How Do Environmental Metal Ions Impact Biofilm Mechanics?

Bacterial biofilms are soft, viscoelastic materials consisting of cells embedded in a self-produced extracellular polymeric substance (EPS). Their mechanical properties play a central role in their persistence and response to environmental challenges; however, quantitative mechanical testing of intact biofilms is challenging due to their fragility. Here, using a custom-built mechanical tester, The Uniaxial Tensile Tester for UltraThin films (TUTTUT), we measure the complete uniaxial stress-strain relationship of Bacillus subtilis pellicle biofilms following incubation with selective metal ions. Exposure to Fe³⁺, Fe²⁺, and Cu²⁺ significantly increased elastic modulus and yield strength while decreasing failure strain. Meanwhile, treatment with Ca²⁺, Cu⁺, and Na⁺ produced only minor, statistically insignificant changes under the same conditions. Fe³⁺-induced stiffening was partially reversed upon the introduction of the chelating agent EDTA, and pH changes alone cause negligible mechanical differences, indicating Fe³⁺-mediated ionic crosslinking of the EPS. These results reveal how environmental cues can regulate the soft matter mechanics of bacterial communities, providing new insight into biofilm stability and failure.