Microbial nitrate reduction electro-assisted by
exo-electrogenic reduction of dioxygen with a
Pseudomonas dominated cathodic biofilm

Timothe Philippon, James A Behan, Frédéric Barrière

► To cite this version:

Timothe Philippon, James A Behan, Frédéric Barrière. Microbial nitrate reduction electro-assisted by exo-electrogenic reduction of dioxygen with a Pseudomonas dominated cathodic biofilm. Electrochimica Acta, 2024, 497, pp.144573-144573. 10.1016/j.electacta.2024.144573. hal-04646054

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# Microbial nitrate reduction electro-assisted by exo-electrogenic reduction of dioxygen with a *Pseudomonas* dominated cathodic biofilm

Timothé Philippon*§, James A. Behan*, Frédéric Barrière*

Université de Rennes, CNRS UMR 6226, Institut des Sciences Chimiques de Rennes, France

*Corresponding authors: timothe.philippon@bce.au.dk (T.P.), james.behan@univ-rennes.fr (J.A.B.), frederic.barriere@univ-rennes.fr (F.B.)

§Current address: Department of Biological and Chemical Engineering - Industrial Biotechnology, Aarhus University, Denmark

## Abstract

A nitrate reducing microbial biocathode was developed through constant polarization at -0.5 V vs SCE using two types of inoculums: a pure culture of *Thiobacillus denitrificans* and water collected from the artificial wetland of Rampillon (France). The results show a clear increase of the nitrate removal efficiency for the Pilots with the natural water although no catalytic nitrate reduction can be evidenced by cyclic voltammetry. Further studies show a catalytic oxygen reduction through exo-electrogenic metabolism and a correlation between the cathode polarization at -0.5 V vs SCE and the nitrate remediation. 16S rRNA gene amplicon sequencing of the biofilm bacterial DNA shows a very large predominance of *Pseudomonas*, a genus that includes many species able to reduce nitrate and/or reduce dioxygen (O₂) using electrons from a cathode. The increased nitrate reduction performance is hypothesized to arise from an indirect bioelectro-assistance that allows the emergence of local anoxic conditions caused by microbial endo-electrogenic pathway for oxygen reduction.

## Introduction

In recent years, the understanding and optimization of exo/endo-electrogenic microbial metabolisms at extracellular solids and electrodes has progressed toward a range of possible bioelectrochemical technological applications. Indeed, while the first Microbial Fuel Cells focused on current generation from oxidation of organic matter by anodic exo-electrogenic bacterial species from, for example, the *Geobacter* and *Shewanella* genera, other applications were tested including oxygen reduction [1], metal recovery [2], hydrogen production [3] or nitrate reduction [4],[5]. The latter has become a growing research field due to the ever-increasing problematic nitrate pollution in developed countries. Nitrate pollution is the consequence of the development of the intensive use of fertilizers in modern agriculture that contaminate underground and in turn domestic water [6].

In order to increase natural denitrification efficiency, ecological engineering has become an important field, dealing for example with the optimization of intermediary natural or artificial water retention areas between the agricultural fields and the aquifer where the water eventually runs off. These artificial retention areas are called constructed wetlands and have become the main focus zone for the optimized depollution of diverse compounds such as pesticides, metals or nitrate [7]–[9].

Among the different methods to improve denitrification efficiency, for instance in constructed wetlands, bio-electrochemical systems are receiving increasing attention due to their exploitation of naturally-occurring bacterial metabolisms found in bacterial species that are present in many different ecosystems. From that perspective, most
bio-electrochemical systems are developed using selective pressure approaches to cultivate microbial communities
possessing the desired exo-electrogenic capacity for denitrification.

