Bacterial-biofilm enhanced design for improved electrocatalytic reduction of oxygen in neutral medium

Electrochimica Acta 213 (2016) 314–323

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Bacterial-biofilm enhanced design for improved electrocatalytic reduction of oxygen in neutral medium Weronika A. Lotowskaa,c , Iwona A. Rutkowskaa , Ewelina Setaa,c, Ewelina Szaniawskaa , Anna Wadasa , Slawomir Seka,c, Adrianna Raczkowskab , Katarzyna Brzostekb , Pawel J. Kuleszaa,c,* a

University of Warsaw, Faculty of Chemistry, Pasteura 1, PL-02-093 Warsaw, Poland University of Warsaw, Faculty of Biology, Miecznikowa 1, PL-02-096 Warsaw, Poland c Biological and Chemical Research Center, University of Warsaw, Zwirki i Wigury 101, PL-02-096 Warsaw, Poland b

A R T I C L E I N F O

Article history: Received 4 March 2016 Received in revised form 18 July 2016 Accepted 20 July 2016 Available online 21 July 2016 Keywords: Yersinia enterocolitica bacterial biofilm Multi-walled carbon nanotubes Co-porphyrin Oxygen and hydrogen peroxide reductions Pt nanoparticles

A B S T R A C T

The specific reactivity and ability of biofilms to form stable polymer-like hydrogel aggregates of microorganisms adhering to common solid (including the glassy carbon electrode) surfaces have been explored here to form systems analogous to electrocatalytic redox-polymer modified electrodes. Growth of biofilms has been demonstrated with use of Yersinia enterocolitica, a robust Gram-negative rod-shaped bacteria known to be resistive to pH changes (4-10) and temperature variations (0–40  C). Charge distribution and propagation within the biofilm have been enhanced by introduction of multi-walled carbon nanotubes. The fact that carbon nanotubes are derivatized with the carboxyl-group containing 4(pyrrole-l-yl) benzoic acid has facilitated the hybrid material integrity and stability, namely through electrostatic attractive interactions between anionic carboxyl sites and positively charged domains of bacterial aggregates. In neutral media, the biofilm-based composite (hybrid) matrices have exhibited themselves electrocatalytic activity during electroreductions of oxygen and hydrogen peroxide (with possibility of its sensing in a broad range of concentrations). By immobilizing additional catalytic (cobalt porphyrin) sites, a truly bifunctional redox-polymer-like electrocatalytic system capable of significantly enhancing oxygen reduction currents has been produced. Apparently, the reduction of oxygen (to hydrogen peroxide) is initiated at cobalt porphyrin centers, and the second step (decomposition of hydrogen peroxide intermediate to water) is pursued at reactive sites (perhaps c-cytochrome) existing within biofilm matrix. Comparative measurements have been performed with the biofilm-supported platinum nanoparticles as well as with such a model catalytic system as platinized carbon nanotubes. The proposed electrode designs are relevant to biosensing and to the development of alternate cathode materials for biofuel cells or biobatteries. ã 2016 Elsevier Ltd. All rights reserved.

1. Introduction There has been growing interest in fabrication of electrocatalytic systems for oxygen and hydrogen peroxide reductions that would be useful in biological media, i.e. neutral solutions. While stable bioelectrocatalytic systems for efficient (four-electron and preferably with low-overpotential) oxygen reduction are of importance to such technologies as biofuel cells and biobatteries, the well-behaved catalytic electrodes for accurate and reliable

* Corresponding author at: University of Warsaw, Faculty of Chemistry, Pasteura 1, Warsaw PL-02-093, Poland. Tel.: +48 22 5526211; fax: +48 22 5526434. E-mail address: [email protected] (P.J. Kulesza). http://dx.doi.org/10.1016/j.electacta.2016.07.117 0013-4686/ã 2016 Elsevier Ltd. All rights reserved.

voltammetric or amperometric detection and determination of hydrogen peroxide are of interest to the development of both biomedical and environmental sensors. Oxygen biocathodes utilizing fungal laccases or bilirubin oxidases are highly specific and often perform better at ambient temperatures than noble metal (e.g. platinum) based catalysts for the oxygen electroreduction in neutral media. The enzymes belong to a group of proteins with the copper active centers, and they can lower the oxygen reduction reaction overpotential both in the absence [1,2] and presence of mediators [3]. Despite significant progress in their practical utilization, high cost and the complex proteic structure of those enzymes often result in lack of stability and poor reproducibility of operation [4].

