From fungi to lactic acid bacteria: Dominant microorganisms govern stage-specific esters biosynthesis in pit microecology of strong-flavor baijiu.

Esters are vital flavor compounds in strong-flavor baijiu (SFB). However, their biosynthetic mechanisms during pit fermentation are not fully understood. This study employed integrated metagenomic and metabolomic analyses to investigate stage-specific microecological dynamics governing ester biosynthesis and identified three distinct fermentation stages. Stage P1 (days 0-15) featured initial ester accumulation driven by fungal dominance (e.g., Saccharomyces, Aspergillus, and Rhizopus) and high α-diversity. Elevated esterase (EST) genes mainly involved to the ethyl acetate and ethyl butyrate synthesis. Stage P2 (15-35 d) exhibited minimal changes in esters content (except ethyl lactate increase linked to 42 EST genes). During this stage, acidogenic microorganisms, primarily Acetilactobacillus (> 64 % relative abundance), Lactobacillus, and Clostridium, proliferated and co-aggregated with methanogens, including Methanosarcina and Methanobacterium. In Stage P3 (35-117 d), Lactobacillus-Acetilactobacillus consortium (> 74 %) exhibited rapid ester accumulation alongside acids and alcohols increases, with high EST abundance for ethyl caproate and ethyl butyrate synthesis. Dominant microorganisms govern the ester synthesis, with catalytic dominance shifting from fungal (Aspergillus, Saccharomyces, etc.) to lactic acid bacteria (Lactobacillus, Acetilactobacillus, etc.) systems across stages. Five physicochemical properties exerted the stage-dependent control: pH and reducing sugars shaped the microbial structure in P1 (R2 = 0.40, 0.46); temperature and ethanol drove community simplification in P2 (R2 = 0.35, 0.64); Acidity and ethanol regulated functional dominance in P3 (R2 = 0.85, 0.64). PLS-SEM confirmed direct inhibition/promotion effects on α/β-diversity. This microecology-guided framework provided references for manipulation of ester profiles via phase-specific environmental control, offering a scientific basis for improving the quality of SFB flavor.