2012 Annual Report
1a.Objectives (from AD-416):
1) Determine the key metabolic, physiologic, transport, genetic, and regulatory mechanisms underlying stress tolerance and adaptation in ethanologenic yeast when they convert lignocellulosic hydrolyzates. .
2)Via directed evolution, genetic engineering, and/or adaptation, create new commercially preferred yeast strains for converting lignocellulose hydrolyzates to ethanol. .
3)In collaboration with Cooperative Research and Development (CRADA) partner(s), optimize fermentation process conditions so as to (1) leverage advantages of stress tolerant strains developed in Objective 2 and (2) minimize the cost of fermenting lignocellulosic hydrolyzates to fuel-grade ethanol.
1b.Approach (from AD-416):
The lignocellulose-to-ethanol process involves pretreatment of biomass to predispose it to chemical and enzymatic hydrolysis, saccharification of sugar polymers to simple sugars, and fermentation of the sugars to ethanol. The hydrolysis product is difficult to ferment because inhibitory byproducts are produced, and the resulting sugar mixture contains both hexose and pentose sugars, the latter not fermentable by traditional brewing yeasts. Needed are improved yeast strains which will ferment both types of sugars and are able to withstand, survive, and function in the presence of inhibitors (including furfural and hydroxymethyl furfural), high ethanol concentration and osmotic pressure, and sufficiently elevated temperatures for simultaneous saccharification-fermentation processes. In the research proposed, fermentation hurdles will be overcome by combining process optimization strategies and strain improvements aided by new molecular biology tools allowing high throughput screening of whole genomes to identify key genes and gene networks involved in stress tolerance and sugar utilization. Products of the research will be stress-tolerant yeasts capable of resisting and detoxifying inhibitors and efficiently fermenting hexose and pentose sugars to ethanol, a genetic blueprint describing tolerance mechanisms and metabolic pathways, and optimal culture conditions and process configurations to lower costs by maximizing yeast stress resistance, ethanol productivity and yield.
The overall goal of this project is to develop stress tolerant microbes for lower cost production of ethanol from renewable biomass. By determining key mechanisms of tolerance, evolving and engineering new strains, and optimizing key process conditions, we have made significant progress during FY12. Research studies on genomic mechanisms of yeast tolerance in Saccharomyces cerevisiae were expanded under a National Research Initiative project in collaboration with New Mexico State University scientists. This past year significant time effort has focused on the in-depth analysis of genomics data to understand the biological relevance of our findings and uncover inhibitor tolerance mechanisms through modeling approaches. The experimental phase of a non-funded cooperative agreement with the Institute of BioEnergy and Bioprocessing Technology, Chinese Academy of Sciences, Qingdao, China has recently finished and provided complete sequencing data needed for a genomic comparison of an adapted inhibitor-tolerant strain and its parent. Collaborative data analysis for a manuscript is ongoing. Our prior studies have indicated that the native yeast pentose metabolism may compete for reductase activities needed to reduce toxic furan aldehyde inhibitors to less toxic alcohols, so it is important to integrate the engineering of pentose and tolerance traits in S. cerevisiae to improve hydrolyzate sugar utilization. Our inhibitor-tolerant evolved S. cerevisiae strain was engineered with xylose isomerase and other enzymes to allow conversion of xylose to ethanol. Then cloning of new xylose transporter genes from the S. stipitis genome into S. cerevisiae allowed us to verify the function of each transporter gene and at the same time develop new advanced strains (patent application to be filed FY12). Evolved tolerant S. stipitis strains were developed which exhibited reduced diauxic lag from glucose to xylose uptake and improved production rate and yield of ethanol from ammonia fiber explosion pre-treated corn stover hydrolyzates at 18% solids, and an invention disclosure on top strains was approved for patent application. Additionally, S. stipitis serial micro-batch and continuous cultures have been challenged with concentrated ethanol and dilute acid switchgrass hydrolyzates to enrich for tolerant variants. This past year over 500 single-cell colonies were recovered from enrichment cultures via selective plating and screened to rank strains based on ethanol production kinetics on concentrated switchgrass hydrolyzates with varied nutrient levels. Culture nutrition was key to performance of both native and inhibitor-tolerant S. cerevisiae and S. stipitis strains on switchgrass hydrolyzates, and commercially useful supplements are being developed to maximize performance. Interestingly, nutrient requirements to maximize performance of adapted versus parent strains were often very different, indicating a need for attention to nutrient supplies during both strain selection and process development.
