Integrating high solids enzymatic hydrolysis and co-culture fermentation to improve ethanol production from deep eutectic solvent pretreated rice straw

Rice straw is an abundant agricultural residue with significant potential for sustainable ethanol production. However, the major bottlenecks in converting rice straw into ethanol are biomass recalcitrance, high enzyme costs, and inefficient utilisation of mixed sugars. In this study, an integrated process combining microwave-assisted ChCl:glycerol pretreatment, optimised high-solids enzymatic hydrolysis at low enzyme loading, and co-culture fermentation was developed to enhance ethanol production from rice straw. High-solids enzymatic hydrolysis conditions were optimised by applying central composite design and response surface methodology. At a solids loading of 9.89%, the highest total sugar (TS) yield of 94.92% was obtained compared to a TS of 43.29% at 30% solids loading. The optimal hydrolysis conditions of 17% (w/v) solids loading, 3 FPU/g cellulose enzyme loading, and 75 h hydrolysis time were predicted by the quadratic model and validated, resulting in 75.7% TS yield. Fermentation of the resulting hydrolysates demonstrated that co-culture fermentation outperformed mono- and sequential cultures, achieving a maximum ethanol concentration of 41.1 g/L, with corresponding yields and volumetric productivity of 0.46 g/g and 1.71 g/L.h, respectively. In comparison, co-culture fermentation of hydrolysates derived from 1% H 2 SO 4 pretreatment resulted in lower ethanol yield (0.35 g/g) and productivity (0.67 g/L.h). Thus, the ability to attain high ethanol titre and yield at reduced enzyme dosage and high solids loading highlights the effectiveness of microwave-assisted deep eutectic solvent pretreatment and co-culture fermentation using Saccharomyces cerevisiae and Candida tropicalis . This integrated strategy provides an innovative approach to advancing lignocellulosic bioethanol production from agricultural residues.