Direct bio-electrocatalysis by multi-copper oxidases: Gas-diffusion laccase-catalyzed cathodes for biofuel cells

Electrochimica Acta 56 (2011) 10767–10771

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Direct bio-electrocatalysis by multi-copper oxidases: Gas-diffusion laccase-catalyzed cathodes for biofuel cells Gautam Gupta, Carolin Lau, Brittany Branch, Vijaykumar Rajendran, Dmitri Ivnitski, Plamen Atanassov ∗,1 Center for Emerging Energy Technologies, Department of Chemical and Nuclear Engineering, Farris Engineering Center, University of New Mexico, Albuquerque, NM 87131, United States

a r t i c l e

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Article history: Received 16 November 2010 Received in revised form 21 January 2011 Accepted 22 January 2011 Available online 28 January 2011 Keywords: Laccase Direct electron transfer Biofuel cell Oxygen reduction Gas diffusion electrode

a b s t r a c t We have studied the bio-electroreduction of oxygen based on direct electron transfer (DET) between laccase and the electrode. Laccase enzymes from two different sources, namely, tree laccase from Rhus vernicifera, and fungal laccase from Trametes hirsuta were used in the study. The gas-diffusion cathode was made using a mixture of teflonized carbon and untreated carbon black, with a nickel mesh that served as a current collector, sandwiched between a hydrophobic gas diffusion layer, and a hydrophilic biocatalytic layer with physically adsorbed laccase enzyme. High current densities: up to 1 mA cm−2 under oxygen (for bio-electrocatalytic oxygen reduction) and increased stability (up to 30 days) has been achieved using teflonized carbon blacks at gas–electrode interface, high surface area carbon black for loading the enzyme. Gas diffusion laccase-catalyzed cathode demonstrates a number of advantageous properties including good adhesion, biocompatibility and high bio-electrocatalytic properties. An open circuit potential (OCP) of 600 mV at pH 7 for tree laccase (R. vernicifera) and 725 mV at pH 5 for fungal laccase (T. hirsuta) at zero current densities were obtained with respect to SHE reference electrode. Tafel plots obtained confirmed different DET characteristics for the two sources of laccase enzymes, which could suggest different mechanism of charge transfer: 4-electron electroreduction of oxygen using fungal laccase and 2electron electroreduction using tree laccase. The performance of the cathode was studied in galvanostatic mode and polarization curves at various conditions are reported including those obtained under air and neat oxygen feed from the gas phase. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The concept of power generation using enzyme-based biofuel cells have been established four decades ago, but only in recent years, the biofuel cells have received broad attention [1–5]. Biofuel cells have an unlimited possibility for fuel sources, a wide variety of biocatalysts, and offer low costs for operation and maintenance compared to conventional fuel cells [1–5]. High reaction rates and mild operating conditions of temperature and pH can be easily achieved using biocatalysts. Enzymatically catalyzed reactions occur at redox potentials close to the theoretical value, consequently enzyme based biofuel cells can be designed orders of magnitudes smaller than microbial fuel cells producing the equivalent amount of power. These attractive features of biofuel cells based on whole enzymes,

∗ Corresponding author. Tel.: +1 505 277 2640; fax: +1 505 277 5433. E-mail address: [email protected] (P. Atanassov). 1 1 ISE member. 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.01.089

along with the recent innovative developments in the field of bioelectrochemistry, nano-technology, and material science stimulate further research in this area with the common goal of developing efficient, miniature, rechargeable biofuel cells that would power the future devices [4–6]. Oxygen, due to its abundance in nature, is the fuel of choice for many fuel cells, including enzymatic biofuel cells. Bio-electroreduction of oxygen at the cathode has been reported based on copper-containing oxidases, examples of which include laccases, ascorbate oxidase, and bilirubin oxidase [7–10]. Other enzymes used for electro-reduction of oxygen are tyrosinase [11] and cytochrome c oxidase [12]. Laccase is a multi-copper oxidase (MCO) that catalyzes the oxidation of various organic and inorganic compounds, such as polyphenols, aromatic amines and ascorbate with the simultaneous reduction of oxygen to water [13]. They are involved in the degradation of lignin, in the removal of potentially toxic phenols arising during lignin degradation [13] and in pigment formation [7]. Tree as well as fungal laccases show in general low substrate specificity and similar intermediates suggest analogous catalytic mechanisms [14]. Due to the smaller molecular size of fungal laccase, which

