**8. Carbon nanotubes for bioelectrooxidation of H2: towards H2/O2 biofuel cells**

We already described above hydrogenases, the enzymes that convert with high specificity and efficiency protons into dihydrogen. Most of these biocatalysts are also efficient in the oxida‐ tion of dihydrogen into protons. Consequently this allows to imagine biofuel cells in which the fuel would be dihydrogen, exactly as in PEM fuel cells. As hydrogenases are able to oxidize dihydrogen with very low overvoltage, the open circuit voltage for the biofuel cell using oxygen at the cathode, is expected to be not far from the thermodynamic one, *i.e.* 1.23 V. Hence, high power densities are expected, provided that a strong and efficient electrical connection be‐ tween hydrogenase and electrode can be achieved. Simple adsorption of hydrogenase was performed in a first step, because it allowed a direct oxidation of dihydrogen without any redox mediators [120]. Catalytic mechanisms associated with dihydrogen oxidation at the active site were largely studied. The effect of strong hydrogenase inhibitors such as oxygen and CO were explored by this mean, leading to nice developments in engineering of more tolerant hydrogenases [121] or use of naturally resistant hydrogenases [122, 123]. However, this immobilization procedure relies on a monolayer of enzyme, which furthermore suffers from quick desorption. Otherwise, multilayer enzymatic films require a redox mediator so that even the last layer far from the electrode could be connected. Other immobilization processes are thus needed, that can favor an enhancement in both the amount of connected hydrogenases as well as their stability, while preserving their functionality.

Carbon nanotube networks constituted technological breakthroughs in that way. All the recent developments using immobilization of hydrogenases onto carbon nanotubes point out improved catalytic currents essentially related to an increase in the active area of the electrode. The respective role of metallic-SWCNTs against semiconducting one was ex‐ plored for dihydrogen oxidation by immobilized hydrogenase [124]. A higher oxidation process was revealed when the nanotube mixture was enriched in metallic SWCNT. The study furthermore suggested no need of oxygenated SWCNTs for efficient anchoring of hydrogenases. The catalytic current enhancement was claimed to be due to an increase in active electrode surface area and an improved electronic coupling between hydrogenase redox active sites and the electrode surface. In most cases, however, CNTs are used as a mixture of metallic and semi-conducting tubes. Oxidation of the mixture yields the defects and functionalities described above in this review. Advantage is gained due to these chem‐ ical functions quite easily generated on the surface of the carbon nanotubes. Electrodes modified by carbon nanotubes are thus expected to offer numerous anchoring sites for stable hydrogenase immobilization. The literature provides a few examples of efficient immobili‐ zation of hydrogenase on carbon nanotubes coatings bearing various functionalities. Both SWCNTs and MWCNTs are used. Notably, more and more articles are devoted nowadays to this domain in hydrogenase research. A bionanocomposite made of the hydrogenase, MWCNTs and a thiopyridine derivative was proved to form stable monolayers when trans‐ ferred by Langmuir-Blodgett method on indium tin oxide electrode surfaces [125]. A great‐ er amount of electroactive hydrogenase towards dihydrogen oxidation was demonstrated to be adsorbed on the Langmuir-Blodgett films. De Lacey and co-workers grew MWCNTs on electrode by chemical vapor deposition of acetylene [65]. A high density of vertically aligned carbon nanotubes was obtained, which were functionalized by electroreduction of a diazonium salt for covalent binding of hydrogenase. High coverage of electroactive en‐ zyme was measured, suggesting that almost all the functionalized CNT surface was acces‐ sible to hydrogenase. Great stabilization of the catalytic current for H2 oxidation was obtained, with no decrease in current density after one month. Another work by Heering and coworkers studied a gold electrode pre-treated by polymyxin then a multilayer of carbon nanotubes [126]. Polymyxin was shown to help in the stable attachment of hydrogenase on the gold electrode. Using adsorption of hydrogenase on a nanotube layer pretreated with polymyxin the current density for H2 oxidation was an order of magnitude higher than at the gold electrode only modified by polymixin. This result was supposed to origin from greater surface area even though only the top of the nanotube layer was supposed to be accessible to the enzyme. The catalytic current was stable with time, at least for two hours under continuous cycling, and several days upon storage under ambient conditions. AFM visualization of hydrogenase immobilized onto polymyxin-treated SWCNT layer on SiO2 revealed that hydrogenase was structurally intact and preferentially adsorbed on the side‐ walls of the CNTs rather than on SiO2 [126].

