Inhibition of Microbiologically Influenced Corrosion of 304 Stainless Steel by Artemisia annua L. in Simulated Seawater

Abstract

The combination of electrochemical, surface, and spectroscopic techniques revealed that Pseudomonas aeruginosa biofilm accelerated corrosion of 304 stainless steel (SS), leading to localized pitting with depths up to 3.75 μm. Such damage did not occur on 304 SS treated with P. aeruginosa in the presence of Artemisia annua L. extract, or in sterile seawater. Introducing A. annua into biotic seawater hindered biofilm development and prevented the formation of porous Fe(III) corrosion products. Instead, a compact Fe₃O₄ layer formed, indicating a shift in corrosion product morphology and stability. ATR-FTIR analysis confirmed phenolic groups from the extract were adsorbed onto the steel interface, supporting the dual inhibitory role of A. annua through both surface modification and antimicrobial action. A. annua extract demonstrated a 74.4 ± 4.4% reduction in MIC-induced corrosion of 304 SS in marine conditions.

1. Introduction

Corrosion is a critical issue that impacts global economies, safety, and resource conservation. The economic losses associated with corrosion, including direct costs for replacing corroded structures and indirect losses such as energy and material waste, amount to billions annually [1]. Around 70% of steel corrosion in marine environment is caused by microbial involvement, requiring a multidisciplinary approach by integrating electrochemical and metallurgical perspectives to better understand and mitigate its effects [2]. A pioneering microorganism in marine biofilm formation and a key contributor to microbiologically influenced corrosion (MIC) is the Gram-negative bacterium Pseudomonas aeruginosa. Electron transfer between the steel surface and biofilm can be direct or shuttle-mediated by metabolic products or inorganic electron mediators [3]. The formation of a biofilm has a harmful effect on the metal substrate, creating a micro-environment that can cause the deterioration of the metal and release of potentially dangerous substances into the marine environment [3]. Another concern is finding protection measures against MIC that will replace hazardous antifouling paints with harmful effects on aquatic life [4]. Given the significant financial and environmental costs associated with corrosion, the shift towards more sustainable practices emphasizes non-toxic methods for MIC protection, particularly natural inhibitors that do not release harmful substances into the marine environment [5].

Aerial stems of plants are the richest source of natural antioxidants, with polyphenols being the most prevalent [6]. Research on the rich profile of active compounds of Artemisia annua L. extracts and their antiviral and antibacterial effects regained attention during the COVID-19 pandemic. Nair et al. found that the synergistic effect of the main components of A. annua can prevent the attachment and entry of SARS-CoV-2 to the host cell [7]. The chemical composition of aqueous or alcoholic extracts of A. annua can vary significantly depending on geographic origin and processing method, unlike the essential oil composition, which shows only minor differences [8]. In our previous findings, an aqueous extract of A. annua contained numerous polyphenols with chlorogenic acid as the most abundant [9]. A. annua collected in southeastern Bosnia and Herzegovina demonstrated corrosion inhibition of Al alloy [9,10] and A36 steel [11] in simulated seawater. The extract was prepared at room temperature, deliberately minimizing energy use and solvent waste often associated with plant extract preparation. Moreover, aqueous extract of A. annua inhibited P. aeruginosa growth in the concentration range of 0.50–2.18 mg mL⁻¹ [11]. Building on this, the present work investigates the effect of A. annua extract on film formation and corrosion behavior of 304 stainless steel in the presence of P. aeruginosa in simulated seawater. The findings reveal that A. annua not only inhibits bacterial growth but also promotes the formation of compact Fe₃O₄ instead of porous oxohydroxides, resulting in improved surface protection and corrosion resistance.

2. Materials and Methods

2.1. Preparation of the Working Electrode

For the corrosion experiments, 304 stainless steel (SS) coupons were used as substrates for biofilm growth. The nominal chemical composition of the alloy was (%): 0.021 C, 0.0692 Si, 1.833 Mn, 0.0320 P, 0.0010 S, 0.0810 N, 18.125 Cr, 8.075 Ni, with Fe as the balance. Prior to testing, the coupons (20 × 20 × 3 mm) were sequentially polished using silicon carbide papers ranging from 360 to 1200 grit, ultrasonically cleaned in 96% ethanol for 60 s, rinsed thoroughly with ultrapure water, and finally sterilized by brief flaming over a Bunsen burner combined with intermittent rinsing in 70% ethanol.

