The discovery of oxygen and carbon monoxide tolerant [NiFe] hydrogenases was the first necessary step toward the definition of a novel generation of hydrogen fed biofuel cells. The next important milestone is now to identify and overcome bottlenecks limiting the current densities, hence the power densities. In the present work we report for the first time a comprehensive study of herringbone carbon nanofiber mesoporous films as platforms for enhanced biooxidation of hydrogen. The 3D network allows mediatorless hydrogen oxidation by the membrane-bound hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. We investigate the key physico-chemical parameters that enhance the catalytic efficiency, including surface chemistry and hierarchical porosity of the biohybrid film. We also emphasize that the catalytic current is limited by mass transport inside the mesoporous carbon nanofiber film. Provided hydrogen is supplied inside the carbon film, the combination of the hierarchical porosity of the carbon nanofiber film with the hydrophobicity of the treated carbon material results in very high efficiency of the bioelectrode. By optimization of the whole procedure, current densities as high as 4.5 mA cm(-2) are reached with a turnover frequency of 48 s(-1). This current density is almost 100 times higher than when hydrogenase is simply adsorbed at a bare graphite electrode, and more than 5 times higher than the average of the previous reported current densities at carbon nanotube modified electrodes, suggesting that carbon nanofibers can be efficiently used in future sustainable H2/O2 biofuel cells.
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