Conversion of corn fiber to ethanol by recombinant E. coli strain FBR3

Escherichia coli strain FBR3 that is an efficient biocatalyst for converting mixed sugar streams (eg, arabinose, glucose, and xylose) into ethanol. In this report, the strain was tested for conversion of corn fiber hydrolysates into ethanol. Corn fiber hydrolysates with total sugar concentrations of 7.5% (w/v) were prepared by reacting corn fiber with dilute sulfuric acid at 145°C. Initial fermentations of the hydrolysate by strain FBR3 had lag times of approximately 30 h judged by ethanol production. Further experiments indicated that the acetate present in the hydrolysate could not solely account for the long lag. The lag phase was greatly reduced by growing the pre-seed and seed cultures on corn fiber hydrolysate. Ethanol yields for the optimized fermentations were 90% of theoretical. Maximum ethanol concentrations were 2.80% w/v, and the fermentations were completed in approximately 50 h. The optimal pH for the fermentation was 6.5. Below this pH, sugar consumption was incomplete and above it, excess base addition was required throughout the fermentation. Two alternative neutralization methods (overliming and overliming with sulfite addition) have been reported for improving the fermentability of lignocellulosic hydrolysates. These methods further reduced the lag phase of the fermentation, albeit by a minor amount. Received 29 September 1998/ Accepted in revised form 20 February 1999     

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.     

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.     

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.     

Ethanol recovery from corn fiber hydrolysate fermentations by pervaporation

Corn fiber, a byproduct of corn wet milling, is an attractive feedstock for biomass ethanol production. Corn fiber was hydrolyzed by dilute sulfuric acid and neutralized by one of two methods: conventional lime treatment or neutralization by strongly basic anion exchange. The anion exchange neutralized (AEN) hydrolysate contained substantially lower levels of the inhibiting compounds furfural, 5-hydroxymethylfurfural, and acetic acid compared to the lime neutralized hydrolysate. In batch fermentations the ethanol yields and final ethanol concentration of the two hydrolysates were similar at 0.32-0.43 g/g and 29-44 g/l, respectively. Sugar consumption in the AEN fermentations was superior. Coupling of a membrane pervaporation unit to a fed-batch fermentation of AEN hydrolysate maintained the ethanol concentration below 25 g/l with complete sugar utilization for approximately 5 days. A concentrated ethanol stream of 17 wt.% ethanol was produced by the pervaporation unit.     

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.     

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.     

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.     

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> BG1L1

Dilute sulfuric acid pretreated corn stover is potential feedstock of industrial interest for second generation fuel ethanol production. However, the toxicity of corn stover hydrolysate (PCS) has been a challenge for fermentation by recombinant xylose fermenting organisms. In this work, the thermophilic anaerobic bacterial strain Thermoanaerobacter BG1L1 was assessed for its ability to ferment undetoxified PCS hydrolysate in a continuous immobilized reactor system at 70°C. The tested strain showed significant resistance to PCS, and substrate concentrations up to 15% total solids (TS) were fermented yielding ethanol of 0.39–0.42 g/g-sugars consumed. Xylose was nearly completely utilized (89–98%) for PCS up to 10% TS, whereas at 15% TS, xylose conversion was lowered to 67%. The reactor was operated continuously for 135 days, and no contamination was seen without the use of any agent for preventing bacterial infections. This study demonstrated that the use of immobilized thermophilic anaerobic bacteria for continuous ethanol fermentation could be promising in a commercial ethanol process in terms of system stability to process hardiness and reactor contamination. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol.     

