulp wood (Populus tremuloides), aspen wood pretreated with dilute acid, aspen lignin, aspen logging residues, corn stalk, corn spathe, corn cob, corn stover, corn stover pellet, corn stover pretreated with dilute acid, and lignin extracted from corn stover. Besides the heating rate, the yield of liquid products was found to be dependent on the final liquefaction temperature and the length of liquefaction time. The major compounds of the liquid products from various origins were identified by GC-MS. The lignin was found to be a good candidate for the liquefaction process, and biomass fractionation was necessary to maximize the yield of the liquid bio-fuel. The results suggest a biorefinery process accompanying pretreatment, fermentation to ethanol, liquefaction to bio-crude oil, and other thermo-conversion technologies, such as gasification. Other biorefinery options, including supercritical water gasification and the effectual utilization of the bio-crude oil, are also addressed.     

Quantitative Sustainability Assessment of Seaweed Biomass as Bioethanol Feedstock

Since terrestrial biomass-based ethanol has environmental and economic vulnerability, seaweed-based bioethanol is emerging as a new biofuel. To investigate the sustainability of seaweeds as bioethanol feedstock, this study quantitatively assesses the energy, freshwater, and fertilizer requirements; land-related carbon balance; and bioethanol productivity of seaweed biomass through comparison with terrestrial biomass. Also, the metal resource potential of seaweeds is assessed because valuable metals can be recovered from seaweed fermentation residue. Compared to corn grain and stover, seaweeds exhibit competitive energy requirements and ethanol productivity. Seaweed cultivation does not incur carbon debt derived from land use change and requires less freshwater than corn grain but more than switchgrass in cultivation and fermentation. Seaweed cultivation also does not require fertilizer application despite the high content of nitrogen and phosphorus. Seaweeds exhibit high resource potential for gold and silver. Therefore, seaweed biomass has high potential as a sustainable bioethanol feedstock.     

低水用量约束条件下的高固体含量纤维乙醇生物加工技术策略

木质纤维素原料生物转化生产纤维乙醇需要使用大量的水和蒸汽,从而使过程能耗和废水排放显著增加,大幅度增加了加工成本。最大限度地降低水和蒸汽用量对过程节能和废水减排并对最终成本控制极为重要。对极限低水用量约束条件下木质纤维素生物转化关键路径进行了实验研究和计算分析,确定了极低水和蒸汽用量的新型预处理技术,实现高效率预处理过程的废水零排放;采用独特的生物脱毒技术,用从自然界筛选的煤油霉菌Amorphotheca resinae ZN1对预处理原料中的抑制物进行了快速生物脱毒;对极限高固体含量下高粘度多相流物系在复杂抑制物胁迫下的酶水解与发酵行为以及放大准则进行了研究;建立了基于Aspen plus平台上的生物质加工物性数据库和严格热力学意义上的全过程流程模拟数学模型,实现了对过程的局部和全局设计与调优。这一综合技术在生物炼制微型工厂中进行了测试,并在纤维素乙醇工业示范装置中得到了应用。该研究结果将为构建具有工业实用价值的节能和清洁化木质纤维素生物转化技术提供依据。     

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.     

Policy Implications of Allocation Methods in the Life Cycle Analysis of Integrated Corn and Corn Stover Ethanol Production

A biorefinery may produce multiple fuels from more than one feedstock. The ability of these fuels to qualify as one of the four types of biofuels under the US Renewable Fuel Standard and to achieve a low carbon intensity score under California’s Low Carbon Fuel Standard can be strongly influenced by the approach taken to their life cycle analysis (LCA). For example, in facilities that may co-produce corn grain and corn stover ethanol, the ethanol production processes can share the combined heat and power (CHP) that is produced from the lignin and liquid residues from stover ethanol production. We examine different LCA approaches to corn grain and stover ethanol production considering different approaches to CHP treatment. In the baseline scenario, CHP meets the energy demands of stover ethanol production first, with additional heat and electricity generated sent to grain ethanol production. The resulting greenhouse gas (GHG) emissions for grain and stover ethanol are 57 and 25 g-CO₂eq/MJ, respectively, corresponding to a 40 and 74 % reduction compared to the GHG emissions of gasoline. We illustrate that emissions depend on allocation of burdens of CHP production and corn farming, along with the facility capacities. Co-product handling techniques can strongly influence LCA results and should therefore be transparently documented.     

