**9. Design of a H2/O2 biofuel cell based on carbon nanotubes-modified electrodes**

H2/O2 biofuel cells did not get much attention before O2 and CO resistant hydrogenases were proved to be efficient for H2 oxidation when immobilized onto electrode surfaces. Even though more and more efficient hydrogenase immobilization procedures are nowadays re‐ ported, few H2/O2 biofuel cells are described. An early study by Armstrong's group in 2006 [134] demonstrated that simple adsorption on graphite electrode of hydrogenase at the anode and laccase (a copper protein for O2 reduction) at the cathode, allowed a wristwatch to run for 24h. Power density of around 5 µW cm-2 at 500 mV was delivered with no mem‐ brane between the two compartments providing hydrogenase was extracted from *Ralstonia metallireducens*. As this is an aerobic bacterium, the result underlined that the H2/O2 biofuel cell could operate only with O2 resistant hydrogenase. In 2010, the same group improved the device by using another O2 resistant hydrogenase from *Escherichia coli* and bilirubin oxidase (BOD), another copper protein more efficient than laccase towards oxygen reduction be‐ cause being able to function at neutral pH [135]. The oxygen reductase was covalently linked to the graphite electrode which had been modified by diazonium salt reduction. The power density was enhanced compared to the former study reaching 63 µW cm-2. But most of all, this work provided a nice understanding of the operating conditions of such H2/O2 fuel cells involving hydrogenase as anode catalyst.

Due to the understanding of how hydrogenases could be efficiently connected at CNTcoated electrodes, a huge step jumped over very recently. First, using covalent attach‐ ment of both O2 resistant hydrogenase and BOD on pyrene derivative functionalized MWCNTs, a membrane-less biofuel cell was designed fed with a non-explosive 80/20 dihy‐ drogen/air mixture [133]. This biofuel cell displayed quite a good stability with time and a much higher power density than reported before. Indeed, an average power density of 119 µW cm-2 was measured. Low solubility of oxygen and weak affinity of BOD for oxy‐ gen was shown to limit the cathodic current. Secondly in our group, a more performant H2/O2 mediatorless biofuel cell was constructed based on one step covalent attachment directly on SWCNTs of an hyperthermophilic O2 resistant hydrogenase at the anode and BOD at the cathode [136] (Figure 7).

membrane-bound hydrogenases have been discovered from aerobic or extremophilic organ‐ isms [128, 129-132]. They have been demonstrated to oxidize H2 in the presence of oxygen and CO. The crystallographic structure of three of them has been resolved, showing that an uncommon [4Fe-3S] cluster proximal to the active site prevents deleterious oxygen attack. Of course, the sensitivity to oxygen, and also to CO, of most hydrogenases known before was a strong limitation for their potential use in biotechnological devices. Therefore these resistant biocatalysts open new avenues towards a biohydrogen economy. No doubt that these researches will increase in the next future. To date, two main studies report the immo‐ bilization of resistant hydrogenase on CNT-modified electrodes. Krishnan *et al.* very recent‐ ly modified MWCNTs by pyrenebutyric acid, and demonstrated it was an efficient platform for stable O2-resistant hydrogenase linkage [133]. In our group, original use of a hyperther‐ mophilic O2- and CO-resistant hydrogenase allowed the increase in the catalytic current for direct H2 oxidation on a large range of temperature up to 70°C. Attempts to enhance the number of electrically connected hydrogenase succeeded by use of coatings of chemically oxidized SWCNTs [123]. Values as high as 1 mA cm-2 were reached depending on the amount of SWCNTs used in the coating (Figure 6). For the lowest amounts of SWCNTs, the increase in the catalytic current was demonstrated to be essentially due to the increase in surface area. However the catalytic current rapidly reached a plateau, although the peak

452 Syntheses and Applications of Carbon Nanotubes and Their Composites

current for the redox probe still increased, suggesting rapid saturation of the surface.

**9. Design of a H2/O2 biofuel cell based on carbon nanotubes-modified**

H2/O2 biofuel cells did not get much attention before O2 and CO resistant hydrogenases were proved to be efficient for H2 oxidation when immobilized onto electrode surfaces. Even though more and more efficient hydrogenase immobilization procedures are nowadays re‐ ported, few H2/O2 biofuel cells are described. An early study by Armstrong's group in 2006 [134] demonstrated that simple adsorption on graphite electrode of hydrogenase at the anode and laccase (a copper protein for O2 reduction) at the cathode, allowed a wristwatch to run for 24h. Power density of around 5 µW cm-2 at 500 mV was delivered with no mem‐ brane between the two compartments providing hydrogenase was extracted from *Ralstonia metallireducens*. As this is an aerobic bacterium, the result underlined that the H2/O2 biofuel cell could operate only with O2 resistant hydrogenase. In 2010, the same group improved the device by using another O2 resistant hydrogenase from *Escherichia coli* and bilirubin oxidase (BOD), another copper protein more efficient than laccase towards oxygen reduction be‐ cause being able to function at neutral pH [135]. The oxygen reductase was covalently linked to the graphite electrode which had been modified by diazonium salt reduction. The power density was enhanced compared to the former study reaching 63 µW cm-2. But most of all, this work provided a nice understanding of the operating conditions of such H2/O2

