# Dual role of marine bacteria Pseudoalteromonas NCIMB 2021 in corrosion of mild steel in artificial seawater

Julien Jaume, Dimitri Mercier, Antoine Seyeux, Vincent Semetey, Sandrine

Zanna, Philippe Marcus

► To cite this version:

Julien Jaume, Dimitri Mercier, Antoine Seyeux, Vincent Semetey, Sandrine Zanna, et al. Dual role of marine bacteria Pseudoalteromonas NCIMB 2021 in corrosion of mild steel in artificial seawater. Corrosion Science, 2025, 255, pp.113076. 10.1016/j.corsci.2025.113076. hal-05356615

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# Dual role of marine bacteria *Pseudoalteromonas* NCIMB 2021 in corrosion of mild steel in artificial seawater

Julien Jaume, Dimitri Mercier, Antoine Seyeux, Vincent Semetey, Sandrine Zanna*,
Philippe Marcus*

PSL Research University, CNRS – Chimie ParisTech, Institut de Recherche de Chimie Paris, 11 rue Pierre et Marie Curie, Paris 75005, France

## ARTICLE INFO

**Keywords:**

- Mild steel
- Pseudoalteromonas marine bacteria
- Localized corrosion
- Microbiologically influenced corrosion
- ToF-SIMS surface analysis
- Electrochimical analysis

## ABSTRACT

The modifications in corrosion resistance and local surface modifications caused by the colonization of *Pseudoalteromonas* NCIMB 2021 bacteria on mild steel in artificial seawater were investigated using electrochemical techniques, Scanning Electron Microscopy and Time-of-Flight Secondary Ion Mass Spectrometry. A dual role of these bacteria on corrosion resistance was observed and characterized. While an improvement in corrosion resistance was obtained in the presence of the bacteria, the colonized surface is locally modified and once the bacteria are removed, the previously colonized areas are exposed, leading to enhanced localized corrosion.

## 1. Introduction

Mild steel is a material of choice for applications in need of high mechanical properties, however its poor corrosion resistance requires preventive actions before exposure to corrosive environments such as marine conditions. With appropriate corrosion prevention methods, mild steel remains an attractive material due to its cost-effectiveness compared to other alloyed steels.

Seawater is particularly aggressive toward materials susceptible to corrosion due to its physicochemical characteristics (chlorides, pH, oxygen, etc.) [1] and its significant population of microorganisms. These microorganisms can lead to microbiologically induced corrosion (MIC), accelerating corrosion reactions. In the case of iron and its alloys, sulfate-reducing bacteria are mostly responsible for MIC [2–5]. Other MIC mechanisms involve Extracellular Electron Transfer (EET), where bacteria, such as Geobacter [6] and Shewanella [7], directly extract electrons from metallic iron, oxidizing it to Fe(II). Shewanella metabolism changes in anoxic environments and like other iron reducing bacteria, accelerates iron corrosion by breaking down the passive oxide layer, reducing insoluble Fe(III) compounds into Fe(II) soluble compounds [8]. Additionally, some bacteria secrete chemical compounds that promote iron corrosion. For example, Acetobacter produces acids (and is also capable of EET) [9], while Clostridium generates both acids

and enzymes that contribute to the degradation of iron surfaces [10,11].

Studying corrosion in marine environments is challenging due to the complexity of this system including a wide variety of conditions with distinct physicochemical properties and microbial populations. However, certain bacteria, such as those from the *Pseudoalteromonas* genus, are ubiquitous in marine environments. Therefore, investigating their influence on corrosion is essential for understanding how materials evolve in marine conditions.

For instance, a study on *Pseudoalteromonas lipolytica* demonstrated that these bacteria accelerate steel corrosion by secreting an electron mediator [12]. However, surface modifications were analyzed after 14 days of immersion using SEM and profilometry, limiting the understanding of the initial stages of microbial colonization.

Considering other species of this genus, research on the impact of *Pseudoalteromonas piscicida* on steel corrosion revealed a dual effect [13]. These bacteria reduced uniform corrosion through biomineralization but promoted pitting corrosion by secreting a pigment facilitating electron transfer. These surfaces were analyzed after 14 days of immersion allowing to conclude on an effect on the biofilm but not for the initial stages of colonization including the initiation of the localized corrosion. Studies on the influence of *Pseudoalteromonas* sp. on mild steel corrosion interestingly concluded on the inhibition of corrosion on mild steel, but lack of detailed surface modification analysis [14,15].

