Location: Bioenergy Research2014 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.
3. Progress Report:
This is the final report and over the life of the project significant progress was made on all three objectives. Relative to Objective 1 to discover tolerance mechanisms, high throughput gene expression tools were developed and applied to study an evolved Saccharomyces cerevisiae strain that was tolerant to both ethanol and furan aldehyde inhibitors. Cofactor balances, pathways, and regulatory genes responsible for inhibitor tolerance were identified. Involved were stronger signaling pathways directing cell functions and expressions of amino acid- and energy-providing metabolisms supporting cell repair and other activities. Under Objective 2 to develop new strains, the inhibitor-tolerant strain of S. cerevisiae that was the subject of the tolerance mechanism studies of Objective 1 was engineered with new genes to add xylose transporters and enzymes for efficient xylose conversion to ethanol (U.S. patent pending). In a second approach, hydrolyzate tolerant strains of the native xylose-fermenting yeast Scheffersomyces stipitis (previously named Pichia stipitis) were developed by directed evolution. S. stipitis is commercially promising because of its demonstrated ability to produce economically recoverable ethanol from xylose under microaerobic conditions. Hydrolyzate-tolerant isolates of S. stipitis were derived during serial transfer to increasing concentrations of multiple base- or acid-pretreated hydrolyzates of cornstover or switchgrass (18-20% solids by weight) and after ethanol-challenged xylose-fed continuous culture. Ranking best isolates performing across diverse conditions of hydrolyzate type and nutrition provided desired features (patent pending): reduced growth and diauxic lags, significantly enhanced fermentation rates and ethanol yield (>40 g/L) at acidic pHs. In a third approach, a new ethanologenic Clavispora strain was discovered (patent pending) and developed. It produces a ß-glucosidase and tolerates hydrolyzate inhibitors at warmer temperatures, which saves on enzyme loading and allows more economical consolidation of yeast and enzyme bioprocesses to reduce the overall cost of ethanol from lignocellulose. Under Objective 3 to optimize processes, ethanol was discovered to completely repress key enzymes for xylose utilization by S. stipitis, a situation leading to long diauxic lags during the fermentation of glucose-xylose mixtures occurring in hydrolyzates. From this knowledge, a useful process strategy arose: grow yeast on xylose released in hydrolyzate pretreatment and then recycle the xylose-induced cells to gain higher fermentation rates and yield during mixed sugar conversion. During furfural exposure, yeast were discovered to not only detoxify furfural to protect from its cellular effects but also to repair damage, a function which would activate amino acids metabolism. Amino acid supplementation was found key to the performance of parent and inhibitor-tolerant S. cerevisiae yeast strains in late-season protein-deplete switchgrass hydrolyzates. For parent and inhibitor-tolerant derivatives of S. cerevisiae, ethanol productivity and yield varied with types and quantities of commercial nitrogen sources of varying cost. Finally, a xylose fermentation simulation model was developed and validated, and its application has indicated optimal bioreactor designs for efficient xylose conversion in hydrolyzates. The new more resilient, more efficient yeast strains and associated process know-how that have been developed under this project are expected to reduce the minimum selling price of ethanol from lignocellulose from $2.15/gal to ~$1.80/gal and may reduce it to as low as an estimated ~$1.35/gal with continued research and optimization under the next project plan as the commercial application of novel strains and associated process strategies is pursued.
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.
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.