Bacterial biofilms actively bolster mechanobiological resilience via matrix production and sporulation

Bacillus subtilis bacteria form multicellular biofilms whose extracellular matrix and developmental programs govern their mechanical resilience and survival under stress. Using oscillatory rheology, hydration assays, and viability measurements, I probed how extracellular polymers and sporulation shape biofilm mechanics and water retention over time. At 24 hours post-inoculation, poly-γ-glutamic acid (PGA) enhanced biofilm hydration and critical strain, whereas extracellular polysaccharides (EPS) slightly reduced hydration without substantially affecting mechanics. In contrast, sporulation decreased water content and softened the biofilm, with spore-deficient mutants exhibiting unusually high storage (G′) and loss moduli (G′′). Interestingly, mechanical phenotypes across strains converged by 72 hours, with G′ and G′′ values approaching those of early unwrapped wild-type biofilms, suggesting a shared endpoint in network remodeling. Using colony-forming unit (CFU) counts, I verified that PGA and EPS have no significant effect on the viability and spore percentages of biofilms at 24 and 72 hours. However, sporulation conferred a striking advantage in viability at 72 hours, despite decreasing G′ and G′′ in younger biofilms. These findings highlight dynamic and stage-dependent roles of PGA, EPS, and sporulation in tuning the biofilm’s physical properties, balancing hydration, mechanical stability, and long-term survival. Together, these results suggest that biofilms employ distinct strategies at different stages of growth—matrix polymers initially buffering against deformation and desiccation, and sporulation later ensuring population persistence. More broadly, this work links biofilm mechanics to survival strategies, pointing toward general design principles for resilient microbial communities and potential avenues for disrupting biofilm robustness in applied settings.