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: julien.jaume@chimieparistech.psl.eu (J. Jaume), dimitri.mercier@chimieparistech.psl.eu (D. Mercier), antoine.seyeux@chimieparistech.psl.eu (A. Seyeux), vincent.semetey@chimieparistech.psl.eu (V. Semetey), sandrine.zanna@chimieparistech.psl.eu (S. Zanna), philippe.marcus@chimieparistech.psl.eu (P. Marcus).</footnote>
In this study, we focused on the *Pseudoalteromonas* NCIMB 2021. This strain is an aerobic marine bacterium with a chemoheterotrophic metabolism [16]. Its growth is significantly enhanced in the presence of divalent cations ($Mg^{2+}$, $Ca^{2+}$) and require $Na^+$ for proper cellular function [16,17].

In this work, we investigated the interactions between *Pseudoalteromonas* NCIMB 2021 bacteria and a mild steel surface during the first 24 h of adhesion using advanced nanoscale chemical surface characterization techniques. This study aims to investigate the surface modifications occurring during the initial stages of microbial colonization, providing insights into the increase of corrosion or the inhibition effects that have been reported with other species.

In our approach we combined *in situ* measurements conducted by electrochemistry and detailed chemical surface analysis using Time-of-Flight Secondary Ions Mass Spectrometry to assess the impact of bacteria on oxide layer modifications. This work includes the elaboration of a new protocol to remove the bacteria without modifying the oxide layer.

## 2. Materials and methods

### 2.1. Mild steel samples

Mild steel S235 (wt% C ≤ 0.17, Mn ≤ 1.14, Cu ≤ 0.55, P ≤ 0.035, S ≤ 0.035, N ≤ 0.012 [18]) samples with a diameter of 1.5 cm were cut and then grounded using SiC abrasive papers (800, 2500, and 4000 grit) followed by polishing with diamond suspensions of 6 µm and 3 µm. The polished samples were cleaned using ultrapure water then ethanol with precision wipes and dried under a nitrogen flow.

### 2.2. Artificial sea water (ASW)

Artificial seawater was the medium for corrosion experiments. Its composition is (g/L): NaCl (24.62), $MgCl_2·6H_2O$ (11.06), $Na_2SO_4$ (4.11), $CaCl_2·2H_2O$ (1.54), KCl (0.78), and $NaHCO_3$ (0.20).

### 2.3. Culture of Pseudoalteromonas NCIMB 2021

The bacterial strain used in this study was *Pseudoalteromonas* NCIMB 2021, which was supplied by NCIMB Ltd. A stock culture was maintained at -80 °C in cryotubes containing a 50/50 mixture of artificial seawater (ASW) and glycerol.

To prepare a bacterial solution, a cryotube was emptied into 200 mL of liquid Difco Marine Broth 2216. The inoculated culture medium was then incubated at 30 °C for 24 h with orbital shaking at 160 rpm. After 24 h of incubation, a 5702 R centrifuge (Eppendorf) was used to pellet the bacteria from the medium at 3000 g for 15 min. Subsequently, they were suspended and diluted in ASW to obtain an optical density of 1, measured at 600 nm using a spectrophotometer Prim Light (SECOMAM). Under these conditions, the bacterial concentration was approximately $10^8$ colony-forming units/mL.

### 2.4. Epifluorescence microscopy

Epifluorescence microscopy observations were performed using a DMRX epifluorescence upright microscope (Leica) equipped with a water immersion x40 objective and a Retiga Exi (Qimaging, Canada). Bacteria were labeled using Syto9 (ThermoFisher), a fluorescent nucleic acid stain. Stacks of images spaced by 1 µm were acquired using ImagePro Plus (Media Cybernetics, United States) and were deconvoluted using AutoQuant (Media Cybernetics, United States). The images were processed using ImageJ software to determine the surface coverage by the bacteria.

### 2.5. Cleaning procedure of the surface

A cleaning procedure was developed to remove bacteria and most organic matter from the surface of the samples after immersion in ASW without or with bacteria. The sample was first rinsed with ultrapure water, then exposed to UV light for 20 min, followed by an ultrasonic cleaning in distilled water for 5 min. The sample was rinsed one final time and dried with nitrogen flow.

### 2.6. SEM observations

Scanning Electron Microscopy (SEM) observations were performed using a Zeiss Supra 40 SEM. Secondary electron images were acquired at a working distance of 10 mm with an accelerating voltage of 15 kV. Prior to imaging, the surface of mild steel samples, previously immersed in ASW with or without bacteria, was gently wiped with precision wipes (Kimtech, Science 7552).

### 2.7. Electrochemical study

To perform electrochemical studies, “BM FC EC” cells (Redox.me) were used. The working electrode was a polished mild steel sample and, using this cell, the exposed surface area was 1.33 $cm^2$. The reference electrode was a saturated calomel electrode, and a platinum wire was used as the counter electrode. Approximately 15 mL of ASW, with or without bacteria, was added at the start of the measurements. This experimental setup was placed in a Faraday cage and connected to a MultiPalmSens4 potentiostat (PalmSens).

