Paulo Pierre-Yves Blanchard

Henrique M Buzzetti

Bridget Davies

Yannig Nedellec

Marcelo Emerson

Andrew Girotto

Alan Le Gross

Yuta Goff

Serge Nishina

Michael Cosnier

Holzinger

Henrique M Paulo

Buzzetti

] Emerson

Marcelo Girotto

Andrew J Gross

Alan Le Goff

Yuta Nishina

Serge Cosnier

Michael Holzinger

Electrosynthesis of Pyrenediones on Carbon Nanotube Electrodes for Efficient Electron Transfer with FAD-dependent Glucose Dehydrogenase in Biofuel Cell Anodes

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Enzymatic glucose biofuel cells have gained wide attention due to the possibility to harvest energy out of complex media like body liquids [START_REF] Shi | [END_REF] or soft drinks. [2] A further advantage is the eco-friendly character of such power generators for low power consuming disposable devices. [3] The challenge for such biological catalysts is to transfer the electrons involved in the catalytic oxidation process to an electrode. Fungal glucose oxidase (GOx) and FAD dependent glucose dehydrogenases (FAD-GDH) are two of the mostly used glucose oxidizing enzymes [4]. However, achieving electron transfer between their catalytic centers and an electrode is quite difficult, but not impossible, since their active sites are deeply buried inside the insulating protein [5]. In this case, electron shuttles, called redox mediators have to be employed, even when a loss of the final cell voltage has to be taken into account. There are many examples of efficient redox mediators for electron transfers with good compromises between electron transfer kinetics at acceptable overpotentials. [6] They are mostly based on quinone derivatives [4] or redox tunable Osmium complexes [6][7]. Beside the optimization of electrocatalyic currents and open circuit potential (OCPs), the stability of operational mediator-enzyme assembly is an important criterion for enzymatic glucose biofuel cell research since the operational lifetime of such power generators is the principal drawback for realistic applications. Pyrene and its derivatives have become prominent for the reliable non-covalent functionalization of carbon nanotubes (CNTs) [8]. They have gained interest for the immobilization of biomolecules on sp2 nanocarbon electrodes, principally for biosensing applications [9]. Pyrene itself has no particular electrochemical signature over a wide potential range. However, it can be electropolymerized at 1 V vs. saturated calomel reference electrode (SCE) in organic solvents like acetonitrile, leading to the appearance of a reversible redox system at E½ = 0.43 V vs. Ag/AgCl that corresponds to a reinforced poly-(oligo-) pyrene layer on carbon nanotube surfaces [10]. Oxidation of pyrene and other aromatic hydrocarbons by photo-or electrochemistry became an efficient method for remediation and depollution of contaminated soils [11]. Barathi et al. [12] electro-oxidized pyrene in phosphate buffer (pH 7) and used the formed oxides to immobilize cytochrome c via copper coordination to construct a hydrogen peroxide biosensor. The authors proposed the formation of 4,5,9,10 pyrenetetrone after electrosynthesis based on the observed coordination capacities and by mass spectrometry. Targeted synthesis routes have been developed to obtain pyrenediones and -tetrones since they are interesting intermediates for the synthesis of polycyclic compounds. In these cases, the isolated products were comprehensively characterized [13]. Furthermore, pyrene oxides provide an interesting negative redox potential at -0.1 V vs. SCE at pH 7 because it is close to the redox potentials of flavin adenine dinucleotide (FAD) and FAD containing redox enzymes (between -0.49 V [14] and -0.277 V [15]). The electrontransfer from such enzymes to the mediator is thermodynamically favorable when the redox potential of the mediator is slightly higher that the potential of the active center of the enzyme. Thus, we envisaged the possibility to electrosynthesize specific quinones for mediated electron transfer with flavin adenine dinucleotide FAD dependent enzymes. We evaluated this approach using FAD-GDH in a glucose bioanode setup and were able to isolate the pyrene derivatives to determine their chemical structure with 1H-NMR spectroscopy.

Results and Discussion Electrosynthesis and characterization of pyrenediones

The carbon nanotube -pyrene modified electrodes, prepared as described in the experimental section, were placed in phosphate buffer solution (0.2 molL-1) at different pH values. The electro-oxidation of pyrene was done by recording cyclic voltammograms between initial potentials of -0.2 V, -0.5 V, -1.0 V and a switching potential of 1.2 V for pH 2, 7, and 12, respectively, at a scan rate of 100 mVs-1. Figure 1A-C shows the evolution of the reversible redox waves at different pH values during 15 cycles corresponding to the two-electron transfer process of pyrenedione, as revealed in Section 2. The irreversible oxidation at around 1 V vs. SCE, corresponding to a definite amount of pyrene, decreases after each cycle. On the second and subsequent scans, a new redox species with reversible behavior is formed with E½ = 0.17 V at pH 2 (Figure 1A), E½ = -0.09 V at pH 7 (Figure 1B), and E½ = -0.37 V at pH 12 (Figure 1C) vs. SCE. The peak currents and the charge under the oxidation and reduction peaks are clearly higher after 15 scans of electro-oxidation at pH 2 than at pH 7 and pH 12. The improved yield of electro-oxidized species by electro-oxidation at acidic pH reflects a proton assisted pyrene integration of the anodic peak area to give the transferred charges, Q, according to the formula: . Where n is the number of involved electrons, F is the Faraday constant, and A is the geometric surface area. For an identic amount of deposited pyrene, when electro-oxidized at pH 2, a surface coverage of -2 was determined while at pH 7, 6.52 × 10-9 mol cm-2 of pyrenedione was formed. The redox peaks correspond to the two-electron transfer process of pyrenedione, as revealed in Section 2. The modified electrodes were characterized by cyclic voltammetry in fresh buffer solution at pH 7 in the range between -0.4 and 0.4 V vs. SCE at different scan rates (Figure 1D) with scan rates varying from 5 to 50 mVs-1. By plotting the maximum currents as a function of the scan rate, a linear dependency is observed, confirming that the electro-oxidized species remains immobilized (inset, Figure 1D). Pyrene functionalization of carbon nanotubes became prominent in the literature because of the stable π-stacking interactions between their aromatic structures. The electro-oxidation of pyrene, disturbing its π system, seems not to affect the stability of these interactions. It should be noted that the presence of carbon nanotubes is necessary for the later use of electrosynthesized pyrenediones. Cyclic voltammetric, or amperometric pyrene electro-oxidation using standard glassy carbon electrodes (GCE) led to non-conclusive results, most likely related to the desorption of oxidized pyrene.

Characterization by 1H-NMR spectroscopy

In order to get more insight into which type of pyrenediones was formed, the electro-oxidized pyrene-MWCNT deposits were removed from 8 different modified electrodes. These deposits were subsequently immersed in 0.75 mL CDCl3 to desorb the formed pyrenedioness from the MWCNTs. After centrifugation, the supernatant that was free of solids was used for the 1NMR studies. Figure 2 shows the measured features between 6.6 and 8.8 ppm. By referring to comprehensively characterized pyrenediones derivatives in the literature [13], a mixture of 1,6-pyrenedione and 1,8-pyrenedione can be identified. 1,8-pyrenedione shows a characteristic singlet (2H, protons H) at 8.65 ppm. Two doublets are assigned to protons D and C of 1,6-pyrenedione at 8.49 and 7.85 ppm, respectively. By integration of the peaks H and D, a ratio of the electrosynthesized 1,6-pyrenedione and 1,8-pyrenedione of 2:1 can be determined. It should be noted that no characteristic peak of 4,5-pyrenedione or 4,5,9,10-pyrenetetrone, or any other pyrene quinones [16], could be resolved [13a]. Based on these ratios and the pH effect, a mechanism for the electro oxidation of pyrene in buffer solution can be proposed according to the mechanism of the oxidative photolysis of pyrene in water [11b]. The first oxidation step initially forms a radical which is trapped by a water molecule while the positively polarized MWCNTs serve as a radical scavenger [17], leading to 1-hydroxy pyrene. The formation of a defect on the MWCN T wall is accepted since the outer wall is already highly defective. The second oxidation step occurs in either the 6 or the 8 position, where the formed aromatic radicals are again trapped by water and scavenged by the MWCNT electrode giving the 1,6-and 1,8-dihydroxy pyrene derivative and water in the overall redox equation. These dihydroxy pyrenes may then be reversibly oxidized in a two electron process by cyclic voltammetry, as observed in Figure 1. The intermittent formation of H+ ions is more effective under acidic conditions, supporting the improved electro synthesis of pyrenediones at pH 2. Figure S1 in the supporting information shows the pH dependency of this redox system which is characteristic for quinones. A more interesting point concerns the position where water is added in the second oxidation step. It seems that the pyrene intermediates are better stabilized if they can keep intact their naphthalene units, obeying Hückel's law, rather than forming phenanthrene units when water traps the radical in the ortho-position. This explains the absence of 4,5 pyrenedione in the extracted solutions. Furthermore, the two electro-oxidation steps at two different positions most likely occur almost simultaneously since no traces of monohydroxy pyrene could be isolated, in contrast to Sigman et al. [11b]. Compared to similar experiments in dry organic solvents, there is no water to trap the formed radicals and induces the polymerization process with other pyrene molecules instead of the described water addition reaction.