Nitrate reduction by electrogenic bacteria using electrons harnessed from an electrode was first discovered in 2007
by Clauwaert *et al*. [10] whose work was followed by many other similar studies. Firstly, the large majority of the
nitrate reducing bio-cathodes are developed through constant polarization of the electrode. The potential applied
varies, but a general 'threshold potential' required for nitrate reduction is consistently found to be around -0.250
V vs SHE [11]–[14]. Among the different bacterial genera capable of exo-electrogenic denitrification are *Thauera*
[15],[16], *Pseudomonas* [17],[18] and notably *Thiobacillus denitrificans*, a species which has its potential of
reduction of nitrate and nitrite described in literature by *Pous et al*. [14] and has been confirmed as an exo-
electrogenic denitrifying species [12],[19],[20]. In practice, syntrophic or synergistic interactions are also thought
to play a key role in denitrification accomplished through mixed biofilm communities, and respiratory machinery
of the microorganisms involved are often capable of reducing both nitrate and molecular dioxygen (O₂). Hence,
further research into denitrification by mixed biofilm communities is essential for the development of optimized
microbial electrochemical systems.

In this study we investigate the exo-electrogenic denitrification activity of both pure cultures of *T. denitrificans*
and mixed biological inoculum sourced from the constructed wetland of Rampillon. This wetland was constructed
in 2010 [21] in order to monitor agricultural pollution including that arising from nitrogen compounds [22].
Bioaugmentation studies using pure cultures together with water from this wetland were carried out in order to
investigate their effects on the development of potential exo-electrogenic denitrifying biofilms. Pilot studies were
carried out in three-electrode electrochemical cells at high nitrate concentrations under polarization with regular
monitoring of the nitrate concentration, followed by DNA extraction and 16S rRNA sequencing of the biofilms in
the best-performing pilots. Results imply a greater denitrification efficiency in mixed biofilms developed using
both *T. denitrificans* and Rampillon wetland inoculum. This cathodic biofilm was found to be dominated by the
*Pseudomonas* genus, which contains many species capable of cathodic reduction of both nitrate and dioxygen. We
attribute this denitrification efficiency to an indirect electro-assistance effect whereby dioxygen reduction by the
biofilm produces local anoxic conditions favourable for nitrate reduction.

**Materials and Methods**

* Pilot structure

Pilots were built using glass structures able to contain 150 mL of liquid topped with four openings that were used
to place a three-electrode set-up. Each Pilot was filled with 150 mL of synthetic incubation medium containing
8.6 mM of NaCl, 0.8 mM of MgSO₄, 1.3 mM of CaCl₂, 0.3 mM of NH₄Cl, 11.9 mM of NaHCO₃, 10.9 mM of
Na₂HPO₄, 4.2 mM of KH₂PO₄, 11.7 mM of NaNO₃ (730 mg.L⁻¹ of NO₃⁻), 7.0 mM of NaNO₂ (294 mg.L⁻¹ de NO₂⁻)
and 1 mL.L⁻¹ of SL-6 trace elements solution. Two graphite rods (Morgan AM&T®, 0.5 cm diameter) were used
as-received as working and counter electrodes alongside a saturated calomel reference electrode. A negative
control pilot contained the same medium and incubation conditions but was not inoculated and did not contain
electrodes. All electrodes were mounted through pierced rubber stoppers in order to preserve anoxic conditions in
the Pilot. The initial pH was found to be 7 and did not vary significantly throughout the entire study.

* Addition of pure culture and natural inoculum
A pure culture of *Thiobacillus denitrificans* (strain AB7, purchased from DSMZ®) was prepared. The culture medium used was N113 recommended by DSMZ® for the growing of *Thiobacillus denitrificans*. The pure culture was incubated at 30°C after argon saturation for 18 days. Then, its $OD_{600}$ was measured at 2.2 and 5 mL of this pure culture was collected and centrifuged. The supernatant was removed and the pellet was suspended in 3 mL of the incubation medium described above. This volume was divided in two 1.5 mL aliquots and added to the incubation medium of the Pilots 1 and 2. For Pilot 2, another inoculation using 1.5 mL of natural water from the natural wetland of Rampillon was made. Pilot 3 contained Rampillon inoculum only, while Pilot 4 contained Rampillon inoculum, but was not subjected to polarization in order to assess the contributions of the natural inoculum to nitrate consumption under non bio-electrochemical conditions. Finally in Pilot 5 (without electrodes), no inoculum was added to serve as a negative control.