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Attractive new concepts of designs for alternate bioelectrocatalytic systems originate from recent advances in microbial fuel cell research. Bacteria have been demonstrated to catalyze oxidations of a wide array of organic compounds via a few, direct or indirect, mechanisms of electron transfer between the electrode and microbial cell. More precisely, the microbe-electrode interactions involve excretion of soluble redox mediators or secondary metabolites [5], electron transfer through membrane-bound cytochromes, or extracellular electron transport occurring via conductive pili (so-called electron conducting nanowires) [6–9]. Mixed cultures of bacteria growing at cathodes of microbial fuel cells have been demonstrated to significantly reduce the overpotential for oxygen reduction presumably by inducing the fourelectron reduction mechanism to water [10–15] rather than less effective two-electron reduction of oxygen to hydrogen peroxide. The oxygen activation mechanisms [16] and the charge distribution dynamics within the bacterial based systems are still object of discussion. In the present work, we consider Yersinia enterocolitica as Gramnegative bacterium [17] capable of growing stable biofilms in the 0 to 45  C temperature range. Among important features for electrocatalytic application is the system’s resistivity to pH changes what is crucial when it comes to studying redox processes involving protons (e.g. oxygen reduction). Y. enterocolitica is known to exist at pH’s from 4 to 10; and it often survives even under truly acidic [18] or harsh environmental conditions [19,20]. Yersinia genome contains genes associated with environmental stress modulations. Consequently, Y. enterocolitica survives diverse environmental insults such as high temperature, hydrogen peroxide (which appears as the undesirable oxygen reduction intermediate), osmolarity and low pH’s [21]. We show here that this bacterium exhibits itself electrocatalytic properties and, by analogy to electroactive bacteria [22,23], it is capable of mediating or inducing the oxygen reduction. Macrocyclic N4-complexes are known to catalyze redox reactions involving such simple diatomic inorganic molecules as O2, H2, and N2. Representative examples include enzymatic processes with sulfite reductase [24], nitrate reductase, cytochrome c oxidase [25], blue copper oxidases, pseudo-catalase [26], photosystem II [27], nitrogenase and hydrogenase [28]. Many macrocyclic N4-complexes of cobalt and iron have often been considered as the reduction electrocatalysts [29], particularly toward the reduction of oxygen [30]. To improve dynamics of charge propagation within threedimensional biofilms (i.e. having thicknesses on the micrometer level), we have introduced multi-walled carbon nanotubes (CNTs) [30–34] that are characterized by good electronic conductivity and mechanical stability. Literature examples included electrocatalytic reductions of hydrogen peroxide, oxygen and carbon dioxide [35– 37]. When combined with metalloporphyrins, the CNT-containing biocatalytic layers exhibited reasonable activity during reduction of oxygen. Nevertheless, the H2O2 intermediate, rather than H2O, was predominantly produced as final product under such conditions [35,38–40]. In this study, to produce a hybrid biofilm-based bioelectrocatalytic film, we have utilized derivatized or functionalized multiwalled carbon nanotubes (CNTs) [41–43], namely CNTs modified with ultra-thin layers of organic, 4-(pyrrole-1-yl) benzoic acid (PyBA), anionic adsorbates [43] capable of exhibiting attractive interactions with positively charged domains of the biological matrix. The fact that our hybrid system has also contained Coporphyrin centers (capable of inducing electroreduction of oxygen), in addition to presence of reactive sites (possibly c-type cytochromes) within the biofilm matrix (capable of catalyzing

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reductive decomposition of the hydrogen peroxide intermediate) has led to enhancement effect in terms of both increasing of the oxygen reduction current densities (important in biosensing) and shifting potentials toward more positive values (important in bioenergetics). In other words, we have extended our previous concept of fabricating the bifunctional bioelectrocatalytic system for oxygen reduction [43] to a new design in which, instead of enzyme, biofilm is used. Importance of the biofilm based matrix toward hydrogen peroxide reduction is also evident from kinetic analysis based on measurements of steady-state currents at different concentrations. Finally, comparative diagnostic experiments have been performed model catalytic Pt nanoparticles. 2. Experimental 2.1. Chemicals and reagents All chemicals were obtained from Sigma–Aldrich and are of highest available purity. Co(III) protoporphyrin IX was from Frontier Scientific; Nafion, multi-walled carbon nanotubes (CNTs) and 4-(pyrrole-1-yl) benzoic acid (PyBA) were from Aldrich. Platinum black was obtained from Alfa Aesar. CNTs were purified as reported elsewhere [44] by exposing them to 12 mol dm 3 HCl solution for 1 h, followed by treatment with 3 mol dm 3 HNO3 for 8 h under reflux conditions. Later, the CNT samples were washed with large amounts of water until pH  7 was reached. Solutions were prepared using triply-distilled subsequentlydeionized (Millipore Milli-Q) water. They were de-aerated (using pre-purified argon), or saturated with oxygen, for at least 10 min prior to the experiments. Argon was also used to keep air-free atmosphere over the solution during measurements. Experiments were conducted at room temperature (20  0.5  C). 2.2. Bacterial culture and biofilm formation Yersinia enterocolitica (Ye9; wild-type, bacterial strain pYV+, serotype O:9) was obtained from the Applied Microbiology Facility, Faculty of Biology, University of Warsaw. The Y. enterocolitica strain Ye9 was originally from the collection existing in National Institute of Public Health National Institute of Hygiene (NIPH NIH), Poland. Identification was also confirmed by phenotypic and genotypic methods based on the analysis of the highly conserved 16 S rRNA (rrs) gene and other biomarkers (virulence genes). Bacteria were cultivated strictly aerobically at 25  C in a standard Luria-Bertani (LB) medium, which was a mixture composed of 10 g of peptone, 5 g of yeast extract and 10 g of sodium chloride per liter. After growing under aerobic conditions (with shaking for 24 h), the bacterial culture appearing in stationary phase constituted the diluted culture of Y. enterocolitica. Then the suspension was diluted (1:50) in the LB medium (until the optical density OD600 = 0.1). Thus prepared cell-suspension was poured over the glassy carbon plate and incubated for 48 hours at 25  C. Later, the resulting films were subjected to drying under argon atmosphere followed by over-coating with Nafion (as described later). Thickness of the biofilm based catalytic layers was estimated to be on the level 3 mm (0.6 mm; based on standard deviation of 8 independent experiments) by using an Alpha-step profilometer (Tencor Corp.). To obtain boundary between the bare and modified substrate during electrodeposition, the glassy carbon slide (electrode substrate) was only partially covered with the diluted cell suspension. The thickness value was approximate because it was difficult to distinguish bare and modified portions at the planar electrode substrate.