Evaluation of pyrenedione for mediated electron transfer with FAD-GDH for catalytic glucose oxidation

The onset potential at pH 7 of around -0.1 V of pyrenedione is in the optimal range for mediated electron transfer with FAD dependent glucose oxidizing enzymes like glucose oxidase or FAD-GDH. We used FAD-GDH from Aspergillus sp for the experiments concerning the wiring efficiency of pyrenedione. The bioelectrodes were prepared by drop-casting 40 µL of a 5 mg mL-1 enzyme solution onto the modified electrodes and incubating the electrodes for 2 h at 3 °C. After rinsing with phosphate buffer solution (0.1 molL-1, pH 7), cyclic voltammograms were recorded in the presence and absence of 200 mmol L-1 glucose at pH 7. Figure 3 shows the voltammetric behaviour of the bioelectrodes prepared using pyrenediones initially electro-oxidized at pH 2 (Figure 3A) and at pH 7 (Figure 3B). In both cases, in the presence of glucose, a drastic increase in current signal with a sigmoidal wave is observed at 20 mV s-1 consistent with effective electrocatalytic oxidation of glucose mediated by the pyrenedione molecules. When pyrene was electro-oxidized at pH 2, the maximum current obtained of 1.7 mA (8.51 mA cm-²) is more than 40% higher than that for the pyrenedione electro-oxidized at pH 7 (1.18 mA, 5.93 mA cm-²). This reflects the improved electro-oxidation yield for the electroactive mediator at lower pH.

Evaluation of the steady-state current at a fixed potential of 0.2 V vs. SCE as a function of glucose concentration revealed a linear increase between 1 mmol L-1 and 30 mmol L-1. The current increases with increasing concentration up to a maximum of 1.2 mA, or 13.5 mA cm-2 at 100 mmol L-1 for the pyrenedione electrode prepared at pH 2 (Figure S2). It is noted that the maximum current performance is almost reached at 50 mmol L-1. The data in Figure S2 shows that the linear range matches perfectly with the required detection range of commercialized glucose biosensors. Further data including the apparent Michaelis Menten constant and sensitivity are available in the supporting information. Figure 3: cyclic voltammograms of electrocatalytic glucose oxidation using FAD-GDH modified pyrene-MWCNT electrodes formed by electro-oxidation of pyrene at A) pH 2 and B) pH 7. The cyclic voltammograms were recorded in phosphate buffer (0.1 mol L-1, pH 7) in the absence (black) and presence (grey) of 200 mmol L-1 glucose at a scan rate of 20 mV s-1 Comparison of the wiring performances for catalytic glucose oxidation with 1,4 naphthoquinone.

Naphthoquinone is one of the most commonly used quinone based mediators for the enzymatic bioanodes of glucose biofuel cells [18]. This is why this type of quinone was chosen as a reference mediator to evaluate the appropriateness of electrosynthesized pyrenedione for efficient electron transfer with FAD-GDH. Figure 4A shows an overlay of the cyclic voltammograms of pyrene quinone and 1,4 naphthoquinone modified MWCNT electrodes recorded in 0.1 molL-1 phosphate buffer pH 7 in the presence and absence of 200 mmol L-1 glucose. With 1,4 naphthoquinone as the mediator, in the absence of glucose, the well-known redox system around -0.15 V vs. SCE with an onset potential of -0.2 V is observed. In presence of glucose, a catalytic current density is observed reaching up to 3.3 mA cm-² at 0.2 V. The redox wave at -0.036 V) with an onset potential of -0.1 V for pyrenedione is slightly more positive but leads to clearly higher current densities up to19 mA cm-² at 0.2 V. It has to be noted that the comparison was made using electrodes with identic surface coverages of electroactive species and by drop-casting the same amount of enzyme Figure 4B compares the recorded catalytic currents at 0.2 V, obtained from cyclic voltammograms, for each mediator as a function of the surface coverage.

pyrenedione clearly provides higher efficiency for the mediated electron transfer with FAD-GDH. Figure 4: A) Cyclic voltammograms of FAD-GDH modified MWCNT electrodes including (black) pyrenedione and (grey) 1,4 naphthoquinone in the absence (dashed lines) and the presence (straight line) of 200 mmol L-1 glucose at pH 7. B) Recorded catalytic currents at 0.2 V vs SCE as a function of the surface coverage of the mediator (black: pyrenedione; grey: 1,4 naphthoquinone) This clear superiority of pyrenedione for mediated electron transfer with FAD-GDH and acceptable low overpotentials was further investigated in terms of stability. Again, cyclic voltammetry was used for this study. Figure 5A shows the first and hundredth cyclic voltammograms recorded for pyrenequinone mediated FAD-GDH bioanodes at 200 mmolL-1 glucose between -0;4 V and 0.4 V vs. SCE.. The electrocatalytic performance decreased such that about 60% of the catalytic current was lost after 100 cycles. In the case of 1,4 naphthoquinone, a loss of more than 90 % was observed after 100 cycles (Figure 5C). The performance losses are generally explained by parasitic parallel reactions that reduces the reversibility of the redox system of quinoic mediators [19]. This is further accentuated with the higher water solubility of naphthohydroquinone compared to pyrenehydroquinone, the reduced forms of the mediators, leading to loss of the mediator in solution. Control experiments recorded with the same electrodes in the absence of glucose show that 47 % of the quinone activity is lost for the pyrenedione electrode after the intense treatment of 100 cycles while the naphthoquinone bioelectrodes lost 77% of its redox activity. This experiment allows us to conclude that about 15 % of the performance loss for both bioelectrodes is related to the decrease of the enzyme activity or enzyme desorption. The pyrenedione bioelectrode showed clearly better cycling stability compared to the naphthoquinone bioelectrode highlighting improved robustness for the electro-oxidised electrode. It has to be noted that that these cyclovoltammetric experiments have to be seen as accelerated aging tests. We studied performance losses over time using glucose oxidase and naphthoquinone where we observed an 80 % decrease of produced power after one-year storage in buffer solution and non-continuous discharges. [20] Figure 5: First and hundredth cyclic voltammogram of pyrenedione and 1,4 naphthoquinone mediated FAD-GDH bioelectrodes -0.4 and 0.4 V at pH 7 (scan rate 20 mVs-1). A) pyrenedione electrode in 200 mmolL-1 glucose. B) pyrenedione bioelectrode without glucose. C) 1,4 naphthoquinone bioelectrode in 200 mmol L-1 glucose. D) 1,4 naphthoquinone bioelectrode without glucose. Performances of the bioanode in a glucose/oxygen biofuel cell setup Finally, the studied bioanodes were assembled with a biocathode operating with direct electron transfer between proptoporphyrin IX modified MWCNTs and bilirubin oxidase (BOD) [21]. The bioelectrodes were placed in an electrochemical cell containing 200 mmol L-1 glucose in oxygen saturated buffer solution Figure 6A shows the obtained power profile of the resulting glucose biofuel cell. All data points were recorded after 30 s discharge to eliminate the capacitive current. A maximum power density of 1.35 mW cm-2 , calculated using the geometric surface area of 0.07 cm², could be determined with an OCV at 0.69 V. Figure 6B demonstrate the performance of this bioanode and its contribution on this power output regarding the limiting performances of the biocathode. The electro-oxydation of pyrene forms an efficient mediator for FAD GDH which surpasses currently used mediators like naphthoquinone in terms of electrocatalytic current densities and stability. Contrary to Barathi et al. [12], we determined the formation of a 2:1 mixture of 1,6-pyrenedione and 1,8-pyrenedione with 1H NMR spectroscopy via pyrene electro-oxidation in aqueous conditions. This approach opens up new possibilities in terms of the electrosynthesis of redox mediators from polyaromatic precursors. The oxidation of pyrene and other polyaromatic molecules is furthermore of interest for remediation and transformation into useful products. The easy synthesis procedure, electron transfer efficiency, stability, and potential to make a wide range of redox mediators makes the aqueous electrosynthesis approach a real alternative to other quinone or Os complex based redox mediators. Experimental Section Glassy carbon electrodes (3 mm diameter) were functionalized in several steps. First, MWCNTs were dispersed in NMP (5 mg mL-1) by sonication until a homogeneous black suspension was obtained. 20 µL of the MWCNT suspension was then deposited on the electrode to obtain a 5μmthick film. After drying, the MWCNT deposits were modified with pyrene by drop casting 20 µL of a 10 mmol L-1 CH2Cl2 pyrene solution. After an incubation/evaporation time over 10 min at room temperature in a fume hood, the electrodes were rinsed several times with H2O and then dried and stored at 5°C until use. Naphthoquinone modified MWCNT electrodes were prepared correspondingly. The bioanodes were formed as described in [21] by dropcasting of 20 µL of a 3mmolL-1 DMF solution of protoporphyrin IX on MWCNT electrodes. After drying, the electrodes were rinsed with acetonitrile. Finally, 20 µL of a 5 mg mL-1 phosphate buffer (0.1 molL-1, pH 7) solution of BOD from Myrothecium verrucaria is deposit and dried at 5°C.

