A chemically defined medium to support the growth of food-relevant Bacillus species

Abstract Aims Most chemically defined media for Bacillus are developed with a focus on an individual species. To broaden the applicability, this study aimed to formulate a chemically defined medium that supports the growth of multiple food-relevant Bacillus species. Specifically, it was the aim to support growth of both food fermentation strains from the Bacillus subtilis clade as well as pathogenic strains from the Bacillus cereus clade. Methods and Results We developed a new chemically defined medium, named Pafoba, using thirteen Bacillus strains: two from the Bacillus cereus clade and eleven strains from the Bacillus subtilis clade, representing seven species. Medium optimisation involved substituting ammonium chloride and sodium chloride with ammonium sulphate and trisodium citrate, enriching glucose, iron, and phosphate concentrations, and applying nutrientomission assays to identify growth requirements. All strains exhibited growth on Pafoba. Ten strains reached a comparable or higher maximum optical density (OD600) on Pafoba medium compared to Brain Heart Infusion broth. Strain-specific nutrient requirements were identified, including a biotin dependency for Bacillus subtilis strain PRO64, and essential amino acid requirements in Bacillus mycoides and Bacillus cereus strains. Conclusions The Pafoba medium supports consistent growth across diverse Bacillus species, making it suitable for both fundamental studies and practical applications such as detection and isolation of Bacillus spp. in food-related contexts.