Highly Efficient and Versatile Anodes for Biofuel Cells Based on

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Highly Efficient and Versatile Anodes for Biofuel Cells Based on Cellobiose Dehydrogenase from Myriococcum thermophilum Federico Tasca,† Lo Gorton,† Wolfgang Harreither,‡ Dietmar Haltrich,‡ Roland Ludwig,§ and Gilbert No¨ll*,† Department of Analytical Chemistry, Lund UniVersity, P.O. Box 124, SE-221 00 Lund, Sweden, DiVision of Food Biotechnology, Department of Food Sciences and Technology, BOKU-UniVersity of Natural Recources and Applied Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria, and Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria ReceiVed: June 10, 2008; ReVised Manuscript ReceiVed: June 23, 2008

A powerful alternative to glucose oxidase as anode material in implantable biofuel cells is presented: Cellobiose dehydrogenase (CDH) from the ascomycete Myriococcum thermophilum (MtCDH) catalyzes the electrochemical oxidation of glucose, lactose, and cellobiose over a broad pH range. Current densities of more than 1 mA · cm-2 can be reached when MtCDH is wired to an Os redox polymer in the presence of single-walled carbon nanotubes and when lactose is used as a substrate at pH 8. In contrast to CDHs from basidiomycete fungi, which oxidize only β-1,4-linked di- and oligosaccharides efficiently, MtCDH is also able to oxidize glucose and other monosaccharides at relatively high turnover rates. The current density toward oxidation of 5 mM glucose under physiological conditions was about 100 µA · cm-2. Outstanding properties of MtCDH are high-temperature stability; a strong discrimination of oxygen turnover (and therefore no H2O2 production) in the presence of alternative electron acceptors; an ability to oxidize a range of carbohydrates, and a working pH from 3 to 10, which allows for combination with a variety of enzyme-based cathodes for oxygen reduction. The performance and stability of a membraneless glucose biofuel cell consisting of an MtCDH-modified anode and a Pt black cathode working under physiological conditions (PBS buffer, pH 7.4, 37 °C) were investigated over a period of 3 days. A maximum voltage of 500 mV, a maximum current density of almost 700 µA · cm-2, and a maximum power density of 157 µW · cm-2 at an operating voltage of 280 mV (under oxygen purging/ nonquiescent conditions) could be obtained with glucose (100 mM) as the substrate. Furthermore, the direct and mediated electron-transfer properties of MtCDH are compared in this work. The electrocatalytic current detected for mediated electron transfer (MET) is much higher and starts at a less positive potential than that for direct electron transfer (DET). The reason is that, in MET, the Os redox polymer is able to collect the electrons from the catalytically active flavin domain, whereas DET requires the oxidation of the heme domain, which has a more positive redox potential. The electrocatalytic current densities for DET and MET are significantly increased in the presence of single-walled carbon nanotubes. Introduction Fuel cells comprise a cathode for the reduction of oxygen (or hydrogen peroxide) and an anode for the combustion of fuels such as hydrogen or alcohols. In biofuel cells, at least one of these processes (anode or cathode reaction) is catalyzed by a biological compound. Current research activities focus on enzymatic1-9 and microbial1,10-12 biofuel cells. Up to now, applications of biofuel cells have been rather limited because the maximum power output is still orders of magnitude lower than for conventional fuel cells, batteries, or solar cells.1 Whereas solar energy probably has the highest potential among all forms of alternative energy for solving future energy problems,13,14 biofuel cells are of great interest for a variety of special applications. They can be used at locations where it would be difficult to routinely exchange traditional batteries,10,11 * To whom correspondence should be addressed. Phone: +46 46 222 0103. Fax: +46 46 222 4544. E-mail: [email protected]. New address: Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. New e-mail: [email protected]. † Lund University. ‡ BOKU-University of Natural Recources and Applied Life Sciences Vienna. § Research Centre Applied Biocatalysis.