The open circuit potential (OCP) was recorded continuously for 24 h when no other electrochemical measurements were being performed. Linear sweep voltammetry (LSV) was carried out every hour. The LSV started from the OCP and ended at -0.020 V/OCP at a scan rate of 0.167 mV/s. The polarization resistance $R_p$ was calculated using the slope of the LSV near the OCP. After 24 h of exposure to ASW, electrochemical impedance spectroscopy (EIS) was conducted with a frequency range from 100 kHz to 0.03 Hz, with 10 measurements per decade and an amplitude of 10 mV.

### 2.8. ICP analysis

An inductively coupled plasma optical emission spectrometer (ICP-OES) Agilent 5900 was used to measure the amount of dissolved iron in the solution. To achieve the highest sensitivity, the plasma was axially viewed and analyzed at a wavelength of 234.35 nm to detect iron.

Sampling was conducted hourly over 24 h of immersion of a mild steel sample in ASW, with and without bacteria. The solution was agitated before each sampling, and the collected samples were filtered through a 0.22 µm filter to remove any bacteria. Fresh solution was added after each sampling to keep a constant solution volume.

### 2.9. ToF-SIMS analysis

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was conducted using a ToF-SIMS 5 spectrometer (IonTof, Germany). The operating pressure of the mass spectrometer was maintained at $10^{-9}$ mbar. The total primary ion flux was less than $10^{12}$ ions.cm$^{-2}$ to ensure static conditions. The primary ion source was a pulsed 25 keV $Bi^+$ source with a current of 1.2 pA, rastering an area of 300 × 300 µm$^2$. High current bunched mode was employed to achieve high mass resolution of $m/\Delta m \sim 7000$ m, limiting the lateral resolution to a few µm. Prior to each measurement, the surface was slightly sputtered during 2 s using a $Cs^+$ ion sputter gun to remove the carbonaceous contamination from the surface. Negative secondary ions were selected due to better sensitivity for detecting oxide species. Data acquisition and postprocessing were conducted with SURFACELAB software version 7.2.

In-depth profiles were obtained by alternating between secondary
ion analysis and sputtering, with the sputtering performed using a 2 keV Cs+ source delivering 100 nA target current and rastering an area of 300 × 300 µm². The Bi⁺ and Cs⁺ ion beams were incident on the sample surface at a 45° angle, arranged appropriately to analyse ions from the center of the sputtered crater (avoiding edge effects). Ionized fragment intensity was plotted against Cs⁺ sputtering time on a logarithmic scale, allowing equal emphasis across signal intensities.

The ToF-SIMS data obtained during the acquisition of depth profiles was also used to reconstruct 3D images of the analyzed surface. In this case, the intensity of a fragment of interest is mapped as a function of its position (x, y, z). The images presented in this paper are 2D projections integrating data from the entire analyzed volume. In the ToF-SIMS 3D top-view mapping, bright yellow regions indicate an enrichment of the selected ions, whereas dark black regions signify the absence of these ions.

## 2.10. Dissolved oxygen measurement

The dissolved oxygen was monitored for 24 h in ASW with and without bacteria using a dissolved oxygen meter DO 700 (Eutech Instruments).

# 3. Results

## 3.1. Bacterial colonization and biofilm observations using epifluorescence microscopy

Fig. 1 shows the surface of an iron sample colonized by Pseudoalteromonas bacteria after 1 h (A) and 24 h (B) of immersion in inoculated ASW. The surface coverage by the bacteria was 2.5 ± 0.3% after 1 h and 8.4 ± 0.5% after 24 h, based on an average obtained from 5 images per immersion time. Dead cells were labeled using propidium iodide, which stains compromised membranes, and almost no coloration was visible.

Using the stacks of images, it is possible to determine the structure of the biofilm using images obtained at different heights. Fig. 2 shows the area observed on Fig. 1-B (24 h immersion) at different height from the surface, revealing the heterogeneity of the biofilm. Most areas are covered by a biofilm with a layer thickness of 5–10 µm, as illustrated by the Fig. 2. Moving further away from the surface, it becomes apparent that in certain regions the biofilm has formed aggregates, some of which reach a height of 20 µm.

## 3.2. Surface reactivity in ASW with and without bacteria

### 3.2.1. Electrochemical study and optical observation

The experiments were conducted on mild steel samples, in artificial

seawater (ASW) with or without bacteria. For each experiment, replicated 4 times, the open circuit potential (OCP) was measured during 24 h with a linear sweep voltammetry (LSV) performed every hour. The polarization resistance $R_p$ was then calculated using the slope of the curve, according to the Stern-Geary equation [19].

