Enzymatic biofuel cells (BFCs) show great potential for the direct conversion of biochemically stored energy from renewable biomass resources into electricity. However, enzyme purification is time-consuming and expensive. Furthermore, the long-term use of enzymatic BFCs is hindered by enzyme degradation, which limits their lifetime to only a few weeks. We show, for the first time, that crude culture supernatant from enzyme-secreting microorganisms (Trametes versicolor) can be used without further treatment to supply the enzyme laccase to the cathode of a mediatorless BFC. Polarization curves show that there is no significant difference in the cathode performance when using crude supernatant that contains laccase compared to purified laccase in culture medium or buffer solution. Furthermore, we demonstrate that the oxygen reduction activity of this enzymatic cathode can be sustained over a period of at least 120 days by periodic resupply of crude culture supernatant. This is more than five times longer than control cathodes without the resupply of culture supernatant. During the operation period of 120 days, no progressive loss of potential is observed, which suggests that significantly longer lifetimes than shown in this work may be possible. Our results demonstrate the possibility to establish simple, cost efficient, and mediatorless enzymatic BFC cathodes that do not require expensive enzyme purification procedures. Furthermore, they show the feasibility of an enzymatic BFC with an extended lifetime, in which self-replicating microorganisms provide the electrode with catalytically active enzymes in a continuous or periodic manner.

145inents of the specimens that had thin films (23a, 23b, and 25b) were rather low. Moreover, even in those cases in which the measured refractive index of the film is close t o that of the base glass (26a, 29b, and 30b) the reflection from the interface of the film and the base glass was sufficiently strong to cancel out the reflection from the film-water interface (see Fig. 1). One would not expect this result if there is one continuous film on the glass. For example, the film on sample 29b has a measured refractive index of 1.615. The reflection a t the film-water interface is about o.7v0, whereas the reflection at the film-glass interface should be much less than O.OOIYo. These two rays could not give destructive interferencc to the extent observed (see Fig. I). The observed destructive interference can take place only if the leached film on the glass consists of two layers. The measurements are made on the larger layer. This layer must be separated from the main body of the glass by a layer which has a refractive index far different from that of the first film and that of the base glass. The work reported here merely indicates the presence of such a layer. It does not supply the information necessary to calculate the thickness or the refractive index of the layer. One may speculate, after considering the theory of reflection from multilayers,14 that its refractive index is less than both the outer film and the base glass and that its thickness is between 100 and 200 a.u. These values are purely tentative, of course.Two factors, (1) the effective destructive interference a t larger thicknesses and (2) the low refractive index of the very thin films, indicate that the acid first disturbs the glass netl 4 Antonin VaSiEek, "Reflection of Light from Glass with Ilouble and Multiple Films," J . Opt. SOC. Am., 37 [8] 623-34 (1947); Ceram. Abstracts, 1948, Sept., p. 201d. work considerably. Then, as its effect penetrates deeper and deeper, the glass constituents readjust themselves in a configuration which differs very little from that of the base glass but in a form more stable with respect to the acid. It is somewhat analogous to the physics lecture demonstration of the weighted wire moving through the ice cake. Although the region of great disturbance is confined to a limited area surrounding the wire, it moves through the ice leaving a reordered ice behind. It appears that the center of the greatly distorted network is confined to a limited region which moves into the glass leaving a readjusted network behind it. Such a process is consistent with the intense reflection at the interface of the film and the base glass, as well as with the relatively high refractive index of the thicker stain films and the low values for the thin films. VI. ConclusionsA method of measuring the thickness and dispersion of leached films on glass has been employed to study the kinetics of glass leaching. The thickness of the leached layer varied according to an equation of the form r = at. T @t2; where 7 is the thickness, t is the t...

Enzymatically catalyzed biofuel cells show unique specificity and promise high power densities, but suffer from a limited lifetime due to enzyme deactivation. In the present work, we demonstrate a novel concept to extend the lifetime of a laccase-catalyzed oxygen reduction cathode in which we decouple the electrode lifetime from the limited enzyme lifetime by a regular resupply of fresh enzymes. Thereto, the adsorption behavior of laccase from Trametes versicolor to buckypaper electrode material, as well as its time-dependent deactivation characteristics, has been investigated. Laccase shows a Langmuir-type adsorption to the carbon nanotube-based buckypaper electrodes, with a mean residence time of 2 days per molecule. In a citrate buffer of pH 5, laccase does not show any deactivation at room temperature for 2 days and exhibits a half-life of 9 days. In a long-term experiment, the laccase electrodes were operated at a constant galvanostatic load. The laccase-containing catholyte was periodically exchanged against a freshly prepared one every second day to provide sufficient active enzymes in the catholyte for the replacement of desorbed inactive enzymes. Compared to a corresponding control experiment without catholyte exchange, this procedure resulted in a 2.5 times longer cathode lifetime of 19 ± 9 days in which the electrode showed a potential above 0.744 V vs. normal hydrogen electrode at 110 μA cm(-2). This clearly indicates the successful exchange of molecules by desorption and re-adsorption and is a first step toward the realization of a self-regenerating enzymatic biofuel cell in which enzyme-producing microorganisms are integrated into the electrode to continuously resupply fresh enzymes.

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