Design and characterization of redox enzyme electrodes: New perspectives on established techniques with application to an extremeophilic hydrogenase

Biofuel cells promise power generation with low cost and almost unlimited biocatalyst supply. This paper reports new perspectives on the characterization of immobilized thermostable (Pyrococcus furiosus) hydrogenase electrodes in working configuration, using a combination of well defined electrochemical and spectrophotometric techniques. Electrodes based on porous pyrolytic carbon paper (PCP) and packed graphite columns (PGC) were fabricated for hydrogenase immobilization, which used a direct (hydrophilic adsorption) technique. Potentiostatic dc polarization, dynamic potentiometry, and electrochemical impedance spectroscopy (EIS) were combined with spectrophotometric detection of enzyme activity in order to characterize the electrodes and differentiate the relative contributions from enzyme loading, charge transfer, and mass transport to the limiting current density, which is a critical consideration for electrodes intended for fuel cell and bioelectrocatalytic application. Dynamic potentiometry proved useful as a rapid screening and characterization procedure for both blank electrodes and the presence of bound hydrogenase post-immobilization. Combining current density data determined via dc polarization (hydrogen oxidation currents of 30 μA cm-2 for PCP electrodes and 90-120 μA cm-2 PGC electrodes at 75°C) with hydrogen mass transport limits determined via modeling and platinum electrode polarization (480 μA cm-2 at 75°C), allowed determination that insufficient active enzyme was present to reach system mass transport limits to hydrogen supply for all electrodes. Additionally, spectrophotometric enzyme determination on the PCP electrode (0.034 units cm-2) was used to derive a theoretical maximum in hydrogen oxidation current (110 μA cm -2, assuming 100% enzyme/support charge transfer efficiency), which when combined with current density data (30 μA cm-2), allowed determination that actual charge transfer efficiency per unit total bound enzyme was significant (28%). The EIS scans on immobilized PGC type electrodes suggest that mass transport might attribute the most to cell impedance at the low frequency regime. These combined results indicated that low bulk enzyme loading was the limiting factor to current density for thermostable hydrogenase electrodes fabricated for the study.