Figure 1 :

 1 Cyclic voltammograms showing the electrosynthesis of pyrene quinone at A) pH 2, B) pH 7, and C pH 12. D) cyclic voltammograms of the obtained product in fresh phosphate buffer (0.1 molL-1, pH 7) at different scan rates from 5 to 50 mV s-1 and (inset) plot of the anodic and cathodic peak currents at varying scan rates.

Figure 2 :

 2 1H-NMR spectrum of the extracted pyrenediones derivatives with zoom on the doublets D and C (* = 13C satellite peaks). The detailed assignments of the peaks: 1,6-pyrenedione 1H NMR (300 MHz, CDCl3) δ 6.72 (d, 2H, J = 7.96 Hz, HA), δ 7.67 (d, 2H, J = 8.80 Hz, HB), δ 7.85 (d, 2H, J = 6.03 Hz, HC), δ 8.49 (d, 2H, J = 5.83 Hz, HD). 1,8-pyrenedione 1H NMR (300 MHz, CDCl3) δ 6.67 (d, 2H, J = 7.88 Hz, HE), δ 7.52 (d, 2H, J = 6.80 Hz, HF), δ 7.69 (d, 2H, J = 7.76 Hz, HG), δ 8.65 (d, 2H, J = 0.80 Hz, HH). Scheme 1: Proposed mechanism for the electrooxidation of pyrene at carbon nanotubes in acidic and basic aqueous buffer solution.

Figure 6 :

 6 A) Power profile (grey) and voltage dependent evolution of the maximum current densities (black) of the constructed glucose biofuel cell in in oxygen saturated phosphate buffer (0.1 mol L-1, pH 7). The current densities were recorded after 30 s discharge at given potentials. B) Bioelectrocatalytic performances of the bioanode with pyrenedione mediator (black) and the BOD-protoporphyrin IX based biocathode (grey) in oxygen saturated phosphate buffer solution (0.1 mmolL-1, pH 7) containing 200 mmol L-1 glucose at 20 mV s-1. Conclusion

Acknowledgements

The authors wish to acknowledge the support from the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG) and from the LabEx ARCANE (ANR-11-LABX-0003-01 and CBH-EUR-GS, ANR-17-EURE-0003). The


Mickaël Rimboud

Mohamed Barakat

Wafa Achouak

Alain Bergel

Marie-Line Délia
email: marieline.delia@ensiacet.fr

Oxygen-reducing microbial cathodes in hypersaline electrolyte

Keywords:

O 2 -reducing biocathodes were designed in hypersaline media (NaCl 45 g/L).

• Current density up to 2.2 A m -2 was reached from potential value of 0.2 V/SCE. • The biocathodes switched between operating two modes: maximum and stabilized. • Gammaproteobacteria strains were essential to the electrocatalytic activity.

• These strains were related to Thiohalobacter thiocyanaticus.

Introduction

Microbial fuel cells (MFC) can convert to electrical power the chemical energy stored in the organic matter dissolved in effluents. This conversion is made possible by microbial biofilms self-assembled on the anode surface that catalyze the oxidation of the organic matter. Performance of MFCs have shown significant progress in the last ten years [START_REF] Santoro | Microbial fuel cells: From fundamentals to applications. A review[END_REF] but the current density produced remains too low to consider extrapolation of the technology to large-sized cells. A major issue is the high internal resistance of the cells due to the low ionic conductivity of the electrolytes that are commonly used [START_REF] Oliot | Ion transport in microbial fuel cells: Key roles, theory and critical review[END_REF]. High internal resistance provokes high ohmic drop, which causes considerable power loss in MFC. An appealing solution has been raised by the use of saline and hypersaline environments and effluents [START_REF] Nam | Enhanced hydrogen generation using a saline catholyte in a two chamber microbial electrolysis cell[END_REF][START_REF] Grattieri | Halotolerant extremophile bacteria from the Great Salt Lake for recycling pollutants in microbial fuel cells[END_REF][START_REF] Grattieri | Microbial fuel cells in saline and hypersaline environments: Advancements, challenges and future perspectives[END_REF]. This kind of media is characterized by two antagonistic effects. The high Reactors consisted of Schott containing 540 mL of medium inoculated with 60 mL of salt marsh sediments. The medium derived from the Starkey medium and consisted of 0.5 g L -1 K 2 HPO 4 , 1 g L -1 NH 4 Cl, 1 g L -1 Na 2 SO 4 , 0.1 g L -1 CaCl 2 ⋅2H 2 O, 2 g L -1 MgSO 4 ⋅7H 2 O, 45 g L -1 NaCl. In some experiments, the medium was complemented with 50 mM of NaHCO 3 to act as an additional inorganic carbon source. This presence of NaHCO 3 will be indicated when appropriate. The pH was fixed at 7.0 in all reactors at the beginning of experiments. Air was continuously provided to the reactors via aquarium pumps equipped with porous frit in order to ensure permanent, gentle aeration. The solution in the reactors was thus maintained at saturation with air. The biocathodes were formed under constant polarization by using a threeelectrode set-up. The working electrodes/cathodes consisted of a 6 cm 2 of carbon felt (0.5 cm thickness) connected to coated titanium wires. Counter-electrodes were platinum meshes of 12 cm 2 connected via platinum wires. Reference electrodes were saturated calomel electrode (SCE, from Biologic, France, +0.240 V vs SHE). All the electrodes were placed in the reactor, with the reference electrodes as close as possible to the working electrode (distance less than 0.5 cm) and the auxiliary electrode at least 10 cm away from the working electrode. All potentials are expressed with respect to SCE reference. Reactors were kept in a stove at 30 • C. The pH was regularly measured in all reactors throughout the experiment, but not regulated.

Electrochemical measurements

Electrochemical measurements were performed under potentiostatic control using a multichannel potentiostat MPG2 from BioLogic, France, equipped with the EC-Lab software. Depending on experiments, working electrodes/biocathodes were polarized via chronoamperometry at +0.1 V or +0.3 V vs SCE and current was recorded vs time. These potential values were chosen with the aim of developing O 2 -reducing-biocathodes with catalytic activity at high potential. At some point during the experiments, the polarization was stopped to let the biocathodes rest at open circuit potential (OCP) during 15 min and after that, cyclic voltammetry was recorded. The potential was scanned from OCP up to +0.5 V, down to -0.6 V and back to OCP. The scan rate was 1 mV s -1 and two successive voltammograms were always recorded.

