Prokaryotic community diversity, assembly processes, and potential metabolic pathways of a hexanoic acid-producing...

Hexanoic acid (HA), a vital platform chemical, can be produced by various mixed cultures using ethanol or ethanol-containing waste. However, systematic research remains lacking on its ecological succession, community assembly, and core synthetic pathways. In this study, an ethanol-based HA-producing synthetic community (SynCom) was constructed from the pit mud using a top-down strategy, and dominated by Clostridium sensu stricto 12 (primarily Clostridium kluyveri, 86%), Clostridium sensu stricto 18 and Pseudoclavibacter. A highly significant positive correlation (P < 0.01) was observed between Clostridium sensu stricto 12 and HA biosynthesis. Ecological analysis revealed that community assembly exhibited significant diversity dynamics during enrichment. While overall assembly was predominantly governed by stochastic processes, colonization of the key HA-producing lineage Clostridium sensu stricto 12 was distinctly driven by deterministic processes. Functional predictions and fermentation profiling suggested a potential associationbetween HA production by SynCom (G4) and the reverse β-oxidation pathway. Under optimized conditions (pH 7.9, 37.6°C, 8.2% inoculum, 77% loading volume), SynCom (G4) achieved a HA yield of 4.69 g/L. This study demonstrated that a deterministically assembled Clostridium sensu stricto 12-dominated in this synthetic community may effectively promote HA production, providing mechanistic insights into microbial community assembly and offering a simplified yet functional model in Baijiu fermentation.