**5. Towards a green H2 economy: carbon nanotubes for enzyme and microbe immobilization**

Direct electron transfer process is preferred to mediated one, because it is not limited by the affinity between the enzyme and the redox mediator, and because it avoids the co-immobili‐ zation of enzyme and mediator. It is furthermore expected to yield the highest power densi‐ ty because enzymes, as biocatalysts, transform their substrate into products with very low overvoltages. However it requires the knowledge of the protein structure and the construc‐

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There are many strategies for efficient enzyme immobilization onto electrochemical interfa‐ ces, including simple physical adsorption, covalent attachment, cross-linking or entrapment in polymers. The objectives are to optimize the immobilization procedure so that the effi‐ ciency of the enzyme and its stability are preserved. Moreover, due to the size of enzymes compared to chemical catalysts, large surface area interfaces baring many anchorage sites

To reach these goals, 3D structures are preferred, and CNT-based electrodes are very popu‐ lar, both SWCNTs and MWCNTs. CNTs can be directly grown onto electrode surface, or ad‐ sorbed on it, or imbedded in polymer coating. In most cases, higher activity was reported for enzymes physically adsorbed onto CNTs [34]. Hydrophobic interactions between the en‐ zyme and the CNT walls and π-π interactions between side walls of CNTs and aromatic rings of the enzyme are thought to be the driving force for direct adsorption of enzymes on CNTs [35]. Electrostatic interaction between the defect sites of CNTs and protonated amino residues of the enzyme plays also a role in the adsorption process [35]. CNTs are quite easily functionalized, allowing covalent, thus stable specific attachment of enzymes. The oxidation in strong acidic solutions at high temperature was demonstrated to remove the end caps and shorten the lengths of the CNTs. The length of the CNTs was shown to be a function of the oxidation duration [36]. Acid treatment also adds oxide groups, primarily carboxylic acids, to the tube ends and defect sites [37]. The control of reactants and/or oxidation condi‐ tions may control the locations and density of the functional groups on the CNTs, which can be used to control the location and density of the attached enzymes [37]. Covalent immobili‐ zation is induced by carbodiimide reaction between the free amine groups on the enzyme

Further chemical reactions can be performed at the oxide groups generated on the oxidized CNTs to functionalize with groups such as amides, thiols, etc…From an electrochemical point of view, the side walls of CNTs were suggested to behave as basal plane of pyrolytic graphite, while their open ends resemble the edge planes [38, 39]. But recent work has dem‐ onstrated that the side wall may be responsible for electrochemical activity [40]. It has been

tion of a tuned electrochemical interface that fits the electron transfer site.

surface and carboxylic groups generated by side wall oxidation of CNTs.

are required to obtain high catalytic currents.

Replacement of chemical catalysts is thus nowadays highly needed in view of the develop‐ ment of a green energy economy. Microorganisms contain many biocatalysts, namely en‐ zymes, which are highly efficient and specific towards various substrate conversions. Given they are produced in large enough quantities, these enzymes could be used as catalysts in biotechnological devices. A mandatory condition to develop heterogeneous catalysis is to succeed in the functional immobilization and in the stabilization of the enzymes on solid supports. The redox active site of enzymes is indeed buried inside the protein moiety so that the enzymatic property can be maintained under environmental stresses. Specific channels are often involved to allow the substrate to reach the active site. Complex but highly organ‐ ized electron transfer chains occur for energetic metabolism. Electron transfer between two physiological partners associated with transformation of the substrate involves specific rec‐ ognition site. The game for a bioelectrochemist that aims to get the highest electron transfer rate for heterogeneous catalysis is to reproduce at the electrode interface the physiological electron transfer recognition process. Given the usual size of an enzyme (5-10 nm), electron transfer cannot occur *via* electron tunneling from the active site to the surface of the enzyme. In some enzymes, electron relays, one being located at the protein surface, act as a conduc‐ tive line for electron shuttling. If the electrode interface is built so that it fits the surface elec‐ tron relay environment, one can expect to favor a direct electrical connection of the enzyme onto the electrode. In case of direct electron transfer failure, an artificial redox mediator that acts as a fast redox system and shuttles electrons between the enzyme and the electrode can be used (Figure 2) [13, 33].

