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Cite this: Chem. Commun., 2015, 51, 7447
Published on 27 March 2015. Downloaded on 21/04/2015 07:50:48.
Received 13th March 2015, Accepted 27th March 2015 DOI: 10.1039/c5cc02166a
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A membraneless air-breathing hydrogen biofuel cell based on direct wiring of thermostable enzymes on carbon nanotube electrodes† ab c ab ab Noe ´mie Lalaoui, Anne de Poulpiquet, Raoudha Haddad, Alan Le Goff,* ab de fg c Michael Holzinger, Se ´bastien Gounel, Michel Mermoux, Pascale Infossi, de c ab Nicolas Mano,* Elisabeth Lojou* and Serge Cosnier
www.rsc.org/chemcomm
A biocathode was designed by the modification of a carbon nanotube (CNT) gas-diffusion electrode with bilirubin oxidase from Bacillus pumilus, achieving high current densities up to 3 mA cm
2
for the
reduction of O2 from air. A membraneless air-breathing hydrogen biofuel cell was designed by combination of this cathode with a functionalized CNT-based hydrogenase anode.
Enzymatic biofuel cells represent a subcategory of fuel cells that propose a renewable alternative to noble-metal-catalyst-based fuel cells.1–4 Enzymes offer high catalytic turnovers associated with high substrate specificity and low overvoltages. Furthermore, exploring the biodiversity allows the identification of enzymes exhibiting peculiar properties, such as resistance to extreme pHs and temperatures or typical inhibitors. These advantages make enzymes a family of biocatalysts that can operate in complex media such as physiological fluids or extreme environments.5 Since enzymes are relatively large catalysts, often bearing a complex intramolecular electron transfer chain, electrodes have to be manufactured to efficiently transfer electrons to the enzyme active site, while providing a large specific area for optimal biocatalyst surface coverage.6 In this respect, carbon nanotube networks have proven to offer ideal properties for efficient direct electron transfer (DET) to the enzyme active site, high conductivity and high specific surface.7 Hydrogen biofuel cells rely on hydrogenases to efficiently oxidize H2 and multicopper oxidases to reduce oxygen.8–12 These enzymes have already shown similar catalytic properties compared to conventional platinum-based electrodes.13 However, to a
Univ. Grenoble Alpes, DCM UMR 5250, F-38000 Grenoble, France. E-mail: alan.le-goff@ujf-grenoble.fr b CNRS, DCM UMR 5250, F-38000 Grenoble, France c BIP, UMR7281 CNRS-AMU, F-13009 Marseille, France. E-mail: [email protected] d CNRS, CRPP, UPR 8641, Pessac, France. E-mail: [email protected] e Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France f Univ. Grenoble Alpes, LEPMI UMR 5279, F-38000 Grenoble, France g CNRS, LEPMI UMR 5279, F-38000 Grenoble, France † Electronic supplementary information (ESI) available: Experimental section and Fig. S1 to S3. See DOI: 10.1039/c5cc02166a
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be able to run efficiently in fuel cell set-ups, some drawbacks have to be overcome. One major issue for hydrogen fuel cell applications has been the sensitivity of hydrogenases towards oxygen. Several strategies have been employed to circumvent this issue in the design of biofuel cells. Mutagenesis have led to the increase in tolerance towards oxygen.14 Protective polymers have also been used to prevent oxygen deactivation of hydrogenases.12,15,16 We have recently developed the direct wiring of the membrane-bound hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. This hydrogenase is particularly tolerant towards oxygen, showing excellent electrocatalytic H2 oxidation in the presence of oxygen over a large range of temperatures.17 It was demonstrated that the rate of reactivation of this hydrogenase was much faster than the rate of inactivation by O2 because of a peculiar geometry of a FeS cluster closed to the active site. A biofuel cell based on this enzyme at the anode and a thermostable BOD at the cathode was designed so that these enzymes worked in separated compartments, in optimal conditions for respective enzymes.18 All hydrogenase-based biofuel cell set-ups have employed restrictive and specific conditions to favorably operate hydrogenases: nonexplosive H2-rich–air-low gas mixture,10,11 bioelectrode compartmentalization8,18 or use of oxygen-protecting redox polymers.12 Recent findings have shown that enzymes such as BOD and laccase could efficiently operate in air-breathing systems, especially developed in fuel cell technology.19,20 These electrodes have the great advantages to circumvent issues with gas solubility and diffusion in the electrolyte by providing the enzyme substrate through the gaseous phase. Here we report a novel strategy, which has been unexplored yet, for protecting hydrogenases from oxygen by the design of an air-breathing cathode based on the direct wiring of the thermostable BOD from Bacillus pumilus on carbon nanotube based electrodes. Oxygen from air is reduced at the cathode while the anode operates in H2 saturated aqueous buffer solution. This system could have important advantages such as (i) allowing oxygen-sensitive hydrogenases to work in anaerobic conditions, (ii) allowing the biofuel cell to work in non-explosive H2–O2 mixtures and (iii) avoid any oxygen reduction at the bioanode.