with fuels that are undesired products or waste,1,12 or during medical treatment when a miniaturized implantable energy supply is required.2,4 Implantable biofuel cells can use glucose as a fuel, which is available in blood at a concentration of about 5 mM. These cells have to work under physiological conditions (pH 7.4, 0.14 M NaCl). Currently, the most promising type of implantable biofuel cells is based on glucose oxidase (GOx) and bilirubin oxidase as catalysts at the anode and cathode.2 Both enzymes are “wired” to Os redox polymer hydrogels that are adsorbed to graphite electrodes. The flexible Os2+/3+ redox centers establish the electrochemical communication between enzyme and electrode.2 In this contribution, cellobiose dehydrogenase (CDH) from the ascomycete Myriococcum thermophilum (MtCDH) was studied as a catalyst for biofuel cell applications as an alternative to GOx. CDHs are highly glycosylated monomeric enzymes containing a larger flavin-associated (dehydrogenase) and a smaller heme (cytochrome) domain and are, so far, the only known extracellular flavocytochrome.15,16 CDHs catalyze the oxidation of di- and oligosaccharides at the C(1) carbon to the corresponding lactones over a broad pH range.16 In addition to β-D-cellobiose and cello-oligosaccharides, which are regarded

10.1021/jp805092m CCC: $40.75  2008 American Chemical Society Published on Web 08/12/2008

Biofuel Cell Anodes Based on MtCDH

Figure 1. Polarization curves for an MtCDH modified spectrographic graphite electrode in the presence (MET) or absence (DET) of Os redox polymer. For electrode preparation, 10 µL of enzyme solution, 10 µL of SWCNT suspension, 3 µL of Os polymer solution (only for MET), and 1 µL of cross-linker solution were used. Lactose (0.1 M) dissolved in 0.1 M citrate buffer at pH 4.5 was used as the substrate.

as the natural substrates, lactose, which has the same β-1,4 glycosyl linkage as cellobiose but differs in the 4-OH position of the galactose moiety, is also oxidized with high catalytic efficiency by all CDHs. In contrast to CDHs from basidiomycete fungi, the ascomycete MtCDH also catalyzes the oxidation of monosaccharides such as glucose and some di- and oligosaccharides, e.g., maltose (consisting of two R-1,4-linked glucose moieties), with high turnover rates.15,16 CDHs belong to a group of enzymes that are able to communicate directly with electrodes without the need for any redox mediator.16-19 When redox mediators are required for electron transfer (ET), a voltage loss usually occurs during biofuel cell applications, because, for thermodynamic reasons, the redox potential of the mediator should be slightly more positive than the redox potential of the oxidizing enzyme at the anode.2In order to investigate the efficiency of both ET processes, the direct and redoxpolymer-mediated electron-transfer (DET and MET) properties of MtCDH were compared. The enzyme was adsorbed onto graphite electrodes in the presence or absence of an Os polymer, which served as an immobilized redox mediator.2,20 Poly(vinylpyridine)-[osmium-(N,N′-methylated-2,2′-biimidazole)3]2+/3+, which has shown highly efficient performance in combination with GOx, was used.2 Single-walled carbon nanotubes (SWCNTs) were added during both types of electrode preparation because of their ability to increase the electrocatalytic current density.21-28

J. Phys. Chem. C, Vol. 112, No. 35, 2008 13669 performance. When MtCDH-modified spectrographic graphite electrodes were prepared in the absence of SWCNTs, almost no catalytic current could be detected for DET. During MET experiments, the current density was up to 1 order of magnitude lower in the absence than in the presence of SWCNTs. Polarization curves of MtCDH-modified anodes were determined by linear sweep voltammetry at low scan rate (e1 mV · s-1) at a temperature of 22 °C unless otherwise stated. To compensate for any background current, the forward scan in the absence of substrate was subtracted from the forward scan in the presence of substrate. The current densities were calculated with respect to the geometric electrode area. Electrochemical measurements were performed with an EG&G potentiostat/galvanostat (model 273 A) using modified electrodes as the working electrode, a saturated calomel reference electrode (SCE), and a platinum foil counter electrode. Unless otherwise stated, argon was purged through the solutions for some minutes prior to the experiments. Spectrographic graphite rods were purchased from the Ringsdorff Werke GmbH, Bonn, Germany (type RW001, 3.05-mm diameter and 13% porosity; http:// www.sglcarbon.com/). These rods were sealed in a Teflon holder. Glassy carbon electrodes (3 mm in diameter) were purchased from BASi, West Lafayette, IN. Homemade pyrolytic graphite electrodes with a square surface are of 3 × 3 mm2 were used. Pyrolytic graphite was obtained as a gift from Mr. Robert Pulley, Minerals Technologies, New York (http:// www.mineralstech.com). SWCNTs were purchased from Nanocyl, Sambreville, Belgium. Poly(vinylpyridine)-[osmium-(N,N′methylated-2,2′-biimidalzole)3]2+/3+ was synthesized as reported elsewhere.29 The activity of MtCDH (EC 1.1.99.18) was 361 U · mL-1 (specific activity, 5.73 U · mg-1); the cultivation and purification of the enzyme has been described previously.16 Turnover rates of MtCDH for electron acceptors (2,6-dichloroindophenol, ε520 ) 6.8 mM-1 · cm-1; 1,4-benzoquinone, ε290 ) 2.24 mM-1 · cm-1) were measured in cuvette-based photometric assays using lactose as the electron donor at the optimum pH (6.0 and 8.0, respectively). The turnover number for each substrate was calculated from the obtained initial rate. Oxygen turnover was measured at pH 6.0 in a batch reaction employing MtCDH (2 U · mL-1), catalase for H2O2 removal (2000 U · mL-1), and lactose as the substrate. After 6, 10, and 24 h, samples were taken from the air-saturated, stirred reaction vessel, and the formation of lactobionic acid was analyzed by HPLC. All turnover rates were calculated using the published molecular mass and specific activity of MtCDH.16