**Figure 2.** Interfacial electron transfer between an enzyme and an electrode can be achieved by direct (left) or mediat‐ ed (right) electron transfer process.

Direct electron transfer process is preferred to mediated one, because it is not limited by the affinity between the enzyme and the redox mediator, and because it avoids the co-immobili‐ zation of enzyme and mediator. It is furthermore expected to yield the highest power densi‐ ty because enzymes, as biocatalysts, transform their substrate into products with very low overvoltages. However it requires the knowledge of the protein structure and the construc‐ tion of a tuned electrochemical interface that fits the electron transfer site.

**5. Towards a green H2 economy: carbon nanotubes for enzyme and**

Replacement of chemical catalysts is thus nowadays highly needed in view of the develop‐ ment of a green energy economy. Microorganisms contain many biocatalysts, namely en‐ zymes, which are highly efficient and specific towards various substrate conversions. Given they are produced in large enough quantities, these enzymes could be used as catalysts in biotechnological devices. A mandatory condition to develop heterogeneous catalysis is to succeed in the functional immobilization and in the stabilization of the enzymes on solid supports. The redox active site of enzymes is indeed buried inside the protein moiety so that the enzymatic property can be maintained under environmental stresses. Specific channels are often involved to allow the substrate to reach the active site. Complex but highly organ‐ ized electron transfer chains occur for energetic metabolism. Electron transfer between two physiological partners associated with transformation of the substrate involves specific rec‐ ognition site. The game for a bioelectrochemist that aims to get the highest electron transfer rate for heterogeneous catalysis is to reproduce at the electrode interface the physiological electron transfer recognition process. Given the usual size of an enzyme (5-10 nm), electron transfer cannot occur *via* electron tunneling from the active site to the surface of the enzyme. In some enzymes, electron relays, one being located at the protein surface, act as a conduc‐ tive line for electron shuttling. If the electrode interface is built so that it fits the surface elec‐ tron relay environment, one can expect to favor a direct electrical connection of the enzyme onto the electrode. In case of direct electron transfer failure, an artificial redox mediator that acts as a fast redox system and shuttles electrons between the enzyme and the electrode can

**Figure 2.** Interfacial electron transfer between an enzyme and an electrode can be achieved by direct (left) or mediat‐

**microbe immobilization**

440 Syntheses and Applications of Carbon Nanotubes and Their Composites

be used (Figure 2) [13, 33].

ed (right) electron transfer process.

There are many strategies for efficient enzyme immobilization onto electrochemical interfa‐ ces, including simple physical adsorption, covalent attachment, cross-linking or entrapment in polymers. The objectives are to optimize the immobilization procedure so that the effi‐ ciency of the enzyme and its stability are preserved. Moreover, due to the size of enzymes compared to chemical catalysts, large surface area interfaces baring many anchorage sites are required to obtain high catalytic currents.

To reach these goals, 3D structures are preferred, and CNT-based electrodes are very popu‐ lar, both SWCNTs and MWCNTs. CNTs can be directly grown onto electrode surface, or ad‐ sorbed on it, or imbedded in polymer coating. In most cases, higher activity was reported for enzymes physically adsorbed onto CNTs [34]. Hydrophobic interactions between the en‐ zyme and the CNT walls and π-π interactions between side walls of CNTs and aromatic rings of the enzyme are thought to be the driving force for direct adsorption of enzymes on CNTs [35]. Electrostatic interaction between the defect sites of CNTs and protonated amino residues of the enzyme plays also a role in the adsorption process [35]. CNTs are quite easily functionalized, allowing covalent, thus stable specific attachment of enzymes. The oxidation in strong acidic solutions at high temperature was demonstrated to remove the end caps and shorten the lengths of the CNTs. The length of the CNTs was shown to be a function of the oxidation duration [36]. Acid treatment also adds oxide groups, primarily carboxylic acids, to the tube ends and defect sites [37]. The control of reactants and/or oxidation condi‐ tions may control the locations and density of the functional groups on the CNTs, which can be used to control the location and density of the attached enzymes [37]. Covalent immobili‐ zation is induced by carbodiimide reaction between the free amine groups on the enzyme surface and carboxylic groups generated by side wall oxidation of CNTs.