2.2. Cultivation and Metabolic Activity of P. aeruginosa

Pseudomonas aeruginosa, strain ATCC 27853 was reactivated and propagated under conditions described in previous studies [10,11]. For the corrosion experiments, simulated seawater broth (ASWB) served as the growth medium, consisting of (g L⁻¹): 4.1575 Na₂SO₄, 11.1211 MgCl₂ × 6H₂O, 0.7902 KCl, 1.5877 CaCl₂ × 2H₂O, 24.9772 NaCl, 0.0587 NaHCO₃, 5.0000 peptone, 1.0000 yeast extract. 304 stainless steel coupons were immersed either in sterile ASWB (abiotic media) or ASWB inoculated with P. aeruginosa (biotic media) and incubated at 37 °C for 3, 7, 14, 21, and 30 days. To assess the influence of A. annua extract, parallel tests were carried out using biotic media supplemented with 1 g L⁻¹ of the extract (inhibited media). These samples were incubated for 14 and 21 days. The starting bacterial concentration in both biotic and inhibited systems was approximately 10⁶ cfu mL⁻¹.

The pH values of ASWB before and after incubation, in all tested conditions (abiotic, biotic and inhibited) were determined using a calibrated pH meter (pH 7110, Xylem Analytics Germany GmbH, Weilheim, Germany) equipped with a glass electrode (SenTix 81, Xylem Analytics Germany GmbH, Weilheim, Germany). Dissolved oxygen levels were quantified using a Multi 3630 IDS digital multimeter paired with an optical sensor (FDO^® 925, Xylem Analytics Germany GmbH, Weilheim, Germany).

All glassware and metallic components were sterilized in a dry heat oven (MOV-212F, Sanyo Electric Co., Ltd., Osaka, Japan) at 160 °C for 2 h, while solutions and culture media were sterilized separately using an autoclave (MLS-3751L, Panasonic, Tokyo, Japan) at 121 °C for 15 min.

2.3. Preparation of A. annua Extract

The antibacterial activity of A. annua aqueous extract against P. aeruginosa was determined previously and published in a separate study [11]. The equal method for preparing the A. annua extract (1 g L⁻¹) was applied.

1.0000 g of dried and finely ground plant material, collected from the southeastern region of Bosnia and Herzegovina, was added to 1 L of simulated seawater broth and left to macerate at room temperature in the dark. After three hours, the mixture was passed through a 0.45 μm membrane filter, and the obtained extract was adjusted with ASWB to reach a final volume of 1 L.

2.4. Electrochemical Tests

To investigate the electrochemical behavior of the films formed on 304 SS under sterile and contaminated simulated seawater conditions 304 SS surfaces were examined by measuring the open circuit potential (E_OCP), electrochemical impedance spectra (EIS), and potentiodynamic polarization (PP) curves. Impedance spectra were collected at E_OCP by applying an AC perturbation of 10 mV (rms) in the frequency range from 10 kHz to 5 mHz. Following the EIS tests, PP curves were recorded within the ±200 mV potential range vs. E_OCP at the scan rate of 0.5 mV s⁻¹.

Electrochemical measurements were conducted in a standard three-electrode electrochemical cell, Model K0235 Flat Cell (Ametek Inc., Berwyn, PA, USA) with simulated seawater as the working electrolyte. The composition of the simulated seawater was: (g L⁻¹): 4.1575 Na₂SO₄, 11.1211 MgCl₂ × 6H₂O, 0.7902 KCl, 1.5877 CaCl₂ × 2H₂O, 24.9772 NaCl, 0.0587 NaHCO₃. A platinum mesh served as the counter electrode, while an Ag | AgCl | 3 M KCl electrode was used as the reference. The exposed surface area of the 304 SS working electrode was 1 cm². To ensure reproducibility and minimize experimental variability, all electrochemical measurements were repeated until consistent results were obtained, which on average required three independent measurements per experimental condition. Electrochemical measurements were performed with Autolab PGSTAT320N (Metrohm AG, Herisau, Switzerland) controlled by Nova 1.5 software. Data processing of obtained results was also executed in Nova 1.5 software for Windows.

2.5. Surface Characterization

The surface films formed on 304 SS coupons after exposure to abiotic, biotic, and inhibited media were characterized ex situ using several complementary techniques: non-contact optical profilometry, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS), attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, and inductively coupled plasma optical emission spectroscopy (ICP-OES).

