Biofilm overproduction enhances gastrointestinal stress tolerance and intestinal fitness in Bacillus subtilis

Microorganisms with health-promoting potential often experience substantial losses in viability and function due to stresses encountered during manufacturing and gastrointestinal transit. In this study, we investigate whether biofilm can be leveraged to enhance microbial resilience and functional performance. Using Bacillus subtilis as a model biofilm-forming bacterium, we examined strains with defined biofilm phenotypes: a biofilm-deficient mutant (tasA eps), a biofilm-overproducing mutant (sinR), and an isogenic wild-type control. These strains were evaluated across multiple functional benchmarks, including survival in simulated gastric and bile juices, thermotolerance, and intestinal bacterial colonization in the Caenorhabditis elegans model. Commercially available strains Lactobacillus rhamnosus GG and Saccharomyces boulardii were included as reference comparators. The biofilm-overproducing B. subtilis sinR strain demonstrated markedly enhanced survival under simulated gastrointestinal conditions and showed increased colonization within the C. elegans intestine. In contrast, the biofilm-deficient tasA eps mutant exhibited severe sensitivity to gastric stress and reduced the intestinal bacterial load. Furthermore, we demonstrate that cell-free B. subtilis biofilm can function as an effective bioencapsulation matrix. When used to encapsulate multiple probiotic strains, the biofilm matrix significantly improved their survival under acidic gastric conditions by neutralizing the environmental pH, indicating its broad potential for probiotic formulations and targeted gastrointestinal delivery. Overall, biofilms are traditionally studied for their roles in infection and antimicrobial resistance; however, their protective and adaptive traits may be repurposed for beneficial use. As an example of this concept, our findings show that B. subtilis biofilms enhance multiple functional and technological traits and highlight biofilm-based strategies as a promising platform for improving beneficial microbial robustness and the delivery of live biotherapeutics.