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Various phenoxazine, phenothiazine, phenazine, indophenol and bipyridilium derivatives were tested for their effectiveness as redox mediators in microbial fuel cells containing Alcaligenes eutrophus, Bacillus subtilis, Escherichia coli, or Proteus vulgaris as the active biological agent, and glucose or succinate as the oxidisable substrate. A ferricyanide-Pt cathode was used. The open-circuit cell e.m.f.'s increased in the order of increasing negative formal redox potentials at pH 7(Ez) of the redox compounds. Several of the redox agents worked well as mediators, maintaining steady currents over several hours, and thionine was found to be particularly effective in maintaining relatively high cell voltages when current was drawn from the cell. A number of the compounds tested did not function well, either because they were incompletely or slowly reduced by the microorganisms or because of their instability. P. vulgaris, with thionine as mediator and glucose as substrate, showed the best performance in a fuel cell. This system was examined in some detail under various conditions of external load to establish the effects of organism concentration, mediator concentration, and substrate addition. Coulombic outputs from these cells were calculated by integration of the current-time plots. Coulombic yields of 30-60% were obtained, on the basis of (theoretical) complete oxidation of added substrate to C02 and water.

Potentiometric and amperometric measurements were made with microbial fuel cells containing E. coli or yeast as the anodic reducing agent and glucose as the oxidizable substrate. The catalytic effects of thionine and resorufin on the anode reaction were investigated. Results on the potentiometry, polarization, and coulombic output of the cells support a mediator-coupled mechanism for the transfer of electrons from the organism to the electrode in preference to a mechanism of "direct" electrochemical oxidation of glucose or its degradation products. Experiments with (14)C-labeled glucose show that when a microbial fuel cell produces a current under load, exogenous glucose is metabolized to produce (14)CO(2). The Coulombic yields of the cells indicate a high degree of energy conversion in these systems.

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