Technique to rank evolved yeast strains for bioconversion of lignocellulose to ethanol incorporating kinetic performance factors influenced by nutrition and hydrolyzate type

Traditional industrial yeasts do not ferment xylose, comprising up to 40% of plant sugars, and are unable to function in concentrated hydrolyzates.  Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment.  To avoid need for costly detoxification procedures, adaptive evolution and isolation techniques were applied to yield derivatives of the native pentose-fermenting strain Scheffersomyces stipitis strain NRRL Y-7124 that are able to more efficiently convert pretreated biomass hydrolyzates to economically recoverable ethanol.  Improved individuals were enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and exposure-induced mutations.  Exposures included enzyme saccharified AFEX-pretreated corn stover and dilute acid-pretreated switchgrass hydrolyzates, ethanol or UV radiation.  Evolution cultures were enriched on selective media using stress gradients to recover most promising isolates through dilution plating.  Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data.  Using the RPI statistical parameter, an overall relative performance average is calculated to allow ranking isolates based on multiple factors, including kinetic characteristics and culture conditions varying in hydrolyzate type, nutrients and inhibitors.  Through application of this technique, derivatives of the parent strain were identified with improved features in diverse 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.