One-Pot Biomass Pretreatment for Ethanol Production by Engineered <i>Saccharomyces cerevisiae</i>

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 (H2SO4 or H3PO4) 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 H3PO4 or H2SO4 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 H3PO4 demonstrated a higher enzymatic hydrolysis efficiency than the case of adjusting pH with H2SO4, 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 H3PO4 generated the minimally soluble precipitate Ca3(PO4)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.