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2.3. Diagnostic measurements Electrochemical measurements were done with CH Instruments (Austin, USA) Model 760E workstation. A standard threeelectrode cell was used for all experiments. A glassy carbon rod was used as the counter electrode. A (KCl-saturated) calomel electrode (SCE) was used as the reference electrode. A glassy carbon slide electrode (1cm  2cm  0.2 cm) was utilized as the working electrode. Polished electrodes were soaked overnight in 1 mol dm 3 HCl to remove metals and other contaminants, washed twice, first with acetone and later with deionized water, to remove organic substances. Before modification, the electrode was then polished with successively finer grade aqueous alumina slurries (grain sizes, 5–0.5 mm) on a Buehler polishing cloth. For SEM imaging, biofilms of Yersinia enterocolitica were grown for 48 h on glassy carbon electrodes. The biofilm sample was coated with 2% glutaraldehyde, then exposed to phosphate buffer for 1 h and, subsequently, dehydrated by using the ethanol gradient procedure involving successive applications of the aqueous ethanol (20%, 40%, 60%, and 80%) solutions and, finally, pure ethanol. Before examination with JOEL 6400 scanning electron microscope, the samples were subjected to air-drying and sputtercoating with gold and palladium. Transmission electron microscopy (TEM) images were obtained with JEM 1400 (JEOL Co., Japan, 2008) equipped with high resolution digital camera (CCD MORADA, SiS-Olympus, Germany). Atomic Force Microscopic (AFM) images were recorded using Dimension Icon (Bruker, Santa Barbara, USA) instrument operating at 22  1  C in a phosphate buffer solution (pH = 6.1). The images were collected in tapping mode using PPP-NCLR probes (Nanosensors, Neuchatel, Switzerland) with nominal force constant in the range from 21 to 98 N m 1. The cantilevers were carefully calibrated before each experiment using thermal tuning method. The amplitude of free oscillations was 30–50 nm whereas the setpoint was kept at ca. 50-70% of the initial amplitude.

reference comparative measurements with a model catalyst for oxygen reduction, namely CNT–supported platinum nanoparticles (at the same loading, 100 mg cm 2), first, an ink of platinum nanoparticles was produced by dispersing 14.0 mg of platinum black in 2.0 cm3 of deionized water followed by sonification for 2 h. Second, a suspension of pristine (bare) CNTs was fabricated by placing 10 mg of CNTs in 2 cm3 of water and subjecting to mixing under magnetic stirring for 1 day. Later, 4.2 mdm3 of the CNT suspension was pipetted onto the glassy carbon substrate; this step was followed by introducing 2 mdm3 of the Pt ink and drying at room temperature for 15 min. Finally, the CNT-supported Pt nanoparticles were stabilized by over-coating with 0.25 mdm3 of the Nafion solution diluted in 2.25 cm3 ethanol. Before the actual electrocatalytic experiments, the catalytic films on electrodes were first pre-treated by potential cycling (in the range from 0.7 to 1.0 V) at 10 mV s 1 for 15 min in the deaerated 0.1 mol dm 3 phosphate buffer (pH = 6.1) electrolyte. Later, the systems of interest were pre-conditioned in the oxygensaturated (or in 0.1 mol dm 3 hydrogen peroxide) phosphate buffer solutions by potential cycling (8 full potential cycles at 10 mV s 1) in the potential range from 0.7 to 1.0 V. As a rule, the working electrode was kept for 20 s (“quiet time”) at the starting potential before representative voltammetric responses were recorded. 3. Results and Discussion 3.1. Morphology and vitality of biofilm based electrocatalytic layers Fig. 1 illustrates scanning electron micrographs (SEMs) of the following bacterial biofilm (Yersinia enterocolitica) based systems deposited on glassy carbon slides: (A) bare (simple) biofilm layer, and (B) the biofilm layer together with platinum nanoparticles. Regardless the uncertainty in imaging of the hydrogel-type

