Progression Toward Terminal Oxidation of Starch via Hybrid Organic–Enzymatic Bioanode for Renewable Carbohydrate Conversion

Abstract

The performance of starch-based biofuel cells is fundamentally limited by inefficient utilization of the fuel's inherent energy density. Sequential oxidation of carbohydrate fuels, such as glucose, the monomeric unit of starch, using minimal catalyst systems has therefore emerged as a promising strategy for improving biofuel cell efficiency. Here, we extend a previously reported three-catalyst, organic-enzymatic glucose oxidation cascade to starch by incorporating αamylase (α-AMY) and amyloglucosidase (AMG), enabling terminal oxidation of starch-derived glucose equivalents to carbon dioxide (CO 2 ) under mild electrochemical conditions. To our knowledge, this work represents the first demonstration of a bioanode capable of terminal starch oxidation using only four enzymes and a single oxidation mediator. Colorimetric assays confirm that α-AMY and AMG efficiently hydrolyze soluble starch, achieving 92.6 ± 8.7% conversion of glucose equivalents within one hour at 60 °C. Subsequent electrochemical oxidation through a modified version of our established cascade employing 4-amino-TEMPO, 2-keto-3deoxygluconate aldolase from Picrophilus torridus (ptKDGA), and oxalate decarboxylase from Bacillus subtilis (OxDC) produced current densities up to 4.21 ± 0.06 A m⁻², corresponding to 74% of glucose current density under identical conditions, with the increase in current density after starch hydrolysis found to be statistically significant at 95% confidence interval.Green Foundation1. Electrochemical oxidation uses electrons as clean reagents, replacing stoichiometric chemical oxidants, reducing waste, and allowing operation under mild aqueous conditions. 2. Terminal cascade oxidation maximizes fuel utilization by accessing the full electron content of carbohydrates, enabling efficient conversion of starch-an abundant, inexpensive biomass-derived feedstock-into energy and CO 2 . 3. This modular hybrid organic-enzymatic system provides sustainability-oriented optimization (e.g., identification of rate-influencing steps, substitution of individual catalysts with more active or robust alternatives, and expansion to additional renewable carbohydrate fuels).