Direct electron transfer to hydrogenase for catalytic hydrogen production using a single-walled carbon nanotube forest

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Abstract

The production of hydrogen through the direct electron transfer from electrode to hydrogenase was successfully performed using hydrogenase combined with a single-walled carbon nanotube forest (SWNT-forest). The SWNT-forest has a unique structure comprising dense and vertically aligned SWNTs with millimeter-scale height. The vertically aligned SWNTs became rearranged into a wall-like architecture after immersing in water. Hydrogenase was spontaneously incorporated within the SWNT-forest and confined on the sidewall of the rearranged architecture. The SWNT-forest was a very stable protein carrier in aqueous solution, and an SWNT-forest integrated with hydrogenase could easily be constructed. With a hydrogenase assembled SWNT-forest we were able to electrochemically produce hydrogen with an electron transfer efficiency exceeding 30% without the use of any chemical mediator. Furthermore, a reverse oxidative reaction of hydrogen was also successfully performed using the same device. Thus, the SWNT-forest can work as an effective and direct electron mediator for the oxidation/reduction reaction of hydrogenase. The usefulness of the SWNT-forest as a beneficial material for electrochemistry was demonstrated, underlining the potential of protein assembled SWNT-forests in fabricating highly efficient biofuel cell devices.

Introduction

Hydrogen as a source of energy has been attracting much public attention as a new resource for overcoming energy shortages and reducing environmental pollution [1]. Therefore, many researchers in several different fields have focused their work on the production of hydrogen. One such particularly challenging field is biological hydrogen production [2], [3]. Efficient biological hydrogen production can be used to partially replace the current methods. Hydrogenase is a key enzyme in hydrogen metabolism in many microorganisms and catalyzes the reversible reaction of hydrogen and protons (H2 ↔ 2H+ + 2e). The immobilization of hydrogenase on electrodes is of great interest as it has potential applications for the development of biocatalysts for water electrolysis and fuel cells [4], [5], [6], [7]. The direct electron transfer of hydrogenase adsorbed on electrodes for hydrogen oxidation has been reported [8], [9], [10], [11]. Although hydrogenase isolated from Thiocapsa roseopersicina has attracted a great deal of attention for practical applications because of its high thermal stability and oxygen tolerance, it requires an appropriate electron mediator, such as ferredoxin, cytochrome c3, or methyl viologen (MV), on the electrode to catalyze the proton to hydrogen [12], [13]. Thus, studies of the direct electron transfer from electrodes to the hydrogenase of T. roseopersicina are important, as they will help us to improve biocatalyst systems using hydrogenase.

Carbon nanotubes have excellent electrical conductivity, thermal conductivity, and mechanical strength and have emerged as versatile materials in nanobiotechnology [14]. Owing to these properties, carbon nanotubes are interesting candidates for the electrodes to which enzymes can be attached. In particular, it has been demonstrated that single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) are excellent surface modifiers for increasing the catalytic current density as well as the stability of hydrogenase [15], [16], [17], [18]. Moreover, randomly shortened SWNTs have been immobilized on gold surfaces to form needle-like and forest-like electrodes with excellent electrochemical behavior [19], and oxidatively shortened SWNTs have been constructed on Nafion/iron oxide-coated pyrolytic graphite electrodes forming an SWNT-forest electrode for use as an enzyme biosensor [20]. These reports suggest that forest-like or needle-like SWNT structures have the possibility of being employed as electrodes in biocatalyst systems and fuel cells using hydrogenase.

Hata and colleagues successfully synthesized dense and vertically aligned SWNTs with heights on the millimeter-scale [21]. This bulk SWNT material (SWNT-forest) retains the intrinsic properties of individual SWNTs, such as the high surface area, flexibility, and high electrical conductivity [22]. Thus, the SWNT-forest might be a useful electrode material for use in hydrogen production systems using the hydrogenase of T. roseopersicina. In this study, we constructed a hydrogenase assembled SWNT-forest, with which we attempted to develop a high performance electrochemical hydrogen production system (Fig. 1A). When the SWNT-forest was immersed in aqueous solution, the SWNTs became rearranged to form a wall-like architecture, in which the hydrogenase molecules were adsorbed and confined in a stable manner. We demonstrated highly efficient hydrogen production without a chemical electron mediator using the hydrogenase assembled SWNT-forest on a GC electrode. This is the first report showing that an SWNT-forest functions as a direct and efficient electron transfer material with hydrogenase.

Section snippets

Materials

An SWNT-forest (5 mm × 5 mm size and about 500 μm height) was synthesized using a method reported previously [21]. Hydrogenase was purified from purple sulfur bacterium T. roseopersicina as described previously [23]. The hydrogen oxidation activity of the hydrogenase was about 110 unit/mg-protein and 1 unit of activity is defined as the reduction of 1 mmol MV2+/min to MV+ in hydrogen-saturated solution (100 mM Tris–HCl buffer, pH 9.0) at 30 °C. Cytochrome c (cyt c) from the heart of a horse was

Affinity of SWNT-forest for solvent

Firstly, we investigated the SWNT-forest for enzymatic electrochemical experiments. The SWNT-forest was removed from the silicon wafer on which it was grown and immobilized on an electrode surface. The forests shrank in the organic solvent and ionic liquid (data not shown), and these shrunken forests could not be re-swollen. It was only in aqueous solution that the bulk SWNT-forest did not shrink (data not shown). Thus, these can be used as an electrochemical device for enzymes in aqueous

Conclusions

In this study, we succeeded in developing a highly efficient biofuel cell device for producing hydrogen using hydrogenase as a biocatalyst and an SWNT-forest as the electrode material without the use of any mediators. This is the first report on the production of hydrogen using hydrogenase without a chemical mediator. The vertically aligned SWNTs became rearranged to form a wall-like architecture in aqueous solution and proteins were stably confined within the SWNT walls. The confined

Acknowledgments

This work was supported by the foundation of AIST.

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