## Electrochemical measurements

All electrochemical studies were made on a Metrohm® PGSTAT 302N potentiostat. Electrodes were characterized by a combination of chronoamperometry and cyclic voltammetry studies under different conditions as specified throughout the text.

## Nitrate determination

The nitrate concentrations were measured using the colorimetric salicylic acid method reported previously by Cataldo *et al.* [23]. The absorbance at 410 nm was measured using a UV-1605 spectrophotometer from Shimadzu®. In order to ensure the precision of this method, a calibration study was made using controlled nitrate concentrations solutions and the margin of error always remained below 5 %.

## Experiment chronology

### 1. Incubation under polarization

In the first part of the experiment, the Pilots were left incubated at ambient temperature (from 14°C to 26°C) and at ambient day light (ca. 12 hours per day) for 30 days. For experiments under polarization, the working electrodes were polarized at -0.5 V vs SCE using an analogue potentiostat. Non-polarized controls included an identical 3-electrode setup left at open circuit. During the 30 days of incubation, nitrate concentrations were regularly measured in the synthetic medium of the Pilots. After 30 days, the working electrodes = were transferred into new electrochemical cells (using the same cell geometry and electrode arrangement) with renewed synthetic medium and fresh graphite counter electrodes for further electrochemical studies.

### 2. Chronoamperometry study

The new synthetic medium was prepared as described before but without any addition of nitrate or nitrite. Chronoamperometry at -0.5 V vs SCE were launched on the working electrodes of Pilots 1 and 2 for 10 h under ambient air. Sodium nitrate was then added to the two Pilots at t= 2 h during the chronoamperometry to a final concentration of 10 mM (620 mg.L⁻¹ of $NO_3^-$).

### 3. Successive polarization experiments
The working electrode of the Pilot 2 (Rampillon inoculum + Pure culture) was finally transferred to a new glass cell with a renewed counter electrode and containing 20 mL of freshly-prepared synthetic medium but with an initial NaNO₃ concentration of 10 mM and no nitrite. This working electrode was then subjected to a succession of phases with and without polarization at -0.5 V vs SCE as shown in Table 1:

<table><caption>**Table 1:** Details of the polarization periods of the working electrode from Pilot 2 (Rampillon inoculum + Pure culture). Nitrate concentration measurements were made regularly by spectrophotometric assay as described in the main text.</caption><thead><tr><th>Polarization</th><th>Polarization at -0.5 V vs SCE</th><th>No polarization</th><th>Polarization at -0.5 V vs SCE</th><th>No polarization</th></tr></thead><tbody><tr><td>Time</td><td>32 hours</td><td>34 hours</td><td>32 hours</td><td>42 hours</td></tr></tbody></table>