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is nearly half that of tree laccase, higher surface concentrations can be achieves. Multi-copper oxidases are in general inhibited at higher pH values due to the formation of copper hydroxo complexes; pKa values of fungal and tree laccase are around pKa 3.5 and 7.4, respectively [14]. Significant differences in redox potentials of laccase from fungal and tree sources make a direct comparison difficult. Especially the fungal enzymes have high thermodynamic potentials and excellent catalytic parameters, which are beneficial for design and development of cathode electrode for biofuel cells [4,15]. A slight difference in the protein sequence complexing the T1 copper side is suggested as explanation: methionine (in most plant laccases) is replaced with leucine (in fungal laccase) which cannot serve as copper ligand [14]. Electrochemical studies of tree R. vernicifera laccase assign redox potentials of +434 mV and +484 mV (vs. SHE) for T1 suggesting a two-electron mechanism, whereas fungal laccases vary between 680 mV and 850 mV depending on pH and fungal source [4,16–19]. Our group recently identified the redox potentials for T. versicolor laccase as 520 mV, 360 mV and 197 mV (vs. Ag/AgCl, pH 5.8) for T1, T2, T3 respectively [10]. The spectroscopic studies and X-ray crystallography reveals that the catalytic unit of the enzyme consists of four copper ions classified into Type 1 (T1), Type 2 (T2), and two Type 3 (T3) ions [7,19–22]. The T1 center provides long-range intra-molecular rapid electron transfer from the substrate, redox mediator or electrode to the trinuclear T2/T3 redox copper center where the oxygen reduction to water takes place via intermolecular electron transfers [7,19–22]. The accepted mechanism includes an electron hopping cascade starting at the highest redox potential copper center T1, over to T3 to the lowest redox potential T2 [4]. In general, electron transfer is classified by two different mechanisms: mediated electron transfer (MET), and direct electron transfer (DET) [4–6,18,23–26]. In MET, low molecular weight, redox-active species, referred to as mediators, are introduced to shuttle electrons between the enzyme active site and the electrode. Application of redox mediators allows a significant decrease in the applied potential and it can minimize interference with coexisting electro-active compounds present in real samples. Common mediators for multi-copper oxidases include 2, 2 - azinobis(3-ethylbenzothiazoline-6-sulfonate) and “wired” osmium redox polymers [8,23,27], but the stability and toxicity of some mediators limit their application. Most of the loss in power comes from the slow decomposition and loss of the redox mediators from the enzyme electrode, as well as the denaturation of the enzyme itself. Therefore, in recent years biofuel cells based on direct electrical communication between redox center of enzyme and electrode have received intense attention [4,22,28–30]. The DET of electrons between the active site of the enzyme and the electrode surface is the most attractive approach due to simplicity of the process, excellent potential for miniaturization, and the high power-output of the system. The enzyme electrode based on DET approach can work in a potential range close to the redox potential of the enzyme itself [22]. The experimental evidence of DET has been reported in the literature for both low molecular weight electron-transfer proteins and enzymes with large, more complicated structures such as laccase, peroxidase, and glucose oxidase on gold and carbon surfaces [4,22,28,29,31–36]. For development of enzyme-based biofuel cells with high power-output, an efficient electrical enzyme-electrode communication is required. Therefore, it is necessary to maximize the driving force (e.g., the potential difference) and to minimize the ohmic resistance losses [37]. Especially the low solubility and small diffusion coefficients of oxygen as substrate for laccase limit the biocatalytic reduction. The latter can be achieved through the development of new electrode materials and appropriate cell

Fig. 1. Schematic representation of the three-phase contact zone in a gas diffusion electrode.

