One-Pot Biomass Pretreatment for Ethanol Production by Engineered Saccharomyces cerevisiae

This study presents a novel and cost-effective approach to biomass pretreatment that addresses the limitations of conventional methods, which often result in high water and chemical usage as well as the production of chemical-laden wastewater. We investigated the integration of metal oxides (specifically CaO and MgO) for biomass pretreatment and mineral acids (H ₂SO ₄ or H ₃PO ₄) for pH adjustment at a high solid loading of 20 wt %. This innovative method allows for direct enzymatic hydrolysis and fermentation of the resulting slurry, effectively eliminating the need for solid-liquid separation and extensive washing. Our findings reveal that hydrolysates from MgO combined with H ₃PO ₄ or H ₂SO ₄ were inhibitory to Saccharomyces cerevisiae, resulting in no ethanol production. In contrast, corn stover that was pretreated with CaO and subsequently adjusted to pH with H ₃PO ₄ demonstrated a higher enzymatic hydrolysis efficiency than the case of adjusting pH with H ₂SO ₄, achieving over 65% glucan conversion and 80% xylan conversion, along with an ethanol concentration of approximately 33 g/L following separate hydrolysis and fermentation. This enhanced performance can be attributed to reduced osmotic stress, decreased salt toxicity, and minimal formation of inhibitors, as CaO neutralized with H ₃PO ₄ generated the minimally soluble precipitate Ca ₃(PO ₄) ₂. Furthermore, employing a semisimultaneous saccharification and fermentation process improved sugar utilization efficiency, resulting in an increased ethanol concentration of 46 g/L. The corn stover fermentation residue (CSFR) contained 93% lignin, predominantly of syringyl and guaiacyl types. This study offers a sustainable and scalable method for producing cellulosic ethanol, significantly lowering chemical and water consumption while achieving a high conversion efficiency.