A metabolic relay for caproic acid production in Huangshui: Cooperative interaction between Clostridium tyrobutyricum and Caproiciproducens.

Microbial chain elongation for medium-chain fatty acid production has gained significant attention. This study elucidated how initial pH and carbon sources regulate microbial caproic acid production from organic waste. By successively subculturing pit mud with Huangshui, lactic acid, or ethanol at different pH values (4.0-6.0), we obtained two enriched consortia: NatCom H (from Huangshui) and NatCom E (from ethanol), both of which produce 10 g/L caproic acid at pH 6.0. Key functional divergence was observed: NatCom H maintained production from pH 5.0 to 6.0, whereas NatCom E was functional only at pH 6.0. Integrated multiomics and bioreactor analyses revealed that this divergence stemmed from distinct microbial interactions. NatCom H was structured around a pH-mediated mutualism between Clostridium tyrobutyricum and acid-sensitive Caproiciproducens species, engaging in sequential substrate conversion. In contrast, NatCom E was dominated solely by Clostridium kluyveri. The hypothesized interaction mechanism was confirmed by isolating the key strains (including two novel Caproiciproducens species) and constructing a synthetic community, which demonstrated that C. tyrobutyricum alleviates acidic stress for Caproiciproducens, enabling caproate synthesis at pH 5.0. These findings reveal a substrate-driven ecological strategy for environmental adaptation and provide a mechanistic framework for engineering robust consortia to convert wastes such as Huangshui into valuable caproic acid.