Population analyses

Biofilms were stripped from the electrode surfaces and resuspended in 15 mL of PBS by ultrasonication (15 min at 80 W, Fisher Scientific FB 15061). The biofilm suspensions were thereafter centrifugated and the solid material collected. DNA extraction was then performed using the extraction kit DNeasy PowerBiofilm from Qiagen (Netherlands). The DNA samples were sent to the Research and Testing Laboratory (RTLab -Lubbok, Texas, USA) for 454 pyrosequencing (Roche) by using 28F (5 ′ -GAG TTT GAT YMT GGC TC-3 ′ ) and 519R (5 ′ -GWA TTA CCG CGG CKG CTG-3 ′ ) primers.

Data were screened and trimmed on the basis of quality scores and bases below a quality threshold of 25 were discarded. Sequences with less than 250 nt and chimeric sequences were removed using UCHIME [START_REF] Dowd | Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)[END_REF][START_REF] Edgar | UCHIME improves sensitivity and speed of chimera detection[END_REF]. The open source software QIIME was used to analyzed the filtered sequences [START_REF] Caporaso | QIIME allows analysis of highthroughput community sequencing data[END_REF], which were clustered into operational taxonomic units (OTUs) at 97% sequence similarity using uclust [START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF]. RDP classifier was used to perform the taxonomic assignment down to the genus level, with a 0.8 bootstrap cut-off, the most abundant sequence of an OTU being considered representative [START_REF] Cole | The ribosomal database project: improved alignments and new tools for rRNA analysis[END_REF]. The Greengenes database (release gg_13_8_otus), included with the RDP classifier, was used for the taxonomic affiliations.

ICP analysis

Iron and Sulphur elements were quantified by ICP analyses both in the native sediment (before experiment, i.e. before aeration and salinity increases the conductivity of the medium and lowers the internal resistance of the fuel cell, on one hand. On the other hand, the high salinity reduces the viability of usual bacterial strains observed under ordinary, non-saline conditions. A recent review described the different works dealing with MFCs designed in various saline (0 to 1% w/v) to hypersaline (>3.5% w/v) environments [START_REF] Grattieri | Microbial fuel cells in saline and hypersaline environments: Advancements, challenges and future perspectives[END_REF].

Several studies have shown the capacity of salt marsh sediments to design powerful bioanodes [START_REF] Rousseau | Halotolerant bioanodes: The applied potantial modulates the electrochemical characteristics, the biofilm structure and the ration of the two dominant genera[END_REF][START_REF] Rousseau | Electrochemical characterization of microbial bioanodes formed on a collector/electrode system in a highly saline electrolyte[END_REF][START_REF] Rousseau | Correlation of the electrochemical kinetics of high-salinitytolerant bioanodes with the structure and microbial composition of the biofilm[END_REF][START_REF] Rousseau | Microbial bioanodes with high salinity tolerance for microbial fuel cells and microbial electrolysis cells[END_REF]. The medium used in these studies was a hypersaline solution, containing up to 60 g.L -1 of NaCl (6.0% w/v). The salt marsh sediment was the source of efficient electroactive halotolerant bacterial strains. The bioanodes were formed under constant polarization at +0.1 V/SCE and with continuous supply of sodium acetate as the substrate. Two main genera, Marinobacter and Desulfuromonas, were identified in the biofilms with a high degree of selection [START_REF] Rousseau | Correlation of the electrochemical kinetics of high-salinitytolerant bioanodes with the structure and microbial composition of the biofilm[END_REF][START_REF] Rousseau | Halotolerant bioanodes: The applied potantial modulates the electrochemical characteristics, the biofilm structure and the ration of the two dominant genera[END_REF]. The highest current densities, up to 85 A m -2 , were obtained with media containing 45 g/L NaCl.

Most of the works devoted to the design of MFCs in hypersaline media [START_REF] Grattieri | Microbial fuel cells in saline and hypersaline environments: Advancements, challenges and future perspectives[END_REF] have focused on the anodic part and used abiotic oxygen-reducing cathodes. These cathodes are generally based on platinum or cobalt porphyrins catalysts. Unfortunately, these catalysts are poorly efficient when implemented in MFC conditions, particularly because they lose most of their catalytic capacity at the pH values at the near-neutral pH values required in MFC. The poor efficiency of abiotic O 2 -reducing cathodes constitutes a major limiting factor in the development of hypersaline MFCs. A recent study, which designed an MFC in a solution containing 62-65 g/L NaCl, has reported the formation of an efficient microbial cathode, which was not ratelimiting with respect to the anode (Roustazadeh [START_REF] Sheikhyousefi | Investigation of different configurations of microbial fuel cells for the treatment of oilfield produced water[END_REF]. In the context of hypersaline media, microbial cathodes may consequently afford a promising alternative to overcome the poor performance of abiotic cathodes. Nevertheless, this topic remains poorly documented.

The purpose of the present work is to check the capacity of salt marsh sediments to form O 2 -reducing microbial cathodes. Salt marsh sediments were chosen as the inoculum because of their capacity to form efficient microbial anodes with maximum performance in media that contained 45 g/L NaCl. The same medium was used here. The microbial cathodes were formed under constant polarization using three-electrode set-ups in order to characterize them accurately by avoiding most of the possible interactions that can occur in MFC. The experimental conditions used were similar to those used previously for the formation of microbial anodes [START_REF] Milner | Evaluation of porous carbon felt as an aerobic biocathode support in terms of hydrogen peroxide[END_REF][START_REF] Milner | Microbial fuel cells with highly active aerobic biocathodes[END_REF], in particular the value of the applied potential was identical, equal to +0.1 V vs SCE. In this way, the O 2 -reducing biocathodes designed and characterized here should be fully suited to be coupled with the bioanodes described previously. It should thus be possible in the next step to design a MFC with both the bioanode and the biocathode working in the same hypersaline electrolyte of conductivity as high as 10.4 S.m -1 . The bacterial communities' structure in the different biocathodes was analyzed by using 16S rRNA metabarcoding to give first insights in hypersaline O 2 -reducing biofilms.

Materials and methods

Sampling of the salt marsh sediments

Salt marsh sediments were taken from salt marshes located near Mediterranean Sea (Salins de Saint Martin, Gruissan, France). The sediments were kept in the lab at room temperature in a 50 L closed container and were used fresh during the two weeks following the sampling to avoid long-term anaerobic evolution that may alter their bacterial composition.

Reactors

e. after 20 days of aeration and electrode polarization).

Samples of known masses of sediments were mineralized in several successive steps. A first dissolution of the samples by 5 mL of nitric acid 69% was realized for 12 h at room temperature, followed by 1 h warming at 95 • C. The dissolution was then completed by an addition of 2 mL of a solution of HCl and HNO 3 69% (50% volume both) and a second warming at 95 • C for two hours. The solutions were filtered after cooling and the volume completed with ultrapure water in order to get 50 mL of final mineral solutions.

The solutions were analyzed using an ICP-OES Ultima 2 from Horiba (Japan). Sulfur quantification was measured at a wavelength of 180.676 nm. Iron quantification was measured at two different wavelengths 234.349 nm and 259.940 nm, as a way to evidence any matrix effect.

Results and discussion

Electrochemical performance

Twelve O 2 reducing-biocathodes numbered from C1 to C12 were designed and characterized. Five parameters were used to compare the biocathodes (Table 1): -the lag time, which is the time required before a significant current density of oxygen reduction was observed; this threshold level was arbitrarily chosen at -0.01 A m -2 ; -the maximum (J max ) and stabilized (J stab ) values of the current density, as illustrated in Fig. 1; -the total charge transferred from the cathode throughout the polarization time; -the average daily charge transferred from the cathode during the polarization, lag time excepted.

