Experimental and Theoretical Insights into Bienzymatic Cascade for Mediatorless Bioelectrochemical Ethanol Oxidation with Alcohol and Aldehyde Dehydrogenases

The efficient utilization of biomass fuels is a critical component of a sustainable energy economy. Via respiration, acetic acid bacteria can oxidize biomass ethanol into acetic acid using membrane-bound alcohol and aldehyde dehydrogenases (ADH and AlDH, respectively). Focusing on the ability of these enzymes to interact directly and electrically with electrode materials, we constructed a mediatorless bioanode for ethanol oxidation based on a direct electron transfer (DET)-type bienzymatic cascade by ADH and AlDH. The three-dimensional structural data of ADH and AlDH elucidated by cryo-electron microscopy were valuable for effectively designing electrode platforms with multi-walled carbon nanotubes (MWCNTs) and pyrene (Py) derivatives. DET-type bioelectrocatalysis by ADH and AlDH was improved by using 1-pyrene carboxylic acid-functionalized MWCNTs. The catalytic current densities for bienzymatic ethanol oxidation were recorded at the bioanodes modified by various ADH/AlDH ratios. The reaction model was constructed by focusing on the competitive adsorption of two enzymes on the electrode surface and the collection efficiency of the intermediately produced acetaldehyde. The power output of an ethanol/air biofuel cell using the bienzymatic bioanode reached 0.48 ± 0.01 mW cm–2, which is the highest value reported for ethanol biofuel cells. In addition, the Faraday efficiency of acetate production by the cell reached 100 ± 4%. This study will lead to efficient conversion of biomass fuels based on a multi-catalytic cascade system.