Dataset Information

Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST).

ABSTRACT: Current technology using corn stover (CS) as feedstock, Ammonia Fiber Expansion (AFEX) as the pretreatment technology, and Saccharomyces cerevisiae 424A(LNH-ST) as the ethanologenic strain in Separate Hydrolysis and Fermentation was able to achieve 191.5 g EtOH/kg untreated CS, at an ethanol concentration of 40.0 g/L (5.1 vol/vol%) without washing of pretreated biomass, detoxification, or nutrient supplementation. Enzymatic hydrolysis at high solids loading was identified as the primary bottleneck affecting overall ethanol yield and titer. Degradation compounds in AFEX-pretreated biomass were shown to increase metabolic yield and specific ethanol production while decreasing the cell biomass generation. Nutrients inherently present in CS and those resulting from biomass processing are sufficient to support microbial growth during fermentation. This platform offers the potential to improve the economics of cellulosic ethanol production by reducing the costs associated with raw materials, process water, and capital equipment.

SUBMITTER: Lau MW

PROVIDER: S-EPMC2635794 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST).

Lau Ming W MW Dale Bruce E BE

Proceedings of the National Academy of Sciences of the United States of America 20090122 5

Current technology using corn stover (CS) as feedstock, Ammonia Fiber Expansion (AFEX) as the pretreatment technology, and Saccharomyces cerevisiae 424A(LNH-ST) as the ethanologenic strain in Separate Hydrolysis and Fermentation was able to achieve 191.5 g EtOH/kg untreated CS, at an ethanol concentration of 40.0 g/L (5.1 vol/vol%) without washing of pretreated biomass, detoxification, or nutrient supplementation. Enzymatic hydrolysis at high solids loading was identified as the primary bottlene ...[more]

PMID: 19164763

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Project description:BackgroundIn a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions and performing comprehensive mass balances will help focus attention on necessary process improvements, and thereby help reduce the cost of biofuel production.ResultsAn on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes, notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEX(TM)), using the same source of corn stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX (26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%), IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of the xylose produced during the pretreatment was removed and not converted to ethanol during fermentation.ConclusionsCompositional analysis of the pretreated biomass solids showed no significant change in composition for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about 90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation.

Project description:BackgroundSimultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover, degradation products resulting from biomass pretreatment impair fermentation of sugars, especially xylose, and can synergize with high temperature stress. One approach to resolve both concerns is to utilize a strain background with innate tolerance to both elevated temperatures and degradation products.ResultsIn this study, we screened a panel of 108 wild and domesticated Saccharomyces cerevisiae strains isolated from a wide range of environmental niches. One wild strain was selected based on its growth tolerance to simultaneous elevated temperature and AFEX™ (Ammonia Fiber Expansion) degradation products. After engineering the strain with two copies of the Scheffersomyces stipitis xylose reductase, xylitol dehydrogenase and xylulokinase genes, we compared the ability of this engineered strain to the benchmark 424A(LNH-ST) strain in ethanol production and xylose fermentation in standard lab medium and AFEX pretreated corn stover (ACS) hydrolysates, as well as in SSCF of ACS at different temperatures. In SSCF of 9% (w/w) glucan loading ACS at 35°C, the engineered strain showed higher cell viabilities and produced a similar amount of ethanol (51.3 g/L) compared to the benchmark 424A(LNH-ST) strain.ConclusionThese results validate our approach in the selection of wild Saccharomyces cerevisiae strains with thermo-tolerance and degradation products tolerance properties for lignocellulosic biofuel production. The wild and domesticated yeast strains phenotyped in this work are publically available for others to use as genetic backgrounds for fermentation of their pretreated biomass at elevated temperatures.

Project description:BackgroundCellulolytic enzymes produced by Trichoderma reesei are widely studied for biomass bioconversion, and enzymatic components vary depending on different inducers. In our previous studies, a mixture of glucose and disaccharide (MGD) was developed and used to induce cellulase production. However, the enzymatic profile induced by MGD is still not defined, and further optimization of the enzyme cocktail is also required for efficient ethanol production from lignocellulosic biomass.ResultsIn this study, cellulolytic enzymes produced by T. reesei Rut C30 using MGD and alkali-pretreated corn stover (APCS) as inducer were compared. Cellular secretome in response to each inducer was analyzed, which revealed a similar enzyme profile. However, significant difference in the content of cellulases and xylanase was detected. Although MGD induction enhanced β-glucosidase production, its activity was still not sufficient for biomass hydrolysis. To overcome such a disadvantage, aabgl1 encoding β-glucosidase in Aspergillus aculeatus was heterologously expressed in T. reesei Rut C30 under the control of the pdc1 promoter. The recombinant T. reesei PB-3 strain showed an improved β-glucosidase activity of 310 CBU/mL in the fed-batch fermentation, 71-folds higher than that produced by the parent strain. Meanwhile, cellulase activity of 50 FPU/mL was detected. Subsequently, the crude enzyme was applied for hydrolyzing corn stover with a solid loading of 20% through separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation, respectively, for ethanol production. Better performance was observed in the SHF process, through which a total of 119.9 g/L glucose was released within 12 h for concomitant ethanol production of 54.2 g/L.ConclusionsThe similar profile of cellulolytic enzymes was detected under the induction of MGD and APCS, but higher amount of cellulases was present in the crude enzyme induced by MGD. However, β-glucosidase activity induced by MGD was not sufficient for hydrolyzing lignocellulosic biomass. High titers of cellulases and β-glucosidase were achieved simultaneously by heterologous expression of aabgl1 in T. reesei and fed-batch fermentation through feeding MGD. We demonstrated that on-site cellulase production by T. reesei PB-3 has a potential for efficient biomass saccharification and ethanol production from lignocellulosic biomass.