In "standard" experimental conditions the biocathodes were formed in the modified Starkey medium that contained NaCl 45 g/L, under constant applied potential of +0.1 V vs SCE. Two other conditions, different from the standard one, were also tested: a medium complemented with NaHCO 3 for the two biocathodes C9 and C10, and a polarization potential of +0.3 V vs SCE for the two biocathodes C11 and C12. The addition of NaHCO 3 was expected to increase the level of inorganic carbon available, in order to stimulate the growth of autotrophic bacterial strains on the cathodes and possibly its electrochemical performances. The polarization was increased to +0.3 V vs SCE in an attempt to develop more efficient biocathodes able to reduce O 2 at the highest possible potential (Desmond-Le [START_REF] Quéméner | Biocathodes reducing oxygen at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF]. We didn't try to form biocathodes at open circuit, i.e. without applied potential, according to a previous work, which showed that this strategy was not efficient to form O 2 -reducing microbial cathodes [START_REF] Rimboud | Different methods used to form oxygen reducing biocathodes lead to different biomass quantities, bacterial communities, and electrochemical kinetics[END_REF].

The biocathodes C1 to C8 were formed in standard conditions. They differed only by the duration of the polarization. Biocathodes C7 and C8 were stopped earlier, after 11.9 days of polarization, in order to compare the bacterial populations inside their electroactive biofilms to the populations of more mature electrodes of 20.1 days and more.

All the biocathodes displayed similar general behavior in term of lag time, stable and maximal current densities and charge transferred (Table 1). They differed only by the way they reached the maximum and stabilized current density values. The shorter duration of the polarization of C7 and C8 explained their lower performance (current densities and charges exchanged). The highest current density value J max , -2.2 A m -2 , was observed with C3. Its stabilized current density value (J stab ) however was lower than that displayed by other biocathodes, notably C2, whose stabilized current density reached -0.91 A m -2 . Regarding the charges transferred by each electrode, the biocathode C6 exchanged up to -953.2 C during the whole experiment time, when biocathode C3, though less performing in term of stabilized current density, exchanged up to -44.4 C on daily basis.

The chronoamperometric record of the two best O 2 -reducing-biocathodes, C3 and C6, are displayed in Fig. 1. As illustrated by the dashed lines, two significant levels of current density were observed. The maximal current density (J max ) reached by the two biocathodes was -2000 and -1400 mA m -2 for C3 and C6, respectively. These maxima were always sustained for several days, showing that they corresponded to specific operating periods of the biocathodes and not to experimental events. After reaching the maxima, reduction current densities started to electrode polarization) and in the residual sediment (after experiment, i.

-0.79 A m -2 was observed at +0.14 V vs SCE. Another signal was detected with a peak at -0.24 V vs SCE. The observation of two distinct signals for oxygen reduction has already been described in previous works realized with compost leachate as inoculum (Desmond-Le [START_REF] Quéméner | Biocathodes reducing oxygen at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF][START_REF] Rimboud | Multiple electron transfer systems in oxygen reducing biocathodes revealed by different conditions of aeration/agitation[END_REF]. In these previous reports, the two electrochemical signals had two different behaviors: the signal at low potential (-0.2 V vs SCE) was enduringly observed whatever the experimental conditions, while the signal at higher potential (+0.1 to +0.2 V vs SCE) was observed only when a higher polarization potential (0 V vs SCE or higher) was applied. The two reduction waves obtained here in hypersaline medium by polarization at +0.1 V vs. SCE were consistent with these previous reports. The values of current density measured at +0.1 V vs. SCE on the voltammetric records were identical to the values of J max and J stab measured under chronoamperometry just before starting the voltammetries. This means that the scan rate of 1 mV s -1 used for the voltammetries was low enough to record the current densities produced by the biocathodes at steady state. The slow-rate voltammetries provided thus with a relevant characterization of the steady-state behavior of the biocathodes. The voltammetry at day 17 showed a great catalysis of the O 2 reduction reaction, with a positive shift of the potential values of around 600 mV. For instance, the value of 2 A m -2 was reached at around -0.40 V vs SCE with the initial clean electrode, while it was obtained from around +0.20 V vs. SCE with the 17-days old biocathode. Similar potential shift of around 600 mV was observed by considering the potential values at which the initial clean electrode and the 17-days old biocathode started to produce current. This considerable positive shift of potential points out the great promise hold by the biocathodes designed here when they operate in their maximum performance mode.

The electrodes polarized at +0.3 V vs SCE, C11 and C12, barely displayed any reduction current densities and their chronoamperometric records remained close to 0. Consistently, the voltammetries did not show any significant catalytic effect. The biocathodes formed at +0.1 V vs SCE showed here remarkable O 2 -reducing capacity, while changing the applied potential to +0.3 V vs. SCE did not lead to any biofilm-based catalysis. As nearly no current was recorded with these biocathodes, their bacterial population was not analyzed thereafter.

The biocathodes formed with the medium supplemented with NaHCO 3 , C9 and C10, demonstrated both longer lag time and lower current density productions compared to the electrodes without carbonates (C1 to C8). The addition of NaHCO 3 then resulted in no benefits in term of current density production.

Discussion of the electrochemical performance in relation to the literature

Getting biocathodes able to reduce oxygen at high potential is crucial for the development of efficient MFCs, therefore high polarization potential were used to growth them. It was stated here that the applied potential of +0.1 V vs SCE was fully suitable to develop these biocathodes and increasing it to +0.3 V vs SCE was not successful. It has previously been observed that biocathodes formed under constant applied potential were more efficient when the applied potential was increased. For instance, increasing the applied potential from -0.4 to +0.1 V vs. SCE significantly increased the current produce by O 2reducing biocathodes formed with compost leachate used as the inoculum (Desmond-Le [START_REF] Quéméner | Biocathodes reducing oxygen at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF]. In contrast, a study using nitrifying sludge as the inoculum reported that changing the applied potential from -0.2 V to +0.4 V vs SCE decreased the catalytic effect, as observed here [START_REF] Rimboud | The current provided by oxygen-reducing microbial cathodes is related to the composition of their bacterial community[END_REF]. The results obtained here confirmed that the applied potential is a key factor for the formation of biocathodes. They also indicate that there is an upper limit to the positive effect of the high values of applied potential, beyond which it is no longer possible to form efficient O 2 -reducing biocathodes. Here, the value of +0.1 vs SCE was fully appropriate, while +0.3 V vs SCE was above the upper limit.

It is also interesting to note that efficient bioanodes have previously been designed in exactly the same medium and at the same applied potential of +0.1 vs SCE [START_REF] Rousseau | Microbial bioanodes with high salinity tolerance for microbial fuel cells and microbial electrolysis cells[END_REF][START_REF] Rousseau | Halotolerant bioanodes: The applied potantial modulates the electrochemical characteristics, the biofilm structure and the ration of the two dominant genera[END_REF]. For the formation of bioanodes, acetate was added as the substrate (electron donor) and the medium was maintained under anaerobic conditions. Here, the unique difference was the absence of acetate and the aerobic conditions. The applied potential was identical. The microbial electrodes formed in the hypersaline media used here have thus the possibility to act as anode or cathode at the same potential of +0.1 V vs SCE, depending only on the presence or the absence of acetate and oxygen in the medium, i.e. the presence/absence of an electron donor or an electron acceptor. These microbial electrodes should consequently be excellent candidates to form so-called bidirectional bioelectrodes [START_REF] Jiang | Bidirectional extracellular electron transfers of electrodebiofilm: Mechanism and application[END_REF].

Cyclic voltammetry of the efficient biocathode revealed a peak potential at +0.22 V vs SCE, which was similar to the one reported by [START_REF] Milner | Microbial fuel cells with highly active aerobic biocathodes[END_REF], [START_REF] Milner | The effect of oxygen mass transfer on aerobic biocathode performance, biofilm growth and distribution in microbial fuel cells[END_REF]. They observed a peak for oxygen reduction at +0.25 V vs Ag/AgCl (+0.22 V vs SCE) [START_REF] Milner | Microbial fuel cells with highly active aerobic biocathodes[END_REF] by using an inoculum coming from aerobic sludge treated by successive electrochemical enrichments. They reported current densities that reached in average -2.04 A m -2 and culminated at -3.5 A m -2 under aeration (O 2 > 7.0 mg L -1 ). This is one of the highest current density reported in the literature for an O 2 -reducing biocathode, which was obtained in common media consisting of phosphate buffer 50 mM whose ionic conductivity can be assessed to lower than 2.0 S m -1 . The biocathodes formed here, were able to rise to similar level of performance (-2.0 A m -2 ) in a hypersaline medium that contained 45 g/L NaCl with conductivity of 10.4 S m -1 , which is around 1.5 times the conductivity of seawater. Salt marsh was consequently an excellent inoculum to form O 2 -reducing biocathodes able to operate in hypersaline conditions. decrease and stabilized (J stab ) around -350 and -850 mA m -2 for C3 and C6, respectively. The same general behavior with two different levels of current densities was observed with the eight biocathodes formed at +0.1 V vs SCE without NaHCO 3 (C1 to C8).

