Age-dependent microbial succession and functional differentiation in pit mud and Zaopei of strong-flavor Baijiu

The roles of pit mud (PM) and Zaopei (ZP) microorganisms in Baijiu flavor formation are well-recognized; however, their functional division across pit ages remains unclear. We investigated the succession of physicochemical properties, microbial communities, functional pathways, and flavor compounds in PM and ZP during strong-flavor Baijiu fermentation. With increasing pit ages, PM exhibited increased pH, moisture, organic matter, available phosphorus, and dissolved organic carbon content, while ZP showed higher acidity, starch, reducing sugar, and ethanol accumulation. Microbial diversity and stability increased with increasing pit ages, showing functional division. In younger pits, Lactobacillus (Firmicutes) dominated ZP, while non-adaptive species were gradually eliminated from PM. In older pits, PM enriched fatty acid-producing bacteria and methanogenic archaea supporting hexanoate biosynthesis, whereas Lactobacillaceae in ZP favored alcohol and ester formation. Functional prediction confirmed that PM specialized in cellulose degradation, reverse β-oxidation, and methane metabolism, while ZP emphasized the Embden-Meyerhof-Parnas pathway, lactate and alcohol metabolism, and fatty acid biosynthesis. Flavor analysis revealed progressive accumulation of esters, acids, and alcohols in ZP, particularly ethyl hexanoate, and hexanoate. These findings demonstrated that PM and ZP exhibited dynamic and complementary microbial functions across different pit ages, providing a theoretical basis for understanding their cooperative roles in Baijiu fermentation. • Pit age differences in Zaopei and pit mud shape microbial functions and pathways. • Combined metagenomic and flavor analyses reveal coordinated microbial activities. • Interactions between PM and ZP jointly enhance Baijiu flavor formation. • Fatty acids accumulated in PM combined with alcohols to form esters in ZP. • Microbial function and characteristic flavor were enhanced with increasing pit ages.