Before characterization, samples incubated for 14 and 21 days under the three experimental conditions were gently removed from the media and rinsed thoroughly with ultra-pure water to eliminate residual salts and loosely attached deposits. A graded dehydration process was then performed by immersing the surfaces sequentially in ethanol solutions of increasing concentration (25%, 50%, 75%, and 96% v/v), each for 10 min.

Three-dimensional surface topographies of the treated electrodes were recorded using a non-contact optical profilometer (ZYGO NewView 7100, Zygo Corporation, Middlefield, CT, USA). From the 3D scans, two-dimensional line profiles were extracted and used for roughness parameter evaluation. All profilometric analyses were performed over the same surface area of 1.092 mm × 1.092 mm.

For SEM-EDS analysis, after dehydration, electrodes were immersed in a 2.5% (v/v) glutaraldehyde solution, left in the dark at 7 °C for 4 h, and coated with a thin layer of copper to increase the electrical conductivity of the surface. The morphology and chemical composition of the surfaces were investigated using SEM with integrated EDS (Phenom ProX, Thermo Fisher Scientific, Waltham, MA, USA).

The chemical composition was also studied directly from treated steel surfaces using FTIR spectrometer (IRAffinity-1S, Shimadzu Corporation, Kyoto, Japan) with a diamond and optical crystal sampling plate (GladiATR10, Shimadzu, Japan).

For elemental analysis of trace metals released into solution, an inductively coupled plasma optical emission spectrometer (iCAP 6500 Duo, Thermo Scientific, Waltham, MA, USA) was used. Short-term immersion tests were conducted by placing electrodes that had been incubated for 21 days in biotic or inhibited media into absolute ethanol for 1 h. After removal, the electrodes were rinsed with ultra-pure water, and the ethanol extract was evaporated in a water bath to a volume below 1 mL before being diluted to a final volume of 10 mL [10,11]. Calibration curves were obtained using certified multi-element standard solutions (Plasma Standard Solution 4, Specture^®, Alfa Aesar, Thermo Fisher Scientific, Haverhill, MA, USA).

3. Results and Discussion

3.1. Metabolic Activity of the Bacteria

Electrochemical properties of the surface film formed on 304 SS in simulated seawater depend on the pH and concentration of dissolved oxygen (CDO). These parameters also indicate bacterial metabolic activity, which can lead to a decrease in CDO and pH values in the medium [10,11]. The CDO after 14 days of incubation of 304 SS in biotic media decreased from an initial 5.53 ± 0.01 mg L⁻¹ to 1.06 ± 0.07 mg L⁻¹, which was two times lower than the concentration recorded for inhibited media (2.07 ± 0.09 mg L⁻¹) for the same incubation period. At the same time, CDO in abiotic media decreased to 4.43 ± 0.33 mg L⁻¹.

Table 1. pH of the medium after exposure of 304 SS to abiotic, biotic and inhibited media for 3, 7, 14 and 30 days.

t (d)	Medium
	Abiotic	Biotic	Inhibited
3	7.16 ± 0.17	6.76 ± 0.04	-
7	7.17 ± 0.12	6.99 ± 0.03	-
14	6.94 ± 0.11	6.51 ± 0.08	6.82 ± 0.12
30	7.45 ± 0.08	7.10 ± 0.19	-

shows the pH values of the media after incubation of 304 SS in abiotic, biotic, and inhibited media. Before the incubation of working electrodes, the pH of the simulated seawater broth (ASWB) was 7.21 ± 0.02. During the early stage of incubation (3–14 days) in the abiotic medium, a slight decrease in pH to 6.94 ± 0.11 was observed. After 30 days, the pH of the abiotic medium increased to 7.45 ± 0.08 (Table 1). In the biotic medium, a more pronounced drop in pH was recorded after 14 days (6.51 ± 0.08), while after 30 days, the pH slightly increased. The observed pH variations in the biotic system are likely related to bacterial metabolic activity, potentially due to planktonic cell growth [10,11], while in the abiotic system, the observed pH variations are more likely attributed to shifts in the carbonate buffer equilibrium of the artificial seawater, and are unlikely to be directly driven by corrosion processes or passive film formation [1,12].

The pH of the inhibited medium after 14 days of incubation was similar to that of the abiotic medium (6.82 ± 0.12).