The biocathodes were further investigated by voltammetry. Characteristic cyclic voltammograms recorded with biocathode C3 are presented in Fig. 2.

A cyclic voltammetry was recorded on day 17 (black line) when the current density was in the J max phase. A peak-shaped signal for oxygen reduction was observed at +0.22 V vs SCE. The peak current density reached -2.8 A m -2 , which was the highest value observed during this study. Another cyclic voltammogram was recorded on day 25 (grey line), when the current density observed under polarization was close to J stab = -0.35 A m -2 . A peak-shaped signal for oxygen reduction of In the context of hypersaline media, the literature has recently reported a halotolerant O 2 -reducing biocathode, which operated in solution containing 62 to 65 g/L NaCl (Roustazadeh [START_REF] Sheikhyousefi | Investigation of different configurations of microbial fuel cells for the treatment of oilfield produced water[END_REF]. It produced 2 A m -2 at around -0.30 V vs SCE (-0.27 vs Ag/AgCl 3 M). Considering the biocathode C3 designed here (Fig. 2), a positive shift of potential of around 500 mV is observed with respect to this previous study. This comparison points out the great promise hold by the present biocathodes. Obviously, the maximum operating period varied in great extent in terms of duration and current density from one biocathode to another. Efforts must now focus on understanding the evolution of these biocathodes between the two J max and J stab levels with the view to optimizing and stabilizing the maximum level. The great improvement observed with the best-performing biocathode with respect to the state of the art justifies future research in this direction.

In all the experiments, the current density switched between two levels, J max and J stab . The duration and magnitude of these levels varied between the experiments. Nevertheless, a similar pattern was observed, with, in a first period, the highest level of current density, up to -2.2 A m -2 , which lasted up to a few days, and then the stabilized level, lower in magnitude, but more enduring, which lasted up to the end of the polarization. The reasons for this behavior remain to be identified. Analysis of the bacterial communities demonstrated rather low differences between young (C7-C8) and old biocathodes (C1-C6). Their composition remained quite similar. Therefore, it seems unlikely that the change in current density production could be linked to an evolution of the bacterial communities within the biofilms over time. The reasons for changeover the value between J max and J stab are probably related to a change in the physiological state of the bacteria, the size of their population and possibly related to interactions between different bacterial populations within the biofilm. The biofilm ageing may also result in an increasing thickness. The bulk medium showed considerable changes, in pH and sulfate concentration notably, which could also impact the production of current and bacterial functions.

Evolution of the bulk medium

pH measurements revealed two different evolutions depending on the presence or not of NaHCO 3 in the medium. In the ten reactors that had not been complemented with NaHCO 3 , the pH decreased from 7.0 at the starting of the experiment to reach a stable value around 6.4 ± 0.3 after 10 days. In the two reactors complemented with NaHCO 3 , the pH increased from 7.0 to reach 8.8 ± 0.1 after 5 days, then decreased slightly and stabilized at 7.6 ± 0.3 after 30 days. In the presence of HCO 3 -, the decomposition into carbon dioxide and hydroxide ions, which was activated by the forced aeration, explains the pH increase observed during the first days. At the same time and for both conditions, the visual aspect of the sediments inside the reactors changed from a dark and black appearance, characteristic of the presence of iron sulfide FeS, to a clear, brown sand appearance (see picture in Supplementary data). ICP measurements were performed on the native, black sediment and the aerated, brown sediment obtained at the end of the experiments (Table 2).

The composition of the sediment at the beginning and at the end of the biocathode formation was different in both iron and sulfur contents. The proportions of iron and sulfur decreased by 34.2% and 70.3%, respectively. This confirms that the change in the sediment aspect was due to the oxidation of iron sulfide. This oxidation released sulfate anions and Fe 2+ cations, which were oxidized, leading to the formation of Fe 3+ ions. Fe 3+ then reacted with water, leading to the precipitation of iron hydroxide Fe(OH) 3, following the equation:

Fe 3+ + 3H 2 O→Fe(OH) 3 + 3H + (1)
This formation of iron hydroxide, which released 3 protons per iron ion explains the pH acidification observed. The precipitation of iron hydroxide may also explain the difference between the proportion of elemental iron and sulfur inside the aerated sediment (34.2 vs 70.3%).

Control experiments were performed with an open-circuit reactor on one side and a reactor treated with 10 µM of chloramphenicol to inhibit any microbial activity on the other side. Both reactors showed a similar evolution in pH and aspects of the sediment. They showed that the evolution of the solution was neither linked to the electrochemical reactions, on the working and counter-electrodes, nor to the microbial activity. The chemical composition of the bulk solutions evolved spontaneously. The oxidation of FeS, the release of Fe 3+ and the decomposition of HCO 3 -, when present, can fully explain the change in solution aspect and the pH evolution. In return, this evolution may affect microbial behavior and thus impact the production of current as a function of time.

Microbial population analyses

The Gini-Simpson and the Shannon diversity indexes corresponding to the eight biofilms are compiled in Table 3.

The Gini-Simpson index represents the probability of interspecific encounter; if null, it indicates that only one species is represented in the sample. Gini-Simpson index ranged from 0.70 with C6 to 0.94 with C1 and C10. Most values of the Gini-Simpson index ranged between 0.84 and 0.94; only biocathode C6 had a significantly lower index of 0.70. Its biofilm microbial community tended to be more dominated by a single species than the other biocathodes. Similar observations were made with the Shannon index, which mostly ranged from 2.82 (C5) to 3.34 (C10) with C6 displaying the lowest value of 2.05. Interestingly, no other significant difference was observed between the different biocathodes whatever the experimental conditions of their formation. Biocathodes C9 and C10 designed in the presence of NaHCO 3 displayed indexes similar to the biocathodes formed without NaHCO 3 . Biocathodes C7 and C8 whose polarization was stopped earlier (11.9 days) also displayed similar indexes than the biocathodes (C1 to C6) polarized during a longer duration (20.1 days and more). It indicates that the diversity of the population inside the electro-active biofilms was already well-established after 11.9 days of polarization.

The most represented bacterial orders on the eight analyzed biocathodes are presented in Fig. 3.

Proteobacteria and Bacteroidetes were the most represented phyla on the eight electrodes. Flavobacteriales were the most abundant among Bacteroidetes and Proteobacteria of the Alpha-and Gamma-classes the most abundant among the Proteobacteria. Rhodobacterales (Alphaproteobacteria), Alteromonadales, Oceanospirillales and an unidentified Gammaproteobacteria dominated among Proteobacteria. Different trends were observed for the biocathodes depending on the experimental conditions. Bacteroidetes (Flavobacteriales and other) and Gammaproteobacteria were found in higher proportion in biocathodes formed with NaHCO 3 in the medium (C9-C10). Alphaproteobacteria (Rhizobiales, Rhodobacterales and other) were more abundant in the biocathodes formed without NaHCO 3 in the medium (C1-C8).

Table 2

Mass of iron and sulfur element measured by ICP in the native and aerated sediment in mg g -1 . Among Gammaproteobacteria, differences were observed between biocathodes formed with or without NaHCO 3 in the medium. Biocathodes designed in absence of NaHCO 3 (C1-C8) were characterized by the presence of an unidentified Gammaproteobacteria, which was not detected on the biocathodes formed in presence of NaHCO 3 (C9-C10). The corresponding sequences numbered from 5.0% of the whole sequences on C1 to 52.6% on C6. The prevalence of this unidentified Gammaproteobacteria on C6 compared to the other cathodes explains the lower values of the diversity index observed with this cathode, as the microbial community was dominated by this single taxa. Alteromonadales and Oceanospirillales dominated on biocathodes designed in presence of NaHCO 3 : Alteromonadales represented 21.2 and 20.4%, Oceanospirillales 34.2 and 5.7%, on C9 and C10 respectively. On the biocathodes designed in the absence of NaHCO 3 and of similar age (C1, C3, C5 and C6), Alteromonadales accounted for a maximum of 12,6% on C1 and Oceanospirillales represented a maximum of 2.2% on C5.