Experimental Section Enzyme-modified electrodes were prepared by adsorbing the enzyme solution (10 µL) in the presence of 10-15 µL of a suspension of SWCNTs, (10 mg · mL-1). Optionally, 2-6 µL of an Os redox polymer solution (10 mg · mL-1) was added. Thereafter, 1-2 µL of poly(ethylene glycol) (400) diglycidyl ether (PEGDGE; 35 mg · mL-1) was gently mixed to the drop at the electrode surface. Also, in the absence of Os redox polymer, the addition of cross-linker improved the stability of the adsorbed enzyme layer (see Figure 1, direct-electron-transfer conditions). The electrodes were then allowed to dry overnight at 4 °C to complete the cross-linking reaction. There was some variation in the absolute activities of individual electrodes because the suspension of SWCNTs used for electrode modification was not completely homogeneous (even after extended sonication) and the amount of adsorbed SWCNTs could vary, whereas minor variation in the ratio between polymer and crosslinker did not have a significant effect on the electrode

Results and Discussion Enzyme-modified electrodes were prepared by adsorbing the enzyme solution in the presence of a suspension of SWCNTs. For MET, an Os redox polymer solution was added. Thereafter, a solution of cross-linking reagent [poly(ethylene glycol) (400) diglycidyl ether, PEGDGE] was added. For DET, the electrodes were prepared in the absence of the Os redox polymer. Details of electrode preparation are described in the Experimental Section. Primarily lactose was chosen as the substrate, because CDH exhibits high activity for this substrate and, in contrast to cellobiose, there is no substrate inhibition.16,19 However, a series of other sugars were also investigated in further experiments. The polarization curves of the CDH-modified electrodes for the oxidation of lactose in the presence and absence of Os redox polymer are presented in Figure 1. The electrocatalytic current detected for MET was much higher than that for DET. Furthermore, MET started at a potential almost 150 mV less

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TABLE 1: Relative Catalytic Activities of an MtCDH/Os Polymer/SWCNT-Modified Glassy Carbon Electrode Toward Oxidation of Lactose, Cellobiose, and Glucose at Different pHs pH 8 pH 6 pH 4.5