The dissolved oxygen concentration was monitored for some experiments.

At the end of immersion, the electrochemical impedance spectroscopy (EIS) was measured, and data were displayed using a Nyquist diagram. According to the time constant, two types of equivalent circuits were used to describe two different behaviors (Fig. 3).

These equivalent circuits include the following elements:

- $R_S$ the solution resistance which is negligible in highly conductive ASW.

- $R_{CT}$ the charge transfer resistance, associated with electron transfer caused by electrochemical reaction.

- $Q_{DL}$ the double layer constant phase element, associated with the accumulated charges at the electrochemical double layer at the interface between the surface sample and the ASW. It is not modeled by a perfect capacitor because of the heterogeneities of the surface. The $\alpha$ parameter ($0 < \alpha < 1$) defines the shift from a perfect capacitor. It is possible to calculate an equivalent capacitor to obtain a value in Farad. In the case of a constant phase element in parallel with a resistor, the equivalent capacitance $C_{eq}$ can be calculated using the following formula [20]:

$$C_{eq} = Q_{DL}^{1} R_{CT}^{1-\alpha}$$

- The Warburg element W is associated with diffusion. This element is in series with the charge transfer charge, as is it a part of the interfacial impedance [21]. This element is used to obtain a correct model, but its parameters will not be considered in this paper.

For both equivalent circuits, $Q_{DL}$ and $R_{CT}$ are connected in parallel because the current can go through the interface of the working electrode by capacitive current caused by the electrochemical double layer, represented by the double-layer capacity or by Faraday current caused by an electrochemical reaction, which requires an electron transfer and so needs to go through the charge transfer resistance.

All measurements and calculated parameters are summarized in the Table 1 at the end of this section.

- **Condition 1:** immersion in ASW with bacteria for 24 h.

Fig. 4 shows the evolution of the OCP and $R_p$ over 24 h of immersion of mild steel samples in condition 1. The Nyquist diagram was obtained from EIS measurements at the end of the immersion period.

<figure><img src="image_3.png" /><figcaption>Fig. 1. Epifluorescence microscopy image of a mild steel surface exposed to artificial sea water and the Pseudoalteromonas bacteria after 1 h (A) and 24 h (B).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Epifluorescence microscopy image of the same area shown in Fig. 1-B, at different heights from the surface (indicated in the top left corner).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Equivalent circuits used to fit the data according to the time constant. Circuit A includes the solution resistance $R_S$, the double layer capacitor $Q_{DL}$ in parallel with the charge transfer resistance $R_{CT}$. Circuit B is similar to A, with a Warburg impedance added in parallel with $Q_{DL}$.</figcaption></figure>

<table><caption>**Table 1**
Measurements and calculated parameters after 24 h of immersion for each condition.</caption><thead><tr><th></th><th></th><th>Condition 1</th><th>Condition 2</th><th>Condition 3</th><th>Condition 4</th></tr></thead><tbody><tr><td rowspan="3">EIS</td><td>OCP V/ SCE</td><td>-0.742 ± 0.002</td><td>-0.784 ± 0.006</td><td>-0.780 ± 0.002</td><td>-0.781 ± 0.007</td></tr><tr><td>R<sub>p</sub> (Ω)</td><td>41,392 ± 9646</td><td>1833 ± 168</td><td>1969 ± 202</td><td>1936 ± 296</td></tr><tr><td>R<sub>CT</sub> (Ω)</td><td>46,168 ± 9965</td><td>1528 ± 133</td><td>1313 ± 119</td><td>1360 ± 188</td></tr><tr><td></td><td>Q<sub>DL</sub> (µF. s<sup>-1</sup>)</td><td>53 ± 10</td><td>631 ± 538</td><td>372 ± 57</td><td>463 ± 143</td></tr><tr><td></td><td>α</td><td>0.83 ± 0.05</td><td>0.76 ± 0.03</td><td>0.77 ± 0.02</td><td>0.73 ± 0.04</td></tr><tr><td></td><td>C<sub>eq</sub> (µF)</td><td>65 ± 16</td><td>459 ± 284</td><td>251 ± 15</td><td>401 ± 164</td></tr></tbody></table>

A rapid decrease in OCP is observed immediately after immersion, followed by a rapid stabilization to reach the steady state after 2 h, around -0.74 V/SCE. The mean value after 24 h was -0.742 ± 0.002 V/SCE.

The $R_p$ increased over 8–12 h and stabilized around 40,000 Ω. The mean $R_p$ value after 24 h reached 41,392 ± 9646 Ω.