A significant difference was also observed depending on the duration of the polarization. Biocathodes C7 and C8 stopped earlier, after 11.9 days of polarization, were richer in Alteromonadales (25.8% on C7) and Oceanospirillales (12.3% on C7) than those stopped later, after 20.1 days of polarization or more. These proportions made them closer to C9 and C10. As counterpart, they were poorer in Alphaproteobacteria other than Rhodobacterales and Rhizobiales than biocathodes C1 to C5. The proportion of the unidentified Gammaproteobacteria on C7 and C8, on the other hand, was not significantly different from the one observed on C1 to C5.

Bacteria for which identification at genus level was possible are listed in table SI 2. Only genera representing >1% on at least one biocathode were listed. Several genera of the marine Roseobacter clade (Rhodobacteraceae) were identified indifferently on the different cathodes: Phaeobacter, Roseovarius, Roseobacter, Labrenzia, Pelagicola and Lutimaribacter. Other genera of the same clade (Roseibacterium, Roseibium, Donghicola…) were also identified at less than 1% on one or more electrodes. Three genera from the order Alteromonadales, Marinobacter, Idiomarina and Pseudoalteromonas were observed on the different electrodes and genus Marinobacter was the most or at least the second most represented genus on most of the biocathodes (from 5.5 to 19.4%), with the notable exception of C5. Two phylogenetically close genera from the family Alcanivoracaceae (order Oceanospirillales), Alcanivorax and Kangiella, were identified in rather high proportion on C7, C9 and C10.

Discussion of the biocathode microbial populations in relation to the literature

Most of the bacterial genera and orders identified in majority inside the different biocathodes are involved in various biogeochemical cycles. As it was the case here, notable proportions of Marinobacter (1.7 to 19.4%) and other phylogenetically related bacteria (other Alteromonadales) have been detected in previous examples of O 2 -reducing biocathodes designed with marine media (seawater, sediments…) [START_REF] Strycharz-Glaven | Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst[END_REF][START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF]Wang et al., 2015aWang et al., , 2015b)). Marinobacter are known as opportunistic colonizers inside electroactive biofilms and have been detected in significant proportions in both anodic [START_REF] Rousseau | Correlation of the electrochemical kinetics of high-salinitytolerant bioanodes with the structure and microbial composition of the biofilm[END_REF][START_REF] Rousseau | Halotolerant bioanodes: The applied potantial modulates the electrochemical characteristics, the biofilm structure and the ration of the two dominant genera[END_REF] and cathodic biofilms [START_REF] Strycharz-Glaven | Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst[END_REF][START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF]Wang et al., 2015b). Some Marinobacter species, like Marinobacter santoriniensis, have a role in bio-geochemical cycles [START_REF] Handley | Marinobacter santoriniensis sp. nov[END_REF]. A recent work with Marinobacter atlanticus suggested a role of trace mineral elements in the activity of electroactive biofilms [START_REF] Onderko | Electrochemical characterization of marinobacter atlanticus strain CP1 suggests a role for trace minerals in electrogenic activity[END_REF]. Marinobacter adhaerens is known to produce high quantity of exopolysaccharides (EPS) and has been evidenced as an usual constituent of the electroactive biofilms grown under chlorinated conditions on the surface of copper alloys [START_REF] Carvalho | The study of marine corrosion of copper alloys in chlorinated condenser cooling circuits: The role of microbiological components[END_REF]. Here, Marinobacter sp. (and Alteromonadales) were detected in higher proportions on the young (C7 and C8) than on the old biocathodes (C1 to C6). This could be related to their heterotrophy: as they require organic carbon for their growth, they would be at the long term disadvantage as no organic carbon source was provided in the medium.

Bacteria from the Roseobacter clade of the family Rhodobacteraceae were also identified in great proportion in the different biofilms. This clade groups versatile bacteria able to grow chemoorganotrophically and/or photoheterotrophically (aerobic anoxygenic phototrophic bacteria) with some species capable of autotrophy. They usually are inorganic sulfur oxidizers and have a great importance in the biogeochemical sulfur and carbon cycles. They are also known to be among the firstly-adherent bacteria during the constitution of marine biofilms [START_REF] Dang | Cross-ocean distribution of Rhodobacterales bacteria as primary surface colonizers in temperate coastal marine waters[END_REF][START_REF] Elifantz | Rhodobacteraceae are the key members of the microbial community of the initial biofilm formed in Eastern Mediterranean coastal seawater[END_REF]. Their presence could therefore be linked both to their affinity to solid matrix to constitute biofilms and to their ability to survive in conditions where the availability of organic matter is low, as in the context of O 2 -reducing biocathodes. Moreover, sulfur cycling by sulfur-reducing bacteria and purple non-sulfur bacteria has been speculated to be involved in the Fig. 3. Most represented bacterial orders in the eight bacterial biofilms. Categories labelled as « other » regrouped less abundant bacterial orders and sequences identified at the immediately higher taxonomic level (for example sequences belonging to order Chromatiales are grouped with sequences classified as Gammaprotobacteria without lower assignation at the order level in "other Gammaproteobacteria").

Conclusion

The use of salt marsh sediment as the inoculum allowed the design of efficient O 2 -reducing biocathodes, which produced current densities up to 2.0 A m -2 for several days, in hypersaline media with ionic conductivity of 10.4 S m -1 . These biocathodes will be fully suitable to be coupled with the bioanodes formed previously in the same medium at the same value of the applied potential of +0.1 V vs SCE. Among the dominating species, one (or more?) Gammaproteobacteria strain(s) related to Thiohalobacter thiocyanaticus were enriched in higher proportion than the other strains, in correlation with the electrochemical efficiency of the biocathodes. catalysis of oxygen reduction in various biocathodes [START_REF] Daghio | Anodic and cathodic microbial communities in single chamber microbial fuel cells[END_REF][START_REF] Rago | Influences of dissolved oxygen concentration on biocathodic microbial communities in microbial fuel cells[END_REF], including biocathode formed in a hypersaline environment [START_REF] Sheikhyousefi | Investigation of different configurations of microbial fuel cells for the treatment of oilfield produced water[END_REF]. The presence of inorganic sulfur oxidizers bacterial genera observed here may be a further confirmation of this catalytic pathway.

CRediT authorship contribution statement

Several sequences related to an unclassified Gammaproteobacteria were found in high proportion (5.0-52.6%) on the efficient biocathodes formed without NaHCO 3 addition. Interestingly, these sequences were absent from the C9 and C10 biocathodes, formed with NaHCO 3 , which were electrochemically inefficient. Moreover, these bacteria were found in dramatically lower proportion in the bulk media compared to on the electrodes, as illustrated by the percentages of sequences affiliated to the unidentified Gammaproteobacteria inside both the electroactive biofilms and the corresponding bulk media, collected in table SI3. As a comparison, the ratios observed between the percentage of Rhodobacterales in the biofilms and in the bulk solution varied between 0.4 and 4.0 for C6 and C3. For Alteromonadales, it varied from 0.3 to 4.9 for C6 and C7. This ratio was considerably higher when looking for the unclassified Gammaproteobacteria, varying between 3.1 and 263 for respectively C1 and C6. It evidences a high enrichment of the unclassified Gammaproteobacteria on the biocathodes and a strong affinity of the latter for the polarized surface. It is therefore very likely that these bacteria played a key-role in the electroactivity of the biocathodes.

Attempt was made to deepen the identification of these bacteria via alignment of the corresponding DNA sequences using the online Blast software. It led to the identification of sequences from the bacterium Thiohalobacter thiocyanaticus as closest relatives (from 91.9 to 98.7%). This species is rather isolated on the phylogenetic tree: it was identified as a Gammaproteobacteria but not classified at lower taxonomic level in the existing orders and families [START_REF] Sorokin | Thiohalobacter thiocyanaticus gen. nov., sp nov., a moderately halophilic, sulfur-oxidizing gammaproteobacterium from hypersaline lakes, that utilizes thiocyanate[END_REF]. To our knowledge, no close genetic relatives to this bacterium are known yet.