lactose

cellobiose

glucose

100% 48% 33%

81% 33% 22%

79% 43% 9%

positive than that for DET. Apparently, the Os redox polymer is able to collect the electrons from the catalytically active flavin domain of CDH, whereas DET requires the oxidation of the heme domain, which has a more positive redox potential.17,30 For CDH from Phanerochaete chrysosporium, the redox potentials of the flavin (106 mV, pH 3; -132 mV, pH 7) and heme (190 mV, pH 3; 130 mV, pH 7) domains have been reported.30 The experiments presented in Figure 1 were performed at pH 4.5 facilitating DET, whereas in previous studies of MtCDH with respect to potential applications in amperometric biosensors, it was observed that MET has an optimum at pH 8.16 Therefore, the catalytic activity of the MtCDH/Os polymer/ SWCNT-modified electrodes was investigated in the range between pH 4.5 and 8, which is of interest for biofuel cell applications in combination with laccase (pH 4-6) or bilirubin oxidase (pH 7-8) modified cathodes for oxygen reduction.2,4 In addition to lactose, cellobiose and glucose were used as substrates. In the examined pH range, the highest current density was detected for the oxidation of lactose with a maximum at pH 8, but the current density at pH 6 was still at a promising level for biofuel cell applications (details are presented in the Supporting Information). Table 1 presents a summary of the ratios of the catalytic activity of an MtCDH-modified glassy carbon electrode toward oxidation of lactose, cellobiose, and glucose at pH 4.5, 6, and 8 relative to the catalytic current for lactose oxidation at pH 8, which was set to 100%. The high catalytic activity toward glucose is of primary importance in context with the development of implantable biofuel cells, but also catalytic activities toward lactose and cellobiose are of potential interest. For further measurements studying the electrocatalytic process depending on pH, pyrolytic graphite (PG) was used as the electrode material. By changing the electrode material from spectrographic graphite or glassy carbon to PG, the absolute current density for MET could be increased. Both edge-planeoriented and basal-plane-oriented PG showed good performance. When PG was used as the electrode material, after reaching a maximum, the current density remained almost constant. The redox potential of the Os polymer is independent of pH.2 Therefore, the onset of the electrocatalytic current is expected to be at the same potential. However, at low pH, the onset of the electrocatalytic current was shifted to more positive values, implying that the redox potential of the flavin (dehydrogenase) domain was in that vicinity. As presented in Figure 2, the onset of the electrocatalytic current for lactose oxidation between pH 7 and pH 4.5 was about -100 mV (or slightly more negative), whereas at pH 3.5, the onset was shifted by about 60 mV in the positive direction. (According to Nernst, a shift of -59 mV per pH unit is expected for flavin oxidation when two protons and two electrons are involved.) Apparently, the electrocatalytic current at pH 4.5 and 3.5 started at a potential close to the redox potential of the flavin domain. In the polarization curves measured at pH 4.5 and 3.5, there was a second onset that might be caused by “wiring” the heme domain (see also Figure 1).

Figure 2. Polarization curves of an MtCDH/Os polymer/SWCNTmodified PG electrode (edge-plane-oriented) in the presence of 0.1 M lactose at different pHs collected at a scan rate of V ) 1 mV · s-1. Additional onsets of the electrocatalytic current at pH 4.5 and 3.5 are marked by arrows.

Figure 3. (A) CVs of an MtCDH/Os polymer/SWCNT-modified PG electrode (basal-plane-oriented) in the presence and absence of 0.1 M lactose (Lac) at pH 8 (0.1 M phosphate buffer) collected at a scan rate of V ) 1 mV · s-1. (B) Polarization curves of the same electrode under physiological conditions (PBS buffer, pH 7.4, 37 °C) in the presence of glucose (Glc) at concentrations of 5 mM and 0.1 M.

In Figure 3A, cyclic voltammograms (CVs) of an MtCDHmodified PG electrode in the presence and absence of lactose at pH 8 are presented. In the CV collected in the absence of substrate, the waves for oxidation and reduction of the Os redox polymer can be seen. The electrocatalytic current starts with the beginning of the oxidation of the Os polymer. The same electrode was applied under physiological conditions (PBS buffer, pH 7.4) at 37 °C in the presence of glucose at concentrations of 5 mM and 0.1 M, as depicted in Figure 3B. The electrocatalytic current at 0.1 M glucose is almost 4 times higher than that at 5 mM glucose. This can be explained by the high Michaelis-Menten constant (KM ) 240 mM) of MtCDH for glucose.16 Whereas, for lactose (KM ) 55 µM), substrate saturation is reached already below a concentration of 0.1 M,16

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Figure 4. Polarization curves of an MtCDH/Os polymer/SWCNTmodified PG electrode (edge-plane-oriented) toward oxidation of 0.1 M lactose (Lac), glucose (Glc), cellobiose (Cel), maltose (Mal), mannose (Man), galactose (Gal), and xylose (Xyl) at pH 7 (0.1 M phosphate buffer). Measurements were performed with the same PG electrode. The current density for 0.1 M lactose oxidation measured at pH 8 with this electrode was 31% higher than that measured at pH 7.

