To elucidate the influence of distinct workshop brewing microecologies on physicochemical factors, microbial community structures, and flavor substance compositions during the stacking and pit fermentation of Jiang-flavor Baijiu, this study investigated the environments and fermented grains across three fermentation rounds in a new workshop (operational time < 3 years) and an old workshop (operational time > 20 years). High-throughput sequencing, high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) were employed, combined with SourceTracker and multivariate statistical analysis. Results revealed that for physicochemical characteristics, the new workshop exhibited a more drastic consumption of reducing sugars and starch in the early stage, with higher levels of total acidity and lactic acid throughout the fermentation cycle compared to the old workshop. The acidity of the middle-layer pit-out grains was 4.17 mmol/10 g in the new workshop and 3.95 mmol/10 g in the old workshop, while the old workshop featured a more gradual organic acid accumulation process and a higher enrichment of acetic acid. Microbially, the old workshop pit mud showed a higher bacterial richness (OTUs = 2 400.00±92.00) in the early phase. During the mid-stacking phase, Bacillus (35%) dominated the bacterial community in the old workshop, whereas Lactobacillus (71%) occupied an absolute dominance in the new workshop. Meanwhile, the relative abundance of Pichia in the old workshop fermented grains remained consistently stable above 70% throughout the cycle, whereas the new workshop environmental fungi showed higher richness on the surfaces of operating tools and the upper layer of empty pits. SourceTracker analysis indicated that the factory ground was the primary bacterial contributor (>50%) for both workshops during the open stacking stage; however, upon entering the sealed pit fermentation stage, the core bacteria in the old workshop mainly originated from the middle layer of empty pits, and its dominant fungi stably sourced from spatial media such as object surfaces and indoor air. In contrast, the microbial community in the new workshop exhibited a strong dependence on specific operating tools and non-pit environments, displaying spatial heterogeneity. Redundancy analysis (RDA) further confirmed that the fungal community in the new workshop was intensively driven by moisture, acidity, and lactic acid, whereas the community succession in the old workshop converged toward a homeostatic state. Driven by these microecological variations, the abundance of flavor substances in the old workshop was higher overall than that in the new workshop, where the total ester content continuously increased from 48.08 μg/g to 385.55 μg/g at the pit-out stage, whereas it peaked at 146.52 μg/g during the mid-fermentation phase and then decreased in the new workshop. Furthermore, the final contents of total alcohols and volatile fatty acids in the new workshop were lower than those in the old workshop. This study clarifies the distinct characteristics of the brewing microecological structures, source tracking functions, and flavor metabolism between the new and old workshops, providing theoretical references for the microecological replication of traditional workshops and the precise regulation of newly established workshops.
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