Strategies for Efficient Fermentation of Biomass Derived Glucose and Xylose to Ethanol using Naturally Occurring Saccharomyces cerevisiae

The bioconversion of lignocellulosic biomass to fuel ethanol requires both hexose and pentose sugars released to be fully utilized for ethanol production to make this process cost effective. The major challenge for using naturally occurring S. cerevisiae in cellulosic ethanol production is its inability to convert xylose, the most abundant pentose sugar in biomass hydrolysate, to ethanol. Instead of using genetically modified microorganism, xylose is exogenously isomerized to xylulose, a ketal isomer of xylose, which can be slowly fermented by yeast. However, isomerization equilibrium predominantly favors xylose. At neutral pH, sodium tetraborate was used to shift the equilibrium towards 90% xylulose formation by in-situ product removal. A hollow fiber membrane fermentor was used for accommodating high density of yeast in order to compensate for low ethanol productivity resulting from low xylulose transport and metabolic fluxes in the non-oxidative pentose phosphate pathway and towards glycolysis. S. cerevisiae was distributed on the shell-side of the fibers whereas media containing sugars was recirculating through the fiber lumen. Continuous fermentation of glucose for 7 days gave consistent ethanol yield and low by-product formation. The bench scale module tested on the fermentation of glucose and pre-isomerized xylulose resulted in 20 g/l/hrs and 0.83 g/l/hrs ethanol productivity, respectively, all with close to theoretical ethanol yield. Fermentation of pre-isomerized 7.5% IL-pretreated poplar hydrolysate and its 2-fold concentrate resulted in 92% and 84% theoretical ethanol yield, respectively, which was comparable to the fermentation of model sugar mixture. To accomplish the isomerization under conditions suitable for sugar fermentation, a borate enhanced co-immobilized enzyme system that can sustain two pH micro-environments has been proposed. Simultaneous isomerization and fermentation (SIF) of 60 g/l glucose and 30 g/l xylose yielded 94% ethanol within 10 hrs based on sugar consumed. However, due to xylitol inhibition only 50% xylose was utilized during the course of SIF. In order to further improve xylulose transport and utilization, S. cerevisiae was adapted on xylulose using chemostat continuous cultivation. The evolved C2 strain exhibited 0.066 g/g dry cell/hrs xylulose consumption rate during the fermentation of 5 g/l glucose and 15 g/l xylulose, 16% higher than its parental strain.