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Fig. 1 (A) Image of the MWCNT film deposited on carbon cloth obtained by laser confocal microscopy; (B) SEM image of a MWCNT film; (C) airbreathing electrode based on the deposition of MWCNTs and BOD on carbon cloth; (D) CV of MWCNT-based air-breathing electrode under argon (dashed line), under air (full line) and under oxygen (dotted line) (10 mV s 1, deaerated phosphate buffer pH 7.2, 45 1C).
For the MWCNT-based biocathode, pristine MWCNTs were deposited by drop-coating on a carbon cloth electrode. Fig. 1A and B show the morphology of a 40 mm-thick MWCNT film deposited on carbon cloth, investigated by confocal laser and SEM microscopy. These images underline the deposition of a homogenous MWCNT film on carbon cloth electrodes. After adsorption of BOD, the biocathode was integrated in an air-breathing chamber (Fig. 1C). Argon was bubbled in the electrolyte to avoid any contribution of dissolved oxygen to the catalytic current. CV scans are displayed in Fig. 1D. High catalytic current are accompanied with onset potentials of 0.4 V, confirming efficient direct wiring of BOD and efficient oxygen reduction catalysis under air-breathing mode. Electrocatalytic currents of up to 3 mA cm 2 at 0 V vs. Ag/AgCl in quiescent conditions were measured at pH 7.2 and 45 1C. By investigating the effect of the MWCNT film thickness by laser scanning microscopy, we did not observe any increase or decrease of the catalytic current between 4- and 40 mm-thick MWCNT films, underlining the excellent DET with the adsorbed BOD.
Fig. 2 (A) CV of MbH1 adsorbed on MWCNT and f-MWCNT GC electrodes (v = 5 mV s 1, pH 7.2, 60 1C, under H2); (B) CV of MbH1 for different f-MWCNT film thickness of 5, 7, 10, 18 mm (v = 5 mV s 1, pH 7.2, 60 1C, under H2, background was subtracted to remove capacitive current contribution).
7448 | Chem. Commun., 2015, 51, 7447--7450
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It is noteworthy that replacing air flux by a pure oxygen flux has negligible effect on the electrocatalytic current (Fig. 1D). This demonstrates the excellent diffusion of oxygen to the BOD providing optimal oxygen concentrations for the enzyme. The resulting biocathodes present higher performances, namely, current density of 3.0(+/ 0.2) mA cm 2 under air, than previously-developed highly-efficient breathing cathodes by P. Atanassov and coworkers20,21 or K. Kano and coworkers.22,23 Pristine CNTs were compared with modified CNTs. SWCNTs and MWCNTs were modified with amino-naphthoic acid, affording f-SWCNTs and f-MWCNTs, using a previouslydeveloped procedure based on the in situ generation of an aryl diazonium salt.24 Reductive behavior of CNT sidewalls triggers the formation of highly-reactive aryl radicals, forming a polyphenylene layer surrounding the CNTs. The main advantage of using diazonium-based functionalization is that high surface coverage of carboxylate groups can be grafted, thanks to the high reactivity of in situ aryl radicals towards CNT sidewalls.24 The presence of the naphthoic-acid groups was evidence by infra-red spectrum, showing the appearance of an intense band at 1715 cm 1 corresponding to CQO bonds of carboxylated groups and a broad band at 3000 cm 1 corresponding to C–H bonds (Fig. S1A, ESI†). The Raman spectra of f-SWCNT are shown in Fig. S1B (ESI†). The excitation wavelength was the 514.5 nm line from an argon ion laser. A noticeable increase is observed in the relative intensity of the D-band at 1350 cm 1 compared to the G-band at about 1590 cm 1 after aryldiazonium functionalization process. The D/G ratio is equal to 0.14 for SWCNT and 0.44 for f-SWCNT. This increase is associated with the formation of defects that break the translational symmetry along the SWNT sidewalls by the covalent addition of aryl radicals, associated with the attachment of naphtoic carboxylate groups. Furthermore, thanks to efficient sidewall functionalization with hydrophilic groups, both f-SWCNT and f-MWCNT exhibit excellent solubility in water of 12 and 37 mg mL 1 respectively (Fig. S1C, ESI†). Naphthoic-acid-modified electrodes have already been used as a DET promoter for the wiring of BOD from Myrothecium verrucaria.25 However, poor DET was observed for BOD from B. pumilus with f-MWCNTs and f-SWCNTs compared to pristine MWCNTs. Maximum current densities of 0.84 mA cm 2 and 0.10 mA cm 2 at 0 V were measured for f-SWCNT and f-MWCNT respectively (Fig. S2A, ESI†). This behavior is likely caused by the presence of hydrophobic domains located at the surface of the BOD, favoring hydrophobic interactions with pristine MWCNT sidewalls. On f-CNTs, the strong dipole moment pointing at the opposite of the T1 copper centre likely hinders the favorable orientation of the BOD towards the T1 copper centre on negatively-charged f-CNTs.18 On the contrary to BOD from B. pumilus, f-MWCNT and f-SWCNT were chosen because of the ability of carboxylate groups to enhance DET of MbH1 on carbon nanotubes.26 CV of the bioanodes based on MWCNT and f-MWCNT performed under H2 bubbling are displayed in Fig. 2A. Under H2, a catalytic current is observed with an onset potential of 0.59 V vs. Ag/AgCl accompanied with a high catalytic bias in favor of hydrogen oxidation. This is in good