Further chemical reactions can be performed at the oxide groups generated on the oxidized CNTs to functionalize with groups such as amides, thiols, etc…From an electrochemical point of view, the side walls of CNTs were suggested to behave as basal plane of pyrolytic graphite, while their open ends resemble the edge planes [38, 39]. But recent work has dem‐ onstrated that the side wall may be responsible for electrochemical activity [40]. It has been

furthermore suggested that the uncovered surface of CNTs promotes the accessibility of the substrate to the enzyme [41]. It is also interesting to note that the open spaces between CNTs are accessible to large species such as entire bacteria [42], opening the way for the develop‐ ment of fuel cells using whole microorganisms instead of purified enzymes. The cost and complexity of CNT manufacturing seem to be still clogging issues in that field.

Abundant literature exists on the ways CNTs are architectured for efficient enzyme immobi‐ lization, including those specific for development of enzymatic fuel cells. Enzymes and pro‐ teins as various as glucose oxidase and dehydrogenase, tyrosinase, laccase and bilirubin oxidase, peroxidase, haemoglobin and myoglobin, *i.e.* flavin, copper or heme containing ac‐ tive sites, have been studied. Whereas direct electron transfer between protein or enzyme and an electrochemical interface has been for long time supposed to be restricted to small proteins (<15kDa) possessing active sites exposed to the surface (it is the case for many cyto‐ chromes as example [43]), the use of CNT-modified electrodes has greatly enhanced the number and kinds of enzymes able to be directly connected to an electrode. Enzymes as large as one hundred kDa, with many cofactors are now considered for direct electron trans‐ fer. Consequently, recent works during the last years focus and report on direct communica‐ tion between enzymes and electrode interface through CNT network. The induced porosity of the film depends on the type of CNTs used. But generally the nanometric size of the CNTs compared to the size of enzymes favors a direct electronic connection of the enzyme whatever its orientation [44]. The physical properties of CNTs, including high electrical con‐ ductivity, explain why CNT layers can be built up on electrodes most often yielding high rate direct electron transfer for enzymatic product transformation. Many researches report on the increase in electroactive surface area by use of CNT coatings that contribute to an in‐ crease in the direct electron transfer process [45-52]. CNTs are usually deposited on electro‐ des as thick films. Alternatively, layer-by-layer (LBL) process induces a quite stable protein film with nice electrocatalytic properties [53-55]. LBL is based on electrostatic interaction be‐ tween oppositely charged monolayers in an alternating assembling. Although CNTs greatly amplify the current response, layer-by-layer architecture suffers from weak stability of the build-up and decrease in electron transfer for the upper layers. Besides vertically aligned CNTs were suggested to act as molecular wires that ensure the electrical communication be‐ tween enzyme and electrode [56-58]. The carboxylic functions induced by acidic treatment of CNTs can be used for further chemical modifications. Amine- [59-61], thionin- [62, 63], di‐ azonium salts [64, 65], pyrene [66, 67] (Figure 3) or other π-π stacking interactions [68] were used to functionalize CNTs. These modifications were demonstrated to be efficient plat‐ forms for enzyme immobilization.

