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A major economic obstacle in lignocellulosic ethanol production is the low sugar concentrations in the hydrolysate and subsequent fermentation to economically distillable ethanol concentrations. We have previously demonstrated a two-stage fermentation process that recycles xylose with xylose isomerase to increase ethanol productivity, where the low sugar concentrations in the hydrolysate limit the final ethanol concentrations. In this study, three approaches are combined to increase ethanol concentrations. First, the medium-additive requirements were investigated to reduce ethanol dilution. Second, methods to increase the sugar concentrations in the sugarcane bagasse hydrolysate were undertaken. Third, the two-stage fermentation process was recharacterized with high gravity hydrolysate. It was determined that phosphate and magnesium sulfate are essential to the ethanol fermentation. Additionally, the Escherichia coli extract and xylose isomerase additions were shown to significantly increase ethanol productivity. Finally, the fermentation on hydrolysate had only slightly lower productivity than the reagent-grade sugar fermentation; however, both fermentations had similar final ethanol concentrations. The present work demonstrates the capability to produce ethanol from high gravity sugarcane bagasse hydrolysate using Saccharomyces pastorianus with low yeast inoculum in minimal medium. Moreover, ethanol productivities were on par with pilot-scale commercial starch-based facilities, even when the yeast biomass production stage was included.


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