time. We postulated that the conductive polymer which was electropolymerized onto CNTs could play a multiple role: enhancement of the electroactive surface area, enhancement of redox mediator units due to phenothiazine monomers entrapped in the polymer matrix, en‐ hancement of hydrogenase anchorage sites. We have already mentioned in this review the advantages of a direct electron transfer over a mediated one for H2 oxidation, including gain in over-potential values, less interferences due to enzyme specificity, absence of redox medi‐ ators that could be difficult to co-immobilize with the enzyme… Functionalized carbon nanotube films were evaluated in our group as platforms for various hydrogenases, that present a very different environment of FeS cluster electron relay. Dihydrogen oxidation was studied at gold electrodes modified with functionalized self-assembled-monolayers [128]. As expected, dihydrogen oxidation process was demonstrated to be driven by electro‐ static or hydrophobic interactions according to the specific environment of the surface elec‐ tron relay. Interestingly, at CNT coatings, although CNTs were negatively charged, direct electrical connection of hydrogenases that present a negatively charged patch around the FeS surface electron relay was observed [44, 123]. In other words, despite unfavourable elec‐ trostatic interactions, direct electron transfer process for dihydrogen oxidation was ach‐ ieved. One important conclusion was that on such CNT films, the nanometric size of the CNTs allows a population of hydrogenases to be directly connected to a neighbouring nano‐ tube, hence allowing direct electron transfer for H2 oxidation, whatever the orientation of

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**Figure 6.** Comparative evolution of the catalytic current for dihydrogen oxidation with the amount of SWCNTs depos‐ ited at a graphite electrode in the case of hydrogenases from *Aquifex aeolicus* (Aa) or *Desulfovibrio fructosovorans*

However, the extreme oxygen sensitivity of hydrogenases used in the former studies yield‐ ed an intensive research towards more resistant enzymes. During the last years, four [NiFe]

(Df). Catalytic currents are measured using voltammetry under H2 at 60 and 25°C for Aa and Df respectively.

the enzyme.

In our laboratory, we immobilized the [NiFe] hydrogenase from a mesophilic anaerobic bac‐ terium (the sulfate reducing bacterium *Desulfovibrio fructosovorans* Df) by adsorption onto SWCNT films [44]. The current for direct H2 oxidation was shown to increase with the amount of SWCNTs in the coating (Figure 6).

Because non-turnover signals were not detected for hydrogenase in these conditions, the in‐ crease in surface area was evaluated using a redox protein as a probe. It was shown that SWCNTs induced one order larger surface area. The same hydrogenase was entrapped in methylviologen functionalized polypyrrole films coated onto SWCNTs and MWCNTs [127]. Although no direct electrical hydrogenase connection was observed, an efficient dihydrogen oxidation through a mediated process occurred. It was concluded that the entrapment of hy‐ drogenase into the redox polymer coated onto CNTs combined the electron carrier proper‐ ties of redox probes, the flexibility of polypyrroles, and the high electroactive area developed by CNTs. The reason why no direct connection could be observed is however not clearly understood yet. In our group we handled immobilization of hydrogenase on a film obtained by electropolymerization of a phenothiazine dye on a SWCNT coating [81]. The phenothiazine dye was shown to be able to mediate dihydrogen oxidation but also to serve as an anchor for the enzyme when adsorbed or when electropolymerized. Higher current density than in the absence of SWCNT was observed. In addition, a wider potential window for dihydrogen oxidation was reached as well as very stable electrochemical signals with time. We postulated that the conductive polymer which was electropolymerized onto CNTs could play a multiple role: enhancement of the electroactive surface area, enhancement of redox mediator units due to phenothiazine monomers entrapped in the polymer matrix, en‐ hancement of hydrogenase anchorage sites. We have already mentioned in this review the advantages of a direct electron transfer over a mediated one for H2 oxidation, including gain in over-potential values, less interferences due to enzyme specificity, absence of redox medi‐ ators that could be difficult to co-immobilize with the enzyme… Functionalized carbon nanotube films were evaluated in our group as platforms for various hydrogenases, that present a very different environment of FeS cluster electron relay. Dihydrogen oxidation was studied at gold electrodes modified with functionalized self-assembled-monolayers [128]. As expected, dihydrogen oxidation process was demonstrated to be driven by electro‐ static or hydrophobic interactions according to the specific environment of the surface elec‐ tron relay. Interestingly, at CNT coatings, although CNTs were negatively charged, direct electrical connection of hydrogenases that present a negatively charged patch around the FeS surface electron relay was observed [44, 123]. In other words, despite unfavourable elec‐ trostatic interactions, direct electron transfer process for dihydrogen oxidation was ach‐ ieved. One important conclusion was that on such CNT films, the nanometric size of the CNTs allows a population of hydrogenases to be directly connected to a neighbouring nano‐ tube, hence allowing direct electron transfer for H2 oxidation, whatever the orientation of the enzyme.