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agreement with previous electrochemical studies of MbH1 on oxidized SWCNTs and carbon nanofibers, underlining that this hydrogenase can afford both hydrophobic and hydrophilic surroundings.27 Interestingly, the shape of both CV from Fig. 2A are different. The DET process on pristine MWCNT film exhibits a classic twisted shape of the CV, which is generally observed for this enzyme on PG electrode or oxidized SWCNT electrodes. This shape is caused by a reversible deactivation of hydrogenases at high overpotential.17 For f-MWCNT, a plateau shape similar to the one observed at carbon nanofibers electrode27 is associated with high maximum current densities of 2.7(+/ 0.2) mA cm 2. This plateau current arises from a high number of enzymes participating to the catalytic current that causes a mass-transport-controlled electrocatalytic current. It has been shown recently that reversible deactivation of hydrogenases at high potential could also be minimized by using redox polymers12 and gas-diffusion electrodes.28,29 These experiments demonstrate the excellent wiring properties of f-MWCNTs for MbH1 compared to pristine MWCNTs. In order to optimize the wired MbH1 surface coverage, we have studied the influence of the f-MWCNT film thickness over the electrocatalytic current for H2 oxidation (Fig. 2B and Fig. S3, ESI†). The CV curves in Fig. 2A and B show the maximum electrocatalytic current for f-MWCNT film thicknesses above 10 mm, reaching 2.7 mA cm 2 for hydrogen oxidation at pH 7.2 at 0 V vs. Ag/AgCl. Since the amount of deposited hydrogenases (20 mL at 5 mM) was kept constant, these experiments show that upon increasing the thickness, the number of enzymes participating to the catalytic oxidation of H2 is increased (Fig. 2B and Fig. S3, ESI†). For pristine MWCNT, a maximum catalytic current densities of 1.4 mA cm 2 was measured, accompanied with deactivation process above 0.3 V (Fig. 2A). f-SWCNT also exhibits excellent DET for MbH1, reaching 1.8 mA cm 2 at 0.25 V (Fig. S2B, ESI†). This intermediary catalytic current density, between MWCNT and f-MWCNT performances, likely arises from the hydrophobic nature of SWCNT,16 corroborated by lower solubility in water, which might counterbalance electrostatic interactions of MbH1 with carboxylate groups. For the membraneless hydrogen biofuel cell set-up, the GC/MWCNT/MbH1 electrode was connected in a two-electrode system in front of the gas-diffusion biocathode. Fig. 3A shows the schematic representation of the membraneless biofuel cell set-up. Bubbling of hydrogen prevents the bioanode from any contact with oxygen from air diffusion through the cathode. Fig. 3B shows the CV scans for the bioanode and the biocathode in this set-up. The biofuel cell was operated at 45 1C for technical and safety reasons. The bioanode is approximatively three-time less active at 451 than at 601 leading to maximum current densities of 1.4(+/ 0.1) mA cm 2. Fig. 3C displays the polarization curve and the power curve performed by successive potentiostatic discharges of 30 s. The non-classical polarization curve shape at low voltages is caused by hydrogenase deactivation, which is triggered by low-potential discharge and limiting behavior of the bioanode. Under bubbling of H2 at 45 1C, maximum power density of 0.72(+/ 0.04) mW cm 2 is obtained at 0.6 V with an
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Fig. 3 (A) Membraneless air-breathing hydrogen biofuel cell; (B) CV of a MWCNT/MbH1 bioanode (v = 5 mV s 1) and a MWCNT/BOD biocathode (v = 10 mV s 1, pH 7.2, 45 1C); (C) power curves and polarization curves of the hydrogen biofuel cell for MWCNT/MbH1 and f-MWCNT/MbH1 bioanode (pH 7.2, 45 1C, an error of 6% was estimated from the performances measured for two biofuel cells); (D) 1000 s potentiostatic discharge (0.8 V) of the biofuel cell based on the f-MWCNT/MbH1 bioanode.