an additional increase in the glucose concentration beyond 0.1 M is expected to further increase the electrocatalytic current. During multicycle CVs of CDH-modified electrodes with substrate in PBS buffer or in phosphate buffer at pH 8 in the absence of NaCl, no difference in stability was observed. The electrodes lost less than 5% of their activity upon 10-20 h of operation. This indicates that, unlike other enzymes of interest for biofuel cell research,2,4 CDH is not inhibited by chloride anions. Furthermore, MtCDH converts a variety of substrates at neutral pH.16 In Figure 4, the responses of CDH to cellobiose, galactose, glucose, lactose, maltose, mannose, and xylose at pH 7 are presented. Aside from glucose, lactose and cellobiose exhibited the highest current density. Whereas catalytic activity toward the oxidation of disaccharides has been described for other types of CDH, MtCDH is the first type for which the ability to catalyze the oxidation of glucose has been described in the context of a biosensor application based on DET.16 In contrast to GOx, MtCDH strongly suppresses oxygen conversion in favor of other electron acceptors, which is of advantage for the development of membraneless biofuel cells leading to enhanced Coulombic efficiency,31 where Coulombic efficiency is defined as the Coulombic output derived from the fuel compared to the total amount of fuel being consumed. As the main undesired side reaction to the electrocatalytic pathway (substrate oxidation at the anode combined with oxygen reduction at the cathode), the substrate could react directly with oxygen at the anode. In this context, the turnover rates for MtCDH with different electron acceptors were determined using photometric techniques and a batch conversion experiment. The turnover number found for oxygen (air saturation at atmospheric pressure) is 0.09 s-1, which is around 200-fold lower than the values for electron acceptors such as 2,6-dichloroindophenol (16.8 s-1) and 1,4-benzoquinone (19.6 s-1). An additional advantage of MtCDH compared to GOx is that it does not result in significant production of H2O2, which could decrease the stability of modified electrodes because of its high reactivity. The current densities measured with MtCDH-modified planar PG electrode surfaces (under linear diffusion conditions) were quite high. By increasing the amount of redox polymer, current densities of more than 1 mA · cm-2 could be obtained at pH 8 for oxidation of lactose. (A representative CV is shown in the Supporting Information, Figure S2.) However, because the concentration of oxygen is only about 0.1 mM in blood and

Figure 5. (A) Polarization curve and (B) dependence of the power density on the operating voltage for a membraneless biofuel cell consisting of an MtCDH/Os polymer/SWCNT-modified PG electrode (basal-plane-oriented) as the anode and a Pt black electrode as the cathode. As the fuel, a 0.1 M solution of glucose in PBS buffer (pH 7.4) at 37 °C was used.

0.2 mM in air equilibrated water at 37 °C,2,4 the maximum power output of biofuel cells is rather limited by the cathode. MtCDH is expected to be highly stable at enhanced temperature, as M. thermophilum is an ascomycete fungus with an optimal growth temperature of 45 °C and an upper growth limit of 53 °C. As a model for a membraneless biofuel cell, a CDH-modified electrode was applied as the anode together with a Pt black electrode as the cathode in a 0.1 M solution of glucose in PBS buffer at 37 °C. Oxygen was gently purged around the cathode. Because the area of the cathode was much larger than that of the anode, the current density of this cell was limited by the anode. After an equilibration time of 5 min, a polarization curve was collected using linear sweep voltammetry (V ) 0.2 mV · s-1) connecting the anode as the working electrode and the cathode as the reference and counter electrode. The polarization curve and the dependence of the power density on the operating voltage are presented in Figure 5. This cell exhibited a maximum voltage (Vmax) of 500 mV, a maximum current density (Jmax) of almost 700 µA · cm-2, and a maximum power density (Pmax) of 157 µW · cm-2 at an operating voltage of 280 mV (under oxygen purging/nonquiescent conditions). A fill factor of 0.45 was calculated by dividing Pmax by the product of Vmax and Jmax. Replacing the Pt cathode by a bilirubin oxidase modified cathode2,4 is expected to increase the voltage of the cell even further. To verify its stability, the biofuel cell was investigated for a period of 3 days by multicycle CVs at a low scan rate (V ) 0.1 mV · s-1) in a potential range between -100 and -200 mV simulating a variable load (see Figure 6). A partial release of