zation of hydrogenase on carbon nanotubes coatings bearing various functionalities. Both SWCNTs and MWCNTs are used. Notably, more and more articles are devoted nowadays to this domain in hydrogenase research. A bionanocomposite made of the hydrogenase, MWCNTs and a thiopyridine derivative was proved to form stable monolayers when trans‐ ferred by Langmuir-Blodgett method on indium tin oxide electrode surfaces [125]. A great‐ er amount of electroactive hydrogenase towards dihydrogen oxidation was demonstrated to be adsorbed on the Langmuir-Blodgett films. De Lacey and co-workers grew MWCNTs on electrode by chemical vapor deposition of acetylene [65]. A high density of vertically aligned carbon nanotubes was obtained, which were functionalized by electroreduction of a diazonium salt for covalent binding of hydrogenase. High coverage of electroactive en‐ zyme was measured, suggesting that almost all the functionalized CNT surface was acces‐ sible to hydrogenase. Great stabilization of the catalytic current for H2 oxidation was obtained, with no decrease in current density after one month. Another work by Heering and coworkers studied a gold electrode pre-treated by polymyxin then a multilayer of carbon nanotubes [126]. Polymyxin was shown to help in the stable attachment of hydrogenase on the gold electrode. Using adsorption of hydrogenase on a nanotube layer pretreated with polymyxin the current density for H2 oxidation was an order of magnitude higher than at the gold electrode only modified by polymixin. This result was supposed to origin from greater surface area even though only the top of the nanotube layer was supposed to be accessible to the enzyme. The catalytic current was stable with time, at least for two hours under continuous cycling, and several days upon storage under ambient conditions. AFM visualization of hydrogenase immobilized onto polymyxin-treated SWCNT layer on SiO2 revealed that hydrogenase was structurally intact and preferentially adsorbed on the side‐

In our laboratory, we immobilized the [NiFe] hydrogenase from a mesophilic anaerobic bac‐ terium (the sulfate reducing bacterium *Desulfovibrio fructosovorans* Df) by adsorption onto SWCNT films [44]. The current for direct H2 oxidation was shown to increase with the

Because non-turnover signals were not detected for hydrogenase in these conditions, the in‐ crease in surface area was evaluated using a redox protein as a probe. It was shown that SWCNTs induced one order larger surface area. The same hydrogenase was entrapped in methylviologen functionalized polypyrrole films coated onto SWCNTs and MWCNTs [127]. Although no direct electrical hydrogenase connection was observed, an efficient dihydrogen oxidation through a mediated process occurred. It was concluded that the entrapment of hy‐ drogenase into the redox polymer coated onto CNTs combined the electron carrier proper‐ ties of redox probes, the flexibility of polypyrroles, and the high electroactive area developed by CNTs. The reason why no direct connection could be observed is however not clearly understood yet. In our group we handled immobilization of hydrogenase on a film obtained by electropolymerization of a phenothiazine dye on a SWCNT coating [81]. The phenothiazine dye was shown to be able to mediate dihydrogen oxidation but also to serve as an anchor for the enzyme when adsorbed or when electropolymerized. Higher current density than in the absence of SWCNT was observed. In addition, a wider potential window for dihydrogen oxidation was reached as well as very stable electrochemical signals with

walls of the CNTs rather than on SiO2 [126].

450 Syntheses and Applications of Carbon Nanotubes and Their Composites

amount of SWCNTs in the coating (Figure 6).

**Figure 6.** Comparative evolution of the catalytic current for dihydrogen oxidation with the amount of SWCNTs depos‐ ited at a graphite electrode in the case of hydrogenases from *Aquifex aeolicus* (Aa) or *Desulfovibrio fructosovorans* (Df). Catalytic currents are measured using voltammetry under H2 at 60 and 25°C for Aa and Df respectively.

However, the extreme oxygen sensitivity of hydrogenases used in the former studies yield‐ ed an intensive research towards more resistant enzymes. During the last years, four [NiFe] 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 current for the redox probe still increased, suggesting rapid saturation of the surface.

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

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**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

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

BOD at the cathode [136] (Figure 7).

waterbaths. (B) Performance of the H2/O2 biofuel cell.