design facilitating hydrophobic gas diffusion pathways as well as hydrophilic, high surface area biocatalytic layers [38–40]. This work presents the achievement of both, an effective electrical communication between redox centers of enzyme and electrode together with a highly efficient oxygen supply through the gas phase using three-dimensional laccase gas-diffusion cathode for oxygen reduction based on DET approach. The key advantage of gas-diffusion laccase electrode is that it can operate under “air-breathing” conditions by utilizing oxygen directly from atmospheric air (Fig. 1). In addition, the gas diffusion electrode allows increasing the current density significantly by increasing the microscopic surface area and the amount of immobilized enzyme molecules [40,41]. 2. Experimental 2.1. Materials Laccase (EC 1.10.3.2 from Rhus vernicifera, 120 U mg−1 ), was purchased from Sigma Chemical Company, St. Louis, MO, USA. Laccase (EC 1.10.3.2 from Trametes hirsuta, previously classified also as Coriolus hirsitus, 40 U mg−1 ) was purchased from SynectiQ Corp., Denville, NJ, USA. Carbon black (VULCAN XC72R) was purchased from Cabot Corp., Billerica, MA, USA. Teflonized carbon was prepared following a previously reported procedure [23]. Nickel gauze (40-mesh woven from 0.13 mm diameter wire) was purchased from Alfa Aesar, a Johnson Matthey Company, Ward Hill, MA, USA. Nylon membrane filter (Nylaflo® , 0.45 ␮m pore, 47 mm diameter) was purchased from Pall Corp., Ann Arbor, MI, USA. All other chemicals were of analytical grade. 2.2. Electrode preparation and instrumentation Fig. 1 shows a schematic illustration of the miniature gasdiffusion electrodes used in this study. The principle design is based on processing a two-layered pressed tablet structure of a hydrophobic carbon black/Teflon® layer: gas-diffusion layer, and a hydrophilic catalytic layer made of carbon black with enzyme immobilized on it. The hydrophobic layer in this study was made of Teflonized® carbon blacks: Vulcan XC 72 with 35 wt.% Teflon® , (designated as XC35) pressed in a 10 cm2 disk tablet. For the catalytic layer a mix of the same materials XC35 (10 wt.%) with untreated carbon blacks was used. It was pressed upon the gas-

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diffusion layer and treated with enzyme solution as follows: 0.6 ␮l of 10 mg ml−1 laccase in the respective buffer solution was spread evenly on the hydrophilic layer and allowed to dry for 30 min at room temperature. The enzyme electrode with the catalytic layer facing “down” was then cut out from the tablet by a modified pipette tip (used as an electrode plastic body) and assembled as shown in Fig. 1 with the current collection through the opening of the pipette tip that served also for air or oxygen supply. The dimensions of the exposed electrode surface area were approximately 0.02 cm2 . This represents the formal (geometrical) surface area of the cutout disk-shaped coupon from the tablet, and all the normalizations were based on it. This method allowed manufacturing of multiple electrodes from a single carbon black two-layered disk of 10 cm2 . Electrochemical measurements were performed in a half-cell format, with the gas-diffusion electrode as “working electrode” using a EG&G potentiostat (Model 263 A) from Princeton Applied Research, Princeton, NJ, USA in a three electrode configuration with a Ag/AgCl sat. KCl reference electrode, and a Pt-wire counter electrode. Electrochemical studies of tree laccase were performed in 100 mM phosphate buffer at pH 7.0. Fungal laccase was studied in 100 mM acetate buffer at pH 5.0.

3. Results and discussion 3.1. Current–voltage behaviors of the laccase catalyzed gas diffusion cathodes The application of the laccase modified gas diffusion electrode as cathode for the biofuel cell has been demonstrated during testing in galvanostatic regime. Fig. 2a shows a typical polarization curves obtained for both fungal (T. hirsuta) and tree (R. vernicifera) laccases in galvanostatic mode. Open circuit potentials (OCP vs. SHE) of +725 mV for fungal laccase at pH 5, and +590 mV for tree laccase at pH 7 was obtained at zero current densities. The higher open circuit potential of fungal vs tree laccase agrees with the reported values between 680 mV and 850 mV in literature and is close to redox potential of the enzyme itself [4,10,16–19]. The catalytic electroreduction potential of oxygen at laccase (T. hirsuta) modified electrode appears at +694 mV at a current density of 100 ␮A cm−2 and reaches +580 mV at 400 ␮A cm−2 (Fig. 2a ). Similarly the catalytic electroreduction potential of oxygen at laccase (R. vernicifera) modified appears at +445 mV at a current density of 100 ␮A cm−2 and reaches +190 mV at 400 ␮A cm−2 (Fig. 2a 䊉). Also, the effect of pH on the electroreduction of oxygen is obvious from the placement of the polarization curves, where fungal laccase at pH 5.0 is placed at higher potential values compared to tree laccase at pH 7.0. Furthermore, there is a steep curving of the tree laccase polarization curve at higher current densities (in the diffusion-limited regime) compared to the fungal laccase, which could be explained by the fact that the tree laccase molecules is twice the size of the fungal laccase [42] with an active site deeply buried within the carbohydrate shell thus limiting the rate of diffusion of oxygen. Tafel plots in Fig. 2b were obtained from polarization data by plotting the overpotential  as a function of current density j. Tafel plots are used in this study as an initial attempt to provide insight on the electron transfer mechanism [43]. The interpretations of Tafel slopes: b = −2.3RT (F˛n)−1 are dependents on the assumptions (or theoretical estimations) of ˛ – the charge transfer coefficient. Usually, n – the numbers of electrons exchanged per molecule are being sought as an indication of the mechanistic path of the reaction under defined stoichiometry. The remaining parameters: R, F, T are gas constant, Faraday constant and temperature, respectively. Fig. 2b shows Tafel plots for both tree and fungal laccases (in both air and oxygen-fed conditions). The difference in catalytic