2.4. Procedures To fabricate multi-walled carbon nanotubes (CNTs) modified with 4-(pyrrole-1-yl) benzoic acid (PyBA) [43], the appropriate suspension was formed by dispersing ca. 50 mg of CNTs (length, 1– 10 mm; outer diameter, 10–30 nm; inner diameter, 3–10 nm) in 5 cm3 of aqueous PyBA solution (50 mg dm 3). The suspension was sonicated for 12 h. Subsequently, it was centrifuged, and the supernatant solution was removed and replaced with fresh PyBA solution. The centrifuging procedure was repeated three times. Later, the PyBA solution was decanted. Then the carbon material was subjected twice to washing and centrifuging with water. Finally, a stable colloidal solution (5 cm3) of PyBA-modified carbon nanotubes (CNTs/PyBA) was obtained. To prepare hybrid biofilm-based systems, the biofilm grown on the glassy carbon electrode (geometric surface area, 1 cm2), was decorated with CNTs/PyBA by introducing an ink (46.5 mdm3) of the respective carbon colloidal suspension (containing 15% ethyl alcohol and stabilized with 0.1% Nafion perfluorinated resin solution at which had been obtained by diluting the commercial 5 wt% alcoholic–aqueous solution of Nafion). To produce a bifunctional electrocatalytic interface for oxygen reduction, the biofim-based material was over-coated (following air-drying and rinsing with buffer) with 15.5 mdm3 of 1 mmol dm 3 Co-porphyrin solution in DMSO (reagent quality). Aqueous inks of platinum nanoparticles (from Sigma-Aldrich) were prepared using the analogous procedure to that described earlier [37,45,46]. The appropriate amount of the ink (suspension) was dropped onto the electrodes surfaces (bare or biofilmmodified) to assure the Pt loading of 100 mg cm 2. To perform

Fig. 1. SEM images of (A) pristine Yersinia enterocolitica biofilm, and (B) the biofilm into which platinum nanoparticles have been introduced. Glassy carbon was used as the substrate.

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aggregates under vacuum conditions (SEM), it is reasonable to expect that the Yersinia enterocolitica biofilm (on glassy carbon) is microstructured (Fig. 1A) and is composed of irregularly distributed pellets exhibiting strong tendency to undergo agglomeration. The characteristic microstructured image is retained following introduction of Pt nanoparticles (Fig. 1B). These particles are known to have diameters on the level 5–8 nm [37,45,46], and they exhibit tendency to undergo agglomeration. Indeed, islands of agglomerates of Pt nanoparticles are clearly visible within pores between the pellets of the bacterial biofilm (Fig. 1B). While the SEM approach permits reliable characterization of such dense inorganic species as Pt nanoparticles, careful examination of bacterial aggregates (biofilms) would require other means of imaging. Therefore, we have performed a series of AFM measurements (Fig. 2) of bacterial (Yersinia entrocolitica) biofilm based films (deposited on glassy carbon electrode surfaces) under ambient hydrated conditions (upon exposure to the phosphate buffer electrolyte). The image of the pristine (fresh) biofilm on the electrode surface (Fig. 2A) is consistent with sub-microstructured morphology of biofilms and existence of the bacterial aggregates. This observation is not surprising because biofilms are well-known to form microcolonies [5–9] and exist as aggregates of microorganisms in which cells (living or non-living) adhere to each other when deposited on various surfaces. More careful examination of certain enlarged portions of the biofilms implies the elongated shape of bacterial cells (Fig. 2B). But the film’s morphology

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somewhat changes (Fig. 2C) upon the film’s dehydration (drying with argon) followed by covering with Nafion (despite “rehydration” by exposing to the phosphate buffer electrolyte). The AFM data (Fig. 2C) suggests that distinguishing between separate bacterial cells is barely feasible now. It is difficult to attribute the observed topographical changes solely to presence of the overcoating layer of Nafion, a polyelectrolyte known to commonly used as a stabilizing agent in enzymatic bioelectrochemistry and in electrocatalysis [41,43,45,46]. The topography of Fig. 2C is basically retained upon introducing carbon nanotubes (CNTs) except that the elongated hair-like structures start to appear due to the presence of CNTs (Fig. 2D). Finally, depositing of Co-porphyrin (as the outer-most layer) does not change morphology of the latter biofilm based system and produces the image as for Fig. 2D (for simplicity not shown here). To comment whether the biofilm based systems are still alive following fabrication, we subjected them to microbiological examination involving re-seeding of the bacterial culture. The samples considered here included (i) a freshly prepared Y. enterocolitica biofilm (a reference model sample) (ii) the biofilm dried and stabilized by overcoating with Nafion, (iii) the biofilm covered with carbon nanotubes and stabilized with Nafion, and (iv) the biofilm covered with carbon nanotubes, decorated with Coporphyrin and stabilized with Nafion. All samples were re-seeded again using the Luria-Bertani Agar (the cultures were re-seeded for 48 h at 26  C). Then the grown bacterial colonies have been

Fig. 2. AFM images of (A) freshly prepared Yersinia enterocolitica biofilm, (B) enlarged two bacterial cells of elongated shape, (C) dehydrated Yersinia enterocolitica biofilm covered with Nafion (it was rehydrated before the AFM measurement), and (D) dehydrated Yersinia enterocolitica biofilm with multiwalled carbon nanotubes and covered with Nafion, (it was rehydrated before the AFM measurement).

