Oriented butyrate production through a novel bacteria-yeast microbiome: batch verification, key electron donor identification, and long-term validation.

Recovering butyrate from organic waste enables its high-value conversion, aligning with the principles of a circular economy. Traditional butyrate fermentation emphasizes carbohydrates and protein degradation, with limited focus on chain elongation (CE). This study, for the first time, systematically evaluated the effects of different Saccharomyces cerevisiae (SC) concentrations (1, 2, 4, 6, and 8 g/L) on ethanol production (a key electron donor) and subsequent CE for butyrate synthesis, identifying 2 g/L as the optimal SC dosage. At this concentration, butyrate production reached 15.41 ± 2.84 g COD/L, which was 2.72 times higher than that of the blank. Metabolic pathway analysis revealed that yeast not only enhanced substrate degradation (＞90 %) but also facilitated the in situ generation and utilization of ethanol. 16S rRNA indicated 54.10 % relative abundance of butyrate-producing bacteria (Clostridium). Long-term tests found that adding SC reversed the halt in production from prolonged distiller yeast inoculum, stabilising output at 15 g COD/L. Metagenomic analysis revealed that SC inoculation primarily enriched Clostridium luticellarii and Clostridium tyrobutyricum. In addition to raising reverse β-oxidation gene abundance, this treatment also enhanced lactate utilization genes, thereby strengthening acetyl-CoA to butyrate conversion. Through further experiments involving different electron donor ratios and long-term operation, this study highlights the critical role of yeast-bacteria synergy in enhancing butyrate synthesis, providing a theoretical foundation and technical strategy for food waste valorization in line with circular economy principles.