Product Analysis of Operating an Ethanol/O-2 Biofuel Cell Shows the Synergy between Enzymes within an Enzymatic Cascade

We report the preparation of mono and bi-enzymatic bioanodes immobilizing dehydrogenase enzymes at the surface of a carbon support aiming to follow the 2 and 4 electron transfer of ethanol oxidation by HPLC. Enzymatic immobilization of ADH and A1DH enzymes at the bioanodes was performed using Nafion membranes modified with tetrabutylammonium salt. The monoenzymatic film with immobilized alcohol dehydrogenase (ADH) provided, at pH 8.50, power densities of 29.5 +/- 3.4 mu W cm(-2); however, addition of the second enzyme (A1DH) aiming to transfer 4 electrons furnished values as high as 186.0 +/- 19.4 mu W cm(-2). These results indicate that as the product of the first oxidation is consumed by the second enzyme it inhibits the first enzyme less thus, furnishing better performance for the bienzymatic electrodes. Long-term electrolysis (12 hours) of ethanol followed by HPLC-UV/RID system confirms our electrochemical finding demonstrating formation of 2 and 4 electrons products, e.g., acetaldehyde (6.6 mmol L-1) and acetate (0.72 mmol L-1) when a two dehydrogenase enzyme system was employed. The faradaic efficiency was above 60% for the bi-enzymatic system. We demonstrate that simultaneous product identification and mass balance analysis is an important tool to follow the synergy between the enzymes within the enzymatic cascade. (C) The Author(s) 2018. Published by ECS. (AU)