Prokaryotic evolution shapes specialized communities in long term engineered pit mud ecosystem

Elucidating the temporal dynamics of complex microbial consortia is crucial for engineering robust microbiome. We investigated prokaryotic evolution in pit mud, a centuries-old engineered environment used in Chinese liquor fermentation. Metagenomic analysis of 120 pit mud samples across different ages revealed a transition from generalist-dominated to specialist-enriched communities. This shift was characterized by decreased hydrolytic potential and increased organic acid metabolism, with key taxonomic changes including declines in Proteiniphilum and Petrimonas, and increases in Methanobacterium and Caproicibacter. The mature specialist community accelerates the short-chain organic acids turnover through syntrophic fatty acid oxidation, methanogenesis, and carbon chain elongation, maintaining ecosystem stability. While nutrient availability primarily shapes early stages community interactions, environmental stress becomes a dominant factor in mature systems. These insights into long-term prokaryotic adaptation provide a foundation for the rational design of resilient, functionally optimized microbial communities for biotechnological applications.