**Figure 3.** Schematic drawing of the build-up of enzyme on SWCNTs *via* π-π interactions.

Many enzymes however cannot be electrically connected to the electrode interface and require redox mediator to electrochemically follow substrate conversion. In that case, elec‐ trode kinetics is mainly dependant on mediator kinetics, so that the choice of the redox mediator mainly impedes the power density. Another issue is that the mediator can be coimmobilized with the enzyme at the electrode, while still being capable of efficient interac‐ tion with the enzyme. CNTs have also been used for building networks enabling coimmobilization of enzymes and redox mediators. In that way, one of the most popular redox entities is osmium polymer which forms hydrogels with enzymes allowing both charge transfer reaction between enzyme and mediators and diffusion of substrate and product [75]. Composite CNT/osmium films were used To immobilize bacteria [76], or enzymes [77]. By optimizing the CNT and polymer amounts, enhanced current responses were obtained linked to a promotion of the electron transfer within the composite. Various phenothia‐ zine derivatives were also used to form nanohybrids with CNTs acting as efficient redox mediator platforms [78-80]. Phenothiazine derivatives strongly adsorb onto CNTs leading to great enhancement of redox dye loading onto the electrode, but also to improved electro‐ chemical sensing devices. Another strategy involves the use of a redox polymer as redox mediator platform. Electropolymerization of the redox conducting polymer onto CNTs en‐ hances the amount of redox units and the electrical conductivity of the coating [81]. An

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Mixing CNTs with surfactant [69-71] was claimed to assist in the dispersion of CNTs while avoiding oxidative functionalization which may disrupt their π-network. Polymer modified CNTs [72, 73] and sol-gel-CNT nanocomposite films [74] were proved to behave as friendly platforms for enzyme encapsulation.

**Figure 3.** Schematic drawing of the build-up of enzyme on SWCNTs *via* π-π interactions.

furthermore suggested that the uncovered surface of CNTs promotes the accessibility of the substrate to the enzyme [41]. It is also interesting to note that the open spaces between CNTs are accessible to large species such as entire bacteria [42], opening the way for the develop‐ ment of fuel cells using whole microorganisms instead of purified enzymes. The cost and

Abundant literature exists on the ways CNTs are architectured for efficient enzyme immobi‐ lization, including those specific for development of enzymatic fuel cells. Enzymes and pro‐ teins as various as glucose oxidase and dehydrogenase, tyrosinase, laccase and bilirubin oxidase, peroxidase, haemoglobin and myoglobin, *i.e.* flavin, copper or heme containing ac‐ tive sites, have been studied. Whereas direct electron transfer between protein or enzyme and an electrochemical interface has been for long time supposed to be restricted to small proteins (<15kDa) possessing active sites exposed to the surface (it is the case for many cyto‐ chromes as example [43]), the use of CNT-modified electrodes has greatly enhanced the number and kinds of enzymes able to be directly connected to an electrode. Enzymes as large as one hundred kDa, with many cofactors are now considered for direct electron trans‐ fer. Consequently, recent works during the last years focus and report on direct communica‐ tion between enzymes and electrode interface through CNT network. The induced porosity of the film depends on the type of CNTs used. But generally the nanometric size of the CNTs compared to the size of enzymes favors a direct electronic connection of the enzyme whatever its orientation [44]. The physical properties of CNTs, including high electrical con‐ ductivity, explain why CNT layers can be built up on electrodes most often yielding high rate direct electron transfer for enzymatic product transformation. Many researches report on the increase in electroactive surface area by use of CNT coatings that contribute to an in‐ crease in the direct electron transfer process [45-52]. CNTs are usually deposited on electro‐ des as thick films. Alternatively, layer-by-layer (LBL) process induces a quite stable protein film with nice electrocatalytic properties [53-55]. LBL is based on electrostatic interaction be‐ tween oppositely charged monolayers in an alternating assembling. Although CNTs greatly amplify the current response, layer-by-layer architecture suffers from weak stability of the build-up and decrease in electron transfer for the upper layers. Besides vertically aligned CNTs were suggested to act as molecular wires that ensure the electrical communication be‐ tween enzyme and electrode [56-58]. The carboxylic functions induced by acidic treatment of CNTs can be used for further chemical modifications. Amine- [59-61], thionin- [62, 63], di‐ azonium salts [64, 65], pyrene [66, 67] (Figure 3) or other π-π stacking interactions [68] were used to functionalize CNTs. These modifications were demonstrated to be efficient plat‐

Mixing CNTs with surfactant [69-71] was claimed to assist in the dispersion of CNTs while avoiding oxidative functionalization which may disrupt their π-network. Polymer modified CNTs [72, 73] and sol-gel-CNT nanocomposite films [74] were proved to behave as friendly

complexity of CNT manufacturing seem to be still clogging issues in that field.