Despite the variation in pH values across all media (ranging from 6 to 8), literature suggests that passivity of 304 SS can be maintained within this range due to the formation of a thin Fe(II)/Fe(III) oxide layer [1,12,13]. However, factors such as limited CDO and microbial activity may influence the growth and stability of the surface film.

3.2. Electrochemical Results

3.2.1. Electrochemical Impedance Spectroscopy

Impedance spectra of 304 SS recorded after incubation in abiotic and biotic media are presented by Nyquist and Bode plots in Figure 1. Results were fitted with the electrical equivalent circuits (EEC) shown in Figure 2. When analyzing the EIS results a constant phase element (CPE) was adopted to compensate for surface heterogeneity [14]. All EIS data obtained for the 304 SS exposed to abiotic and biotic media were described with a two-time constant model [R(Q₁[R₁(Q₂R₂)])] (Figure 2a), except for the 304 SS incubated in an abiotic medium for 30 days which was described by EEC [R(Q₁R₁)(Q₂R₂)] (Figure 2b). Here, R is the ohmic resistance of the electrolyte which was 16.6 Ω cm² for all measurements. Q₁ and R₁ represent the capacitance and resistance of the film formed on the 304 SS surface in abiotic and biotic media. Q₂ and R₂ represent the capacitance of the electrical double layer, that is, the charge transfer resistance at the electrode/electrolyte interface. EEC values are shown in

Table 2. The values of EEC obtained by analyzing the impedance spectra for 304 stainless steel from Figure 1.

Media	t	Q1 × 105	n1	R1	Q2 × 105	n2	R2
	d	Ω−1 sn cm−2		kΩ cm2	Ω−1 sn cm−2		kΩ cm2
Abiotic	3	5.57	0.807	13.54	33.18	0.976	31.40
	7	5.83	0.773	16.53	33.97	0.969	49.40
	30	3.65	0.888	51.32	30.10	0.972	126.82
Biotic	3	6.47	0.775	9.65	16.44	0.962	20.67
	7	4.13	0.799	4.73	3.40	0.904	22.93
	30	1.95	0.865	20.49	1.22	0.897	53.30

.

From the EIS of 304 SS incubated in abiotic (Figure 1a,b) and biotic media (Figure 1c,d) it can be seen that the total impedance of the examined systems elevated with the increasing exposure time. Yet, in the presence of bacterium P. aeruginosa, the film resistance R₁ and charge transfer resistance R₂ of 304 SS during the early incubation period (3 d) decreased by around 30% compared to values obtained without the bacteria (Table 2). Moreover, these values further declined with increasing exposure time, with a decrease of 60% observed after 30 days of incubation. The latter was associated with the development of a P. aeruginosa biofilm on the 304 SS surface that reduced the capacitances of the surface film Q₁ and electrical double layer Q₂ (Table 2) and resulted in the development of a more heterogeneous surface layer as recognized by a decrease in n values in the presence of bacteria (Table 2) [3,10,11].

The influence of the plant A. annua extract on the development of a surface film on the 304 SS exposed to simulated seawater in the presence of P. aeruginosa during 14 days of incubation is shown by Nyquist and Bode plots in Figure 3. The results of EIS measurements for 304 SS after 14 days of exposure to the sterile and inhibited medium were represented with the two-time constants model [R(Q₁R₁)(Q₂R₂)] (Figure 2b), while the results for the biotic medium were represented with the EEC [R(Q₁[R₁(Q₂R₂)] (Figure 2a) [15]. The interpretation of the EECs is as described earlier, so Q₁ and R₁ refer to the capacitance and the resistance of the film formed on the electrode surface in abiotic, biotic, and inhibited media. The double-layer capacitance Q₂ in parallel with a charge transfer resistance R₂ represents the interface between electrolyte and bare electrode. The inhibition efficiency (IE) of A. annua toward microbiological corrosion of 304 SS induced by P. aeruginosa in simulated seawater was calculated using the following expression [15]:

$$IE= {\displaystyle \frac{R_p^{*}-R_p }{R_p^{*}}}\times 100.$$

(1)

Here, R_p and $R_p^{*}$ are the polarization resistances (R_p = R₁ + R₂) recorded after incubating the 304 SS coupons in inoculated seawater without and with the addition of A. annua, respectively [16]. The values of EECs, calculated R_p and IE values are summarized in

Table 3. The values of EEC obtained by analyzing the impedance spectra for 304 stainless steel from Figure 3.

