The relationship between microbial diversity and the physicochemical characteristics of pit mud of Strong-flavor Baijiu

Baijiu, a traditional Chinese distilled spirit and the world's most consumed liquor, derives its distinctive quality and complex flavor profile, particularly in strong-aroma types, primarily from the unique microbial ecosystem within pit mud (PM), the fermented clay lining of fermentation pits. To gain a deeper understanding of the role microorganisms play in shaping the quality of baijiu, this study comprehensively examined the microbial communities and physicochemical characteristics of PM sourced from various ages (20, 30, 40, and 50 years) and distinct spatial layers (upper, middle and bottom), as well as explored the correlations between these factors. PM aging drives microbial community simplification characterized by declining α-diversity yet increasing ASV richness, forming a "high richness/low evenness" succession pattern that favors functional specialists (Caproiciproducens, Aminobacterium). Vertical stratification creates distinct microenvironments: anaerobic lower layers accumulate Ammonium nitrogen (NH₄⁺-N) and available phosphorus (AvP > 150 mg/100 g), enriching Methanobacterium and Priceomyces for hexanoate precursors; oxygen-transition middle layers support peak prokaryotic diversity and Lactobacillus-mediated niche facilitation for Caproicibacterium; aerobic upper layers enrich Firmicutes for short-chain fatty acid synthesis. Cross-regional conservation of patterns (e.g., depth-dependent AvP zonation) contrasts with regionally adaptive Lactobacillus distribution. Critically, microbial consortia interactions govern flavor outcomes, Hydrogenispora/Methanobacterium syntrophy promotes hexanoate generation, while dynamic succession from Lactobacillus (young pits) to hexanoic acid bacteria (aged pits) determines metabolic balance. This study establishes Mortierellomycota as a biomarker for pit age (≤ 30 years) and proposes targeted maintenance of anaerobic/AvP thresholds and oxygen-mediated community control to optimize flavor compound synthesis in Baijiu fermentation ecosystems. Yet, the operational viability of these insights necessitates further validation.