Microenvironmental Gradients Drive Spatial Stratification of Saccharifying Microbial Communities and Enzyme Activity in Strong-Flavor Daqu Fermentation

Daqu, a representative solid-state fermentation product, produces saccharifying enzymes to degrade sorghum starch into fermentable sugars for ethanol synthesis. Spatial heterogeneity in Daqu drives community assembly. However, its regulatory role in enzyme-driven saccharification remains unclear. By integrating metagenomics and PacBio full-length sequencing, this study investigated how microenvironmental gradients across distinct Daqu layers (QP (surface layer), HQ (middle layer), QX (center layer)) shape saccharifying microbiota and activity. Saccharifying activity exhibited a declining surface-to-center gradient (e.g., QP: 870.9 ± 21.2 U/mL > HQ: 631.2 ± 16.4 U/mL > QX: 296.5 ± 16.1 U/mL on day 30, p < 0.05), paralleled by divergence in microenvironments. Metagenomics identified α-amylase and α-glucosidase as key saccharifying enzymes, primarily encoded by fungi; their abundance was inhibited by heat and humidity, yet promoted by acidity. Enzymatic validation confirmed higher saccharifying activity in QP and HQ core microbes (e.g., Lichtheimia ramosa: 43.16 ± 1.97 U/mL) than in QX (e.g., Paecilomyces variotii: 14.27 ± 1.25 U/mL). Network analysis revealed Lactobacillaceae are closely linked with saccharifying communities. This study establishes microenvironmental gradients as critical regulators of spatial saccharification in Daqu, informing strategies to optimize microbial consortia for baijiu production.