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bioRxiv · 10.1101/2025.07.14.664712

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

Canoy, T. S.·Wiedenbein, E. S.·McPhillips, C.·Jespersen, L.·Roder, H. L.·Nielsen, D. S.
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Abstract

The Bacillus genus contains many members with food significance, including the food-grade Bacillus subtilis clade often used in fermentations and the pathogenic Bacillus cereus clade. Chemically defined media for Bacillus species are crucial tools to allow detailed investigations of the influence of specific nutrients on growth and also improve reproducibility and consistency of experiments. Previous studies have focused on the development of defined media for single species, while the aim of this study was to develop a chemically defined medium that supports the growth of multiple food relevant Bacillus species. The new medium, Pafoba, was tested using two pathogenic strains of the Bacillus cereus clade and eleven strains of the Bacillus subtilis clade representing seven different clade members. All thirteen Bacillus strains were able to grow on Pafoba, of which ten displayed a similar or higher maximum OD600 on Pafoba medium compared to rich medium (Brain Heart Infusion broth). Detailed analysis revealed a biotin requirement for Bacillus subtilis strain PRO64, and the necessity of including essential amino acids for Bacillus weihenstephanensis and Bacillus cereus strains. In conclusion, the chemically defined Pafoba medium provides a controlled and reproducible growth environment for fundamental studies and is suitable for detection and enumeration of a broad range of Bacillus spp. related to food processing and safety. ImportanceBacillus species are important in both food fermentation and food safety. While members of the Bacillus subtilis clade are used in the production of fermented foods, those in the Bacillus cereus group are associated with foodborne illness. This study presents a chemically defined medium that supports the growth of multiple food-relevant Bacillus species, enabling precise control over nutrient composition. Knowledge of Bacillus cereus metabolism under defined conditions is essential to support efforts in food safety, risk assessment, and the development of targeted intervention strategies. Likewise, an understanding of Bacillus subtilis metabolism under defined conditions is essential to optimize fermentation processes, improve product consistency, and enhance functional food development. By providing a standardized and reproducible growth environment, the medium developed in this study will facilitate research that advances both microbial food safety and the controlled use of beneficial Bacillus strains in food production.

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Canoy, T. S., Wiedenbein, E. S., McPhillips, C., Jespersen, L., Roder, H. L., Nielsen, D. S.. 2025-07-15. A chemically defined medium to support the growth of food-relevant Bacillus species. https://doi.org/10.1101/2025.07.14.664712

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