Prospects of thermotolerant Kluyveromyces marxianus for high solids ethanol fermentation of lignocellulosic biomass.

Simultaneous saccharification and fermentation (SSF) is effective for minimizing sugar inhibition during high solids fermentation of biomass solids to ethanol. However, fungal enzymes used during SSF are optimal between 50 and 60 °C, whereas most fermentative yeast, such as Saccharomyces cerevisiae, do not tolerate temperatures above 37 °C. Kluyveromyces marxianus variant CBS 6556 is a thermotolerant eukaryote that thrives at 43 °C, thus potentially serving as a promising new host for SSF operation in biorefineries. Here, we attempt to leverage the thermotolerance of the strain to demonstrate the application of CBS 6556 in a high solids (up to 20 wt% insoluble solid loading) SSF configuration to understand its capabilities and limitations as compared to a proven SSF strain, S. cerevisiae D5A. For this study, we first pretreated hardwood poplar chips using Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) to remove lignin and hemicellulose and to produce cellulose-enriched pretreated solids for SSF. Our results demonstrate that although CBS 6556 could not directly outperform D5A, it demonstrated similar tolerance to high gravity sugar solutions, superior growth rates at higher temperatures and higher early stage ethanol productivity. We discovered that CBS 6556's membrane was particularly sensitive to higher ethanol concentrations causing it to suffer earlier fermentation arrest than D5A. Cross-examination of metabolite data between CBS 6556 and D5A and cell surface imaging suggests that the combined stresses of high ethanol concentrations and temperature to CBS 6556's cell membrane was a primary factor limiting its ethanol productivity. Hence, we believe K. marxianus to be an excellent host for future genetic engineering efforts to improve membrane robustness especially at high temperatures in order to achieve higher ethanol productivity and titers, serving as a viable alternative to D5A.