A major challenge to fermentation of lignocellulosic biomass is achieving 5% or greater ethanol titers in the product stream for economic downstream distillation because of limited fermentation efficiencies and mixing difficulties at high biomass solids loadings. Nevertheless, a recently developed co-solvent enhanced lignocellulosic fractionation (CELF) pretreatment technology that uses an acidic aqueous tetrahydrofuran (THF) solution to produce highly digestible glucan-rich solids has achieved higher ethanol titers for simple batch simultaneous saccharification and fermentation (SSF) than possible with previous batch fermentations at low enzyme loadings. Despite poor initial mixing in batch SSF, CELF pretreated corn stover realized 57 g/l ethanol and a 90.5% of theoretical yield in 9 days with 11% glucan at an enzyme loading of only 5 mg protein/g glucan. However, because the maximum glucan loading is limited to about 11% in batch operations by the glucan and moisture content of the solids after pretreatment plus the need to supplement the yeast and media, fed batch systems promise to boost ethanol titers even further, thereby saving on downstream distillation costs. Furthermore, different feeding strategies could result in possible savings of enzymes or time compared to simple batch processes.
Based on previous results with high solids loading of batch SSF for which an increase in solids loadings did not reduce ethanol yields or increase fermentation times, the aim of this study was to investigate whether fed batch systems can increase ethanol titers at enzyme loadings of ≤5 mg protein/g glucan while maintaining the high ethanol yields. In one such strategy, only pretreated solids were fed to dilute the effective enzyme loading to 5 mg protein/g glucan. In a second strategy, both pretreated solids and enzymes were fed to maintain the enzyme loading of 5 mg protein/g glucan. More specifically, for the first strategy, a fermentation initiated with a solids loading of 11% glucan and an enzyme loading of 15 mg protein/g glucan was fed with four additions of wet pretreated solids at one or two-day intervals for a total glucan content of 15-16%, translating into an overall enzyme loading of 5 mg protein/g glucan. For the second strategy, a fermentation initiated with a solids loading of 11% glucan and an enzyme loading of 5 mg protein/g glucan was fed with four similar additions of wet pretreated solids as well as 5 mg protein/g glucan, also translating into an overall enzyme loading of 5 mg/g glucan and a glucan content of 15-16%. Comparisons of these two strategies provide insights into pathways to increase ethanol concentrations while keeping enzyme loadings and costs low.