Profiling prokaryotic community in pit mud of Chinese strong-aroma type liquor by using oligotrophic culturing

Pit mud microbiota plays a key role in flavour production for Chinese strong-aroma type liquor. However, the pit mud microbiota cannot be cultured in laboratory. In this study, an oligotrophic medium with acetate as carbon source was used to enrich pit mud microbiota. The 16S rRNA gene amplicon sequencing was applied to examine the microbial dynamics of the enrichment consortia. Both methanogens and bacteria were simultaneously enriched. Euryarchaeota, Bacteroidetes and Firmicutes were the top 3 enriched phyla, and 31 genera were successfully enriched. More specifically, 11 genera (65%) out of the 17 dominant genera in pit mud were successfully enriched, including Petrimonas, Proteiniphilum, Anaerocella, Hydrogenispora, Methanosarcina, Fermentimonas, LNR_A2-18, Sedimentibacter, Lutispora, Syntrophomonas and Aminobacterium. Furthermore, 20 rare genera in the analyzed pit mud samples were also enriched. Aceticlastic Methanosaeta and Methanosarcina were found to be dominant methanogens in the enrichment consortia. Metagenomic sequencing was then applied to the enriched microbial consortia to explore the metabolic potentials of pit mud microbes. Aceticlastic methanogenesis pathway of Methanosaeta was reconstructed. Furthermore, 26 high-quality metagenome-assembled genomes (MAGs) were obtained based on the metagenomic binning analysis. Moreover, nutrients in pit mud were found to be crucial to sustain the methanogenesis of the enriched microbial consortia. These results suggested that the enrichment approach by using oligotrophic culturing can effectively cultivate the pit mud microbiota. Combined with metagenomics, the oligotrophic culturing will be greatly helpful to decipher the community composition and metabolic potentials of pit mud microbiota.