One-compartment biofuel cells without separators have been constructed, in which D-fructose dehydrogenase (FDH) from Gluconobacter sp. and laccase from Trametes sp. (TsLAC) work as catalysts of direct electron transfer (DET)-type bioelectrocatalysis in the two-electron oxidation of D-fructose and four-electron reduction of dioxygen as fuels, respectively. FDH adsorbs strongly and stably on Ketjen black (KB) particles that have been modified on carbon papers (CP) and produces the catalytic current with the maximum density of about 4 mA cm^–2 without mediators at pH 5. The catalytic wave of the D-fructose oxidation is controlled by the enzyme kinetics. The location and the shape of the catalytic waves suggest strongly that the electron is directly transferred to the KB particles from the hemec site in FDH, of which the formal potential has been determined to be 39 mV vs. Ag|AgCl|sat. KCl. Electrochemistry of three kinds of multi-copper oxidases has also been investigated and TsLAC has been selected as the best one of the DET-type bioelectrocatalyst for the four-electron reduction of dioxygen in view of the thermodynamics and kinetics at pH 5. In the DET-type bioelectrocatalysis, the electron from electrodes seems to be transferred to the type I copper site of multi-copper oxidases. TsLAC adsorbed on carbon aerogel (CG) particles with an average pore size of 22 nm, that have been modified on CP electrodes, produces the catalytic reduction current of dioxygen with a density of about 4 mA cm^–2, which is governed by the mass transfer of the dissolved dioxygen. The FDH-adsorbed KB-modified CP electrodes and the TsLAC-adsorbed CG-modified CP electrodes have been combined to construct one-compartment biofuel cells without separators. The open-circuit voltage was 790 mV. The maximum current density of 2.8 mA cm^–2 and the maximum power density of 850 µW cm^–2 have been achieved at 410 mV of the cell voltage under stirring.

Biofuel cells are a next-generation energy device because they use renewable fuels with high energy density and safety. We have developed passive-type biofuel cell units, which generate a power over 100 mW (80 cm³, 39.7 g). Our biofuel cell, in which two-electron oxidation of glucose and four-electron reduction of O₂ occurs at pH 7 in mediated bioelectrochemical processes under quiescent conditions, accomplished the maximum power density of 1.45 ± 0.24 mW cm⁻² at 0.3 V. This performance was achieved by introducing three technologies: (1) Enzymes and mediator are densely entrapped on carbon-fiber electrodes with the enzymatic activity retained, (2) the concentration of buffer in electrolyte solution was optimized for the immobilized enzymes, and (3) the cathode structure was designed to supply O₂ efficiently. The cell units with a multi-stacked structure successfully operate a radio-controlled car (16.5 g), which demonstrates the potential of biofuel cells in practical applications.