Tracing Protons within Electrochemically Active Biofilms via Real-Time pH Mapping

Accurately characterizing the proton's spatiotemporal distribution is critical for elucidating proton/electron generation and transfer mechanisms in electroactive biofilms (EABs). This study employed ratiometric fluorescence sensing for nondestructive, real-time pH mapping in current-producing EABs. The distribution of protons in EABs is determined by both their generation, which is electron donor-dependent, and their transfer, mediated by concentration gradients and buffering effects. Real-time pH mapping evidenced that under low organic conditions (≤0.4 g/L NaAc) in a PBS-free system, proton diffusion driven solely by concentration gradients prevented internal acidification (pH ≥ 6). However, elevated organics (0.8 g/L NaAc) triggered excessive proton accumulation exceeding transfer capacity, resulting in pronounced acidification (pH < 6) and subsequent electroactivity suppression. It also showed that while employing PBS maintained a neutral pH (∼7) for sustained current generation, it concurrently diminished the intrinsic proton concentration gradient, impairing proton diffusion efficiency. Finally, the electron flux was further derived in two ways: converted from the stoichiometric relationship with proton flux (J_{e, cal}) and calculated from the measured current (J_{e, test}) by applying proton flux, and a better linear fitting correlation was achieved when there was no PBS. This evidences the possibility to trace electron transfer through monitoring protons.