Abstract
Biofilms formed by Bacillus subtilis confer protection against environmental stressors through extracellular polysaccharides (EPS) and sporulation. This study investigates the roles of these biofilm components in resistance to hydrogen peroxide, a common reactive oxygen species source and disinfectant. Using wild-type and mutant strains deficient in EPS or sporulation, biofilm colonies were cultivated at various maturation stages and exposed to hydrogen peroxide. EPS-deficient biofilms exhibited reduced resilience, particularly in early stages, highlighting the structural and protective importance of the matrix. Mature biofilms demonstrated additional protective mechanisms, potentially involving TasA protein fibers and/or the biofilm surface layer (BslA). In contrast, sporulation showed limited contribution to hydrogen peroxide resistance, as survival was primarily matrix-dependent. These findings underscore the necessity of targeting EPS and other matrix components in anti-biofilm strategies, suggesting that hydrogen peroxide-based disinfection could be enhanced by combining it with complementary sporicidal treatments. This study advances our understanding of biofilm resilience, contributing to the development of more effective sterilization protocols.