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calculated, and the following results indicating a degree of reseeding were obtained: (i) 14.9  105 (ii) 15.9  102 (iii) 4.2  102, and (iv) 0 (in cfu/cm3 units). By assuming that a freshly prepared Y. enterocolitica biofilm is a model sample with the maximum number of bacterial colonies which could be obtained after reseeding, the results were recalculated into percent-values (i) 100, (ii) 0.11, (iii) 0.3, and (iv) 0%. It can be concluded that vitality of the pre-treated (modified) biofilm samples is negligible. Most likely, the drying step in argon followed by over-coating with Nafion (in addition to introducing of carbon nanotubes or Co-porphyrin species) leads to killing bacterial cells. Furthermore, as demonstrated in Fig. 2A–D, the above observations are accompanied by morphological changes. Despite rehydration (exposure to the phosphate buffer electrolyte), the once dried (Nafion-stabilized) bacterial cells are irreversibly agglomerated to form dense biofilm deposits on electrode surfaces. 3.2. Diagnosis of electrocatalytic properties of pristine biofilms Our initial voltammetric experiments aimed at identifying electrochemical properties of the pristine Yersinia enterocolitica biofilm (deposited on such an inert electrode substrate as glassy carbon) and at probing the system’s electrocatalytic properties toward reduction of oxygen (Fig. 3A) and hydrogen peroxide (Fig. 3B). The latter processes proceed at bare glassy carbon electrodes with large overpotentials in neutral media (for simplicity not shown here), namely with onsets of reduction waves starting typically below 0.6 V. It is apparent from the data of Fig. 3A and B, that the biofilm exhibits some catalytic reactivity during reductions of O2 and H2O2. Here, the electroreduction currents for oxygen and hydrogen peroxide start to appear at

potentials equal to 0.1 and 0.25 V, respectively. But high electroreduction currents are observed at rather low potentials (below 0.2 and 0.4 V), obviously too negative to be attractive for bioenergetic applications (biofuel cells, biobatteries). The drawnout shapes of cyclic voltammograms recorded during the electrocatalytic reductions (Fig. 3A and B) are indicative of possible limitations originating either from the system’s reactivity or dynamics of charge propagation. On the other hand, the bioelectrocatalytic system is well-behaved as demonstrated by the fact that the hydrogen peroxide reduction currents increase linearly with the reactant concentration (Fig. 3C). Indeed, the dependence of the rising chronoamperometric currents on the increasing concentration is linear in the concentration range from 0.1 to 0.9 mmol dm 3 H2O2 (Inset to Fig. 3C). The currents level off at concentrations higher than 1.4 mmol dm 3; and it can be estimated that the maximum current density of 1.05 mA cm 2 is then obtained. Although we do not have unequivocal evidence for the presence of heme prosthetic groups (bacterial c-cytochromes) or terminal reductases in bacterial periplasm [6–8], the observed electrocatalytic activities of pristine Yersinia enterocolitica biofilms (Fig. 3) may reflect existence of such catalytic centers. The appearance of a small background peak (in absence of oxygen) at potentials lower than 0.2 V (solid line in Fig. 3A) could be attributed to electroactivity of heme groups. On the other hand, population (concentration) of the N4-coordinated iron sites in the biofilm would be rather low. Under such conditions, electron transfers via cell-membrane-bound cytochromes would not be effective due to existence of too large distances for electron self-exchange and direct communication with the electrode surface [47,48]. But mobility and availability of counterions within the biofilm’s open

Fig. 3. Voltammetric reduction of (A) oxygen (in the O2-saturated electrolyte) and (B) hydrogen peroxide at the Yersinia enterocolitica biofilm on glassy carbon. Solid lines show background responses in the de-aerated electrolyte, 0.1 mol dm 3 phosphate buffer (pH = 6.1). Dashed, dotted and dashed-dotted lines refer (in B) to concentrations of H2O2 equal to 1, 2 and 5 mmol dm 3. Scan rate, 10 mV s 1. (C) The dependence of steady-state amperometric current responses on concentration (recorded following successive additions of 0.1 mmol dm 3 hydrogen peroxide to the deoxygenenated buffer). Applied potential, 0.8 V. Inset to (C) illustrates changes of amperometric currents with time of injection of hydrogen peroxide (growing H2O2-concentration).

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water channels existing in between microbial colonies [49] is expected to be high enough to support redox transitions. The effective operation at the electrocatalytic redox polymer type films requires fast charge distribution within the layer and mobility of charge compensating ions, in addition to presence of the active sites [40,47,48,50,51]. Having in mind this strategy, carbon nanostructures or redox mediators should be incorporated into the biofilm layers to facilitate transfers of electrons at the catalytic interface. 3.3. Electrocatalysis at biofilm-based hybrid-systems Fig. 4A shows cyclic voltammetric responses recorded at the PyBA-modified CNT-containing biofilm in presence (dashed line) and absence (solid line) of oxygen in the electrolyte (phosphate buffer at pH = 6.1). The results imply increase of the electrocatalytic activity toward oxygen reduction following introduction CNTs/ PyBA (TEM image is shown in Inset to Fig. 4A). Upon consideration of data of Figs. 4 A and 3 A, the background-subtracted oxygenreduction net-currents characteristic of linear scan (forward) voltammetric responses have been plotted and presented in Fig. 4B. The hybrid layer composed of bacterial biofilm and CNTs/ PyBA has exhibited much larger electrocatalytic currents (dashed line) than the pristine biofilm (solid line). Judging from the relatively moderate electrocatalytic response of CNTs/PyBA during the oxygen reduction (dotted line), the enhancement phenomenon cannot be simply attributed to reactivity of carbon nanotubes (although they are known to have some activity depending on their pre-treatment and presence of iron or cobalt impurities [41]). The result (Fig. 4B, dashed line) should be rationalized in terms of the