Conversion of corn milling fibrous co-products into ethanol by recombinant Escherichia coli strains K011 and SL40

Corn hulls and corn germ meal were both evaluated as feedstocks for production of ethanol for biofuel. Currently, these fibrous co-products are combined with corn steep liquor and the fermentation bottoms (if available) and marketed as cattle feed. Samples were obtained from wet and dry corn mills. The corn hulls and germ meal were evaluated for starch and hemicellulose compositions. Starch contents were 12 to 32% w/w and hemicellulose (arabinoxylans) contents were 23 to 64% w/w. Corn fibrous samples were hydrolysed, using dilute sulphuric acid, into mixed sugar streams containing arabinose, glucose and xylose. Total sugar concentrations in the hydrolysate varied from 8.4 to 10.8% w/v. The hydrolysates were fermented to ethanol using recombinant E. coli strains K011 and SL40. Ethanol yields were 0.38 to 0.41g ethanol produced/g total sugars consumed and fermentations were completed in 60h or less. However, residual xylose was detected for each hydrolysate fermentation and was especially significant for fermentations using strain SL40. Strain K011 was a superior ethanologenic strain compared with strain SL40 in terms of both ethanol yield and maximum productivity.     

玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化

木质纤维素高浓度还原糖水解液的获得是纤维乙醇产业化发展的方向。在发酵工业领域,分批补料法是实现这一目标的重要研究途径。本研究采用分批补料法对获得高浓度玉米秸秆酶解还原糖的条件进行了优化。以稀硫酸预处理的玉米秸秆为原料,考察了液固比、补加量与补加时间对分批补料糖化的影响。结果表明,秸秆高浓度酶解液条件的初始物料为20％ (重量/体积),木聚糖酶220 U/g (底物),纤维素酶6 FPU/g (底物),果胶酶50 U/g (底物),在24 h、48 h后分批补加8％预处理后的物料,同时添加与补料量相应的木聚糖酶20 U/g (底物),纤维素酶2 FPU/g (底物),72 h后,最终糖化结果与非补料法相比,还原糖浓度从48.5 g/L提高到138.5 g/L,原料的酶解率最终达到理论值的62.5％。试验结果表明补料法可以显著提高秸秆水解液还原糖浓度。     

Bioconversion of corn stover derived pentose and hexose to ethanol using cascade simultaneous saccharification and fermentation (CSSF)

A cascade type of fermentation, designated the cascade simultaneous saccharification and fermentation (CSSF), was studied to convert corn stover derived pentose and hexose to ethanol with reduced enzyme input. In detail, each step of CSSF utilizes two sequential SSF phases operating on pentose and hexose, i.e., pentose conversion using xylanase, endo-glucanase, and recombinant Escherichia coli (KO11) with minimal glucose conversion in the first phase SSF, and hexose conversion in the second phase SSF using cellulase, β-glucosidase, and Saccharomyces cerevisiae (D(5)A). In this cascade scheme, multiple stages of 1st and 2nd phase SSF were performed in series; enzymes are recycled from the fermentation broth of the last stage for the use of the next stage. This bioconversion process yielded up to 60% of the theoretical maximum ethanol yield based on the total sugars in untreated corn stover, while enzyme loadings were reduced by 50% (v/v) and the final ethanol concentration reached 27 g/l.     

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.     

A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover

Two different process configurations, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF), were compared, at 8% water-insoluble solids (WIS), regarding ethanol production from steam-pretreated corn stover. The enzymatic loading in these experiments was 10 FPU/g WIS and the yeast concentration in SSF was 1 g/L (dry weight) of a Saccharomyces cerevisiae strain. When the whole slurry from the pretreatment stage was used as it was, diluted to 8% WIS with water and pH adjusted, SSF gave a 13% higher overall ethanol yield than SHF (72.4% versus 59.1% of the theoretical). The impact of the inhibitory compounds in the liquid fraction of the pretreated slurry was shown to affect SSF and SHF in different ways. The overall ethanol yield (based on the untreated raw material) decreased when SSF was run in absence on inhibitors compared to SSF with inhibitors present. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the case of SHF. However, the SHF yield achieves in the absence of inhibitors was still lower than the SSF yield achieves with inhibitors present.     

