Skip to main content
ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #418412

Research Project: GENOMICS AND ENGINEERING OF STRESS TOLERANT MICROBES FOR LOWER COST PRODUCTION OF ETHANOL FROM LIGNOCELLULOSE

Location: Bioenergy Research

2014 Annual Report


4. Accomplishments
1. Novel Scheffersomyces stipitis strains reduce the price of ethanol from biomass hydrolyzates. Traditional yeasts used to produce ethanol from grains are unable to utilize xylose, the second most abundant sugar in hydrolyzates of lignocellulose. Toxic fermentation inhibitors generated during biomass pretreatment are problematic to all yeasts. S. stipitis is a native pentose-fermenting yeast with strong aptitude for industrial conversion of lignocellulosic plant biomass to ethanol. So in order to utilize the natural pentose fermenting attribute to greater effect in hydrolyzates, Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois, repetitively cultured S. stipitis in hydrolyzates along with ethanol-challenged continuous culture to force targeted evolution. Ranking performance on diverse hydrolyzate types and nutrient supplementations identified robust isolates able to perform in enzyme hydrolyzates of either base- or acid-pretreated cornstover or switchgrass. Improved features of novel strains include: reduced lag time preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and rapid economically recoverable ethanol at acidic pHs. As a result of the improved features, the new strains allow a $0.31/gal ethanol savings in selling price compared to the parent strain, an accomplishment that advances our progress toward national goals for renewable fuels to stimulate the rural economy, preserve the environment and reduce dependence on foreign oil.

2. Computer simulations of ethanol production from xylose by Scheffersomyces stipitis reveal paths to low cost ethanol from plant biomass. Lignocellulosic plant biomass is an abundant, renewable feedstock for production of low cost fuel-grade ethanol. However, a major technical hurdle to realizing this vision is the fermentation of the sugar xylose, which comprises ~40% of lignocellulose. Xylose is not fermented by traditional yeasts, but S. stipitis ferments it to economically harvestable concentrations of ethanol. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois, formulated a kinetic model for this yeast that describes growth and ethanol production as functions of ethanol, oxygen, and xylose concentrations. The model was validated for various oxygen-limited growth conditions including batch, cell recycle, batch with in situ ethanol removal, and fed-batch. It accurately predicts the time courses of yeast biomass, dissolved oxygen, ethanol, and sugar as functions of the progressing fermentation process in common reactor designs. The new model will expedite the design of improved processes for producing ethanol with xylose utilization. Simulation results show optimization routes to reducing the selling price of ethanol from $2.18/gal to $1.35/gal, furthering progress toward national renewable fuels goals.

3. Novel yeast strain consumes cellobiose, reducing cellulose to ethanol costs. Enzymatic hydrolysis of biomass and the lack of robust biocatalysts are major hurdles that limit sustainable cellulosic biofuels production at a large scale. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois, patented a new yeast strain NRRL Y-50464 to facilitate a more economical consolidated bioprocess of simultaneous saccharification and fermentation (SSF). While producing ethanol from cellulose, this new strain produced sufficient ß-glucosidase enzyme activity to break down cellobiose into glucose, eliminating the need to add ß-glucosidase supplement. Additionally, the new strain tolerates major fermentation inhibitors and the warmer temperatures required to support the consortium of sugar-releasing enzyme activities key to SSF processing and rapid ethanol production. The new technology allowed enzyme cost and consolidated process efficiencies providing an estimated savings of ~$0.35/gal in the selling price of ethanol to allow a minimum selling price of ~$1.80/gal compared to current technology allowing $2.15/gal. This accomplishment addresses mission goals of agriculture as an energy producer that enhance rural economic development and preserve the environment.

4. Novel genes underlying yeast tolerance and detoxification of inhibitors in hydrolyzates. Yeast in situ detoxification relies on multiple gene functions and interactions; however, not all the functions of the genes involved in tolerance are known. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois, characterized new genes encoding a new enzyme that reduces and detoxifies many different inhibitory aldehydes encountered during advanced biofuels production from lignocellulosic materials. The new enzymes work seamlessly with cofactors to reduce toxicity of aldehydes, and can be applied using genetic engineering to tailor yeast to optimize ethanol productivity. Identification and characterization of the new genes for enhanced yeast tolerance has aided our understanding of the mechanisms of yeast tolerance and advanced the creation of next-generation biocatalysts for advanced biofuels production using genetic engineering and systems biology. This accomplishment addresses mission goals of agriculture as an energy producer to reduce dependence on foreign oil, enhance rural economic development and preserve the environment.


Review Publications
Slininger, P.J., Dien, B.S., Lomont, J.M., Bothast, R.J., Ladisch, M.R., Okos, M.R. 2014. Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose. Biotechnology and Bioengineering. 111(8):1532-1540.