OCV of 0.95 V. Power performances were also measured for pristine MWCNT bioanodes. As expected from lower maximum current densities for pristine MWCNT bioanodes, the maximum power density only reaches 0.34 mW cm 2. A 15 min discharge at a fixed voltage of 0.8 V for the f-MWCNT-based biofuel cell, displayed in Fig. 3D, underlines the excellent operating stability upon discharge of the biofuel cell. We report a novel and unexplored strategy for the elaboration of an efficient membraneless air-breathing/H2 biofuel cell. The new design consists in assembling a highly-efficient airbreathing biocathode based on the direct wiring of BOD from Bacillus pumilus on MWCNT and a bioanode based on the direct wiring of O2-tolerant hydrogenases from A. aeolicus on carboxylated MWCNTs. After integration in the hydrogen/air biofuel cell, the biocathode scavenges the O2 from the anodic solution allowing the hydrogenase to operate in pure hydrogen flow without the need of a membrane. The biofuel cell achieves excellent power output which falls in the mW cm 2 range of recently-developed hydrogen biofuel cells.10,12,18 Since most hydrogenases are oxygen-sensitive, this type of system is well-adapted for the integration of a large variety of these enzymes for the future increase of stability and power output of hydrogen enzymatic biofuel cells. All authors gratefully acknowledge funding from the Agence Nationale de la Recherche through the project CAROUCELL (ANR-13-BIOME-0003-02). ALG, NL, RH, MH and SC gratefully acknowledge the financial support from the ANR Investissements d’avenir-Nanobiotechnologies 10-IANN-0-02 program for the PhD funding of N. Lalaoui, the support from the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG) and from the LabEx ARCANE (ANR-11-LABX-0003-01). NM and SG thank la ´gion Aquitaine for financial support and ANR through the Re project RATIOCELLS (ANR-12-BS08-0011-01). They also thank the GDR CNRS 3540 ‘‘Biopiles’’ for partial financial support.
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Notes and references 1 S. C. Barton, J. Gallaway and P. Atanassov, Chem. Rev., 2004, 104, 4867–4886. 2 J. A. Cracknell, K. A. Vincent and F. A. Armstrong, Chem. Rev., 2008, 108, 2439–2461. 3 W. Gellett, M. Kesmez, J. Schumacher, N. Akers and S. D. Minteer, Electroanalysis, 2010, 22, 727–731. 4 I. Willner, Y.-M. Yan, B. Willner and R. Tel-Vered, Fuel Cells, 2009, 9, 7–24. 5 A. Zebda, S. Cosnier, J.-P. Alcaraz, M. Holzinger, A. Le Goff, C. Gondran, F. Boucher, F. Giroud, K. Gorgy, H. Lamraoui and P. Cinquin, Sci. Rep., 2013, 3, 1516. 6 A. de Poulpiquet, A. Ciaccafava and E. Lojou, Electrochim. Acta, 2014, 126, 104–114. 7 S. Cosnier, M. Holzinger and A. Le Goff, Front. Bioeng. Biotechnol., 2014, 2, 45. 8 A. Ciaccafava, A. De Poulpiquet, V. Techer, M. T. Giudici-Orticoni, S. Tingry, C. Innocent and E. Lojou, Electrochem. Commun., 2012, 23, 25–28. 9 A. de Poulpiquet, D. Ranava, K. Monsalve, M.-T. Giudici-Orticoni and E. Lojou, ChemElectroChem, 2014, 1, 1724–1750. 10 S. Krishnan and F. A. Armstrong, Chem. Sci., 2012, 3, 1015–1023. 11 L. Xu and F. A. Armstrong, RSC Adv., 2014, 5, 3649–3656. 12 N. Plumere, O. Ruediger, A. A. Oughli, R. Williams, J. Vivekananthan, S. Poeller, W. Schuhmann and W. Lubitz, Nat. Chem., 2014, 6, 822–827. 13 J. W. Tye, M. B. Hall and M. Y. Darensbourg, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 16911–16912. 14 P.-P. Liebgott, A. L. de Lacey, B. Burlat, L. Cournac, P. Richaud, ´ger M. Brugna, V. M. Fernandez, B. Guigliarelli, M. Rousset, C. Le and S. Dementin, J. Am. Chem. Soc., 2010, 986–997.