Media	Q1 × 105	n1	R1	Q2 × 105	n2	R2	Rp	IE
	Ω−1 sn cm−2		kΩ cm2	Ω−1 sn cm−2		kΩ cm2	kΩ cm2	%
Abiotic	25.26	0.885	38.50	5.80	0.940	80.59	119.09	–
Biotic	2.97	0.811	6.53	3.78	0.886	24.68	31.21	–
Inhibited	0.13	0.895	43.36	6.53	0.925	60.24	103.6	69.9

.

Figure 3. Nyquist (a) and Bode plots (b) of impedance spectra recorded on 304 SS after incubation in abiotic (blue), biotic media (red), and inhibited media (green) for 14 days.

Figure 3. Nyquist (a) and Bode plots (b) of impedance spectra recorded on 304 SS after incubation in abiotic (blue), biotic media (red), and inhibited media (green) for 14 days.

The detrimental impact of bacteria on the film development of 304 SS in simulated seawater was evident from the EIS recorded after 14 days of incubation in abiotic, biotic, and inhibited media. The addition of P. aeruginosa to simulated seawater resulted in the development of a film with the resistance of R₁ = 6.5 kΩ cm², which was almost six to seven times lower than the values recorded for the abiotic (38.5 kΩ cm²) and inhibited media (43.4 kΩ cm²), respectively (Table 3). Additionally, in the presence of bacteria, the charge transfer resistance R₂ decreased from 80.6 kΩ cm² (abiotic medium) to 24.7 kΩ cm² (biotic medium). Conversely, in the presence of both the bacteria and A. annua, R₂ increased to 60.2 kΩ cm², indicating a notable improvement compared to the biotic seawater alone. The increase in resistance values after adding A. annua plant extract to the biotic seawater was associated with the development of a protective film and the adsorption of phenolics on the 304 SS surface. The adsorption of extract molecules onto the 304 SS surface resulted in a reduction in electric double layer thickness, as evidenced by the increased Q₂ parameter in relation to that observed in the biotic seawater [14]. According to EIS data, the inhibition efficiency of A. annua toward microbiological corrosion of 304 SS induced by P. aeruginosa in artificial seawater was 70%.

3.2.2. Potentiodynamic Polarization

Potentiodynamic polarization (PP) curves recorded after 14 days exposure of 304 SS coupons to abiotic, biotic and inhibited seawater are given in Figure 4. Corrosion parameters obtained from data presented in Figure 4 are shown in Table 4. The corrosion rates r_corr of treated samples were calculated with the following equation:

$$r_{corr}= {\displaystyle \frac{j_{corr} M}{zF}}.$$

(2)

Here, j_corr is the corrosion current density deduced from PP curves, M is the molar mass of Fe(III) oxide, z is the number of exchanged electrons and F is the Faraday constant. Corrosion current densities obtained from PP curves for biotic (j_corr) and inhibited media ($j_{\mathrm{corr}}^{*}$) were also used in evaluation of inhibition efficiency (IE) of A. annua according to Equation (3):

$$IE= {\displaystyle \frac{j_{corr }- j_{corr}^{*}}{j_{corr}}}.$$

(3)

The results obtained by PP measurements are presented in Table 4.

Figure 4. Potentiodynamic polarization curves of 304 SS recorded in artificial sweater after exposure to abiotic (◼), biotic (▼), and inhibited media (●) for 14 days.

Figure 4. Potentiodynamic polarization curves of 304 SS recorded in artificial sweater after exposure to abiotic (◼), biotic (▼), and inhibited media (●) for 14 days.

Table 4. Corrosion parameters obtained by analyzing the experimental results from Figure 4.

Media	Ecorr	jcorr	rcorr	IE
	V	μA cm−2	μA cm−2 h−1	%
Abiotic	–0.249	0.039	0.078	–
Biotic	–0.158	0.201	0.399	–
Inhibited	–0.150	0.043	0.085	78.7

Table 4. Corrosion parameters obtained by analyzing the experimental results from Figure 4.