According to EIS measurements, the calculated charge transfer resistance was 46,168 ± 9965 Ω. The associated capacitance is on the order of µF, close to a standard double-layer capacitance value [22]. The equivalent circuit A (Fig. 3) was used to fit the data.

The dissolved oxygen concentration dropped sharply to 1.3 mg/L after 1 h, compared to 7.2 mg/L in ASW without bacteria, and then stabilized around 0.5 mg/L after 2 h.

To conduct a macroscopic observation, a sample that was immersed in ASW with bacteria for 24 h was wiped with precision wipes and exposed to air for 2 days after immersion. Optical observations (Fig. 5) revealed that the sample exhibited localized corrosion.

– **Condition 2:** pre-exposure to ASW with bacteria (i.e. as condition 1), cleaning, then immersion in ASW without bacteria for another 24 h.

Fig. 6 shows the evolution of the OCP and $R_p$ over 24 h of immersion of mild steel samples in condition 2. The Nyquist diagram was obtained
<figure><img src="image_6.png" /><figcaption>Fig. 4. (A) Open Circuit Potential (OCP – red curve) and Polarization Resistance ($R_p$ – blue dots) measured over 24 h of immersion for mild steel samples in ASW with bacteria. The oxygen concentration is noted in green. (B) The Nyquist diagram (orange dots) was obtained from EIS measurements at 24 h of immersion.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. Optical observation of a mild steel sample exposed to ASW with bacteria for 24 h, wiped with precision wipes and left to air for 2 days.</figcaption></figure>

from EIS measurements at the end of the immersion period.

After removing bacteria, a slower decrease of OCP is observed compared to condition 1. The OCP stabilizes around –0.78 V/SCE after 8–12 h of immersion. The mean value after 24 h is –0.784 ± 0.006 V/SCE, slightly lower than for condition 1.

The $R_p$ increased slightly at the beginning and stabilized after 4–8 h around 2000 Ω. The mean $R_p$ after 24 h is 1833 ± 168 Ω, also lower than for condition 1.

Comparing the equivalent circuits before (condition 1) and after cleaning (condition 2), it is noticeable that the charge transfer resistance has decreased to 1528 ± 133 Ω and the capacitance has increased. The high double-layer capacitances (on order of mF) indicate a significant amount of charge present at the interface. This charge accumulation is related to a large specific surface area caused by the degradation of the electrode. The equivalent circuit A (Fig. 3) was used to fit the data.

– **Condition 3:** immersion in ASW without bacteria for 24 h.

Fig. 7 shows the evolution of the OCP and $R_p$ over 24 h of immersion of mild steel samples in condition 3. The Nyquist diagram was obtained from EIS measurements at the end of the immersion period.

The OCP stabilizes around –0.78 V/SCE after 8–12 h of immersion. The mean value after 24 h is –0.780 ± 0.002 V/SCE.

The resistance increased slightly at the beginning and stabilized after 4–8 h around 2000 Ω. The mean $R_p$ after 24 h is 1969 ± 202 Ω.

The calculated charge transfer resistance is 1313 ± 119 Ω and the double-layer capacitances are of the order of mF indicating a significant amount of charge present at the interface. This charge accumulation is related to a large specific surface area caused by the degradation of the electrode and the precipitation of corrosion products on the electrode. The Warburg element supports this assumption by modeling the diffusion of the corrosion products. The equivalent circuit B (Fig. 3) was used

<figure><img src="image_8.png" /><figcaption>Fig. 6. (A) Open Circuit Potential (OCP – red curve) and Polarization Resistance ($R_p$ – blue dots) measured for mild steel samples pre-exposed to ASW with bacteria, cleaned and immersed in ASW without bacteria for another 24 h. (B) The Nyquist diagram (orange dots) was obtained from EIS measurements at 24 h of immersion.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** (A) Open Circuit Potential (OCP – red curve) and Polarization Resistance ($R_p$ – blue dots) measured over 24 h of immersion for mild steel samples in ASW without bacteria. The measured oxygen concentration is noted in green. (B) The Nyquist diagram (orange dots) was obtained from EIS measurements at 24 h of immersion.</figcaption></figure>

to fit the data.

The values obtained in this condition are similar to the values obtained in condition 2 but the impedance differs indicating a different electrochemical behavior.

The oxygen concentration close to the surface sample during the experiment was 7.2 mg/L at the beginning and then stabilized around 6.7 mg/L during the immersion.

To conduct a macroscopic observation, a sample that was immersed in ASW for 24 h, was wiped with precision wipes and exposed to air for 2 days after immersion. Optical observations (Fig. 8) revealed that the sample underwent uniform corrosion.

- **Condition 4:** pre-exposure to ASW without bacteria (i.e., as for condition 3), cleaning, then immersion in ASW without bacteria for another 24 h.