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ChemComm 15 A. A. Karyakin, S. V. Morozov, O. G. Voronin, N. A. Zorin, E. E. Karyakina, V. N. Fateyev and S. Cosnier, Angew. Chem., Int. Ed., 2007, 7244–7246. 16 J. Baur, A. Le Goff, S. Dementin, M. Holzinger, M. Rousset and S. Cosnier, Int. J. Hydrogen Energy, 2011, 12096–12101. 17 M.-E. Pandelia, V. Fourmond, P. Tron-Infossi, E. Lojou, P. Bertrand, ´ger, M.-T. Giudici-Orticoni and W. Lubitz, J. Am. Chem. Soc., C. Le 2010, 132, 6991–7004. 18 A. de Poulpiquet, A. Ciaccafava, R. Gadiou, S. Gounel, M. T. GiudiciOrticoni, N. Mano and E. Lojou, Electrochem. Commun., 2014, 42, 72–74. 19 N. Mano and L. Edembe, Biosens. Bioelectron., 2013, 50, 478–485. 20 C. Lau, E. R. Adkins, R. P. Ramasamy, H. R. Luckarift, G. R. Johnson and P. Atanassov, Adv. Energy Mater., 2012, 2, 162–168. 21 Y. Ulyanova, S. Babanova, E. Pinchon, I. Matanovic, S. Singhal and P. Atanassov, Phys. Chem. Chem. Phys., 2014, 16, 13367–13375. 22 I. Asano, Y. Hamano, S. Tsujimura, O. Shirai and K. Kano, Electrochemistry, 2012, 80, 324–326. 23 K. So, S. Kawai, Y. Hamano, Y. Kitazumi, O. Shirai, M. Hibi, J. Ogawa and K. Kano, Phys. Chem. Chem. Phys., 2014, 16, 4823–4829. 24 J. L. Bahr and J. M. Tour, Chem. Mater., 2001, 3823–3824. 25 J. A. Cracknell, T. P. McNamara, E. D. Lowe and C. F. Blanford, Dalton Trans., 2011, 40, 6668–6675. 26 F. Oteri, A. Ciaccafava, A. de Poulpiquet, M. Baaden, E. Lojou and S. Sacquin-Mora, Phys. Chem. Chem. Phys., 2014, 16, 11318–11322. 27 A. de Poulpiquet, H. Marques-Knopf, V. Wernert, M. T. GiudiciOrticoni, R. Gadiou and E. Lojou, Phys. Chem. Chem. Phys., 2013, 16, 1366–1378. 28 K. So, Y. Kitazumi, O. Shirai, K. Kurita, H. Nishihara, Y. Higuchi and K. Kano, Chem. Lett., 2014, 43, 1575–1577. 29 K. So, Y. Kitazumi, O. Shirai, K. Kurita, H. Nishihara, Y. Higuchi and K. Kano, Bull. Chem. Soc. Jpn., 2014, 87, 1177–1185.
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COMMUNICATION
Cite this: Chem. Commun., 2015, 51, 7447
Published on 27 March 2015. Downloaded on 21/04/2015 07:50:48.
Received 13th March 2015, Accepted 27th March 2015 DOI: 10.1039/c5cc02166a
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A membraneless air-breathing hydrogen biofuel cell based on direct wiring of thermostable enzymes on carbon nanotube electrodes† ab c ab ab Noe ´mie Lalaoui, Anne de Poulpiquet, Raoudha Haddad, Alan Le Goff,* ab de fg c Michael Holzinger, Se ´bastien Gounel, Michel Mermoux, Pascale Infossi, de c ab Nicolas Mano,* Elisabeth Lojou* and Serge Cosnier
www.rsc.org/chemcomm
A biocathode was designed by the modification of a carbon nanotube (CNT) gas-diffusion electrode with bilirubin oxidase from Bacillus pumilus, achieving high current densities up to 3 mA cm
2
for the
reduction of O2 from air. A membraneless air-breathing hydrogen biofuel cell was designed by combination of this cathode with a functionalized CNT-based hydrogenase anode.