Thiohalobacter thiocyanaticus is a strictly chemo-autotrophic, halophilic sulfur-oxidizing bacterium isolated from the sediment of a saline lake [START_REF] Sorokin | Thiohalobacter thiocyanaticus gen. nov., sp nov., a moderately halophilic, sulfur-oxidizing gammaproteobacterium from hypersaline lakes, that utilizes thiocyanate[END_REF]. It performed inorganic carbon (CO 2 ) fixation via the Calvin-Benson cycle and the Rubisco enzyme. These characteristics are in accordance with the nature of the medium and inoculum used here. They are also in accordance with characteristics observed with bacteria already identified as "electro-autotrophic" (able to grow by fixing inorganic carbon using electrons supplied by a polarized electrode as sole energy source) [START_REF] Ishii | From chemolithoautotrophs to electrolithoautotrophs: CO2 fixation by Fe(II)-oxidizing bacteria coupled with direct uptake of electrons from solid electron sources[END_REF]Wang et al., 2015b;[START_REF] Eddie | Metatranscriptomics supports the mechanism for biocathode electroautotrophy by[END_REF][START_REF] Rimboud | Electroautotrophy of Thioalkalivibrio nitratireducens[END_REF]. We can therefore postulate that the unclassified Gammaproteobacteria identified in the cathodic biofilms could be related to Thiohalobacter thiocyanaticus and a possible electro-autotrophic bacterium, which would have key-roles both as catalyst for O 2 reduction and as inorganic carbon fixing agent for the biofilm maintenance. It would be relevant to isolate this bacterium and test its ability to form biocathodes in order to validate our hypothesis.

Fig. 1 .

 1 Fig. 1. Evolution of the current density as a function of time under polarization at +0.1 V vs SCE. Values of maximal (J max ) and stabilized (J stab ) current density are materialized by dashed lines. A) biocathode C3, B) biocathode C6.

Fig. 2 .

 2 Fig. 2. Representative cyclic voltammograms recorded with biocathode C3. Dashed line: control voltammogram recorded at t = 0, before the development of an electroactive biofilm on the cathode; black line: voltammogram recorded when J = J max on day 17; grey line: voltammogram recorded when J = J stab on day 25. The polarization potential applied in chronoamperometry is materialized by the vertical dashed line at +0.1 V vs SCE.

Mickaël Rimboud :

 Rimboud Conceptualization, Methodology, Investigation, Writing -original draft, Writing -review & editing. Mohamed Barakat: Validation, Investigation. Wafa Achouak: Validation, Writing -review & editing, Supervision. Alain Bergel: Conceptualization, Writingoriginal draft, Writing -review & editing. Marie-Line Délia: Conceptualization, Methodology, Validation, Investigation, Supervision.

Table 1

 1 Characteristics of the twelve O 2 -reducing biocathodes. The last column indicates whether or not the microbial population of the biocathode was analyzed by 16S-rRNA pyrosequencing.

		Duration	Lag	J max	J stab	Total	Daily	DNA
		Time (days)	Time (days)	(mA m -2 )	(mA m -2 )	charge (C)	charge (C day -1 )
	C1	31.1	2.5	-1100	-860	-871.7	-30.5	Yes
	C2	20.1	3.3	-1500	-910	-766.6	-33.1	No
	C3	31.1	2.8	-2000	-350	-920.2	-44.4	Yes
	C4	17.9	4.7	-800	-630	-252.1	-19.1	No
	C5	24.9	2.1	-1000	-600	-637	-27.9	Yes
	C6	24.9	2.7	-1400	-850	-953.2	-42.9	Yes
	C7	11.9	3.3	-310	-250	-80.4	-9.3	Yes
	C8	11.9	2.3	-700	-400	-200.6	-20.9	Yes
	C9*	30.7	12.4	-610	-590	-293.0	-16.0	Yes
	C10*	30.7	15.1	-65	-18	-21.5	-1.4	Yes
	C11**	24.9	12.5	-20	+3	+2.7	+0.2	No
	C12**	24.9	_	+2	+4	+5.2	+0.2	No

* Reactor medium contained 50 mM NaHCO 3 as additional inorganic carbon source.

** Potential was poised at +0.3 V/SCE, instead of +0.1 V/SCE.

Table 3

 3 Gini-Simpson and Shannon indexes for the eight biofilms analyzed by pyrosequencing.

	Indexes	C1	C3	C5	C6	C7	C8	C9	C10
	Gini-Simpson	0.94	0.90	0.87	0.70	0.93	0.86	0.84	0.94
	Shannon	3.28	2.94	2.82	2.05	3.27	2.88	2.54	3.34

Mickaël Rimboud 1 , Mohamed Barakat 2 , Wafa Achouak 2 , Alain Bergel 1 , Marie-Line Délia 1 *.

Acknowledgments

This work supported by funding from the French state, managed by the Agence Nationale de la Recherche (ANR), within the framework of the Koropokkuru project (ANR-14-CE05-0004). We gratefully thanks M.-L. de Solan Bethmale (Laboratoire de Génie Chimique) for achieving ICP analysis.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.biortech.2020.124165.

Supplementary Information

Figure SI 1.

Picture of the native, black (right) and aerated, brown (left) sediment. The native sediment was the salt marsh sediment as sampled from the natural spot (Gruissan, France). The aerated sediment was obtained from the native sediment after 20 days of aeration in the experimental reactor.

Figure SI 2

Table of the relative abundance of the different bacterial population on the eight ORbiocathodes at genus level.

Only genera represented at least 1 % on at least one biocathode are presented. For each biocathode, the proportion of the five more represented genera are highlighted in shades of red. Genera related to Orders Rhodobacterales, Alteromonadales and Oceanospirillales are highlighted in yellow. Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Roseovarius 3.6 3.3 3.0 0.9 2.3 1.2 2.9 3.2

Phylum

Proteobacteria Alphaproteobacteria Sneathiellales Sneathiellaceae Sneathiella 0.7 0.2 1.7 0.3 0.6 0.1 0.0 0.0 Proteobacteria Betaproteobacteria Nitrosomonadales Nitrosomonadaceae Nitrosomonas 1.0 0.3 0.6 0.3 0.2 0.1 0.5 0.3 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae Marinobacter 7.9 5.5 1.7 7.3 19.4 10.7 9.9 8.4

Proteobacteria Gammaproteobacteria Alteromonadales Idiomarinaceae Idiomarina 0.2 0.0 1.1 0.0 3.9 1.9 7.4 1.8

Proteobacteria Gammaproteobacteria Alteromonadales Pseudoalteromonadaceae Pseudoalteromonas 0.0 0.0 0.1 0.0 0.1 0.0 0.1 4.6

Proteobacteria Gammaproteobacteria Cellvibrionales Halieaceae Haliea 0.0 0.0 0.1 0.5 1.4 0.5 0.8 3.6

Proteobacteria Gammaproteobacteria Chromatiales Ectothiorhodospiraceae Thiorhodospira 0.1 0.5 0.0 0.0 0.0 0.0 1.4 2.8

Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax 0.4 0.2 0.4 0.2 0.6 1.4 1.1 4.5

Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Kangiella 0.2 0.1 1.2 0.4 6.7 1.0 38.3 0.6

Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae Halomonas 0.0 0.1 0.0 0.0 0.1 0.1 0.2 1.1

Proteobacteria Gammaproteobacteria Oceanospirillales Oceanospirillaceae Marinobacterium 0.1 0.2 0.5 0.1 4.6 0.5 0.0 0.0 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas 0.0 0.0 0.1 0.0 0.0 0.0 1.1 1.9

Figure SI 3

Table of the relative abundance of three different bacterial orders (Unidentified Gammaproteobacteria, Rhodobacterales and Alteromonadales) inside the electroactive biofilm and the corresponding bulk medium for five of the OR-biocathodes.

The ratios between the two relative abundances inside the biofilm and the bulk medium are also indicated for each order and OR-biocathode.