## DNA extraction and 16S rRNA gene amplicon sequencing

The working electrodes from the best-performing denitrifying pilots, Pilots 1 and 2, were scratched with a sterile spatula in order to collect the biofilms and perform a DNA extraction of the biofilms in order to study their communities through 16S rRNA gene amplification and sequencing. DNA extraction was carried out using the DNeasy® PowerSoil® Kit from Qiagen® following the instructions of the manufacturer. After DNA extraction, the concentration in pure DNA for each of the two samples was determined using the Nanodrop® hardware and software which showed sufficient DNA concentrations for both samples. Archaeal and bacterial hyper variable region V4-V5 of the 16S rRNA gene was amplified and then sequenced according to a protocol described previously [24],[25] with some modifications. V4-V5 of the 16S rRNA gene was amplified by PCR with fusion primers 515F (5'- Ion A adapter-Barcode-GTGYCAGCMGCCGCGGTA-3') [26] and 928R (5'-Ion trP1 adapter- CCCCGYCAATTCMTTTRAGT-3') [27], which include a barcode and sequencing adapters according to the recommendations of the "Ion Amplicon Library Preparation (Fusion Method)". PCR mixture contained 1X SuperFi Buffer, 0.2 mM of each dNTP, 0.5 µM of each primer, 1 U Platinum SuperFi DNA Polymerase (Invitrogen), and 10-20 ng template DNA in a 50 µL reaction volume. Amplification was performed as follows: 30 s at 98 °C, 30 cycles of 10 s at 98 °C, 10 s at 50 °C, 30 s at 72 °C, followed by final extension of 5 min at 72 °C. PCR products were purified using Solid Phase Reversible Immobilization (SPRI) magnetic beads (Mag-Bind TotalPure NGS magnetic beads, Omega Bio-Tek) according to the manufacturer's instructions, with a bead/amplicon ratio of 1.2, and were eluted in 45 µL TE Buffer (10mM Tris-HCl pH 8.0, 1 mM EDTA). Purified amplicons were quantified using D1000 ScreenTape and 4200 TapeStation or DNA 1000 Kit and 2100 Bioanalyzer (Agilent Technologies), following the manufacturer's instructions. Then all amplicons were pre-diluted to 500 pM in molecular grade water and equimolarly pooled. The pool was then diluted to 100 pM for sequencing. Briefly, to prepare template-positive Ion Sphere Particles (ISPs) containing clonally amplified DNA by emulsion PCR, the library was diluted to 26 pM and set up on the Ion OneTouch 2 Instrument (Life Technologies) using the Ion PGM Hi-Q View OT2 Kit (Life Technologies) and following the manufacturer's protocol. These templated ISPs were then purified on the Ion OneTouch ES (Life Technologies) according to the manufacturer's instructions. Sequencing was performed on Ion Torrent Personal Genome Machine using Ion 316 Chip V2 (Life Technologies) and Ion PGM Hi-Q View Sequencing Kit (Life Technologies). Sequencing data was processed by the Torrent Suite Software. The software filtered out low quality and polyclonal sequence reads, and quality filtered data was exported as FastQ file.
Bio-informatic treatment

The obtained 16S rRNA library in FastQ format was then treated using the FROGS pipeline [28] in order to pre-
process sequences (remove those not containing the two primers or with too much unattributed nucleotides),
cluster, eliminate chimera, filter the low abundance (keeping only those appearing more than 0.005 % in the whole
dataset.) and finally, attribute the sequences left using the silva 132 16S database [29]. The sample collected from
the biofilm on the working electrode from Pilot 1 showed only 1500 sequences which is considered too low for a
reliable study. The fact that a high total DNA concentration was found following the extraction but a limited
number of sequences were obtained after amplification and sequencing of the 16S rRNA gene suggests that most
of the biofilm in Pilot 1 was composed of eukaryotic organisms. For the sample collected from Pilot 2 (Rampillon
inoculum + Pure culture), the final number of sequences was around 35 000, which is considered large enough for
a solid analysis of the bacterial communities.
Results

Evolution of the nitrate concentration during the 30 days of incubation

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Evolution of the nitrate concentrations in the synthetic medium during the 30 days of incubation of the three pilots. Pilot 1 (Rampillon lake water inoculum + *Thiobacillus denitrificans* Pure culture) in blue, Pilot 2 (*Thiobacillus denitrificans* Pure culture) in orange which both include a three-electrodes set-up with polarization at -0,5 V vs SCE of their working electrodes. Pilot 3 in red contained Rampillon water inoculum only, while Pilot 4 (black) also contained Rampillon inoculum but was not subjected to polarization. Pilot 5 (grey) contained no biological inoculum or electrodes.</figcaption></figure>

When analyzing the evolution of the nitrate concentration evolution over time in **Figure 1**, we first note that the negative control (no polarized electrodes or inoculum) shows stable values across the duration of the experiment. Both Pilots 1 and 2 show a steady decrease of the nitrate concentrations over time. When comparing the two Pilots, we observe an improved nitrate removal for the Pilot 2, which includes mixed inoculum from the natural water from Rampillon. This pilot consumed 25 mg of NO₃⁻ per day on average compared to Pilot 1 which contained only the pure culture of Thiobacillus as inoculum (19 mg of NO₃⁻ per day on average). Pilot 3 and 4, containing Rampillon inoculum only, shows a slight decrease in measured nitrates compared to the negative control. This is noteworthy as Pilot 4 was not subjected to polarization, suggesting a comparable performance under ‘Rampillon-only’ conditions regardless of the electrode potential.

