Packing Density Governs Tobacco Quality Through Microbial Community Assembly and Metabolic Reprogramming

Packing density regulates the microenvironment of tobacco (Nicotiana tabacum L.) fermentation and may thereby influence microbial activity and product quality. However, its effects on microbial community assembly and quality formation remain poorly understood. This study aimed to clarify how packing density affects flue-cured tobacco quality by shaping microbial communities, functional potential, and ecological interactions. Here, we investigated the effects of three packing densities (60%, 70%, and 80%) on chemical components, aroma compounds, microbial community structure, functional potential, co-occurrence networks, and assembly mechanisms of flue-cured tobacco (cv. Piaohe No. 2) after 10 days of fermentation. Moderate density (70%) achieved the most balanced chemical profile, with appropriate nicotine retention, potassium/chlorine ratio, and sugar/nicotine balance. T70 also exhibited the highest levels of total esters, total ketones, and β-ionone, key contributors to fruity, floral, and woody aromas. Microbial analysis revealed that T70 supported the highest diversity and was characterized by the enrichment of aroma-related bacterial taxa, including Bacillus and lactic acid bacteria, as well as the fungal genus Pichia. In contrast, T60 favored aerobic nicotine degraders, whereas T80 selected for obligate anaerobes associated with off-odor production. Functional predictions and network analysis showed that T70 upregulated fatty acid and carotenoid biosynthesis pathways and exhibited the highest modularity, indicating a compartmentalized, functionally complementary community. Neutral model fitting revealed increasing stochasticity with density, with T70 displaying a mixed assembly regime. Collectively, our findings show that packing density influences tobacco quality by regulating microbial community composition, functional potential, network interactions, and assembly processes. These results provide a scientific basis for optimizing packing density in tobacco processing.