Temperature-mediated shift from competitive to facilitative interactions between lactic acid bacteria and bacillus species in daqu fermentation: Insights from metagenomics, dual RNA-seq, and coculture analysis

Abstract

Daqu, a pivotal starter that defines the flavor profile and quality of Baijiu, undergoes dynamic temperature changes during its production, significantly influencing the microbial community structure and function. Although the importance of fermentation temperature in shaping microbial biodiversity is well-recognized, its impact on microbial interaction dynamics and the underlying mechanisms remains poorly understood. This study integrates metagenomics, dual RNA-seq, and coculture experiments to elucidate temperature-dependent microbial interactions during Daqu fermentation. Metagenomic analysis revealed that lactic acid bacteria (LAB) and Bacillus are dominant genera with distinct thermal preferences that nevertheless coexist throughout the fermentation process. Elevated temperature stress was found to enhance positive microbial interactions within the Daqu ecosystem. Dual RNA-seq analysis uncovered temperature-responsive gene expression patterns associated with oxidative stress, metabolic capacity, and environmental information processing in representative LAB and Bacillus strains. Guided by these multi-omics findings, co-culture assays demonstrated a temperature-dependent shift in microbial interaction modes. At 30 °C, Lactococcus lactis secretes lactic acid that inhibits the growth of Bacillus subtilis, whereas at 50 °C, B. subtilis alleviates oxidative stress in L. lactis by producing cobalamin, thereby enabling short-term rescue and sustained coexistence over serial transfers. These findings provide critical insights into the temperature-driven modulation of microbial interactions, enhancing the precision and manageability of the Daqu fermentation process.