Physicochemical properties and molecular adaptations of Bacillus velezensis Huang in response to ethanol stress.

Bacillus species are dominant microorganisms in traditional fermented food production and play a key role in shaping the flavor profiles of alcoholic beverages. The notable stress resistance of Bacillus velezensis suggests its potential utility in food fermentation. However, the ethanol tolerance of B. velezensis and its underlying mechanisms remain poorly understood. In this study, B. velezensis Huang, isolated from Huangjiu fermentation, was employed to investigate its survival and adaptive responses under ethanol stress. B. velezensis Huang exhibited high ethanol tolerance, sustaining growth at 6 % ethanol. Ethanol stress significantly inhibited growth during the early phase and disrupted membrane integrity, as evidenced by substantial leakage of intracellular nucleic acids and proteins and altered cell morphology. Under ethanol exposure, the activities of catalase (CAT) and superoxide dismutase (SOD) increased significantly, indicating their role in oxidative stress defense. In addition, metabolite levels-particularly those related to membrane lipids-were markedly reduced after 8 h at 6 % ethanol, suggesting that ethanol exposure altered membrane composition and affected cellular metabolism. Several genes associated with lipid biosynthesis, including PlsX and PlsC, were differentially expressed under ethanol stress, consistent with the observed reduction in membrane lipid content. Notably, supplementation with exogenous L-acetylcarnitine-a metabolite elevated under ethanol stress and known to participate in lipid metabolism-significantly promoted the growth of B. velezensis Huang under stress conditions. These findings demonstrate that lipid biosynthesis pathways are active in the ethanol stress response and are essential for maintaining membrane homeostasis. This study provides insight into the physicochemical adaptations and molecular mechanisms underlying ethanol tolerance in B. velezensis Huang and supports its application in Huangjiu fermentation.