Fig. 9 shows the evolution of the OCP and $R_p$ over 24 h of immersion of mild steel samples in condition 4. The Nyquist diagram was obtained from EIS measurements at the end of the immersion period.

The OCP stabilizes around –0.78 V/SCE after 8–12 h of immersion. The mean value after 24 h is –0.781 ± 0.007 V/SCE.

The resistance increased slightly at the beginning and stabilized after 4–8 h around 2000 Ω. The mean $R_p$ after 24 h is 1936 ± 296 Ω.

The calculated charge transfer resistance is 1360 ± 188 Ω and the

double-layer capacitances are of the order of mF indicating a significant amount of charge present at the interface. This charge accumulation is related to a large specific surface area caused by the degradation of the electrode and the precipitation of corrosion products on the electrode. The Warburg element supports this assumption by modeling the diffusion of the corrosion products. The equivalent circuit B (Fig. 3) was used to fit the data.

The electrochemical behavior and calculated values are similar to condition 3.

### 3.2.2. ICP analysis of dissolved iron

Fig. 10 presents the cumulative iron released for mild steel samples in ASW, with and without bacteria, measured by ICP during 24 h of immersion. The exposed surface area was identical to that used for the electrochemical experiments.

In the presence of bacteria, the iron concentration increases gradually and stabilizes after 18 h of immersion, whereas in the absence of bacteria, it rises more rapidly and does not reach stabilization, with a final iron concentration after 24 h six times higher than in the presence of bacteria. These findings confirm a protective effect of bacteria on corrosion of steel.

### 3.2.3. Scanning electron microscopy (SEM) observations

A mild steel sample was immersed for 1 h in artificial seawater (ASW) with and without *Pseudoalteromonas* bacteria. Following immersion, the surface was gently wiped with precision wipes to remove any residual bacteria from the sample exposed to ASW containing bacteria. Scanning Electron Microscopy (SEM) observations, shown in Fig. 11, were conducted immediately after wiping and again after two days of exposure to ambient air.

In the presence of bacteria, visible residues remained on the surface after immersion. These formations, close to the size of the bacteria, evolved over two days of exposure to air, resulting in the formation of corrosion products. In contrast, when bacteria were absent from the ASW, the surface remained smooth, without noticeable asperities or visible changes even after two days in air.

### 3.3. ToF-SIMS analysis of samples exposed to bacteria

A mild steel sample was immersed 24 h in ASW containing bacteria. The biofilm was then removed according to the cleaning procedure described in 2.4 and the sample was left in air for two days.

The spectrometer was set in imaging mode (providing sufficient lateral resolution), and the cumulative mass spectra obtained over the entire analyzed depth allowed us to generate 3D chemical maps,

<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Optical observation of a mild steel sample exposed to ASW for 24 h, wiped with precision wipes and left to air for 2 days.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 9.** (A) Open Circuit Potential (OCP – red curve) and Polarization Resistance ($R_p$ – blue dots) measured for mild steel samples pre-exposed to ASW without bacteria, cleaned and immersed in ASW for another 24 h. (B) The Nyquist diagram (orange dots) was obtained from EIS measurements at 24 h.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 10.** Cumulative iron concentration in ASW with or without bacteria over 24 h of immersion of a mild steel sample (same exposed surface as in electro-chemical experiments).</figcaption></figure>

presented as 2D projections integrating data from the entire analyzed volume (Fig. 12). The selected ions, ($CN^-$ + $CNO^-$), $Fe^{2+}$, $FeOH^-$, $FeO_2$, $Cl^-$ and $FeOCl^-$, are characteristic of organic matter (proteins and amino acids [23–25]), metallic iron, iron hydroxide, iron oxides, chlorides, and iron oxychloride, respectively. While most of the surface is covered by a thin oxide layer, as indicated by intense $FeO_2$ and $FeOH^-$ on the whole surface, highly corroded areas are identified according to the corresponding maps (Fig. 12). The cross-section view extracted in the vicinity of a highly corroded area revealed the local penetration of oxidized species, corresponding to pit formation. This pattern was consistently observed in other corroded areas.

Region Of Interest (ROI) were defined in Fig. 12, corresponding to the highly corroded regions and outside highly corroded regions. To allow a better characterization and understanding of the in-depth distribution of chemical species, depth profiles were extracted from ROI (Fig. 13). As the sputtering time increases, the analysis reaches deeper into the sample.

In both profiles, the $Fe^{2+}$ signals, characteristic of the metallic substrate, exhibit significantly different behaviors. While the intensity of these signal's peaks after only 100 s of sputtering outside the heavily corroded areas, it takes approximately 800 s to reach maximum intensity in the highly corroded regions. This observation is consistent with the cross-sectional map shown in Fig. 13 and further confirms the local formation of pits.

