The bioanode of the biofuel cell is the electrode at which the fuel is utilized by enzymes to produce electrons and protons, which are then utilized by enzymes of biocathodes to reduce O2 to H2O. Alcohol-based enzymatic systems that have been chosen most frequently for the bioanode involve NAD+-dependent alcohol dehydrogenase (ADH), which oxidizes alcohols to aldehydes. This enzyme can be employed with aldehyde dehydrogenase to further oxidize the aldehyde. Lit­erature reports of alcohol biofuel cells are limited to only two alcohol-based enzymatic schemes. They are for methanol and ethanol and are shown in Figure 12.6. Methanol is oxidized to formaldehyde by alcohol dehydrogenase and then the formaldehyde is oxidized to formate by formaldehyde dehydrogenase. The formate is completely oxidized to carbon dioxide by formate dehydrogenase. The ethanol system involves oxidizing ethanol to acetaldehyde by alcohol dehydro­genase and then oxidizing the acetaldehyde to acetate by aldehyde dehydrogenase.

All of these enzymatic systems require NAD+ as a coenzyme/cofactor and the reduced form (NADH) is the hydrogen source at the electrode surface. How­ever, NADH has a high overpotential at most typical electrode surfaces (platinum, carbon, etc.), so an electrocatalyst layer is necessary to decrease the potential and increase the power output. The problem with electrocatalyst layers is that they are not as conductive as carbon or most metals and they add an extra complexity to the system that makes forming high surface area bioanodes difficult. A variety
of electrocatalysts have been used, but at this stage, there is no optimal electro­catalyst. Palmore and coworkers have employed a diaphorase/benzyl viologen system that has shown good thermodynamics properties, but poor lifetimes [13]. Minteer and coworkers have employed methylene green as the electrocatalyst layer due to its optimal electrocatatlytic properties. Poly(methylene green) pre­pared via electropolymerization has been shown to be an electrocatalyst for NADH [23].

The second problem with an NAD+-dependent bioanode is the instability of the NAD+/NADH couple in the membrane. When ethanol is oxidized to acetate, the NAD+ is converted to NADH. It is simple to electrostatically immobilize NAD+ in the bioanodes membrane, but NAD+ has a short lifetime in solution and a limited lifetime in the membrane. NAD+ is only stable in solution for a few hours, but it can be stabilized for up to 45 days in the membrane. Dehydrogenase enzymes are stable for much longer (>6 months) in the membrane, so it is necessary to employ a coenzyme that is stable for at least as long as the enzyme is stable.

Akers et al. have tested ethanol-based biofuel cells fabricated using bioanodes containing NAD+-dependent ADH immobilized in a modified Nafion® membrane as discussed above and cathodes formed from ELAT electrodes with 20% Pt on Vulcan XC-72 (E-Tek). These bioanodes can function for greater than 30 days [23]. The test cell contains an anode solution of 1.0 mM ethanol in pH 7.15 phosphate buffer and a cathode solution containing pH 7.15 phosphate buffer saturated with dissolved oxygen. The two solutions are separated by a Nafion® 117 membrane. The ethanol-based biofuel cells have had open-circuit potentials ranging from 0.61 to 0.82 V at 20°C and have maximum power densities of 1.12 mW/cm2 [23]. This is a 16-fold increase in power density versus the state-of-the — art biofuel cell developed by Heller and co-workers [24]. The milestone that was required to further develop a biofuel cell is to eliminate the need for the electro­catalyst layer, poly (methylene green). This will be done by replacing NAD+- dependent ADH with PQQ-dependent ADH.