Fig. 2. (a) Current–voltage behavior of the fungal laccase (, ) and tree laccase (䊉) based gas diffusion cathodes. Influence of oxygen on the polarization curve obtained for fungal laccase in air () and oxygen () stream conditions. (b) Tafel plots obtained from the polarization data (a) for fungal laccase (, ) and tree laccase (䊉). (c) Eoxy −Eair curve for fungal laccase.

behavior can also be seen from the polarization dependences in Fig. 2a at low current densities. The Tafel slope for oxygen reduction reaction (ORR) catalyzed by T. hirsuta is approximately 30 mV per decade (under neat oxygen and under air feed). The corresponding Tafel slope for an electrode catalyzed by a tree laccase from R. vernicifera is estimated to be more than twice in value: 70 mV per decade. Similar Tafel slopes of 30 mV/dec have been observed for adsorbed laccase (from a similar fungi T. versicolor) on carbon [44]. Alternative multi-copper oxidase, CueO, has yielded a Tafel slope of 67 mV/dec [45], whereas via redox-polymer “wired” multi-copper oxidases like BOD or laccase show Tafel slopes of 120 mV/dec [46] or 45–95 mV/dec [47], respectively. These quite diverse reports leave a little room for reconciliation and thus a preliminary hypothesis can be drawn only, based on the results reported here for tree and fungal laccases examined under similar (yet not identical) conditions. One can suggest that if 30 mV/decade, observed for fungal laccase is a slope corresponding to a 4-electron ORR mechanism (as suggested in [44] and references therein), thus a 60 mV/decade (or larger, but close), observed for tree laccase should be attributed to a 2-electron reduction pathway. Such hypothesis indeed should assume ˛ away from 0.5 and similar for both laccases. This work was dedicated to the introduction of the gas-diffusion electrode concepts to enzyme-catalyzed systems. Such electrodes allowed us to observe significant differences in the catalytic behavior of fungal and tree laccases. The electrode structure, however is far away from ideal for fundamental electrocatalysts studies of enzymecatalyzed ORR and additional, rotating ring disk electrode studies are needed to elucidate the mechanism and register a potential intermediates of oxygen reduction. Only such a study, combined

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Fig. 3. Operational stability of the cathode based on fungal laccase (T. hirsuta) at 100 ␮A cm−2 current load (upper) and tree laccase (R. vernicifera) at 60 ␮A cm−2 (lower).

