Abstract: In this study, a dominant caproic acid-producing strain (CPB), designated XB2, was isolated from pit mud of baijiu using enrichment and traditional anaerobic isolation techniques. It was identified as Caproicibacterium lactatifermentans based on 16S rRNA gene sequencing and whole-genome sequencing. Comparative genomic analysis revealed that in addition to possessing a complete reverse β-oxidation (RBO) pathway, strain XB2 is the first reported Caproicibacterium strain carrying the full complement of key enzyme genes in both the fatty acid biosynthesis (FAB) pathway and the L-threonine degradation pathway for propionate synthesis. Transmission electron microscopy (TEM) revealed that strain XB2 exhibited an elliptical to fusiform morphology, differing significantly from the short-rod shape of its phylogenetic relatives. Fermentation results indicated carbon source-dependent metabolism: when glucose was used as the substrate, caproate was synthesized at a low concentration (0.23 g/L), whereas using lactate as the substrate increased butyrate production by 0.59 g/L. Under conditions with limited electron acceptors, propionate was produced at 0.21 g/L, suggesting the activation of the L-threonine pathway. This study elucidates the morphology and intraspecific metabolic diversity of C. lactatifermentans. It challenges the conventional view that CPBs rely solely on the RBO pathway for caproic acid synthesis, providing a new perspective for understanding functional redundancy and adaptability of microbial communities in pit mud of baijiu. Furthermore, it establishes a theoretical foundation for optimizing baijiu flavor profiles through the precise regulation of microbial metabolism.

Key words: nongxiangxing baijiu; Caproicibacterium lactatifermentans XB2; reverse β-oxidation pathway; fatty acid biosynthesis pathway; propionic acid; L-threonine degradation pathway