To explain the apparent enhanced nitrate removal in Pilot 1 compared to Pilot 2, we present two potential hypotheses: the increase in denitrification is exo-electrogenic in nature (e.g. due to the presence of *Thiobacillus denitrificans*) with an enhancement in this activity due to the presence of others microorganisms brought by the addition of the natural water from Rampillon. The contribution of these microorganisms is indirect and is attributed to local O₂ consumption by exo-electrogens capable of O₂ reduction in the mixed cathodic biofilm. Alternatively,
the increase of denitrification is performed by non exo-electrogenic denitrifying bacteria also introduced by the
Rampillon natural water addition. The apparent weak denitrification response of Pilots 3 and 4, which contained
only Rampillon inoculum, suggests that this is unlikely to be the case.

It would appear as if *T. denitrificans* is certainly responsible for the denitrification activity in Pilot 1, as it is the
sole micro-organism present in the medium. This would explain why this pilot showed significant denitrifying
activity despite the sequencing results suggesting insufficient prokaryotic DNA for 16S rRNA community
analysis. However, without sufficient DNA for community analysis, this denitrifying activity cannot be
definitively attributed to any particular prokaryotes. Pilot 2 – which showed superior denitrification activity –
contained both *T. denitrificans* and natural inoculum from the Rampillon water addition. This enhancement may
be explained by *T. denitrificans* activity as well as the aforementioned syntrophic interactions with other micro-
organisms from the constructed wetlands to increase the overall denitrifying activity. These hypotheses are
explored more in the following sections which discuss these results in light of the DNA analysis.

* Chronoamperometry study of electrodes from both pilots

In order to investigate whether or not the mechanism of enhanced denitrification is exo-electrogenic, we subjected
both inoculated pilots to further chronoamperometry studies under polarisation. After being transfer in a renewed
synthetic medium, the working electrodes of Pilot 1 and 2 were studied via chronoamperometry at the fixed
potential of -0.5 V vs SCE.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Current evolution of the working electrodes from Pilots 1 (*T. denitrificans*) and the working electrode from Pilot 2 (*T. denitrificans*, + water from Rampillon lake, T+R) in a renewed synthetic medium without nitrate or nitrite at the beginning of the chronoamperometry. The electrodes are polarized at -0.5 V vs SCE and 10 mM of NO₃⁻ are added at t = 2 h.</figcaption></figure>
**Figure 2** shows current evolution at -0.5 V vs SCE for both electrodes from Pilot 1 and Pilot 2. As shown in the Figure, no increase in the cathodic current is observed after nitrate addition at t= 2 h. This result suggests that either nitrate reduction isn't performed at all on the electrode (and hence the observed nitrate removal in **Figure 1** is non exo-electrogenic) or it is performed at a more negative potential (i.e., at values more negative than -0.5 V vs SCE). The second observation of note is that the greater cathodic current is observed in Pilot 2, which contained both natural water inoculum from Rampillon and the *T. denitrificans* pure culture, even prior to nitrate addition. Indeed, we measure a stable current density around -0.45 mA.cm⁻² whereas Pilot 1 (*Thiobacillus denitrificans* pure culture only) shows a lower absolute cathodic current density around only -0.17 mA.cm⁻².

Considering the lack of an appreciable response to nitrate addition, this constant reductive current is most likely attributed to oxygen reduction from ambient air present in the medium. We first opted to investigate the capacity of both Pilots for nitrate reduction at a more negative potentials than -0.5 V vs SCE using cyclic voltammetry studies in the potential range from +0.4 to -0.8 V vs SCE.

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Cyclic voltammetry of the working electrode incubated with a) Rampillon inoculum and *T. denitrificans* pure culture and b) *T. denitrificans* only in renewed medium before the chronoamperometry study shown in **Figure 2** (dashed line) and after the chronoamperometry (solid line) under argon saturation. Scan rate: 1 mV.s⁻¹.</figcaption></figure>

Results are reported in **Figure 3**. In both **Figure 3a** and **3b** we find no evidence of exo-electrogenic catalytic reduction of nitrate in either the pure *T. denitrificans* pilot or in the pilot in presence of both *T. denitrificans* and natural inoculum, even at significantly lower potentials than -0.5 V vs SCE. This implies that the difference in denitrification efficiency between the two pilots evident in **Figure 1** cannot be attributed to contributions from exo-electrogenic microorganisms, whether *T. denitrificans* or otherwise. Similar results were found for the Rampillon-inoculated Pilots 3 and 4 (Supporting Information **Figure S1**), with no apparent exo-electrogenic nitrate reduction observed for both polarized and unpolarized electrodes from these pilots.