442 Syntheses and Applications of Carbon Nanotubes and Their Composites

forms for enzyme immobilization.

platforms for enzyme encapsulation.

Many enzymes however cannot be electrically connected to the electrode interface and require redox mediator to electrochemically follow substrate conversion. In that case, elec‐ trode kinetics is mainly dependant on mediator kinetics, so that the choice of the redox mediator mainly impedes the power density. Another issue is that the mediator can be coimmobilized with the enzyme at the electrode, while still being capable of efficient interac‐ tion with the enzyme. CNTs have also been used for building networks enabling coimmobilization of enzymes and redox mediators. In that way, one of the most popular redox entities is osmium polymer which forms hydrogels with enzymes allowing both charge transfer reaction between enzyme and mediators and diffusion of substrate and product [75]. Composite CNT/osmium films were used To immobilize bacteria [76], or enzymes [77]. By optimizing the CNT and polymer amounts, enhanced current responses were obtained linked to a promotion of the electron transfer within the composite. Various phenothia‐ zine derivatives were also used to form nanohybrids with CNTs acting as efficient redox mediator platforms [78-80]. Phenothiazine derivatives strongly adsorb onto CNTs leading to great enhancement of redox dye loading onto the electrode, but also to improved electro‐ chemical sensing devices. Another strategy involves the use of a redox polymer as redox mediator platform. Electropolymerization of the redox conducting polymer onto CNTs en‐ hances the amount of redox units and the electrical conductivity of the coating [81]. An interesting construction has also been obtained by immobilization of physiological cofac‐ tor onto CNT layers *via* π-π interactions, then immobilization of the enzyme [82]. The cova‐ lent coupling between the enzyme and its natural cofactor which was immobilized onto CNTs was proved to be efficient towards mediated substrate catalysis. This overview of multiple architectures involving enzymes and CNTs highlights the deep efforts engaged in the last years for efficient biocatalyst immobilization that open avenues towards biotechno‐ logical devices.

Grafting of hydrogenase onto gold electrode modified by thiolated Self-Assembled-Mono‐ layer [87] allowed efficient proton reduction into dihydrogen in aqueous buffer solutions. Hydrogenase is also considered as a promising biocatalyst for photobiological production of dihydrogen when coupled to a photocatalyst [88]. Hybrid complexes of hydrogenases with TiO2 nanoparticles [89, 90] were studied for H2 production. The optimized system was shown to produce H2 at a turnover frequency of approximately 50 (mol H2) s−1 (mol total hy‐ drogenase)−1 at pH 7 and 25 °C, even under the typical solar irradiation of a northern Euro‐ pean sky. Cd-based nanorods [91, 92] were recently studied. The CdS nanorod/hydrogenase complexes photocatalyzed reduction of protons to H2 at a hydrogenase turnover frequency of 380-900 s-1 and photon conversion efficiencies of up to 20% under illumination at 405 nm. Cd-based complexes allowed photoproduction of dihydrogen for a couple of hours, but still

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Although a very attractive way, little work has been done towards enhancement of green hydrogen production using CNTs. Three studies from the same group reported however catalytically active hydrogenase-SWCNT biohybrids [93, 94]. Surfactant-suspended SWCNTs were shown to spontaneously self-assemble with hydrogenase. Photolumines‐ cence excitation and Raman spectroscopy showed that SWCNTs act as molecular wires to make electrical contact with at least one of the FeS electron relay. Hydrogenase was demon‐

suffer from quick inhibition of hydrogenase.