Media	Ecorr	jcorr	rcorr	IE
	V	μA cm−2	μA cm−2 h−1	%
Abiotic	–0.249	0.039	0.078	–
Biotic	–0.158	0.201	0.399	–
Inhibited	–0.150	0.043	0.085	78.7

As can be seen from Table 4 the highest corrosion current density of 0.201 μA cm⁻² was recorded after 14 days of incubation of 304 SS in biotic medium, while the lowest value of 0.039 µA cm⁻² was achieved for abiotic medium, which was close to values obtained for inhibited medium (0.043 μA cm⁻²). Consequently, with the addition of P. aeruginosa alone, corrosion rate increased (0.399 μA cm⁻² h⁻¹), while the addition of A. annua decreased corrosion rate to 0.085 µA cm⁻² h⁻¹ which was close to 0.078 μA cm⁻² h⁻¹ recorded for abiotic media. The IE of A. annua against microbiological corrosion of 304 SS in seawater was 79% which was in agreement with data deduced from EIS results.

3.3. Surface Characterization

3.3.1. Optical Profilometry

Figure 5 presents both 3D and 2D surface profiles of 304 SS electrodes following a 21-day incubation in abiotic, biotic, and inhibited environments.

The presence of bacteria on treated 304 SS electrodes resulted in noticeable surface mutilation, manifested as pitting corrosion in the linear (2D) profiles (Figure 5b). The depth of pits on the presented 2D profile reaches up to 3.75 μm. Such damage was not recorded on the electrodes exposed to abiotic (Figure 5a) nor inhibited medium (Figure 5c). Exposure to the biotic medium resulted in a surface layer with an average roughness value of ~1.555 μm. In contrast, after incubation of 304 SS in an abiotic medium, only a few small pits were observed and a surface film with an average roughness of ~0.096 μm was formed (Figure 5a). The presence of A. annua in the biotic solution led to minimized surface defects and the formation of a smoother and more consistent film, with a roughness average close to ~0.092 μm.

3.3.2. SEM-EDS Analysis

The morphology of 304 SS surfaces following exposure to abiotic, biotic, and inhibited conditions was examined using scanning electron microscopy (SEM), as shown in Figure 6.

Localized surface irregularities observed in the SEM image of 304 SS surface after 14-day incubation in abiotic medium (Figure 6a), suggested the onset of pitting corrosion. Small dark spots and uneven depressions are visible on the surface, indicating early pit nucleation. Although the pits are not fully developed at this stage, these morphological features demonstrated that the material is not immune to chloride attack. During the 14-day incubation of 304 SS in a biotic medium, P. aeruginosa formed biofilm on the steel surface (Figure 6b). Biofilm formation induced microgalvanic coupling between the cathodic and anodic areas on the 304 SS surface, where the anodic partial reaction dominated the electrochemical processes. This led to accelerated and localized metal degradation, as shown in Figure 6b. The surface of the 304 SS was not smooth, where the dark areas visible under the biofilm indicated localized dissolution of the material.

Conversely, adding A. annua to the biotic seawater mitigated the impact of bacteria and aggressive chloride ions on the formation and growth of the protective film (Figure 6c). The damaged area is significantly reduced compared to the surfaces of the 304 SS incubated in biotic (Figure 6b) and abiotic media (Figure 6a). In the case of an inhibited medium, the dissolution of the material started on the damages caused by the mechanical preparation of the surface. In addition, only a few bacterial cells were recorded on the 304 SS after incubation in the inhibited medium, as opposed to the advanced colony developed on the 304 SS surface in the biotic medium. The elemental analysis of corrosion-related deposits on 304 SS, formed during exposure to biotic and inhibited media, was performed using SEM-EDS and is summarized in

Table 5. Energy-dispersive X-ray spectroscopy (EDS) analysis of elemental composition of the 304 SS surface after 14 days of incubation in abiotic, biotic, and inhibited media, corresponding to SEM images shown in Figure 6.

Element	Biotic Medium		Inhibited Medium
	Atomic (%)	Mass (%)	Atomic (%)	Mass (%)
Fe	46.84	63.60	52.54	66.00
O	23.17	9.01	19.55	7.04
C	5.46	1.59	2.16	0.58
Cr	11.77	14.88	13.12	15.35
Ni	4.78	6.82	5.56	7.34
N	5.97	2.03	4.77	1.50
Mn	0.99	1.33	1.16	1.43
Si	0.55	0.37	0.65	0.41
P	0.32	0.24	0.26	0.18
S	0.15	0.12	0.23	0.16

.

