Exploring Properties of a Hyperthermophilic Membrane-Bound Hydrogenase at Carbon Nanotube Modified Electrodes for a Powerful H 2 /O 2 Biofuel Cell Anne De Poulpiquet, a Alexandre Ciaccafava, a Katarzyna Szot, b Baptiste Pillain, a Pascale Infossi, a Marianne Guiral, a Marcin Opallo, b Marie-ThØrse Giudici-Orticoni, a Elisabeth Lojou* a a UnitØ de BioØnergØtique et IngØnierie des ProtØines, UMR7281-FR3479, CNRS, AMU, Marseille, France tel: + 33 491164524, fax: + 33 491164097 b Institute of Physical Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224 Warszawa, Poland *e-mail: lojou@imm.cnrs.fr Received: July 27, 2012 Accepted: November 6, 2012 Published online: January 15, 2013 Abstract Hydrogenases are the key enzymes for hydrogen metabolism in many microorganisms. Due to the high efficiency and specificity they developed for H 2 oxidation, they have been recently used as biocatalysts for an efficient H 2 /O 2 biofuel cell. In this work we explore new properties of an O 2 -, CO- and T8-resistant hydrogenase. Enzyme immobili- zation on carbon nanotube modified electrodes is studied and optimized for long-term stabilization of the direct hy- drogen catalytic oxidation process. The role of co-immobilized redox mediator is finally discussed in view of H 2 /O 2 fuel cell performance enhancement. Keywords: Hydrogenase, Carbon nanotube, Biofuel cell, Immobilization, Electrochemistry DOI: 10.1002/elan.201200405 Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/elan.201200405. 1 Introduction Microorganisms couple enzymatic reactions via a mem- brane quinone pool with generation of a proton gradient for the synthesis of ATP, their energy source [1,2]. This energetic pathway can be considered as an “in vivo fuel cell”, and led to the concept of biofuel cells as a new val- uable source for our energy needs. Biofuel cells function like chemical low temperature fuel cells (proton exchange membrane (PEM) fuel cell), coupling the oxidation of a fuel at the anode with the reduction of an oxidant at the cathode, but using enzymes instead of expensive and scarce platinum catalysts [3]. There are numerous advan- tages of enzymatic biofuel cells. Biocatalysts are wide- spread and biodegradable. They exhibit high efficiency and specificity for their substrates that might allow de- signing fuel cells without separator. A large variety of fuels and oxidants can be used as many enzymes which differ by their natural abundant substrates are nowadays characterized. Finally, biofuel cells can deliver power under mild working conditions typical for enzymes, namely at mild pH and temperatures. However, some ex- tremophilic enzymes operate at extreme acidic or basic pH, as well as at high temperatures (up to 90 8C) or high pressures, offering the possibility of developing biofuel cell devices for special applications requiring extreme working conditions [4]. So far sugar/O 2 is the most common fuel/oxidant couple to power biofuel cells, essentially because of its abundance in nature and essential role in living metabo- lism [5–7]. Glucose oxidase and copper proteins, such as laccase and bilirubin oxidase (BOD), were largely studied as biocatalysts for glucose oxidation and O 2 reduction, re- spectively. All over the last ten years, thanks to great re- search in engineering of enzymes, electrochemical interfa- ces and cell design, power densities as high as 1 mW cm À2 were recently reached [8–10]. The step for biofuel cell credibility was jumped and some emerging applications are nowadays reported [11–13]. A few years ago, a new type of biofuel cells, based on an enzyme specific for dihydrogen oxidation, the hydro- genase, was developed [14]. This H 2 /O 2 biofuel cell is di- rectly inspired from PEM fuel cell, and is expected to yield the highest massic energy output. Various types of hydrogenases are available, differing by the metal content of their active site, their activity towards H 2 uptake or evolution, and their localization in bacterial cells. All [NiFe] hydrogenases from the group 1 [15] share a similar structure, composed of two subunits with a molecular weight around 100 kDa. The large subunit harbors the active [NiFe] centre where dihydrogen is cleaved. In the TOPICAL CLUSTER Electroanalysis 2013, 25, No. 3, 685 – 695  2013 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 685 Full Paper