Regional physicochemical filtering shapes microbiome-metabolome coupling at the Huangshui interface during...

Huangshui (HS), the liquid fraction that accumulates at the bottom of fermentation pits, serves as a key ecological interface linking microbial diversity and metabolism between fermented grains and pit mud during fermentation. Although HS is increasingly recognized as a reservoir of microorganisms and flavor precursors, how regional heterogeneity shapes its microbiome-metabolome coupling remains unclear.

HS samples derived from nine representative Nongxiangxing Baijiu (NXB) production regions were compared using physicochemical characterization, amplicon sequencing, volatile metabolomics, and ecological association analysis.

The results showed marked regional differences in acidity, nitrogen availability, mineral nutrient content, and organic acid composition, indicating distinct fermentation microenvironments. Lactic acid dominated the acid pool in all samples, whereas short- and medium-chain fatty acids varied substantially among regions, suggesting differences in carbon flux allocation and chain elongation activity. Bacterial communities were dominated by Lactobacillus, methanogenic archaea, and Caproiciproducens, whereas fungal communities were enriched with fermentative yeasts (Pichia, Saccharomyces, and Kazachstania). A total of 162 volatile organic compounds were identified, with esters as the predominant aroma class, showing clear regional differentiation. Furthermore, integrated correlation and network analyses indicated that regional physicochemical factors acted as ecological filters, shaping microbial guild assembly and volatile metabolite patterns.

Lactobacillus emerged as a central taxon associated with acid-ester balance, whereas methanogenic and chain-elongating taxa were linked to carbon redistribution and medium-chain fatty acid formation. Further, these findings support the revised view of HS as an active metabolic interface, highlighting its potential for origin discrimination and precise fermentation control in NXB production.