However even if denitrification is not directly bio-electrochemical in nature, contributions of exo-electrogens to the facilitation of nitrate reduction cannot be directly ruled out. Indeed, some contributions of non-denitrifying exo-electrogens are suggested by the greater (i.e. more negative) cathodic current density value in the case of the electrode from Pilot 2 (T+R) compared to the second electrode from Pilot 1 (T) during the chronoamperometry study as described on **Figure 2**. Considering that these experiments were carried out in ambient air, we propose
that the cathodic currents observed could be linked to the reduction of oxygen rather than nitrate. The oxygen reduction reaction (ORR, E° = 0.817 V vs SHE at pH 7) at bare graphite electrodes is extremely sluggish, generally attainable only with large overpotentials comparable to the values applied in this study for nitrate reduction. It is also well-known that biocatalysis of the ORR is possible by cathodic exo-electrogenic biofilms. Given that all pilots here were preserved in sealed glass cells which were *not* initially anoxic, we propose that the initial biofilm development in both pilots resulted in the development of oxygen reducing bio-cathodes.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Cyclic voltammetry of Biocathode from Pilot 1 incubated with natural water from Rampillon and pure culture of *Thiobacillus denitrificans* (T+R, dashed line) and Biocathode from Pilot 2 incubated with pure culture of *Thiobacillus denitrificans* (T, dotted line) compared to bare graphite electrode (line) in Phosphate buffer (0.1 M of K₂HPO₄ and 0.1 M of KH₂PO₄) under oxygen saturation. Reference electrode is Saturated Calomel Electrode and the scan rate is 1 mV.s⁻¹.</figcaption></figure>

To test this hypothesis, we carried out cyclic voltammetry studies of biocathodes derived from both pilots under conditions of oxygen saturation in controlled buffer (0.2 M phosphate buffer, pH 7.2). For reference, a bare graphite electrode is included as a negative control. The results are summarized in **Figure 4**. Here the onset of ORR is observed on Pilot 2 at values close to +0.15 V vs SCE with a maximum cathodic current density below -0.8 mA.cm⁻². Pilot 1 shows significantly lower onset potentials for ORR closer to -0.3V vs SCE, comparable to that of bare graphite.

These results suggest that the bacterial biofilms obtained in both pilots are active in the exo-electrogenic reduction of O₂. We note that the trend in ORR activity is coherent with the cathodic current densities observed during the chronoamperometry study in **Figure 2**, and with the extent of denitrification observed in **Figure 1**. Considering that no direct exo-electrogenic nitrate reduction is performed on either of the incubated electrodes (**Figure 3a** and **3b**) while nitrate removal is observed during the one-month incubation under polarization (**Figure 1**) we propose that the enhanced denitrification observed in Pilot 2 can be indirectly attributed to ORR bio-cathode activity.
To further explore this hypothesis, bio-cathodes from Pilots 1 and 2 were put in a fresh aerated synthetic medium with no nitrite and 10 mM of NaNO₃ submitted to a succession of polarization phases at -0.5 V vs SCE interrupted with unpolarized phases with regular nitrate concentration measurements being made in parallel.