To gain deeper insight, it is interesting to examine the distribution of ions characteristic of chlorides and organic matter (proteins and amino acids) in the profiles. When considering the $Cl^-$ and $FeOCl^-$ profiles, it is noteworthy that these species show intense signals at the surface, both within and outside the heavily corroded areas. While outside the

corroded areas the signals decrease immediately indicating that $Cl^-$ species are only adsorbed on the oxide layer surface, they remain intense, until the metallic substrate (around 800 s of sputtering), in the corroded regions, indicating that iron oxychlorides are formed inside the pits during the corrosion process.

The combined signals of $CN^-$ and $CNO^-$ ions follow the same trends as $Cl^-$ and $FeClO^-$. These signals have higher intensity in the pits formed in the highly corroded areas. The stronger signal from organic matter is attributed to the previous presence of bacteria, before cleaning, loosely bound to corrosion products in regions where pit formation occurred.

The $FeOH^-$/ $FeO_2$ ratio was then calculated inside the oxide layer for both areas, after 50 s of sputtering. Although it is 0.10 outside the pits, it increases to 0.38 in the strongly corroded areas. This indicates that the oxide composition differs in the two regions and is enriched in Fe hydroxide inside the pits.

## 4. Discussion

The influence of Pseudoalteromonas NCIMB 2021 on mild steel corrosion was investigated by comparing two conditions. For the first one, bacteria in ASW colonized the surface and for the second one, the bacteria that had previously adhered to the surface were removed. This approach allowed us to compare the consequences of bacterial colonization in the presence or the absence of bacteria on the surface.

After 24 h immersion in ASW containing bacteria, approximately 10 % of the mild steel surface was colonized, forming a heterogeneous biofilm characterized by a bacterial carpet scattered with localized aggregates reaching tens of microns in height (Fig. 1 and Fig. 2).

Electrochemical analysis revealed a significant reduction of the surface degradation compared to immersion in ASW, as evidenced by the increased polarization and charge transfer resistance along with a decreased double-layer capacitance (Table 1). This protective effect of bacteria was confirmed by a six-fold reduction in dissolved iron measured by ICP (Fig. 10). The observed increase in average polarization resistance (25–35-fold) in the presence of bacteria suggests that the corrosion inhibition cannot be solely attributed to a reduction in active surface area due to bacterial coverage.

Monitoring the dissolved $O_2$ concentration is a key point to understanding the role of the bacteria on corrosion mechanisms. Since oxygen reduction is the primary cathodic reaction in this system, the oxygen concentration at the interface directly influences the corrosion rate [26]. Within the first hour of immersion in ASW with bacteria, the dissolved oxygen concentration near the surface decreased to 0.5 mg/L while it stabilized around 6.7 mg/L in ASW without bacteria. This rapid oxygen depletion, attributed to its consumption by bacteria, suggests a mechanism of corrosion inhibition based on the limitation of the cathodic
<figure><img src="image_13.png" /><figcaption>**Fig. 11.** SEM observation of the mild steel surface after 1 h of immersion in artificial seawater, with (ASW + B) or without (ASW) bacteria. The sample was then wiped with precision wipes, then observed immediately and again after 2 days of exposure to air, in the same area.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Fig. 12.** TOF-SIMS 3D chemical maps obtained on a mild steel surface after 24 h immersion in ASW with bacteria, cleaning to remove bacteria and 2 days in air. The Region Of Interest (ROI) colored in blue, corresponding to highly corroded areas, was defined using the FeO<sub>2</sub> high intensity signal. A cross-section was extracted from the recorded data (along with the green dotted line).</figcaption></figure>

oxygen reduction reaction.

The observed differences in open circuit potential (OCP) during the initial hours can be partially explained by these variations in oxygen availability. In both conditions, initial oxide layer dissolution driven by

oxygen reduction leads to a decrease in OCP. However, in the presence of bacteria, rapid oxygen consumption leads to a faster reaching of steady state. Conversely, higher oxygen concentrations in ASW without bacteria prolong oxide dissolution and the approach to steady state,
<figure><img src="image_15.png" /><figcaption>**Fig. 13.** ToF-SIMS profiles reconstructed from 3D images after selection of ROI corresponding to inside and outside highly corroded areas, as defined in Fig. 12.</figcaption></figure>

until the reactants are hindered by the new oxide layer.

Additionally, the rapid deposition of a conditioning film composed of organic matter, such as the fast and irreversible adsorption of extracel-lular polymeric substances [27], can influence the OCP.