Fuel ethanol from corn residue prehydrolysate by a patented ethanologenicEscherichia coli B

Summary The hemicellulose component of corn cobs was completely hydrolyzed by treatment with dilute sulphuric acid. HPLC analysis showed the prehydrolysate to contain 36g/L xylose; 7g/L arabinose; 6g/L glucose and 4.2g/L acetic acid. The patented genetically engineered ethanologenEscherichia coli B (ATCC 11303 pLOI 297) exhibited high performance characteristics with a synthetic model medium in which the pH was controlled at 7 in order to minimize sensitivity to acetic acid. With the synthetic medium, fermentation was completed in 36h and the process yield was 0.49g/g, equivalent to 96% max. theoretical conversion efficiency. However, with the supplemented corn cob prehydrolysate medium, about 20% of the sugar remained unfermented after 8 days, with a conversion efficiency of <40%. Treatment of the prehydrolysate with calcium hydroxide did not result in a significant improvement in fermentation performance. Based on the poor yield, it is concluded that, under the test conditions employed, this patented ethanologen does not qualify as a potential process biocatalyst for the production of fermentation fuel ethanol from corn residue hemicellulose hydrolysate prepared with dilute sulphuric acid.     

Conversion of hydrolysates of corn cobs and hulls into ethanol by recombinantEscherichia coli B containing integrated genes for ethanol production

Summary Hemicellulose and residual starch in corn hulls from wet milling and hemicellulose in corn cobs were hydrolyzed by incubation in dilute sulfuric acid at 140°C to 160°C. These hydrolysates were efficiently fermented to ethanol by a genetically engineered derivative ofE. coli B, strain KO11. Fermentation of com hull hydrolysate was complete after 48 h with a final ethanol concentration of 38 grams per liter. Fermentation of corn cob hydrolysate was essentially complete after 24 h due to a lower concentration of sugars and higher levels of inocula. In both cases, ethanol produced was equivalent to 100% of the maximum theoretical yield (0.51 grams ethanol/gram sugar) based on momoner sugar content. ThusE. coli B strain KO11 appears to be an excellent candidate for the efficient production of ethanol from hydrolysates of corn residues.     

Removal of enzymatic and fermentation inhibitory compounds from biomass slurries for enhanced biorefinery process efficiencies

Within the biorefinery paradigm, many non-monomeric sugar compounds have been shown to be inhibitory to enzymes and microbial organisms that are used for hydrolysis and fermentation. Here, two novel separation technologies, polyelectrolyte polymer adsorption and resin-wafer electrodeionization (RW-EDI), have been evaluated to detoxify a dilute acid pretreated biomass slurry. Results showed that detoxification of a dilute acid pretreated ponderosa pine slurry by sequential polyelectrolyte and RW-EDI treatments was very promising, with significant removal of acetic acid, 5-hydroxymethyl furfural, and furfural (up to 77%, 60%, and 74% removed, respectively) along with >97% removal of sulfuric acid. Removal of these compounds increased the cellulose conversion to 94% and elevated the hydrolysis rate to 0.69 g glucose/L/h. When using Saccharomyces cerevisiae D₅A for fermentation of detoxified slurry, the process achieved 99% of the maximum theoretical ethanol yield and an ethanol production rate nearly five-times faster than untreated slurry.     

Grain sorghum stillage recycling: Effect on ethanol yield and stillage quality

Stillage obtained from ethanol production of grain sorghum was separated into two fractions: thin stillage and wet solids. A portion of the thin stillage was recycled as cooking water in subsequent fermentation runs using both bench- and full-scale ethanol production plants. When thin stillage replaced 50-75% of the cooking water, large increases occurred in solids content, COD, and EC of the resulting thin stillage. It was found that while the volume of thin stillage requiring treatment or disposal was reduced, there was little reduction in the total pollutant load. Stillage rcycling had little effect on the quality of the stillage wet solids fraction. At the high levels of stillage recycle used, ethanol yield was reduced after three to five runs of consecutive recycling.