Enzymatic biofuel cells represent a subcategory of fuel cells that propose a renewable alternative to noble-metal-catalyst-based fuel cells.1–4 Enzymes offer high catalytic turnovers associated with high substrate specificity and low overvoltages. Furthermore, exploring the biodiversity allows the identification of enzymes exhibiting peculiar properties, such as resistance to extreme pHs and temperatures or typical inhibitors. These advantages make enzymes a family of biocatalysts that can operate in complex media such as physiological fluids or extreme environments.5 Since enzymes are relatively large catalysts, often bearing a complex intramolecular electron transfer chain, electrodes have to be manufactured to efficiently transfer electrons to the enzyme active site, while providing a large specific area for optimal biocatalyst surface coverage.6 In this respect, carbon nanotube networks have proven to offer ideal properties for efficient direct electron transfer (DET) to the enzyme active site, high conductivity and high specific surface.7 Hydrogen biofuel cells rely on hydrogenases to efficiently oxidize H2 and multicopper oxidases to reduce oxygen.8–12 These enzymes have already shown similar catalytic properties compared to conventional platinum-based electrodes.13 However, to a
Univ. Grenoble Alpes, DCM UMR 5250, F-38000 Grenoble, France. E-mail: alan.le-goff@ujf-grenoble.fr b CNRS, DCM UMR 5250, F-38000 Grenoble, France c BIP, UMR7281 CNRS-AMU, F-13009 Marseille, France. E-mail: [email protected] d CNRS, CRPP, UPR 8641, Pessac, France. E-mail: [email protected] e Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France f Univ. Grenoble Alpes, LEPMI UMR 5279, F-38000 Grenoble, France g CNRS, LEPMI UMR 5279, F-38000 Grenoble, France † Electronic supplementary information (ESI) available: Experimental section and Fig. S1 to S3. See DOI: 10.1039/c5cc02166a
This journal is © The Royal Society of Chemistry 2015
be able to run efficiently in fuel cell set-ups, some drawbacks have to be overcome. One major issue for hydrogen fuel cell applications has been the sensitivity of hydrogenases towards oxygen. Several strategies have been employed to circumvent this issue in the design of biofuel cells. Mutagenesis have led to the increase in tolerance towards oxygen.14 Protective polymers have also been used to prevent oxygen deactivation of hydrogenases.12,15,16 We have recently developed the direct wiring of the membrane-bound hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. This hydrogenase is particularly tolerant towards oxygen, showing excellent electrocatalytic H2 oxidation in the presence of oxygen over a large range of temperatures.17 It was demonstrated that the rate of reactivation of this hydrogenase was much faster than the rate of inactivation by O2 because of a peculiar geometry of a FeS cluster closed to the active site. A biofuel cell based on this enzyme at the anode and a thermostable BOD at the cathode was designed so that these enzymes worked in separated compartments, in optimal conditions for respective enzymes.18 All hydrogenase-based biofuel cell set-ups have employed restrictive and specific conditions to favorably operate hydrogenases: nonexplosive H2-rich–air-low gas mixture,10,11 bioelectrode compartmentalization8,18 or use of oxygen-protecting redox polymers.12 Recent findings have shown that enzymes such as BOD and laccase could efficiently operate in air-breathing systems, especially developed in fuel cell technology.19,20 These electrodes have the great advantages to circumvent issues with gas solubility and diffusion in the electrolyte by providing the enzyme substrate through the gaseous phase. Here we report a novel strategy, which has been unexplored yet, for protecting hydrogenases from oxygen by the design of an air-breathing cathode based on the direct wiring of the thermostable BOD from Bacillus pumilus on carbon nanotube based electrodes. Oxygen from air is reduced at the cathode while the anode operates in H2 saturated aqueous buffer solution. This system could have important advantages such as (i) allowing oxygen-sensitive hydrogenases to work in anaerobic conditions, (ii) allowing the biofuel cell to work in non-explosive H2–O2 mixtures and (iii) avoid any oxygen reduction at the bioanode.
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Fig. 1 (A) Image of the MWCNT film deposited on carbon cloth obtained by laser confocal microscopy; (B) SEM image of a MWCNT film; (C) airbreathing electrode based on the deposition of MWCNTs and BOD on carbon cloth; (D) CV of MWCNT-based air-breathing electrode under argon (dashed line), under air (full line) and under oxygen (dotted line) (10 mV s 1, deaerated phosphate buffer pH 7.2, 45 1C).
For the MWCNT-based biocathode, pristine MWCNTs were deposited by drop-coating on a carbon cloth electrode. Fig. 1A and B show the morphology of a 40 mm-thick MWCNT film deposited on carbon cloth, investigated by confocal laser and SEM microscopy. These images underline the deposition of a homogenous MWCNT film on carbon cloth electrodes. After adsorption of BOD, the biocathode was integrated in an air-breathing chamber (Fig. 1C). Argon was bubbled in the electrolyte to avoid any contribution of dissolved oxygen to the catalytic current. CV scans are displayed in Fig. 1D. High catalytic current are accompanied with onset potentials of 0.4 V, confirming efficient direct wiring of BOD and efficient oxygen reduction catalysis under air-breathing mode. Electrocatalytic currents of up to 3 mA cm 2 at 0 V vs. Ag/AgCl in quiescent conditions were measured at pH 7.2 and 45 1C. By investigating the effect of the MWCNT film thickness by laser scanning microscopy, we did not observe any increase or decrease of the catalytic current between 4- and 40 mm-thick MWCNT films, underlining the excellent DET with the adsorbed BOD.