**Figure 4.** Structure of an oxygen-tolerant [NiFe] hydrogenase.

#### **6. Carbon nanotubes for biological production of dihydrogen**

Apart from replacement of noble metal catalysts in fuel cells, a new green technology for production of dihydrogen is required. It currently relies on steam reforming of hydrocar‐ bons under high temperature and pressure conditions, which starts from fossil fuels, thus producing greenhouse gases. Dihydrogen production *via* water electrolysis appears as a re‐ newable solution given that the energy input comes from a renewable source, ideally solar energy. Many bacteria gain energy by the oxidation of dihydrogen assisted by a number of complex mechanisms. Various species evolve H2 under anaerobic conditions. This is also a human being process since bacteria in our digestive tract produce H2, though not detectable because immediately recycled by other bacteria. Photosynthetic organisms such as microal‐ gae and cyanobacteria are very efficient in water splitting [83]. They possess photosensitiz‐ ers for photon capture and charge separation, and enzymes for water oxidation to oxygen and water reduction to dihydrogen. This chemical activity relies on the expression of very efficient enzymes, called hydrogenases [84], which catalyze with high turn-over (one mole‐ cule of hydrogenase produces up to 9000 molecules of H2 per second at neutral pH and 37°C) and low overvoltage the conversion of protons into dihydrogen and the oxidation of dihydrogen. The sequences of 450 hydrogenases are now available. Hydrogenases differ in size, structure, electrons donors. They also differ by their position in the cell (soluble in the periplasm, membrane-bound), and by their activity preferentially towards H2 oxidation or protons reduction. Hydrogenase active site is composed of non noble metals such as iron and nickel, unlike platinum catalyst necessary for the chemical electrolysis of water. Three distinct classes can be split which differ from the type of metal content in the active site: [NiFe], [FeFe] and [Fe] hydrogenases. [NiFe] and [FeFe] hydrogenases possess dinuclear ac‐ tive centers which are connected through thiolate bridges. [NiFe] hydrogenase (Figure 4) is the most usual hydrogenase in microorganisms. It is composed of two subunits. The larger subunit harbors the [NiFe] active site. The small subunit contains FeS clusters. Electrons are transferred to the active site along these FeS clusters distant less than 10 Å that act as a con‐ ductive line. [FeFe] hydrogenases are monomeric. In addition to the active site they contain additional domains which accommodate FeS clusters.

In order to use these biocatalysts for green dihydrogen production, two main research do‐ mains are currently concerned: the understanding of the catalytic mechanisms of H2 produc‐ tion, and the optimization of enzyme immobilization. Adsorption onto graphite electrodes [85, 86] was largely used to study the mechanisms by which hydrogenases produce H2. Grafting of hydrogenase onto gold electrode modified by thiolated Self-Assembled-Mono‐ layer [87] allowed efficient proton reduction into dihydrogen in aqueous buffer solutions. Hydrogenase is also considered as a promising biocatalyst for photobiological production of dihydrogen when coupled to a photocatalyst [88]. Hybrid complexes of hydrogenases with TiO2 nanoparticles [89, 90] were studied for H2 production. The optimized system was shown to produce H2 at a turnover frequency of approximately 50 (mol H2) s−1 (mol total hy‐ drogenase)−1 at pH 7 and 25 °C, even under the typical solar irradiation of a northern Euro‐ pean sky. Cd-based nanorods [91, 92] were recently studied. The CdS nanorod/hydrogenase complexes photocatalyzed reduction of protons to H2 at a hydrogenase turnover frequency of 380-900 s-1 and photon conversion efficiencies of up to 20% under illumination at 405 nm. Cd-based complexes allowed photoproduction of dihydrogen for a couple of hours, but still suffer from quick inhibition of hydrogenase.

**Figure 4.** Structure of an oxygen-tolerant [NiFe] hydrogenase.

interesting construction has also been obtained by immobilization of physiological cofac‐ tor onto CNT layers *via* π-π interactions, then immobilization of the enzyme [82]. The cova‐ lent coupling between the enzyme and its natural cofactor which was immobilized onto CNTs was proved to be efficient towards mediated substrate catalysis. This overview of multiple architectures involving enzymes and CNTs highlights the deep efforts engaged in the last years for efficient biocatalyst immobilization that open avenues towards biotechno‐