Dry biorefining maximizes the potentials of simultaneous saccharification and co‐fermentation for cellulosic ethanol production

Despite the well‐recognized merits of simultaneous saccharification and co‐fermentation (SSCF) on relieving sugar product inhibition on cellulase activity, a practical concomitance difficulty of xylose with inhibitors in the pretreated lignocellulose feedstock prohibits the essential application of SSCF for cellulosic ethanol fermentation. To maximize the SSCF potentials for cellulosic ethanol production, a dry biorefining approach was proposed starting from dry acid pretreatment, disk milling, and biodetoxification of lignocellulose feedstock. The successful SSCF of the inhibitor free and xylose conserved lignocellulose feedstock after dry biorefining reached a record high ethanol titer at moderate cellulase usage and minimum wastewater generation. For wheat straw, 101.4 g/L of ethanol (equivalent to 12.8% in volumetric percentage) was produced with the overall yield of 74.8% from cellulose and xylose, in which the xylose conversion was 73.9%, at the moderate cellulase usage of 15 mg protein per gram cellulose. For corn stover, 85.1 g/L of ethanol (equivalent to 10.8% in volumetric percentage) is produced with the overall conversion of 84.7% from cellulose and xylose, in which the xylose conversion was 87.7%, at the minimum cellulase usage of 10 mg protein per gram cellulose. Most significantly, the SSCF operation achieved the high conversion efficiency by generating the minimum amount of wastewater. Both the fermentation efficiency and the wastewater generation in the current dry biorefining for cellulosic ethanol production are very close to that of corn ethanol production, indicating that the technical gap between cellulosic ethanol and corn ethanol has been gradually filled by the advancing biorefining technology.     

玉米秸秆酸解副产物对重组酿酒酵母6508-127发酵的影响

将木质纤维素类生物质如玉米秸秆等用稀酸水解预处理,在半纤维素水解为单糖的同时,水解液中还会产生一些可能对后续发酵有影响的副产物。本实验分别考查了在玉米秸秆稀酸水解液中检测出的乙酸、甲酸、香草醛、糠醛和羟甲基糠醛对重组木糖发酵菌株S. cerevisiae 6508-127生长和发酵的影响。结果表明,甲酸和乙酸对菌体生长的抑制强于乙醇生成,且甲酸的抑制程度远大于乙酸;2g/L香草醛可使菌体生长延滞期明显延长,而在较低浓度（≤1.2g/L）此现象不明显。糠醛在0.5-1.5g/L范围内对菌体生长有抑制作用,但使乙醇得率提高;羟甲基糠醛在0.2g/L浓度存在就使乙醇得率有明显降低,但使生物量得率提高;研究中还发现,糠醛、羟甲基糠醛和香草醛可被S. cerevisiae 6508-127代谢。     

Detoxification of corn stover prehydrolyzate by trialkylamine extraction to improve the ethanol production with Pichia stipitis CBS 5776

In order to realize the separated ethanol fermentation of glucose and xylose, prehydrolysis of corn stover with sulfuric acid at moderate temperature was applied, while inhibitors were produced inevitably. A complex extraction was adopted to detoxify the prehydrolyzate before fermentation to ethanol with Pichia stipitis CBS 5776. The best proportion of mixed extractant was 30% trialkylamine-50% n-octanol -20% kerosene. Detoxification results indicated that 73.3% of acetic acid, 45.7% of 5-hydroxymethylfurfural and 100% of furfural could be removed. Compared with the undetoxified prehydrolyzate, the fermentability of the detoxified prehydrolyzate was significantly improved. After 48 h fermentation of the detoxified prehydrolyzate containing 7.80 g/l of glucose and 52.8 g/l of xylose, the sugar utilization ratio was 93.2%; the ethanol concentration reached its peak value of 21.8 g/l, which was corresponding to 82.3% of the theoretical value.     