Fig. 2 (A) CV of MbH1 adsorbed on MWCNT and f-MWCNT GC electrodes (v = 5 mV s 1, pH 7.2, 60 1C, under H2); (B) CV of MbH1 for different f-MWCNT film thickness of 5, 7, 10, 18 mm (v = 5 mV s 1, pH 7.2, 60 1C, under H2, background was subtracted to remove capacitive current contribution).
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It is noteworthy that replacing air flux by a pure oxygen flux has negligible effect on the electrocatalytic current (Fig. 1D). This demonstrates the excellent diffusion of oxygen to the BOD providing optimal oxygen concentrations for the enzyme. The resulting biocathodes present higher performances, namely, current density of 3.0(+/ 0.2) mA cm 2 under air, than previously-developed highly-efficient breathing cathodes by P. Atanassov and coworkers20,21 or K. Kano and coworkers.22,23 Pristine CNTs were compared with modified CNTs. SWCNTs and MWCNTs were modified with amino-naphthoic acid, affording f-SWCNTs and f-MWCNTs, using a previouslydeveloped procedure based on the in situ generation of an aryl diazonium salt.24 Reductive behavior of CNT sidewalls triggers the formation of highly-reactive aryl radicals, forming a polyphenylene layer surrounding the CNTs. The main advantage of using diazonium-based functionalization is that high surface coverage of carboxylate groups can be grafted, thanks to the high reactivity of in situ aryl radicals towards CNT sidewalls.24 The presence of the naphthoic-acid groups was evidence by infra-red spectrum, showing the appearance of an intense band at 1715 cm 1 corresponding to CQO bonds of carboxylated groups and a broad band at 3000 cm 1 corresponding to C–H bonds (Fig. S1A, ESI†). The Raman spectra of f-SWCNT are shown in Fig. S1B (ESI†). The excitation wavelength was the 514.5 nm line from an argon ion laser. A noticeable increase is observed in the relative intensity of the D-band at 1350 cm 1 compared to the G-band at about 1590 cm 1 after aryldiazonium functionalization process. The D/G ratio is equal to 0.14 for SWCNT and 0.44 for f-SWCNT. This increase is associated with the formation of defects that break the translational symmetry along the SWNT sidewalls by the covalent addition of aryl radicals, associated with the attachment of naphtoic carboxylate groups. Furthermore, thanks to efficient sidewall functionalization with hydrophilic groups, both f-SWCNT and f-MWCNT exhibit excellent solubility in water of 12 and 37 mg mL 1 respectively (Fig. S1C, ESI†). Naphthoic-acid-modified electrodes have already been used as a DET promoter for the wiring of BOD from Myrothecium verrucaria.25 However, poor DET was observed for BOD from B. pumilus with f-MWCNTs and f-SWCNTs compared to pristine MWCNTs. Maximum current densities of 0.84 mA cm 2 and 0.10 mA cm 2 at 0 V were measured for f-SWCNT and f-MWCNT respectively (Fig. S2A, ESI†). This behavior is likely caused by the presence of hydrophobic domains located at the surface of the BOD, favoring hydrophobic interactions with pristine MWCNT sidewalls. On f-CNTs, the strong dipole moment pointing at the opposite of the T1 copper centre likely hinders the favorable orientation of the BOD towards the T1 copper centre on negatively-charged f-CNTs.18 On the contrary to BOD from B. pumilus, f-MWCNT and f-SWCNT were chosen because of the ability of carboxylate groups to enhance DET of MbH1 on carbon nanotubes.26 CV of the bioanodes based on MWCNT and f-MWCNT performed under H2 bubbling are displayed in Fig. 2A. Under H2, a catalytic current is observed with an onset potential of 0.59 V vs. Ag/AgCl accompanied with a high catalytic bias in favor of hydrogen oxidation. This is in good
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agreement with previous electrochemical studies of MbH1 on oxidized SWCNTs and carbon nanofibers, underlining that this hydrogenase can afford both hydrophobic and hydrophilic surroundings.27 Interestingly, the shape of both CV from Fig. 2A are different. The DET process on pristine MWCNT film exhibits a classic twisted shape of the CV, which is generally observed for this enzyme on PG electrode or oxidized SWCNT electrodes. This shape is caused by a reversible deactivation of hydrogenases at high overpotential.17 For f-MWCNT, a plateau shape similar to the one observed at carbon nanofibers electrode27 is associated with high maximum current densities of 2.7(+/ 0.2) mA cm 2. This plateau current arises from a high number of enzymes participating to the catalytic current that causes a mass-transport-controlled electrocatalytic current. It has been shown recently that reversible deactivation of hydrogenases at high potential could also be minimized by using redox polymers12 and gas-diffusion electrodes.28,29 These experiments demonstrate the excellent wiring properties of f-MWCNTs