Inter-species interactions driven by different pit mud supplements determine the features of community assembly and flavor synthesis in Zaopei during anaerobic solid-state fermentation

Abstract

In Strong-flavor Baijiu (SFB) brewing, pit mud (PM) and its age directly affect flavor quality. Here, we established the laboratory-scale microcosms with three PM treatments (no PM, old PM (OPM), new PM (NPM)) coupled with genome-scale metabolic models (GEMs) to investigate the underlying mechanisms of PM-driven differences in Zaopei anaerobic solid-state fermentation (ASSF). Results showed that OPM and NPM supplements prevented the production of non-typical SFB aromatics. They increased the complexity of the Zaopei community but exhibited distinct modulation patterns, and the community and flavor features across different PM supplements were largely driven by microbial interactions. For community assembly, OPM enhanced determinism during mid-late ASSF while NPM increased stochasticity. For flavor synthesis, OPM mainly increased gene abundance of the reverse β-oxidation pathway whereas NPM primarily altered that of the fatty acid biosynthesis pathway. For microbial interactions, OPM promoted more synergistic interactions between OPM-derived and Daqu-derived (fermentation starter) microbiota. GEMs predicted that Daqu-derived Thermoactinomyces vulgaris would act as the metabolic hub in OPM-supplemented Zaopei, providing essential nutrients to OPM-derived Methanoculleus bourgensis and grain-derived Pseudomonas asiatica. In contrast, in NPM-supplemented Zaopei, cross-feeding among OPM-derived Ligilactobacillus acidipiscis, Mesorhizobium terrae, and grain-derived Ralstonia thomasii could be the metabolic center, without any Daqu-derived taxa. These results facilitate Zaopei ASSF optimization and deepen insights into traditional fermentation mechanisms.