Engineering Bacillus amyloliquefaciens with impaired nitrate reduction: Microbiome modulation and nitrogenous-hazard mitigation during cigar tobacco fermentation and in water microcosms

Nitrite is a critical precursor of the carcinogenic tobacco-specific nitrosamines (TSNAs), which pose significant risks to human health. The formation and accumulation of these hazardous compounds are primarily driven by microbially mediated nitrate reduction during the processing of nitrogen-rich organic materials. In this study, an engineered nitrate reductase-deficient strain, Bacillus amyloliquefaciens HZ∆ narG, was constructed to impair nitrate-to-nitrite conversion. In laboratory-controlled culture assays, HZ∆ narG decreased nitrite by 88% and resulted in apparent decreases of 60% and 67% in NNN and NNK, respectively. During cigar tobacco fermentation, compared with the uninoculated control, HZΔ narG treatment reduced nitrite and total TSNAs by 94% and 72%, respectively. Bacterial community analysis revealed that HZΔ narG inoculation increased community diversity and altered community composition. Correlation analysis showed that the relative abundance of Bacillus was negatively associated with nitrite and TSNAs levels. Furthermore, in tobacco-contaminated water microcosms, co-inoculation with HZΔ narG and a culturable tobacco-derived microbial consortium reduced nitrite and total TSNAs by 85% and 30%, respectively, compared with the uninoculated control. Overall, this study demonstrates the potential of targeted microbial inoculation strategies to effectively limit the accumulation of nitrite and TSNAs, providing a promising approach for improving the safety of fermented products.