Due to the understanding of how hydrogenases could be efficiently connected at CNTcoated electrodes, a huge step jumped over very recently. First, using covalent attach‐

**electrodes**

fuel cells involving hydrogenase as anode catalyst.

**Figure 7.** (A) Schematic representation of H2/O2 biofuel cell with O2 resistant hydrogenase at the anode and bilirubin oxidase (BOD) at the cathode. Each half cell, separated by a Nafion® membrane, is independently thermoregulated with waterbaths. (B) Performance of the H2/O2 biofuel cell.

Taking advantage of temperature, the biofuel cell delivered power densities up to 300 µW cm-2 at 0.6V with an OCV of 1.1V, which is the highest performance ever reported. Further‐ more, promising stability of the biofuel cell during 24h of continuous use lets us consider this device as an alternative power supply for small portable applications. The analysis of the fuel cell parameters during polarization, allows us to define the potential window in which the fuel cell fully operates. Interestingly, in Armstrong's group [135] and in our group, different approaches on the settings of biofuel cell working conditions, led to similar observations of an unexpected increasing anodic potential. This high oxidizing potential generates an inactive state of hydrogenase active site. It is worth noticing that this hydroge‐ nase inactivation occurred under anaerobic conditions in our group while it was under aero‐ bic conditions in Armstrong's group. Consequently, dramatic loss in power densities was observed. By applying negative potential to the anode, and thus providing electrons to the active site, we were unable to reactivate hydrogenase. Another protocol used by Armstrong, consisted to add a second hydrogenase coated anode, unconnected to the system but present in the anodic half-cell which was consequently unaffected by the oxidizing potential but still in presence of O2. This second anode, under H2 oxidation was used as an electron supplier and connected to the first electrode. This procedure reactivated hydrogenase and allowed full recovery of OCV. It is of relevant interest to overcome hydrogenase inactivation in H2/O2 biofuel cell.

tion and substrate diffusion. Carbon fibers, mesoporous carbon templates could be used to build very interesting new electrochemical interfaces. This diversity in potential carbon ma‐ terials for efficient enzyme immobilization would be a key step to go through the difficulties linked to CNTs, *i.e.* effective cost for separation and purification as well as possible toxicity. Finally, to avoid the membrane between the cathodic and anodic compartments, and build a miniaturized biofuel cell, unusual cell designs, such as microfluidic or flow-through sys‐ tems, are likely to open new avenues. All these future developments will certainly require a multidisciplinary approach, coupling electrochemists with biochemists and physicists, and coupling methods such as electrochemistry and spectrometry, electrochemistry and molecu‐ lar genetics or electrochemistry and materials chemistry. This multidisciplinary willingness will help in the elucidation of the interactions between enzymes and nanostructured materi‐ als at the nanoscale and yield innovative nanobiotechnological approaches and applications.

Carbon Nanotube-Enzyme Biohybrids in a Green Hydrogen Economy

http://dx.doi.org/10.5772/51782

455

We gratefully acknowledge the contribution of Marielle Bauzan (Fermentation Plant Unit, IMM, CNRS, Marseille, France) for growing the bacteria, Dr Marianne Guiral, Dr Marianne Ilbert and Pascale Infossi for fruitful discussions. This work was supported by research

Bioénergétique et Ingénierie des Protéines, CNRS - AMU - Institut de Microbiologie de la

[2] Grove, W. (1838). On a new voltaic combination. *Philosophical Magazine and Journal of*

[3] Charlou, J. L., Donval, J. P., Konn, C., Ondréas, H., Fouquet, Y., Jean-Baptiste, P., & Fourré, E. (2010). High production and fluxes of H2 and CH4 and evidence of abiotic hydrocarbon synthesis by serpentinization on ultramafic-hosted hydrothermal sys‐ tems on Mid-Atlantic Ridge. Rona P., Devey C., Dyment J. Murton B. Editors,. *"Di‐*

**Acknowledgements**

**Author details**

Méditerranée, France

**References**

grants from CNRS, Région PACA and ANR.

Anne De Poulpiquet, Alexandre Ciaccafava, Saïda Benomar,

Marie-Thérèse Giudici-Orticoni and Elisabeth Lojou\*

\*Address all correspondence to: lojou@imm.cnrs.fr

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