Apart from replacement of noble metal catalysts in fuel cells, a new green technology for production of dihydrogen is required. It currently relies on steam reforming of hydrocar‐ bons under high temperature and pressure conditions, which starts from fossil fuels, thus producing greenhouse gases. Dihydrogen production *via* water electrolysis appears as a re‐ newable solution given that the energy input comes from a renewable source, ideally solar energy. Many bacteria gain energy by the oxidation of dihydrogen assisted by a number of complex mechanisms. Various species evolve H2 under anaerobic conditions. This is also a human being process since bacteria in our digestive tract produce H2, though not detectable because immediately recycled by other bacteria. Photosynthetic organisms such as microal‐ gae and cyanobacteria are very efficient in water splitting [83]. They possess photosensitiz‐ ers for photon capture and charge separation, and enzymes for water oxidation to oxygen and water reduction to dihydrogen. This chemical activity relies on the expression of very efficient enzymes, called hydrogenases [84], which catalyze with high turn-over (one mole‐ cule of hydrogenase produces up to 9000 molecules of H2 per second at neutral pH and 37°C) and low overvoltage the conversion of protons into dihydrogen and the oxidation of dihydrogen. The sequences of 450 hydrogenases are now available. Hydrogenases differ in size, structure, electrons donors. They also differ by their position in the cell (soluble in the periplasm, membrane-bound), and by their activity preferentially towards H2 oxidation or protons reduction. Hydrogenase active site is composed of non noble metals such as iron and nickel, unlike platinum catalyst necessary for the chemical electrolysis of water. Three distinct classes can be split which differ from the type of metal content in the active site: [NiFe], [FeFe] and [Fe] hydrogenases. [NiFe] and [FeFe] hydrogenases possess dinuclear ac‐ tive centers which are connected through thiolate bridges. [NiFe] hydrogenase (Figure 4) is the most usual hydrogenase in microorganisms. It is composed of two subunits. The larger subunit harbors the [NiFe] active site. The small subunit contains FeS clusters. Electrons are transferred to the active site along these FeS clusters distant less than 10 Å that act as a con‐ ductive line. [FeFe] hydrogenases are monomeric. In addition to the active site they contain

In order to use these biocatalysts for green dihydrogen production, two main research do‐ mains are currently concerned: the understanding of the catalytic mechanisms of H2 produc‐ tion, and the optimization of enzyme immobilization. Adsorption onto graphite electrodes [85, 86] was largely used to study the mechanisms by which hydrogenases produce H2.

**6. Carbon nanotubes for biological production of dihydrogen**

444 Syntheses and Applications of Carbon Nanotubes and Their Composites

additional domains which accommodate FeS clusters.

logical devices.

Although a very attractive way, little work has been done towards enhancement of green hydrogen production using CNTs. Three studies from the same group reported however catalytically active hydrogenase-SWCNT biohybrids [93, 94]. Surfactant-suspended SWCNTs were shown to spontaneously self-assemble with hydrogenase. Photolumines‐ cence excitation and Raman spectroscopy showed that SWCNTs act as molecular wires to make electrical contact with at least one of the FeS electron relay. Hydrogenase was demon‐ strated to be strongly attached to the SWCNTs and to mediate electron injection into nano‐ tubes. The displacement of the surfactant by hydrogenase to gain access to the SWCNTs was strongly suggested by photoluminescence studies. Furthermore, Raman studies of charge transfer complexes between hydrogenase and either metallic (m) or semiconducting (s) SWCNTs revealed a difference in oxygen deactivation of hydrogenase according to the SWCNT species. m-SWCNTs most probably interact with hydrogenase to produce a more oxygen-tolerant species. The study further suggested that purified m-SWCNTs or s-SWCNTs, rather than mixed preparation, would be more suitable for hydrogenase-SWCNTs biohybrids. The formation of these catalytically active biohybrids in addition with the intrin‐ sic properties developed by CNT networks on electrodes certainly accounts for the im‐ proved dihydrogen production observed in the following studies. Kihara *et al.* immobilized hydrogenase on a SWCNT-forest with a unique dense structure of vertically aligned milli‐ metre-scale height SWCNTs [95]. Hydrogenase was demonstrated to spontaneously assem‐ ble between adjacent nanotubes. The maximum rate of dihydrogen production was reported to be 720 nmol/min/(mg hydrogenase) and the electron transfer efficiency was estimated to be 32%. It is two thousand fold higher than reported before using the same hydrogenase on Langmuir-Blodgett film [96]. Nevertheless, one key point in the development of biotechno‐ logical devices is the long term stability of enzymes. If these biological catalysts are very effi‐ cient *in vivo*, they often suffer from weak stability when extracted from their physiological environment. Enzyme encapsulation in silica-derived sol-gel materials has been demonstrat‐ ed to stabilize many enzymes. This procedure was applied to hydrogenase [97]. The majori‐ ty of hydrogenase was shown to be entrapped in the gel and protected against proteolysis. Hydrogenase/sol-gel pellets retained 60% of the specific mediated activity for H2 production displayed by hydrogenase in solution. The gel-encapsulated enzyme retained its activity for long periods, *i.e.* 80% of the activity after four weeks at room temperature. Notably, by dop‐ ing the hydrogenase-containing sol-gel materials with MWCNTs Zadvorny *et al.* demon‐ strated a 50% increase in dihydrogen production [98]. Furthermore stabilization of hydrogenase was proved through encapsulation process.