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Figure 6. Polarization curves for a membraneless biofuel cell consisting of a PG electrode (basal-plane-oriented) modified with MtCDH/Os polymer/SWCNT as the anode and a Pt black electrode as the cathode collected at a scan rate of V ) 0.2 mV · s-1. As the fuel, a 0.1 M glucose solution in PBS buffer (pH 7.4) at 37 °C was used. The curves were measured after 5 min of equilibration (upper black curve) and after 3 days of performance (lower black curve). In the interim, multicycle CVs were collected at a scan rate of V ) 0.1 mV · s-1 in the potential range between -100 and -200 mV in order to simulate a variable load. The multicycle CVs measured for the time periods 0-12, 24-36, and 48-60 h are shown in blue, and those for the periods 12-24, 36-48, and 60-72 h are shown in red.

the protein-containing layer from the electrode surface due to mechanical stress (by bubbling oxygen through the solution) was observed, which was probably the main reason for the limited stability. Whereas the current density decreased by about 40% within 1 h and another 10% in the next 12 h, the decrease during the second and third days was only about 4% of the initial value within 12 h. Conclusions A biofuel cell anode using a new and versatile sugar-oxidizing dehydrogenase as the catalyst for the anode reaction is presented. For MtCDH wired to an Os redox polymer in the presence of SWCNTs, current densities of more than 1 mA · cm-2 could be reached when lactose was used as the substrate. In contrast to CDHs from basidiomycete fungi, which oxidize only cellobiose, cello-oligosacharides, and lactose, CDH from the ascomycete Myriococcum thermophilum is also able to oxidize glucose and other carbohydrates efficiently. The current densities toward the oxidation of glucose measured with planar electrodes in quiescent solution under physiological conditions were about 100 µA · cm-2 at 5 mM (which is about the glucose concentration in blood) and almost 500 µA · cm-2 at 100 mM. For GOx wired to the same low-potential Os polymer, current densities of 1.1 mA · cm-2 at a glucose concentration of 15 mM and 1.3 mA · cm-2 at 32 mM have been reported.2,29,32 However, these values cannot be compared directly with the results presented in this work because they were measured at miniaturized fiber electrodes2,29 (leading to cylindrical diffusion) or at rotating disk electrodes.32 In addition to a high current density, the MtCDHbased anode combines a number of advantages and is therefore highly qualified for applications in membraneless biofuel cells, including implantable biofuel cells using glucose as the substrate. MtCDH is stable at enhanced temperature, it strongly suppresses oxygen conversion in favor of other electron acceptors, no H2O2 is produced, glucose and other saccharides are converted with high efficiency, and the enzyme is catalytically active in the range between pH 3 and 10, allowing for combination with a large variety of enzyme-based cathodes for oxygen reduction. To study its performance as a biofuel anode,