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improvement of charge distribution within the bacterial biofilm permitting better utilization of catalytic (e.g. heme-type) sites. Provided that the heme-containing proteins exist and are active (as in carbon nanotube supported peroxidase enzymes [43]), it is not surprising that the hybrid film composed of the biofilm and CNTs/ PyBA has also produced pretty well-defined voltammetric responses and sizeable electrocatalytic currents during reduction of hydrogen peroxide (Fig. 4C). Furthermore, the voltammetric responses are fairly well-defined, and the peak currents are the H2O2-concentration dependent (for simplicity working curves are not shown here). Among important properties of Fig. 4B voltammetric responses characteristic of the biofilm based systems are their very good reproducibility (standard deviation within 5%; based on 10 consecutive experiments performed the same day) and the reasonable long-term stability of responses recorded occasionally over the period of two weeks (standard deviation, 12%; based on 8 independent experiments). Furthermore, even after 14 days, the biofilm based systems can work reproducibly (within 7-8%) during repetitive measurements. Though the current densities recorded in Fig. 4 could be of interest to analytical sensing [52,53], the oxygen electroreduction potential (Fig. 4A) is too negative to be of any utility in bioenergetics, e.g. in biofuel cells [41] or in biobatteries [42]. A successful approach to shift the oxygen reduction potential toward more positive values was designed as bifunctional bioelectrocatalytic system [43] in which the catalyst initiating the oxygen reduction reaction (Co-porphyrin) was combined with an active matrix (peroxidase enzyme) capable of the effective reductive decomposition of hydrogen peroxide. Having in mind this concept,

Fig. 4. Voltammetric reduction of (A) oxygen (in the O2-saturated electrolyte) and (C) hydrogen peroxide at the Yersinia enterocolitica biofilm containing carbon nanotubes (CNTs/PyBA). Solid lines (in A and C) show background responses in the de-aerated electrolyte, 0.1 mol dm 3 phosphate buffer (pH = 6.1). Dashed, dotted and dashed-dotted lines refer (in C) to concentrations of H2O2 equal to 1, 2 and 5 mmol dm 3. (B) Background-subtracted voltammetric responses for the oxygen reduction at the pristine biofilm (solid line), CNTs/PyBA (dotted line), and the biofilm together with CNTs/PyBA (dashed line). Inset to (A) illustrates TEM of CNTs/PyBA. Electrode substrate, glassy carbon. Scan rate, 10 mV s 1.

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Fig. 5. Voltammetric reduction of (A) oxygen (in the O2-saturated electrolyte) and (B) hydrogen peroxide at the optimum Yersinia enterocolitica based hybrid biofilm containing carbon nanotubes (CNTs/PyBA) and over-coated with Co-porphyrin. Solid lines show background responses in the de-aerated electrolyte, 0.1 mol dm 3 phosphate buffer (pH = 6.1). Dashed, dotted and dashed-dotted lines refer (in B) to concentrations of H2O2 equal to 1, 2 and 5 mmol dm 3. Electrode substrate, glassy carbon. Scan rate, 10 mV s 1. (C) The dependence of steady-state chronamparometric current responses (determined after 100 s) on concentration (recorded following successive additions of hydrogen peroxide to the deoxygenenated buffer). Applied potential, 0.2 V. Inset to (C) illustrates representative chronoamperometric responses recorded in deoxygenated phosphate buffer (pH = 6.1) in presence of (a) 1, (b) 0.5, and (c) 0.25 mmol dm 3 H2O2 (dotted, dashed, and solid lines, respectively).

we have fabricated the superior hybrid system by over-coating the CNTs/PyBA-containing bacterial biofilm (as for Fig. 4A) with Coporphyrin catalyst. The oxygen reduction voltammetric peak (Fig. 5A) is shifted now more than 300 mV relative the analogous behavior observed in the absence of Co-porphyrin (Fig. 4A). It is noteworthy that the resulting oxygen reduction peak current (at 0.9 V) is fairly reproducible, 1.15 (0.10) mA cm 2 (uncertainty is based on standard deviation from 8 consecutive experiments). But the enhancement effect cannot be correlated simply with the activity of Co-porphyrin because, contrary to the present system utilizing the bacterial biofilm matrix, the oxygen reduction electrocatalytic currents were reported to be low when measured at Co-porphyrin supported onto CNTs/PyBA [43]. Apparently, coexistence of Co-porphyrin and active biofilm sites, together carbon nanotube carriers, leads to the synergistic effect. The fact, that the hybrid system composed of Co-porphyrin, bacterial biofilm and CNTs/PyBA has produced the high hydrogen peroxide reduction currents at fairly positive potentials (Fig. 5B), implies the importance of the following chemical (H2O2 reductive decomposition) reaction as well. This factor would be dominating in explaining the observed positive potential shift if rate of the following chemical reaction [54,55], namely the H2O2 reductive decomposition, is relatively high [56]. 3.4. Kinetic analysis of H2O2-reduction at the optimum biofilm based film To comment on the dynamics of the hydrogen peroxide reduction at the optimum hybrid catalyst (as for Fig. 5B), a series