**7. Carbon nanotubes for biofuel cells: an attractive green alternative**

tions across the cell membrane exist.

**Figure 5.** Schematic representation of an enzymatic biofuel cell.

Beside researches towards decrease in chemical catalyst amount and discovery of less ex‐ pensive catalysts (as alloys for example), a new concept emerged early in 1964 by Yahiro *et al.* [102]. A fuel cell was constructed using usual O2 reduction at platinum modified elec‐ trode in the cell cathodic compartment, but using glucose as a fuel in the anodic compart‐ ment. The innovative idea was the use of an enzyme specific for fuel oxidation instead of platinum. For glucose oxidation, glucose oxidase was tested as the anodic catalyst. The fuel cell delivered 30 nA cm-2 at 330 mV…a very low power density indeed but the proof of con‐ cept of biofuel cell was born. Generally speaking these biofuel cells function as fuel cells but used enzymes instead of noble metals as catalysts (Figure 5). They are referred as enzymatic biofuel cells. Microorganisms can also be used as catalysts, defining microbial fuel cells. Mi‐ crobial biofuel cells use the metabolism of microorganisms under anaerobic conditions to oxidize fuel [103-104]. Although a promising concept, little is known yet about the mecha‐ nisms by which fuel is oxidized at the anode. The involvement of nanowires, electron trans‐ fer mediators, either membrane-bound or excreted, is supposed to be responsible for the cell current. Enzymatic biofuel cells are however more efficient because no mass transfer limita‐

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The advantages of enzymatic biofuel cells over fuel cells are multiple. Biocatalysts are wide‐ spread, then *a priori* inexpensive, and biodegradable. Enzymes are highly efficient and spe‐ cific to their substrates. The substrate specificity decreases reactant cross-over, and might theoretically allow to design fuel cells with no membrane between the anodic and cathodic compartments. Both costs are reduced and the design is simplified. A large variety of fuels and oxidants can be used to feed the biofuel cells, as opposed to the poor available fuels and oxidant in classical fuel cells (dihydrogen, methanol, oxygen). Indeed, many enzymes are nowadays characterized which differ by their natural abundant substrates. Dihydrogen, but also various inexpensive sugars can thus be used as efficient fuels at the anode. Further‐ more, the involvement of cascades of enzymes can enhance the cell performance because of the summation of the electrons from each enzymatic reaction [105]. Finally, biofuel cells can

One alternative for green hydrogen production is to synthesize metal complexes that mimic the active site of enzymes. Huge work has been done in that field in order to obtain bioins‐ pired models that could produce H2 as efficiently as hydrogenase, while being much more stable [99]. The most performing complex involves mononuclear nickel diphosphine com‐ plex. This complex is inspired from the active sites of both [NiFe] and [FeFe] hydrogenases and displays remarkable catalytic proton reduction in organic solvent [100]. Le Goff *et al.* took benefice from this complex and from the results obtained by immobilization of hydro‐ genase on CNT networks [44]. The authors successfully immobilized the nickel complex on‐ to carbon nanotube networks by covalent coupling [101]. Such construction was demonstrated to be very efficient for dihydrogen production in aqueous solution, evolving dihydrogen with overvoltage less than 20 mV and exceptional stability.