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Evolution of the nitrate concentration in 20 mL of renewed synthetic medium with an initial concentration 10 mM of NaNO₃. A three electrodes setup is fitted in the medium including the oxygen bio-cathode from Pilot 2 (T+R). The bio-cathode is submitted to a succession of polarization phases at -0.5 V vs SCE and non-polarized phases at the open circuit potential (OCP) as shown on the graph and detailed in Table 1 (Material and Methods).</figcaption></figure>

As evident in **Figure 5**, it is possible to correlate the denitrification efficiency with the presence or absence of polarization of the bio-cathode from Pilot 2. Indeed, denitrification can only be readily observed while the electrode is polarized; in the first period of 32 hours of polarization (highlighted in green in **Figure 5**) a total of just under 2 mM NO₃⁻ is removed, which represent 2.48 mg of NO₃⁻ for the entire Pilot. The second phase of polarization (highlighted in orange in **Figure 5**) shows a nitrate removal of 2.3 mM (2.85 mg of NO₃⁻ for the entire Pilot) for 32 hours. While the electrode is left at OCP between each polarization phase, no significant reduction in the nitrate concentration is observed. This strongly suggests that electrode polarization is required for the bio-cathode to reduce nitrate. Considering that no reduction could be evidenced via cyclic voltammetry for this electrode when nitrate is added (**Figure 3**), this nitrate removal does not arise from direct electron uptake from the cathode, but instead must arise indirectly as a result of the electrode polarization and consequent oxygen reduction.
In order to confirm that this denitrifying activity related specifically to the cathodic biofilm, we characterized the electrode from Pilot 2 before and after removing a portion of the biofilm by scraping for DNA extraction and sequencing.

<figure><img src="image_7.png" /><figcaption>**Figure 6.** Cyclic voltammetry in phosphate buffer (0.1 M of K₂HPO₄ and 0.1 M of KH₂PO₄). Comparison of the Biocathode (T+R) under ambient air before and after biofilm removal for DNA extraction. The scan rate is 1 mV.s⁻¹.</figcaption></figure>

The results are reported in **Figure 6**. Most notably, the clear drop in cathodic current and the almost total disappearance of the reduction peak at -0.2 V vs SCE strongly suggests that O₂ reduction at the cathode was carried out by the biofilm and hence this O₂ reducing capacity was lost after biofilm removal. To attribute this bioelectrochemical response to specific bacterial communities within the biofilm 16S rRNA gene amplification and sequencing was then carried out on these samples.
<figure><img src="image_8.png" /></figure>

**Figure 7.** Representation under Krona pie chart of the composition of the bacterial community from the biofilm collected at the surface of the electrode from Pilot 2 (incubated with natural water from Rampillon and *Thiobacillus denitrificans* pure culture). The treatment was made using the Galaxy bioinformatic platform and the blast was made against the SILVA 123 database. The number of sequences obtained after treatment was around 35 000. The identification was stopped at the genus level and only the genera with a relative abundance ≥ 3% are shown.

**Figure 7** shows a very large predominance of the *Pseudomonas* genus in the collected biofilm at the surface of the electrode incubated in Pilot 2 with 71 % of relative abundance. Such a high predominance of a single genus inside a biofilm that grew from an uncontrolled biological inoculum suggests the presence of a strong selective pressure for its development under the incubation conditions employed. This is supported by 16S amplicon sequencing of the Rampillon water inoculum which showed a relative abundance of the *Pseudomonas* genus of only 0.5 %.

It is also notable that, for Pilot 2, *T. denitrificans* pure culture was not able to develop a biofilm at the electrode surface under our incubation conditions. This observation is coherent with our electro-chemical study that showed no particular reaction to nitrate and oxygen presence on cyclic voltammetry for the electrode from either Pilot. On another hand, we also noticed that nitrate removal was observed in the Pilot 1 during the month of incubation (**Figure 1**) and a visible biofilm developed from which sufficient DNA could be extracted in order to proceed with sequencing. Nonetheless not enough sequences were obtained after amplification and sequencing of the 16S RNA gene. These two observations could be considered as contradictory but can be explained by the development of eukaryotic organisms such as algae, which may perform nitrate removal through assimilation [30], at the electrode surface. Hence, while we cannot rule out a role of *T. denitrificans* in the denitrifying activity of Pilot 1 specifically, we are confident based on the electrochemical results that a direct exo-electrogenic denitrification mechanism does not operate for either Pilot under our experimental conditions.
Discussion and Conclusions

The results that we obtained show a very particular form of polarization-induced denitrification without the direct
bioelectrochemical reduction of NO3-. Focusing on the best-performing Pilot, which used natural inoculum from
the NO3-rich artificial wetland of Rampillon, this denitrification capacity developed over the course of the initial
30-day polarization period (Figure 1). We also observed (Figure 5) that cathodic polarization is a pre-requisite
for denitrification. As evidenced in Figure 3, there is no direct electrochemical nitrate reduction by cyclic
voltammetry studies, but rather a bioelectrochemical oxygen-reducing capacity observed in aerated and
oxygenated media as reported in Figure 4. We directly attributed this cathodic response to the biofilm by post-
mortem voltammetry studies after biofilm extraction (Figure 6) which revealed a very large predominance of the
Pseudomonas genus by 16S rRNA sequencing.