for MbH1 compared to pristine MWCNTs. In order to optimize the wired MbH1 surface coverage, we have studied the influence of the f-MWCNT film thickness over the electrocatalytic current for H2 oxidation (Fig. 2B and Fig. S3, ESI†). The CV curves in Fig. 2A and B show the maximum electrocatalytic current for f-MWCNT film thicknesses above 10 mm, reaching 2.7 mA cm 2 for hydrogen oxidation at pH 7.2 at 0 V vs. Ag/AgCl. Since the amount of deposited hydrogenases (20 mL at 5 mM) was kept constant, these experiments show that upon increasing the thickness, the number of enzymes participating to the catalytic oxidation of H2 is increased (Fig. 2B and Fig. S3, ESI†). For pristine MWCNT, a maximum catalytic current densities of 1.4 mA cm 2 was measured, accompanied with deactivation process above 0.3 V (Fig. 2A). f-SWCNT also exhibits excellent DET for MbH1, reaching 1.8 mA cm 2 at 0.25 V (Fig. S2B, ESI†). This intermediary catalytic current density, between MWCNT and f-MWCNT performances, likely arises from the hydrophobic nature of SWCNT,16 corroborated by lower solubility in water, which might counterbalance electrostatic interactions of MbH1 with carboxylate groups. For the membraneless hydrogen biofuel cell set-up, the GC/MWCNT/MbH1 electrode was connected in a two-electrode system in front of the gas-diffusion biocathode. Fig. 3A shows the schematic representation of the membraneless biofuel cell set-up. Bubbling of hydrogen prevents the bioanode from any contact with oxygen from air diffusion through the cathode. Fig. 3B shows the CV scans for the bioanode and the biocathode in this set-up. The biofuel cell was operated at 45 1C for technical and safety reasons. The bioanode is approximatively three-time less active at 451 than at 601 leading to maximum current densities of 1.4(+/ 0.1) mA cm 2. Fig. 3C displays the polarization curve and the power curve performed by successive potentiostatic discharges of 30 s. The non-classical polarization curve shape at low voltages is caused by hydrogenase deactivation, which is triggered by low-potential discharge and limiting behavior of the bioanode. Under bubbling of H2 at 45 1C, maximum power density of 0.72(+/ 0.04) mW cm 2 is obtained at 0.6 V with an
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Fig. 3 (A) Membraneless air-breathing hydrogen biofuel cell; (B) CV of a MWCNT/MbH1 bioanode (v = 5 mV s 1) and a MWCNT/BOD biocathode (v = 10 mV s 1, pH 7.2, 45 1C); (C) power curves and polarization curves of the hydrogen biofuel cell for MWCNT/MbH1 and f-MWCNT/MbH1 bioanode (pH 7.2, 45 1C, an error of 6% was estimated from the performances measured for two biofuel cells); (D) 1000 s potentiostatic discharge (0.8 V) of the biofuel cell based on the f-MWCNT/MbH1 bioanode.
OCV of 0.95 V. Power performances were also measured for pristine MWCNT bioanodes. As expected from lower maximum current densities for pristine MWCNT bioanodes, the maximum power density only reaches 0.34 mW cm 2. A 15 min discharge at a fixed voltage of 0.8 V for the f-MWCNT-based biofuel cell, displayed in Fig. 3D, underlines the excellent operating stability upon discharge of the biofuel cell. We report a novel and unexplored strategy for the elaboration of an efficient membraneless air-breathing/H2 biofuel cell. The new design consists in assembling a highly-efficient airbreathing biocathode based on the direct wiring of BOD from Bacillus pumilus on MWCNT and a bioanode based on the direct wiring of O2-tolerant hydrogenases from A. aeolicus on carboxylated MWCNTs. After integration in the hydrogen/air biofuel cell, the biocathode scavenges the O2 from the anodic solution allowing the hydrogenase to operate in pure hydrogen flow without the need of a membrane. The biofuel cell achieves excellent power output which falls in the mW cm 2 range of recently-developed hydrogen biofuel cells.10,12,18 Since most hydrogenases are oxygen-sensitive, this type of system is well-adapted for the integration of a large variety of these enzymes for the future increase of stability and power output of hydrogen enzymatic biofuel cells. All authors gratefully acknowledge funding from the Agence Nationale de la Recherche through the project CAROUCELL (ANR-13-BIOME-0003-02). ALG, NL, RH, MH and SC gratefully acknowledge the financial support from the ANR Investissements d’avenir-Nanobiotechnologies 10-IANN-0-02 program for the PhD funding of N. Lalaoui, the support from the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG) and from the LabEx ARCANE (ANR-11-LABX-0003-01). NM and SG thank la ´gion Aquitaine for financial support and ANR through the Re project RATIOCELLS (ANR-12-BS08-0011-01). They also thank the GDR CNRS 3540 ‘‘Biopiles’’ for partial financial support.
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