with a flux control analysis can provide a path for reconciliation of ORR mechanistic parameters similar to that achieved for Pt-based electrocatalysts in recent years [48–50]. 3.2. Influence of oxygen on the current–voltage behavior of the cathodes According to [38], the transport of oxygen within enzyme modified electrode is a significant rate-limiting step. In this case, the diffusion-controlled rate of enzymatic reaction can be significantly accelerated in three-dimensional system through the use of porous electro-conductive complex of nanoparticles with binding BOD molecules [38,40,41]. The use of metal and carbon nanoparticles in gas-diffusion electrode is advantageous due to their high electrochemically active surface. The acceleration of the diffusion-controlled rate of enzymatic reaction on the solid–solution interface is accomplished by conducting enzymatic reaction in a porous, highly dispersed electrode material and by introducing the oxygen at the gas–electrode interface (teflonized carbon). The influence of oxygen concentration on the polarization curve for fungal laccase is as shown in Fig. 2a (ambient air, oxygen stream ). The potential difference Eoxy − Eair curve is shown in Fig. 2c. There is a small; yet, constant difference of 75 mV potential between the two curves, until the load of 300 ␮A cm−2 is applied to the electrode. This constant regime determines the kinetic and ohmic regime of the working electrode. The diffusion regime dominates at higher current densities, and an increase in potential difference is obtained, under air or under oxygen. Current densities up to 1 mA cm−2 can be obtained at a potential of 573 mV (SHE), under oxygen-saturated conditions. These results clearly determine the advantage of a 3-dimensional interface. 3.3. Stability of the gas diffusion cathodes The operational stability of both fungal and tree laccase is as shown in Fig. 3. A load of 100 ␮A cm−2 and 60 ␮A cm−2 was applied on a fungal laccase and tree laccase modified gas diffusion cathodes, respectively for 20 h. The only comparable measurements in literature were done with osmium–redox polymer “wired” laccase form T. hirsuta and reveal a current density of 19 ␮A cm−2 at 620 mV load, pH 7 [18], supporting the efficiency of our presented gas diffusion design. The storage stability data for fungal laccase is as shown in Fig. 4. Polarization curves were obtained during a time period of 1 month, where the gas diffusion electrodes were stored at 4 ◦ C in the refrigerator at high humidity. The OCP of the modified electrodes is 725 mV and 719 mV (SHE) at day 1 and day 30. The polarization performance at low current densities of the gas-

Fig. 4. Storage stability of the cathode based on fungal laccase obtained at Day 1 (), after Day 10 (䊉), and after Day 30 () stored at 4 ◦ C in the refrigerator.

diffusion electrodes remains relatively unchanged after 30 days of storage. Most degradation (lower polarization) of the electrodes is associated with their performance at higher current densities indicating to a non-enzymatic mechanism of performance loss. Electrode polarization of high current densities is usually associated with transport losses, thus one can assume that the stability of the hydrophobic/hydrophilic interfaces in the gas-diffusion electrodes play substantial role in its durability as a enzyme-catalyzed cathode for biofuel cells. 4. Conclusions Multi-copper oxidases were successfully integrated into gasdiffusion electrodes of hydrophobic type. In this two-layered structures the enzymes were immobilized by physical adsorption onto the carbon blacks comprising the hydrophilic catalytic layer. The substrate, molecular oxygen, was fed from the opposite site and introduced either as an air or neat oxygen gas fed. Polarization of such electrodes demonstrates cathodic current corresponding to ORR catalyzed by enzymes with no mediators or surface modifiers: under conditions of direct electron transfer. The open circuit potential of such electrodes corresponds to the DET of ORR and is close to the redox potential of the T1 copper site of the corresponding multi-copper oxidase. Laccase efficiently catalyzes the electroreduction of molecular oxygen with a minimal overpotential (0.8 V under direct oxygen supply, vs. SHE), which is close to the thermodynamic equilibrium O2 /H2 O potential of +1.23 V (vs. SHE, Pt catalyzed ORR at pH 0). A direct electron communication is established with the highly porous carbon electron yielding high current densities up to 1 mA cm−2 with a minimal drop in potential. This paper reports on significantly different Tafel slopes for fungal laccase of approximately 30 mV/decade and tree laccase of 70 mV/decade, indicating the possibility for substantially different mechanism of ORR catalyzed by different multi-copper oxidases. These differences can be interpreted as drastic as 4-electron mechanism for fungal laccases vs. 2-electron ORR mechanism for tree ones. Integration of fungal laccase-catalyzed gas diffusion cathodes with glucose-oxidizing anodes in a biofuel cell configuration is in progress in our laboratory. Acknowledgments The Office of Naval Research HSI Grant (N00140210169) supported the onset of this project. Completion of this work was supported in part by the Air Force Office of Scientific Research MURI Award to UNM: Fundamentals and Bioengineering of Enzymatic Fuel Cells (FA9550-06-1-0264).

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