New yeast produces enzymes which reduce its cellulose to ethanol cost. Renewable ethanol biofuel production from agricultural biomass requires chemical pretreatment then hydrolysis by costly cellulase and ß-glucosidase enzymes to break down the cellulose polymers into simple sugars for microbial fermentation. Due to the temperature difference between optimal enzyme hydrolysis and yeast fermentation, a two-step procedure is often needed that increases cost and complication of the bioconversion process. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, IL, discovered and developed a new ethanologenic yeast strain NRRL Y-50464 (patent pending) which produces a native ß-glucosidase and which also tolerates inhibitory byproducts of biomass pretreatment and high temperatures supporting enzymatic hydrolysis. Using this novel yeast strain without addition of ß-glucosidase enzyme, cellulosic ethanol production from an industrial corncob residue was demonstrated in a one-step process with simultaneous saccharification and fermentation. Eliminating the addition of external ß-glucosidase reduces the enzyme cost of cellulosic ethanol production, and higher temperature fermentation allows more economical consolidation of yeast and enzyme bioprocesses. Lower cost production of renewable ethanol from agricultural biomass promises to support the rural economy and to reduce United States dependence on foreign petroleum.
Nutrients key to tolerant yeast development and fermentation of switchgrass hydrolyzates. Unlike gasoline which is a fossil fuel, ethanol biofuel is renewable and can be produced through biological conversion of cellulosic agricultural biomass, including new energy crops such as switchgrass. Inhibitory compounds generated during dilute acid hydrolysis pretreatment of lignocellulosic biomass interfere with the conversion process. Tolerant yeast strains have recently been developed by scientists of the Agricultural Research Service in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, IL, who applied evolutionary approaches in the presence of switchgrass hydrolyzate inhibitors. New evolved strains (patents pending) were selected which showed excellent hydolyzate survival with enhanced fermentation rates exhibiting nearly lag-free transition from glucose to xylose utilization and economically recoverable ethanol accumulations. This new technology will support lower cost production of renewable ethanol from agricultural biomass, reduce United States dependence on foreign petroleum and stimulate the rural economy.
Ma, M., Liu, Z., Moon, J. 2012. Genetic engineering of inhibitor-tolerant Saccharomyces cerevisiae for improved xylose utilization in ethanol production. Bioenergy Research. 5:459-469.
Slininger, P.J., Schisler, D.A. 2012. Biological control agents for suppression of post-harvest diseases of potatoes: strategies on discovery and development. In: Dhanasekaran, N., Panneerselvam, A., editors. Fungicides for Plant and Animal Diseases. Rijeka, Croatia: In-Tech. p. 141-166.
Liu, Z., Weber, S.A., Cotta, M.A., Li, S. 2012. A new beta-glucosidase producing yeast for lower-cost cellulosic ethanol production from xylose-extracted corncob residues by simultaneous saccharification and fermentation. Bioresource Technology. 104:410-416.
Zhang, L., Li, J., Li, S., Liu, Z. 2011. Challenges of cellulosic ethanol production from xylose-extracted corncob residues. BioResources. 6(4):4302-4316.
Schisler, D.A., Slininger, P.J., Boehm, M.J., Paul, P.A. 2011. Co-culture of yeast antagonists of Fusarium head blight and their effect on disease development in wheat. Plant Pathology Journal. 10:128-137. DOI: 10.3923/ppj.2011.128.137.
Moon, J., Liu, Z. 2012. Protein engineering of GRE2 from Saccharomyces cerevisiae for enhanced detoxification of 5-hydroxymethylfurfural. Enzyme and Microbial Technology. 50:115-120.
Liu, Z., Weber, S.A., Cotta, M.A. 2013. Isolation and characterization of a ß-glucosidase from a Clavispora strain with potential applications in bioethanol production from cellulosic materials. Bioenergy Research. 6:65-74.
Liu, Z. 2011. Unification of gene expression data for comparable analyses under stress conditions. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels. Microbiology Monographs No. 22. Berlin Heidelberg: Springer-Verlag. p. 279-299.
Liu, Z. 2011. Genomics of yeast tolerance and in situ detoxification. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels. Microbiology Monographs No. 22. Berlin Heidelberg: Springer-Verlag. p. 1-28.
Hughes, S.R., Bischoff, K.M., Gibbons, W.R., Bang, S.S., Pinkelman, R., Slininger, P.J., Qureshi, N., Liu, S., Saha, B.C., Jackson, J.S., Cotta, M.A., Rich, J.O., Javers, J. 2012. Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars. Journal of Industrial Microbiology and Biotechnology. 39(1):163–173.
Ma, M., Liu, Z. 2012. Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels: Systems Biology. Microbiology Monographs 22. Berlin, Germany: Springer-Verlag. p. 77-115.
Matsushika, A., Liu, Z., Sawayama, S., Moon, J. 2011. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae. In: Liu, Z.L., editor. Microbial Stress Tolerance for Biofuels. Microbiology Monographs No. 22. Berlin Heidelberg: Springer-Verlag. p. 137-160.