of steady-state current diagnostic measurements at different concentrations of H2O2 have been performed. Fig. 5C illustrates dependencies of steady-state chronoamperometric currents on concentration in the range from 0.1 to 2.3 mmol dm 3. Representative chronoamperograms recorded at 0.2 V for three different concentrations are shown in Inset to Fig. 5C. Among important issues is that the steady-state plateau currents have been developed in all cases. As a rule, the current value (point in Fig. 5C) has been determined after 100 s from the chronoamperometric data. The current responses have been linearly dependent on concentration up to ca. 0.6 mmol dm 3 of hydrogen peroxide. This result is consistent with the pseudo-first-order kinetic pattern. At concentrations higher than 0.6 mmol dm 3 of H2O2, deviation from linearity is observed, and the current values tend to level off to reach the constant response. The latter situation is in agreement with the pseudo-zero-order kinetics with respect to concentration. The saturation current (that is equal to ca. 5.6 mA cm 2) is the maximum electrocatalytic current density, jmax (that can be viewed as the kinetic parameter). For practical reasons (e.g. to comment about the system’s kinetics), it makes sense to take also into account concentration, C1/2jmax, at which current reaches the half value of the maximum extrapolated current (i.e. j1/2max). For the system studied here, the C1/2jmax concentration has been found to be approximately equal to 0.4 mmol dm 3 H2O2. At this characteristic C1/2jmax concentration, the electrocatalytic system is still well-behaved because the respective catalytic currents are in the range of the approximately linear dependencies on concentration. Although interpretation and practical meaning are different, the whole approach proposed here resembles the well-known

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Michaelis-Menten kinetics analysis proposed together with the Lineweaver-Burk equation for enzymatic kinetics in bioelectrochemistry [57–59]. In both cases, rates of catalytic reactions (here expressed in terms of steady-state electrocatalytic currents) rise linearly as concentrations increase and then begin to level off and approach maximum at higher concentrations. Additional kinetic information can be obtained by referring to fundamentals of electrochemical kinetic analysis [54,55], and by considering the fact that our electrocatalytic systems yield (after 60 s) steady-state currents, they are kinetically slow enough to be described as totally dependent on interfacial charge transfer rates rather than diffusional mass transport, one can refer to the classic kinetic equation, j = nFkbC*, where j refers to the steady-state current density (here we consider j1/2max recorded at C1/2jmax and equal to 2.8  10 4 A cm 2), n is a number electrons involved (here n = 2 for the reduction of H2O2 to H2O), F is Faraday constant (96500C mol 1), kb is the rate constant for the reduction in heterogeneous units (cm s 1), and C* stands for the concentration of a reactant (here we use C1/2jmax = 4  10 7 mol cm 3 H2O2). Thus the heterogeneous rate constant kb = 5  10 3 cm s 1 can be estimated. Although the latter value has approximate meaning, it implies the reasonably fast electrode kinetics. The result supports our view that, relative to the slow (first) step of the oxygen reduction at Co-porphyrin [43], the fast H2O2 reductive decomposition could become of importance in the overall oxygenreduction mechanism and, thus, explain the already mentioned peak-current increase and its positive potential shift during oxygen reduction (compare Figs. 5A and 4A). 3.5. Comparison to model catalytic systems for oxygen electroreduction Fig. 6 illustrates the background-subtracted voltammetric reduction currents illustrating reduction of oxygen at the model system composed of Pt nanoparticles dispersed over pristine multi-walled carbon nanotubes (solid line) relative to the characteristics of the optimum hybrid system (as for Fig. 5A) utilizing CNTs/PyBA and Co-porphyrin supported onto the bacterial biofilm (dashed line). First, care has been exercised to activate carbon-nanotube supported platinum nanoparticles (Inset A to Fig. 6) by potential cycling in sulfuric acid to yield the voltammetric response characteristic of pre-cleaned platinum

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surface (Inset B to Fig. 6). Further diagnostic experiments have been performed in phosphate buffer at pH = 6.1. The carbonnanotube-supported platinum catalyst is considered as the model one because it has produced the relatively largest and the most reproducible O2-reduction peak-currents in comparison to the performance of the analogous systems fabricated by supporting Pt over Vulcan XC–72 R carbon black, Norit SX2 activated carbon or even gold nanoparticles [60] (for simplicity, these results are not shown here). Careful comparison of the voltammetric responses (Fig. 6) implies almost identical peak current values. In other words, the optimum biofilm-based hybrid system exhibits the electroreduction current efficiency similar to that characteristic of the model platinum catalyst. It can also be rationalized that, in both cases, the reduction of oxygen proceeds predominantly according to the four-electron (rather than two-electron) mechanism, as postulated for the Pt-based catalytic systems [61]. The fact that the O2-reduction peak potentials somewhat differ in both cases (compare solid and dashed lines in Fig. 6) reflects distinct activation mechanisms of oxygen favoring (i.e. operating at more positive potentials) for platinum rather than the Co-macrocyclic complex. Other porphyrin or phtalocyanine complexes (e.g. of Fe or Mn) will be a subject of future studies. When compared to the performance of multi-copper oxidase enzymes such as laccase, bilirubin oxidase or copper efflux oxidase (known as being capable to reduce oxygen directly to water) [41,42,62–65], the optimum biofilm based system (Fig. 5A) drives the oxygen reduction at less positive potentials (drawback when it comes to potential applications in biofuel cells) but, in practice, it does produce higher electrocatalytic currents (advantageous for amperometric biosensing or even air-zinc biobatteries [42]). With respect to the possible oxygen reduction mechanisms at biofilm cathodes, recent important works addressing simultaneous determination of oxygen and hydrogen peroxide intermediate in river and sea water should be mentioned here [66,67]. Our optimum biofilm based system (Fig. 5A) is bifunctional in nature: by analogy to our previous work utilizing horseradish peroxidase and Co-porhyrin [43], the present system seems to be characterized by the capability of inducing oxygen reduction (to hydrogen peroxide) at the metalloporphyrin sites followed by the reductive H2O2-intermediate decomposition at the CNT-containing biofilm matrix. Indeed, the performance of the biofilm based catalyst toward hydrogen peroxide reduction (Fig. 5B and C) is analogous to that of the horseradish-peroxidase-enzyme based system [43]. Therefore the obtained oxygen reduction currents have been comparable to those observed at the model Pt catalyst (Fig. 6) favoring four-electron reduction of O2 to H2O. 3.6. Evaluation of biofilm-based matrix for supporting catalytic platinum nanoparticles