Project description:The production of value-added chemicals alongside biofuels from lignocellulosic hydrolysates is critical for developing economically viable biorefineries. Here, the production of propionic acid (PA), a potential building block for C3-based chemicals, from corn stover hydrolysate is investigated using the native PA-producing bacterium Propionibacterium acidipropionici.A wide range of culture conditions and process parameters were examined and experimentally optimized to maximize titer, rate, and yield of PA. The effect of gas sparging during fermentation was first examined, and N2 was found to exhibit improved performance over CO2. Subsequently, the effects of different hydrolysate concentrations, nitrogen sources, and neutralization agents were investigated. One of the best combinations found during batch experiments used yeast extract (YE) as the primary nitrogen source and NH4OH for pH control. This combination enabled PA titers of 30.8 g/L with a productivity of 0.40 g/L h from 76.8 g/L biomass sugars, while successfully minimizing lactic acid production. Due to the economic significance of downstream separations, increasing titers using fed-batch fermentation was examined by changing both feeding media and strategy. Continuous feeding of hydrolysate was found to be superior to pulsed feeding and combined with high YE concentrations increased PA titers to 62.7 g/L and improved the simultaneous utilization of different biomass sugars. Additionally, applying high YE supplementation maintains the lactic acid concentration below 4 g/L for the duration of the fermentation. Finally, with the aim of increasing productivity, high cell density fed-batch fermentations were conducted. PA titers increased to 64.7 g/L with a productivity of 2.35 g/L h for the batch stage and 0.77 g/L h for the overall process.These results highlight the importance of media and fermentation strategy to improve PA production. Overall, this work demonstrates the feasibility of producing PA from corn stover hydrolysate.

Project description:BackgroundCorn stover is lignocellulosic biomass that has potential to be used as raw material for bioethanol production. In the current research, dilute ammonia pretreatment was used to improve the accessibility of corn stover carbohydrates to subsequently added hydrolytic enzymes. Some carbohydrates, however, were still present after enzymatic hydrolysis and fermentation. Hence, this research was aimed to characterise the recalcitrant carbohydrates, especially the oligosaccharides that remained after hydrolysis and fermentation of dilute ammonia-pretreated corn stover (DACS).ResultsAbout 35% (w/w) of DACS carbohydrates remained after enzymatic hydrolysis and fermentation of the released monosaccharides. One-third of these recalcitrant carbohydrates were water soluble and composed of diverse oligosaccharides. By using UHPLC-MS n , more than 50 oligosaccharides were detected. Glucurono-xylooligosaccharides (UAXOS) with a degree of polymerisation (DP) less than 5 were the most abundant oligosaccharides. The (4-O-methyl) glucuronosyl substituent was mostly attached onto the terminal xylosyl residue. It was shown that the glucuronosyl substituent in some UAXOS was modified into a hexenuronosyl, a glucuronamide or a hexenuronamide residue due to the dilute ammonia pretreatment. Another group of abundant oligosaccharides comprised various xyloglucan oligosaccharides (XGOS), with a DP 5 annotated as XXG as the most pronounced. In addition, disaccharides annotated as xylosyl-glucose with different ? linkages as well as larger carbohydrates were present in the fermentation slurry.ConclusionsAround one-third of the 35% (w/w) recalcitrant DACS carbohydrates remained as water-soluble saccharides. In this study, more than 50 recalcitrant oligosaccharides were detected, which mostly composed of xylosyl and/or glucosyl residues. The most pronounced oligosaccharides were UAXOS and XGOS. Hence, ?-glucuronidase and ?-xylosidase were suggested to be added to the enzyme mixture to degrade these oligosaccharides further, and hence the fermentation yield is potentially increased.

Dataset Information

Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST).

Publications

Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST).

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