Constructing simplified microbial consortia that couple lactic acid and ethanol utilization to highly produce caproic acid from liquor-making wastewater.

Converting biodegradable carbon in wastewater into medium-chain fatty acids (MCFAs) through stable microbiota is highly attractive. In this study, we utilized a top-down approach for constructing MCFA-producing microbial consortia. Specifically, an enrichment and plating-screening strategy employing lactic acid and ethanol as selective carbon sources was applied to isolate simplified caproic acid-producing microbial consortia from liquor-making pit mud. The representative microbial consortium SimpCom3 demonstrated high level of caproic acid production (14.62 ± 0.48 g/L) in a semi-synthetic medium, significantly outperforming consortium SimpCom1 (5.96 ± 0.11 g/L) and consortium SimpCom2 (9.63 ± 0.16 g/L). This performance of microbial consortium SimpCom3 was attributed to its ability to co-utilize lactic acid and ethanol, produce fewer odd-chain fatty acids byproducts, and maintain pH self-regulation between 6.45 and 8.29. Metagenomic analyses revealed the dominance of Clostridium kluyveri (30.69 %-50.46 %), C. butyricum (6.71 %-13.98 %) and C. tyrobutyricum (37.11 %-58.07 %) in consortium SimpCom3, which synergistically converted lactic acid and ethanol to caproic acid via reverse β-oxidation. Stable performance over 56 days of cyclic-batch fermentation processes confirmed the robustness of consortium SimpCom3. When applying consortium SimpCom3 to unsterilized liquor-making wastewater in a fermenter with a fed-batch approach, 22.13 g/L caproic acid was produced with 66.38 % selectivity, and microbial dynamics analysis demonstrated the consortium's high adaptability to real wastewater. Metabolic analysis based on high-quality assembly metagenomes (HQ-MAGs) revealed a novel cooperative metabolism: cross-feeding between Clostridium kluyveri (which utilizes ethanol and produces caproic acid) and lactate-utilizing butyrate producers maintained consortium stability and enhanced caproic acid production. Crucially, the decarboxylation of lactic acid counteracted acidification caused by ethanol-driven caproic acid synthesis, enabling self-regulated pH stability within the simplified microbiome system. Together, this study presents a simplified microbial consortium construction method for caproic acid production from liquor-making wastewater, overcoming the limitations of synthetic co-cultures and enhancing the viability of chain-elongation biorefineries in wastewater treatment.