Fig. 6. Background-subtracted voltammetric currents for the oxygen reduction at platinum nanoparticles (loading, 100 mg cm 2) supported onto bare carbon nanotubes (solid line) and at the optimum (as for Fig. 5A) biofilm-based hybrid catalyst (dashed line). Electrode substrate, glassy carbon. Scan rate, 10 mV s 1. Insets illustrate (A) TEM image, and (B) cyclic voltammetric response (recorded in deaerated 0.5 mol dm 3 H2SO4) of the carbon-nanotube-supported platinum nanoparticles.

There have been recent reports describing electrocatalytic utility of bio-templated architectures of noble metal nanoparticles and nanostructured composite cathodes with respect to the reductions of carbon dioxide [37] and oxygen [68]. Consequently, we have also considered here the biofilm-enhanced design utilizing Pt nanoparticles. As illustrated in Insets to Fig. 7A, bare Pt nanoparticles have been examined by (i) TEM, as well as by (ii) voltammetric potential cycling in sulfuric acid: the resulting response is characteristic of the clean platinum. Later, the representative cyclic voltammetric responses of platinum nanoparticles have been recorded in phosphate buffer (pH = 6.1), first, without (Fig. 7A) and, second, together with (Fig. 7B) the bacterial biofilm. Experiments have been performed in absence (solid line) and presence (dashed line) of oxygen. To comment on the platinum activity, the voltammetric experiments have been performed in the extended potential range to include the hydrogen evolution region.

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distribution within the layer of bacterial aggregates. The resulting carbon-nanotube-supported biofilm was demonstrated to function as the active polymer-like matrix for Co-porphyrin sites. Our diagnostic experiments at different concentrations of H2O2, support a view that the effect of the fast following chemical (H2O2-reductive-decomposition) reaction could be the dominating factor in explaining the observed positive potential shift observed during the oxygen reduction. The fact, that the optimum hybrid biofilm-based catalytic system produced the oxygen reduction peak current comparable to that observed at the model platinum containing catalyst, would imply the efficient four-electron-type reduction mechanism. Acknowledgements This work was supported by the National Science Center (Poland) under Maestro Project 2012/04/A/ST4/00287. The AFM measurements were carried out at the Biological and Chemical Research Center, University of Warsaw, established within the project co-financed by European Union from the European Regional Development Fund under the Operational Program Innovative Economy, 2007-2013. References

Fig. 7. Cyclic volatmmetric responses of platinum nanoparticles deposited (A) alone and (B) in combination with the Yersinia entrocolitica bacterial biofilm on glassy carbon. Voltammograms have been recorded at 10 mV s 1 in 0.1 mol dm 3 phosphate buffer (pH = 6.1) in absence (dashed line) and presence (solid line) of oxygen. Insets to (A) illustrate (i) TEM image, and (ii) cyclic voltammetric response (recorded in deaerated 0.5 mol dm 3 H2SO4) of platinum nanoparticles. Inset to (B) compares the background-subtracted currents plotted for the oxygen reduction in absence (solid line) and presence (dashed line) of the biofilm.

It is apparent from the data of Fig. 7 that the bacterial (Yersinia enterocolitica) biofilm tends to inhibit the catalytic activity of platinum nanoparticles toward both the hydrogen evolution and the oxygen electroreduction. This effect is particularly apparent when the background-subtracted currents have been plotted in the oxygen reduction region in absence (solid line) and presence (dashed line) of the biofilm are compared (Inset to Fig. 7B). Apparently, the bio-organic portions of the biofilm matrix passivate surfaces of Pt nanoparticles thus precluding the adsorptive activation of the oxygen molecule. This inhibiting phenomenon was obviously not operative in the case of such a molecular-type catalyst as macrocyclic Co-porphyrin considered earlier in this work. 4. Conclusions We showed here that bacterial (Yersinia enterocolitica) biofilm (despite the fact that the pre-treated (modified) biofilm samples were not vital) exhibited itself electrocatalytic properties toward reduction of oxygen and hydrogen peroxide under mild conditions (phosphate buffer at pH = 6.1). Although we did not have any direct evidence for the presence of heme or cyctochrom c groups in the biofilm matrix, in view of the recent report [69], their existence could not be excluded. The catalytic features were enhanced upon introduction of carbon nanotubes (CNTs/PyBA). Among their important characteristics was their ability to facilitate charge

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