Active pH regulation facilitates biofilm development in minimally buffered environments

Biofilms provide individual bacteria many advantages, yet dense cellular proliferation can also create intrinsic metabolic challenges including excessive acidification. Because such pH stress is commonly masked in buffered laboratory media, it remains unclear how biofilms cope with minimally buffered natural environments. Here, we report Bacillus subtilis biofilms overcome this intrinsic metabolic challenge through an active pH regulation mechanism. Specifically, we find that biofilms can modulate their extracellular pH to the preferred neutrophile range, even when starting from acidic and alkaline initial conditions, while planktonic cells cannot. We associate this behavior with dynamic interplay between acetate and acetoin biosynthesis and show that this mechanism is required to buffer against biofilm acidification. Furthermore, we find that buffering-deficient biofilms exhibit dysregulated biofilm development when grown in minimally buffered conditions. Our findings reveal an active pH regulation mechanism that could be targeted to control biofilm growth.