Tasca et al. the MtCDH/Os polymer/SWCNT electrode was combined with a Pt black electrode as the cathode. This model of a biofuel cell working under physiological conditions with glucose as the substrate was monitored for 3 days. For biofuel cell applications in implantable biofuel cells, it would be appropriate to combine an MtCDH-modified anode with bilirubin oxidase modified cathodes, which are well established.2,33,34 Additionally, the DET and MET of MtCDH are compared in this work. The electrocatalytic current detected for MET is much higher than that for DET, and MET starts at a less positive potential than DET. Electrocatalytic oxidation by means of redox mediators usually requires a more positive potential than DET, because the redox potential of the mediator should be slightly more positive than the redox potential of the oxidizing enzyme. This is not the case for MtCDH, because the Os redox polymer is able to collect the electrons from the catalytically active flavin domain, whereas DET requires the oxidation of the heme domain, which has a more positive redox potential. The electrocatalytic current densities for both DET and MET are significantly increased upon the introduction of SWCNTs. This result might also be of interest for the development of amperometric biosensors based on CDH. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft DFG (DFG Postdoctoral Fellowship No 740/1-1 and Ru¨ckkehrstipendium No 740/3-1), the Swedish Research Council (Projects 621-2004-4476 and 621-2007-4124), and the Austrian Science Fund (Project L395-B11). G.N. thanks Mr. Robert Pulley, Minerals Technologies (http://www.mineralstech.com), for donating free samples of pyrolytic graphite. Supporting Information Available: Information concerning the catalytic activity of an MtCDH/Os polymer/SWCNTmodified electrode at different pHs and the maximum current density. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Bullen, R. A.; Arnot, T. C.; Lakeman, J. B.; Walsh, F. C. Biosens. Bioelectron. 2006, 21, 2015. (2) Heller, A. Phys. Chem. Chem. Phys. 2004, 6, 209. (3) Minteer, S. D.; Liaw, B. Y.; Cooney, M. J. Curr. Opin. Biotechnol. 2007, 18, 228. (4) Barton, S. C.; Gallaway, J.; Atanassov, P. Chem. ReV. 2004, 104, 4867. (5) Willner, B.; Katz, E.; Willner, I. Curr. Opin. Biotechnol. 2006, 17, 589. (6) Pothukuchy, A.; Mano, N.; Georgiou, G.; Heller, A. Biosens. Bioelectron. 2006, 22, 678. (7) Kamitaka, Y.; Tsujimura, S.; Setoyama, N.; Kajino, T.; Kano, K. Phys. Chem. Chem. Phys. 2007, 9, 1793. (8) Willner, I.; Baron, R.; Willner, B. Biosens. Bioelectron. 2007, 22, 1841. (9) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1181. (10) Lovley, D. R. Nat. ReV. Microbiol. 2006, 4, 497. (11) Lovley, D. R. Curr. Opin. Biotechnol. 2006, 17, 327. (12) Rabaey, K.; Verstraete, W. Trends Biotechnol. 2005, 23, 291. (13) Armaroli, N.; Balzani, V. Angew. Chem., Int. Ed. 2007, 46, 52. (14) Kamat, P. V. J. Phys. Chem. C 2007, 111, 2834. (15) Zamocky, M.; Ludwig, R.; Peterbauer, C.; Hallberg, B. M.; Divne, C.; Nicholls, P.; Haltrich, D. Curr. Protein Pept. Sci. 2006, 7, 255. (16) Harreither, W.; Coman, V.; Ludwig, R.; Haltrich, D.; Gorton, L. Electroanalysis 2007, 19, 172. (17) Stoica, L.; Dimcheva, N.; Haltrich, D.; Ruzgas, T.; Gorton, L. Biosens. Bioelectron. 2005, 20, 2010. (18) Stoica, L.; Ludwig, R.; Haltrich, D.; Gorton, L. Anal. Chem. 2006, 78, 393. (19) Stoica, L.; Ruzgas, T.; Ludwig, R.; Haltrich, D.; Gorton, L. Langmuir 2006, 22, 10801.

Biofuel Cell Anodes Based on MtCDH (20) Heller, A. Curr. Opin. Chem. Biol. 2006, 10, 664. (21) Yan, Y.; Zheng, W.; Su, L.; Mao, L. AdV. Mater. 2006, 18, 2639. (22) Zhu, L.; Yang, R.; Zhai, J.; Tian, C. Biosens. Bioelectron. 2007, 23, 528. (23) Gao, F.; Yan, Y.; Su, L.; Wang, L.; Mao, L. Electrochem. Commun. 2007, 9, 989. (24) Wang, J. Electroanalysis 2005, 17, 7. (25) Granot, E.; Basnar, B.; Cheglakov, Z.; Katz, E.; Willner, I. Electroanalysis 2006, 18, 26. (26) Joshi, P. P.; Merchant, S. A.; Wang, Y.; Schmidtke, D. W. Anal. Chem. 2005, 77, 3183. (27) Tasca, F.; Gorton, L.; Harreither, W.; Haltrich, D.; Ludwig, R.; No¨ll, G. J. Phys. Chem. C 2008, 112, 9956.

J. Phys. Chem. C, Vol. 112, No. 35, 2008 13673 (28) Tasca, F.; Gorton, L.; Wagner, J. B.; No¨ll, G. Biosens. Bioelectron. 2008, 24, 272. (29) Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2003, 125, 4951. (30) Igarashi, K.; Momohara, I.; Nishino, T.; Samejima, M. Biochem. J. 2002, 365, 521. (31) Ikeda, T.; Kano, K. Biochim. Biophys. Acta 2003, 1647, 121. (32) Mano, N.; Mao, F.; Heller, A. J. Electroanal. Chem. 2005, 574, 347. (33) Tsujimura, S.; Kamitaka, Y.; Kano, K. Fuel Cells 2007, 7, 463. (34) Tsujimura, S.; Kano, K.; Ikeda, T. J. Electroanal. Chem. 2005, 576, 113.

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