Salinity-driven evolution of psychrotolerant Bacillus velezensis S_51 strain reveals metabolic adaptations to osmotic stress and enhanced glycine betaine production

Abstract Soil salinisation limits agricultural productivity by disrupting microbial homeostasis and plant–microbe interactions. Osmoprotectants, including glycine betaine, play a crucial role in protecting microbial cells against osmotic stress by stabilising cellular structures and maintaining osmotic balance. To optimise microbial production of glycine betaine, adaptive laboratory evolution (ALE) was applied to Bacillus velezensis S_51 under increasing sodium chloride (NaCl) concentrations, providing insight into the underlying metabolic and genomic adaptations. After 10 sequential passages (500 generations), the evolved strain exhibited an adaptive shift in growth preference towards higher salinity from 0% to 4% (0.68 M) NaCl, indicating niche specialisation. This adaptation was accompanied by a pronounced increase in osmoprotection, with intracellular glycine betaine levels rising by ~262-fold relative to the ancestral strain, reaching to 882.25 ± 68.58 µM g⁻¹ DM. Metabolic profiling revealed a non-uniform reorganisation of substrate utilisation, with differentiation between ancestral (P1) and evolved (P10) strains largely driven by a limited subset of compounds (~62% contribution of key substrates). The evolved strain showed a ~ 4–5-fold increase in the number of effectively utilised carbon (C) and nitrogen (N) sources, with selective enhancement of specific amino acids (e.g., L-serine, L-aspartic acid) and reduced utilisation of several carbohydrates and hexose phosphates, indicating redistribution of substrate utilisation profiles under osmotic constraints. Functional trait analysis showed increased biofilm formation and elevated indole-3-acetic acid (IAA) production (from 0.1 to 0.13 µg mL⁻¹), while siderophore production remained stable. Genomic analysis identified mutations in key regulatory genes, including kapB , spo0A , and comP , suggesting an association between phenotypic shifts and regulatory network reconfiguration. Together, these results demonstrate that ALE drives coordinated metabolic, physiological, and regulatory adaptation, enhancing microbial fitness under saline conditions and supporting its application in saline soil biotechnologies.