This last finding is very coherent with what can be found in literature. Indeed the ability of some species from the
*Pseudomonas* genus to perform exo-electrogenic oxygen reduction is well known and documented [31]–[33]. The
specific metabolic mechanisms that are involved in the reduction remain unclear but the *Pseudomonas* genus has
been described for its ability to synthetize specific electron shuttles such as phenazines [34] although some other
studies tends to confirm that direct contact is sometime necessary for the reduction of oxygen to happen [32]. From
this perspective, we can conclude that the incubation conditions employed under polarization with the water from
Rampillon as an inoculum favored the development of oxygen-reducing biocathodes dominated with
*Pseudomonas* species.

The last remaining point concerns the connection between the catalysis of the exo-electrogenic reduction of oxygen
by the *Pseudomonas* biofilm and the nitrate reduction. The influence of the oxygen on the denitrification
metabolism of the Pseudomonas species is well known and has already been described in the literature [35],[36].
This influence had been described as reversible inhibition of the denitrification metabolism of *Pseudomonas*
species such as *Pseudomonas aeruginosa*. Coupling with the ability of reducing oxygen using cathodic exo-
electrogenic metabolism, we can consider the possibility of the emergence of local anoxic conditions when the
electrode is polarized at -0,5 V vs SCE. Indeed, with a three-electrode arrangement, the polarization of the working
electrode provides a continuous flow of electrons from the counter electrode at the poised potential. In this way,
through its cathodic metabolism, *Pseudomonas* species could exploit those electrons in order to perform oxygen
reduction coupled with autotrophic organic matter synthesis as has been documented for many *Pseudomonas*
species previously [37]–[39]. The reduction of oxygen by the biofilm could then cause a local oxygen depletion
which could result in anaerobic conditions inside the biofilm. This absence of oxygen could then lead to the
activation of the non exo-electrogenic denitrification metabolism of this or other species of the mixed biofilm.
<figure><img src="image_9.png" /><figcaption>**Scheme 1.** Description of a possible indirect denitrification mechanism induced by an exo-electrogenic oxygen-reducing biocathode.</figcaption></figure>

**Scheme 1** summarises this hypothesis, which would explain the observed correlation between polarization, nitrate removal (Figure 5) and the catalysis of the oxygen reduction (Figure 4). Indeed, while many studies describe direct nitrate reduction through cathodic exo-electrogenic uptake of electrons [9],[10],[5],[40], to the best of our knowledge this electro-assistance mechanism has not yet been reported in the literature. This study therefore highlights the possible indirect role of exo-electrogenic biofilms in the promotion of denitrification. Further experiments on this system are necessary in order to more fully comprehend this electro-assistance mechanism, for example through the application of co-cultures of known oxygen-reducing exo-electrogens with denitrifying bacteria. Such studies, currently ongoing in our group, will help to elucidate whether this electro-assistance phenomenon relates to alterations in the metabolism of a single species from oxygen reduction towards denitrification, or if it can instead be attributed to syntrophic interactions between specific species within a mixed biofilm community.

**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.
Acknowledgments

This study was supported by the French National Research Agency (ANR-17-CE04-0004). This project has
received funding from the European Union's Horizon 2020 research and innovation programme under the Marie
Skłodowska-Curie grant agreement No 899546 (to J. A. B.). T. P. acknowledges the Région Bretagne for funding
under grant ARED 651 NoNitrat. The authors thank Julien Tournebize and Cédric Chaumont (INRAe Anthony,
France) for sending samples of Rampillon inoculum.

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