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

Abstract

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.

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound d-Fructose Dehydrogenase with Structural Bioelectrochemistry

“…UQ 10 was quantified as 1% of the molar amount of rFDH, indicating that UQ 10 bound weakly and partially to the enzymes. This is consistent with the relatively low resolution of the densities, and similar results have been confirmed in the case of ADH and AlDH from G. oxydans . Furthermore, we confirmed a clear cavity around the FAD catalytic site in the top view of the structure (Figure C).…”

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-bound D-Fructose Dehydrogenase with Structural Bioelectrochemistry

“…This is consistent with the relatively low resolution of the densities, and similar results have been confirmed in the case of ADH and AlDH from G. oxydans. 63 Furthermore, we confirmed a clear cavity around the FAD catalytic site in the top view of the structure (Figure 1C).…”

Enzyme Cascade Electrode Reactions with Nanomaterials and Their Applicability towards Biosensor and Biofuel Cells

“…Later, Adachi et al demonstrated that by immobilizing the enzymes on MWCNTs functionalized with 1-pyrene carboxylic acid, ADH/ALDH bienzymatic bioanode can be produced. Recently, high electricity was generated by an ethanol/air BFC using this bioanode, which showed a maximum current density of 2.69 ± 0.09 mA cm −2 and a maximum power output of 0.48 ± 0.01 mW cm −2 at 0.366 ± 0.004 V [28], as shown in Figure 1d. Chansaenpak et al fabricated a BFC using a graphite electrode, which was modified with MWCNTs, as the cathode [35].…”

“…Similarly, the hydrogenase and reductase-loaded MWCNT for H 2 -driven NADH production is illustrated [27] (c), and the role of MWNCT in constructing a biofuel is depicted (d) (reprinted with permission from Ref. [28], Copyright 2023, ACS).…”