Multi-Omics Reveals Microbial Roles and Metabolic Functions at the Spatiotemporal Niche in Pit Mud

Background Popular distilled Chinese strong-flavoured liquor (CSFL) is produced by solid fermentation in ground pits. Pit mud (PM), as a habitat for microbes, plays an important role in the production of CSFL. However, understanding the taxonomic composition, metabolic potential, and functional diversity of core microbes of PM in spatiotemporal niche remains a major challenge. Results Using a multi-omic approach of high-throughput full-length 16S rRNA, ITS sequencing, and metagenomics, we identified bacteria such as Caproiciproducens, Clostridium, Lactobacillus, Bellilinea, Petrimonas, Proteiniphilum, and Prevotella and the archaea Methanobrevibacter and Methanobacterium as the core microbiota in 30-, 100-, and 300-year-old cellars. Significant correlations between microbial communities and environmental factors showed that lactic, caproic, butyric, and acetic acids were the core driving forces for microbiota succession in different spatial locations and were mainly correlated with Caproiciproducens, Clostridium, Lactobacillus, Methanobrevibacter, and Methanobacterium. A total of 982 metabolites were detected using GC/MS and LC/MS, mainly including amino acids, peptides, and fatty acids, and correlations were shown between seven microorganisms and 12 amino acids and fatty acid metabolites. The crucial genes of flavour-relevant and substrate degradation pathways mainly included nine microorganism orders, with the most important being Clostridiales, Bacteroidales, and Methanobacteriales. However, only Clostridiales and Bacillales were potentially involved in alcohol–aldehyde–carboxylate–ester metabolism. Conclusions The 30-, 100-, and 300-year-old cellars provided an ideal opportunity to understand the effect of cellar age on the microbial composition and functional diversity of the microorganisms in PM. Our detailed metagenomic analyses across the continuously used cells extend the known diversity of microorganisms involved in flavour generation and substrate degradation over a wide range of environmental conditions. The results indicate that Clostridiales and Bacteroidales are major microbiota orders for flavour generation and substrate degradation pathways, while archaea also play an important role in cooperation with bacteria in various flavour generation pathways. This study helps elucidate the core microbiota composition, different metabolic roles of microorganisms, and the formation mechanism of PM partial functions, thus providing a basic theory to support the regulation of Baijiu production.