Multi-in situ Electrochemical Impedance Analysis of Biofuel Cell Performance

Electrochemical impedance spectroscopy (EIS) provides valuable insight into the interfacial kinetics and mass-transport behavior of an electrochemical system. However, conventional steady-state EIS insufficiently captures transient processes in enzymatic biofuel cells (EBFCs), in which electrode/electrolyte interactions evolve dynamically. In this study, we demonstrate an multi-in-situ impedance method applicable to glucose/O₂ EBFCs that employ MgO-templated carbon electrodes. Chronopotentiometry revealed that lower current densities lead to higher glucose utilization efficiencies, which reflects the balance between the rates of substrate diffusion and surface reactions. In-situ impedance analysis further differentiated electrode-specific degradation in which the cathode exhibited progressively increasing charge-transfer resistance attributable to enzyme and mediator leaching, whereas the anode displayed non-monotonic resistance changes linked to overpotential-driven kinetics. Equivalent circuit modeling confirmed that the cathodic overpotential is responsible for accelerating charge-transfer processes, leading to smaller semicircular features in the Nyquist plot over time. These results highlight the utility of multi-impedance measurements in identifying the performance-limiting factors in an EBFC under operational conditions. This approach provides mechanistic insight into enzyme stability, mediator retention, and substrate transport, and serves as a diagnostic tool for the rational design of next-generation bioelectrochemical energy devices.