Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol

Authors: Slininger, Patricia - Pat; Shea Andersh, Maureen; Thompson, Stephanie; Dien, Bruce; Kurtzman, Cletus; DECOSTA SOUSA, LEONARDO - Michigan State University; BALAN, VENKATESH - Michigan State University

Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/10/2016
Publication Date: 10/24/2016
Citation: Slininger, P.J., Shea-Andersh, M.A., Thompson, S.R., Dien, B.S., Kurtzman, C.P., Sousa, L.D., Balan, V. 2016. Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol. Journal of Visualized Experiments. 116:1-15. doi: 10.3791/54227.

Interpretive Summary: Lignocellulosic biomass, in the form of switchgrass or waste corn stover, is an abundant, non-food, non-feed renewable feedstock that is potentially useful for the production of low cost fuel-grade ethanol. One challenge of making biofuels from lignocellulose is the production of economically recoverable ethanol from the mixture of free sugars present in the hydrolyzates after the biomass is broken down. The chemical pretreatment required to open the structure of plant biomass to enzymatic attack results in solutions rich in sugars but also laden with toxic byproducts that inhibit fermentation. Traditional industrial yeasts do not ferment pentose sugars, which comprise about one-third of available sugars from biomass, nor are they able to colonize, survive or ferment the toxic concentrated hydrolyzates needed to produce economically viable ethanol concentrations. Adaptive evolution and isolation techniques were designed and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified, undetoxified hydrolyzates and to accumulate over 40 g/L ethanol. Repeated culturing of the native pentose-fermenting yeast Scheffersomyces stipitis NRRL Y-7124 in concentrated hydrolyzates, combined with controlled ethanol exposure, was used to force targeted evolution.The result was a group of new robust S. stipitis strains adapted by natural selection to perform in industrially important biomass hydrolyzates and to rapidly produce economically recoverable concentrations of ethanol for use in biofuel. The new strains will be useful to the biofuels industry and to other scientists studying yeast genetics, metabolism and physiology and designing improved processes and technologies for the production of ethanol biofuel. As a result, it furthers our progress toward national priorities of achieving energy independence, strengthening our rural economy, and preserving our environment.

Technical Abstract: Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol and other bio-products via the processing steps of pretreatment, enzyme hydrolysis and microbial conversion. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. While detoxification methodologies can render hydrolyzates fermentable, they are too costly and generate waste disposal liabilities. Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified, undetoxified hydrolyzates and to accumulate over 40 g/L ethanol. Single or multiple selection pressures were applied and tried in series or in parallel to achieve the desired strain improvements. The potential for enrichment relied primarily on natural genetic diversity of the S. stipitis population, and mutations induced by inhibitor exposures. However, UV mutagenesis was also shown to be a successful approach to inducing genetic diversity. Final evolution cultures were dilution plated to harvest predominant isolates while intermediate populations frozen in glycerol at various stages of evolution were enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates screened on various hydrolyzate types were statistically ranked using a novel procedure involving dimensionless RPI transformations of the xylose uptake rate and ethanol yield data to compare the overall performances of isolates across culture conditions and kinetic characteristics. Since hydrolyzates vary in nutritional richness, nutrient supplementation was used to modulate the dynamic range of culture responses across the isolates tested. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.