Sporulation kinetics of Bacillus subtilis under pH shift conditions: impact on spore properties and gene expression

ABSTRACT Bacterial spores are highly resistant to environmental stresses, which limits the efficiency of decontamination processes and increases the risk of spoilage and foodborne illness. While pH is a key factor influencing sporulation efficiency and spore properties, current predictive models are typically based on static conditions, limiting their relevance to food processing environments where pH can fluctuate dynamically. In this study, we investigated the effect of a single-step pH shift on Bacillus subtilis sporulation using an integrated modeling experimental approach. Cultures were grown in bioreactors at pH 7.0 and subjected to acidification shifts to within the growth-permissive range at pH 5.5 (pH 7.0–5.5) or below the growth limit at pH 4.0 (pH 7.0–4.0). A growth-sporulation model was applied to describe kinetics, and spores were evaluated for heat resistance and germination. Gene expression analysis was performed to monitor transcriptional responses related to sporulation, stress adaptation, heat resistance, and germination. The results showed that the shift to pH 5.5 led to delayed but successful sporulation, with prolonged expression of sporulation genes. By contrast, a shift to pH 4.0 completely blocked sporulation, with key genes becoming transcriptionally silent. While spores obtained at pH 7.0 or pH 7.0–5.5 conditions showed similar heat resistance, those from the pH 7.0–5.5 condition germinated more efficiently in L-alanine suspension. By combining gene expression analysis with sporulation kinetics, this study contributes to a better understanding of the physiological responses underlying model parameters. The approach provides a preliminary basis for future modeling of more complex environmental conditions, including multi-step pH fluctuations relevant to food processing. IMPORTANCE Sporulation in Bacillus subtilis plays a critical role in food spoilage, as spores are highly resistant to environmental stresses. Environmental factors, particularly pH, significantly affect spore properties such as heat resistance and germination. While most studies focus on static pH, real-world food environments often experience fluctuating pH due to processes such as fermentation, microbial activity, and treatment processes. This gap in knowledge limits predictive microbiology models aimed at managing spore-forming bacteria in food. This study addresses this by examining how pH shifts influence B . subtilis sporulation and alter spore properties. By combining experimental data with gene expression analysis of key genes. This research provides a baseline for predicting sporulation under more realistic and dynamic environmental conditions encountered in food processing.