Despite its efficient consumption by the *Pseudoalteromonas*, some oxygen can reach the surface, considering that these strictly aerobic bacteria are still alive on the surface after 24 h. However, local large biofilm aggregates gather many bacteria in a small area (Fig. 2), which can lead to an anoxic zone underneath.

Following removal of the biofilm after 24 h bacterial exposure, the sample previously covered by bacteria exhibited reduced electro-chemical resistance after a new immersion in ASW compared to the pre-exposure condition. The protective effect provided by bacteria does not persist after their removal and supports the mechanism of corrosion inhibition through cathodic depolarization.

SEM (Fig. 11) and ToF-SIMS (Fig. 12) surface analyses, following bacteria removal and exposure to air, revealed localized alterations in previously colonized areas. Residual organic and chloride compounds remained on the surface despite rigorous cleaning, including UV light exposure and ultrasonic cleaning. These areas also exhibited localized formation of iron oxyhydroxides and chloride-rich corrosion products, suggesting a corrosive environment. The size of these locally corroded regions corresponded to the dimensions of prior bacteria aggregates indicating a link between localized oxygen depletion within aggregates and subsequent corrosion.

The anoxic regions within the biofilm aggregates become anodic relative to the surrounding regions, more cathodic. While the low oxygen concentration limits the overall corrosion rate in these anodic areas, localized oxide breakdown occurs, facilitating chloride penetration and subsequent reaction with the iron substrate.

This breakdown may have been facilitated by bacterial metabolic byproducts and local pH variations [28–31], which made it easier for chloride ions to penetrate the surface and subsequently react with iron (Fig. 13) [32,33].

Upon air exposure, the chloride-enriched zones with a defective oxide layer react with atmospheric moisture and oxygen resulting in further localized attack [34]. SEM observations (Fig. 11) revealed the formation of corrosion products within one hour of air exposure. These compounds contribute to the degradation of the underlying oxide layer, leading to preferential corrosion zones. Following bacterial colonization and then cleaning, the surface becomes sensitized to localized corrosion.

The comparative analysis of mild steel corrosion in the presence and absence of *Pseudoalteromonas* NCIMB 2021 revealed distinct corrosion processes. Electrochemical measurements indicated higher corrosion rates in ASW compared to ASW containing bacteria, but similar rates to the cleaned, previously colonized samples. However, while ASW induced a more uniform corrosion, as confirmed by the absence of localized corrosion products after 1 h of air exposure (SEM), bacterial colonization resulted in localized corrosion, a difference also observed by optical microscopy (Fig. 5 and Fig. 8).

The samples were left in air to observe the effect of atmospheric exposure of the sample after the removal of bacteria. This would correspond to a practical case of hull cleaning operations in a dry dock.

Beyond a better understanding of the effect of *Pseudoalteromonas* NCIMB 2021 on the corrosion of mild steel, this study highlights the importance of investigating early-stage bacterial-induced surface modifications and their role in corrosion processes, which can have dual effects. Advanced analytical techniques in surface science provide complementary insights into the initial surface transformations during bacterial colonization, essential for understanding longer-term processes and thicker biofilm development.

## 5. Conclusion

The effect of *Pseudoalteromonas* NCIMB 2021 on surface modifications and corrosion of mild steel have been investigated, using a combination of techniques. Bacterial colonization significantly reduced the overall corrosion rate, an effect attributed to oxygen consumption limiting cathodic reduction.

This protective effect remains as long as bacteria are present, but after removal of the biofilm, an increased susceptibility to localized corrosion is observed in previously colonized surface areas. These areas exhibited chloride enrichment and localized oxide breakdown, leading to enhanced corrosion upon air exposure. Comparatively, in ASW without *Pseudoalteromonas* sp. the corrosion is very rapidly generalized.

This work emphasizes the dual role of bacteria in affecting mild steel corrosion. While bacterial colonization can reduce the corrosion rate, it also induces surface modifications that promote localized corrosion upon bacterial removal. These findings highlight the importance of understanding the surface modifications that occur during the initial stages of biofouling and corrosion processes.
CRediT authorship contribution statement

**Antoine SEYEUX:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. **Dimitri MERCIER:** Writing – review & editing, Methodology, Formal analysis, Conceptualization. **Sandrine ZANNA:** Writing – review & editing, Validation, Supervision, Investigation, Conceptualization. **Vincent SEMETEY:** Writing – review & editing, Methodology, Formal analysis, Data curation. **Philippe MARCUS:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization. **Julien JAUME:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

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.

Acknowledgements

This work was funded by the Office of Naval Research Global (contract N°62909-23-1-2019). Région Ile-de-France is acknowledged for partial funding of the ToF-SIMS instrument.

Data availability

Data will be made available on request.

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