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

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

446 Syntheses and Applications of Carbon Nanotubes and Their Composites

hydrogenase was proved through encapsulation process.

dihydrogen with overvoltage less than 20 mV and exceptional stability.

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

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‐ tions across the cell membrane exist.

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

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 deliver power under soft working conditions, as enzymes usually perform their enzymatic reactions at mild pH and temperature. Nevertheless, some extremophilic enzymes operate in extreme acidic or basic pH, as well as at high temperatures (around 90°C) or high pres‐ sure, offering the possibility to develop biofuel cell devices for special applications requiring extreme working conditions [106]. The applications of biofuel cells are still in their infancy. They are mainly thought to power small portable devices. Remarkable progress has been re‐ ported for implantable biofuel cells during the last year to power drug pumps, glucose sen‐ sors, vision devices [107-109].

Gox at electrode interfaces is still controversial. Due to the peculiar structure of Gox, a dimer with flavin adenine dinucleotide active site buried within a thick and isolated protein shell, it is understandable that electrical connection of Gox could be unexpected. A recent work concluded that CNTs were capable to electrically connect Gox, but this connection was un‐

Carbon Nanotube-Enzyme Biohybrids in a Green Hydrogen Economy

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

449

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

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‐

fruitful for glucose catalytic oxidation [110].

**cells**

The most common redox couple that has been used in biofuel cells is sugar/O2, essentially because of sugar and O2 abundance in nature and their essential role in living metabolism. In particular, glucose is an important metabolite and a source of energy for many living or‐ ganisms. In that field, CNTs have been widely used, both at the anode and cathode. Glu‐ cose/O2 biofuel cell is thus a very pertinent investigation field to investigate the role of CNTs. A view of some typical results is presented in Table 2.


Gox: Glucose oxidase; GDH: Glucose dehydrogenase; BOD: Bilirubin oxidase; ABTS: 2, 2'-azino-bis(3-ethylbenzothia‐ zoline-6-sulfonate) diammonium; CDH: cellobiose dehydrogenase.

#### **Table 2.** Performances of glucose/O2 fuel cells.

Data highlight that kinetics of bioelectrochemical reactions, thus power density, largely de‐ pends on the experimental conditions, *i.e.* enzyme and mediators, T°, pH, concentration of substrate, electrolyte and type of electrode construction. Highest values are obtained with mediatorless fuel cells, reaching power densities upper than 1 mW cm-2 which is sufficient to power small electrical devices. It appears that direct connection of copper enzymes, namely laccase or BOD, for oxygen reduction at the cathode can be quite easily obtained with the help of CNT network. Direct connection of enzymes for glucose oxidation is conversely hardly observed, even on CNT coatings. From literature examination direct connection of Gox at electrode interfaces is still controversial. Due to the peculiar structure of Gox, a dimer with flavin adenine dinucleotide active site buried within a thick and isolated protein shell, it is understandable that electrical connection of Gox could be unexpected. A recent work concluded that CNTs were capable to electrically connect Gox, but this connection was un‐ fruitful for glucose catalytic oxidation [110].

deliver power under soft working conditions, as enzymes usually perform their enzymatic reactions at mild pH and temperature. Nevertheless, some extremophilic enzymes operate in extreme acidic or basic pH, as well as at high temperatures (around 90°C) or high pres‐ sure, offering the possibility to develop biofuel cell devices for special applications requiring extreme working conditions [106]. The applications of biofuel cells are still in their infancy. They are mainly thought to power small portable devices. Remarkable progress has been re‐ ported for implantable biofuel cells during the last year to power drug pumps, glucose sen‐

The most common redox couple that has been used in biofuel cells is sugar/O2, essentially because of sugar and O2 abundance in nature and their essential role in living metabolism. In particular, glucose is an important metabolite and a source of energy for many living or‐ ganisms. In that field, CNTs have been widely used, both at the anode and cathode. Glu‐ cose/O2 biofuel cell is thus a very pertinent investigation field to investigate the role of

> **Mediators Anode / Cathode**

Gox / Laccase Ferrocene / - 15 [111] GDH / BOD PQQ / - 23 [82] Gox / Pt Ferrocenecarboxaldehyde / - 51 [112] GDH/ BOD Poly(brilliant cresyl blue) / - 54 [113] GDH / laccase Azine dies / - 58 [114] Gox / Pt Benzoquinone / - 77 [52] Gox / Laccase Ferrocene / ABTS 100 [115] Gox / BOD Ferrocene methanol / ABTS 120 [116] GDH / Laccase - / - 131 [117] CDH / Pt Os complex / - 157 [118] Gox / Laccase - / - 1300 [119]

Gox: Glucose oxidase; GDH: Glucose dehydrogenase; BOD: Bilirubin oxidase; ABTS: 2, 2'-azino-bis(3-ethylbenzothia‐

Data highlight that kinetics of bioelectrochemical reactions, thus power density, largely de‐ pends on the experimental conditions, *i.e.* enzyme and mediators, T°, pH, concentration of substrate, electrolyte and type of electrode construction. Highest values are obtained with mediatorless fuel cells, reaching power densities upper than 1 mW cm-2 which is sufficient to power small electrical devices. It appears that direct connection of copper enzymes, namely laccase or BOD, for oxygen reduction at the cathode can be quite easily obtained with the help of CNT network. Direct connection of enzymes for glucose oxidation is conversely hardly observed, even on CNT coatings. From literature examination direct connection of

**Power density**

**µW cm-2 Ref**

CNTs. A view of some typical results is presented in Table 2.

448 Syntheses and Applications of Carbon Nanotubes and Their Composites

zoline-6-sulfonate) diammonium; CDH: cellobiose dehydrogenase.

**Table 2.** Performances of glucose/O2 fuel cells.

sors, vision devices [107-109].

**Enzymes Anode / Cathode**