Enhanced bioenergy recovery from rapeseed plant in a biorefinery concept

The present study investigated the utilization of the whole rapeseed plant (seed and straw) for multi-biofuels production in a biorefinery concept. Results showed that bioethanol production from straw was technically feasible with ethanol yield of 0.15 g ethanol/g dry straw after combined alkaline peroxide and stream pretreatment. The byproducts (rapeseed cake, glycerol, hydrolysate and stillage) were evaluated for hydrogen and methane production. In batch experiments, the energy yields from each feedstock for, either methane production alone or for both hydrogen and methane, were similar. However, results from continuous experiments demonstrated that the two-stage hydrogen and methane fermentation process could work stably at organic loading rate up to 4.5 gVS/(Ld), while the single-stage methane production process failed. The energy recovery efficiency from rapeseed plant increased from 20% in the conventional biodiesel process to 60% in the biorefinery concept, by utilization of the whole rapeseed plant for biodiesel, bioethanol, biohydrogen and methane production.     

Evaluation of pelleting as a pre-processing step for effective biomass deconstruction and fermentation

Densification of bulky forages by pelleting reduces their transportation, handling, and storage costs. Because of high shearing force and frictional heating during the pelleting process, it is hypothesized that pelleting of lignocellulosic biomass could also partially deconstruct its complex structure and facilitate bioethanol production. In this study, pelleted wheat straw, corn stover, big bluestem, and sorghum stalk were evaluated for sugars and ethanol production, and compared with those of unpelleted biomasses. Mass recovery after alkali pretreatment increased by 14%, 11%, 2%, and 5%, respectively, in unpelleted biomasses. Lignin content reduced significantly more in pelleted samples for all types of biomass, except sorghum stalk. Volumetric productivity of enzymatic hydrolysis was 23%, 21%, 20% and 12% higher, respectively, in pelleted samples; ethanol yield on the basis of released sugars did not differ significantly between pelleted and unpelleted samples. These results indicate that the pelleting process led to better enzymatic hydrolysis of pretreated biomasses without affecting the quality of sugars for fermentation. However, overall yield of ethanol from the raw biomass was not significantly higher in pelleted biomasses because of higher mass loss during pretreatment process. In our study, we propose a schematic for complete utilization of various byproducts for enhanced economic viability.     

Production of ethanol from corn stover hemicellulose hydrolyzate using <Emphasis Type="Italic">Pichia stipitis</Emphasis>

Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37–0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.     

Integrated Economic and Environmental Assessment of Cellulosic Biofuel Production in an Agricultural Watershed

SWAT watershed model simulated biomass yield and pollutant loadings were integrated with associated economic costs of farm production and transport to study two dedicated energy crops, switchgrass and Miscanthus, and corn stover, as feedstocks for a cellulosic biorefinery. A multi-level spatial optimization (MLSOPT) framework was employed to get spatially explicit cropping plans for a watershed under the assumption that the watershed supplies biomass to a hypothetical biorefinery considering both the biochemical and the thermochemical conversion pathways. Consistent with previous studies, the perennial grasses had higher biomass yield than corn stover, with considerably lower sediment, nitrogen, and phosphorus loadings, but their costs were higher. New insights were related to the tradeoffs between cost, feedstock production, and the level and form of environmental quality society faces as it implements the Renewable Fuel Standard. Economically, this involved calculating the farthest distance a biorefinery would be willing to drive to source corn residue before procuring a single unit of perennial grasses from productive agricultural soils.     

选育耐受复合抑制剂酿酒酵母提高乙醇产量

能够耐受纤维素预处理中抑制剂的酿酒酵母对高效、经济生产纤维素乙醇至关重要。利用诱变结合驯化工程选育了一株可耐受复合抑制剂(1.3g/L糠醛、5.3g/L乙酸及1.0g/L苯酚)的工业酿酒酵母YYJ003。在pH 4.0的含有抑制剂的培养基中,耐受菌株乙醇产率是原始菌株的7.8倍,糠醛转化速率提高了5倍。在pH 5.5的复合抑制剂条件下,YYJ003发酵时间(16h)比野生菌株发酵时间(22h)缩短6h。在pH 4.0的未脱毒的玉米秸秆水热法预处理水解液中YYJ003的乙醇产率达到0.50g/g(乙醇/葡萄糖),乙醇产速达到4.16g/(L·h),而对照菌株无乙醇产出。