A higher quantity of alloying elements (Cr, Ni and Mn) was detected on the 304 SS surface incubated in the inhibited medium compared to the biotic medium, which indicates that in the presence of A. annua the self-passivating Cr₂O₃ layer remained protected from the aggressive biotic medium. With the addition of A. annua, the ratio of Fe/O elements on the 304 SS surface also increased (Table 5), which could be related to a higher quantity of Fe₃O₄ in the surface film [11]. The above is also noticeable in a decrease in the surface roughness of 304 SS (Table 3) incubated in the inhibited seawater (Figure 5c) compared to the biotic medium (Figure 5b).

3.3.3. ATR-FTIR Analysis

FTIR spectra of the 304 SS surface recorded after 14 days of incubation of the working electrodes in biotic and inhibited media are shown in Figure 7.

The spectrum of 304 SS incubated in a biotic medium showed peaks corresponding to the vibrations of Fe–O and Fe–O–H bonds (700 to 400 cm⁻¹), associated with Fe(III) hydroxides and oxyhydroxides [17,18]. This is further supported by the appearance of bands corresponding to the –OH group vibrations of water molecules (3300 cm⁻¹), indicating the presence of Fe(III) oxyhydroxides on the surface of 304 SS after incubation in the biotic medium [19]. Peaks detected at wave numbers around 1640 cm⁻¹ and 1530 cm⁻¹ in the FTIR analysis of inhibited samples revealed characteristic bands corresponding to the asymmetric COO– stretching and symmetric COO– stretching [20]. The middle peak around the wave number 1090 cm⁻¹ was attributed to C–O stretching vibrations [21]. The presence of these vibration bands on the 304 SS surface incubated in the inhibited medium suggested the adsorption of chlorogenic acid molecules onto the metal surface. As detailed in our earlier publication [9], HPLC analysis of A. annua aqueous extract showed that the most abundant polyphenol in the extract was chlorogenic acid. At the pH of inhibited media (6.82 ± 0.12) after 14-day incubation, chlorogenic acid was ionized (pKa 3.42 [22]) as confirmed by the appearance of asymmetric and symmetric COO– stretching (Figure 7). The ionized form of chlorogenic acid has a higher charge density, which might enhance interactions with bacterial cell walls, particularly if the bacterial surface is oppositely charged [23], as is the case with Gram-negative P. aeruginosa. These interactions can disrupt cell membrane and lead to cell death [23]. Moreover, due to the increase in electron density of chlorogenic acid, stronger electrostatic interactions are enabled with the charged metal surface, resulting in a formation of an adsorption layer that served as a protective interface preventing interaction between the 304 SS surface and marine corrosive agents.

3.3.4. ICP-OES Analysis

The concentrations of Fe and Cr released after 1 h immersion tests of treated 304 SS electrodes determined by ICP-OES are shown in

Table 6. ICP-OES analysis of Fe and Cr concentrations and calculated corrosion rates of 304 SS after 21 days of incubation in biotic and inhibited media.

Media	Concentration (μg L−1 cm−2)		rcorr	IE
	Fe	Cr	μg cm−2 h−1	%
Biotic	10.21 ± 0.63	3.72 ± 0.25	0.102	–
Inhibited	2.62 ± 0.12	3.71 ± 0.31	0.026	74.5

. Corrosion rates r_corr of 304 SS electrodes after 21-day incubation in biotic and inhibited media were calculated using the following equation:

$$r_{\mathrm{corr}}={\displaystyle \frac{W}{At}},$$

(4)

where W is the mass of Fe in µg, A is the surface of the electrode exposed to the solvent (4 cm²), and t is the duration of the immersion test (1 h) [9,16].

The inhibition efficiency (IE) of A. annua against MIC of 304 SS caused by P. aeruginosa was also calculated independently using Equation (5):

$$IE= {\displaystyle \frac{r_{\mathrm{corr}} - r_{\mathrm{corr}}^{*}}{r_{\mathrm{corr}}}}\times 100.$$

(5)

Here r_corr and $r_{\mathrm{corr}}^{*}$ are corrosion rates of 304 SS for biotic and inhibited media, respectively [16]. The results of measurements and calculations are presented in Table 6.

A brief immersion test (1 h) of pretreated 304 SS electrodes revealed that Fe dissolution in the inhibited medium was approximately four times lower than in the biotic medium. In contrast, the levels of Cr, a constituent of the steel alloy, remained similar in both environments (Table 6). ICP-OES analysis suggests that the surface layer developed in the presence of P. aeruginosa and A. annua was more stable under tested conditions compared to that formed in the absence of the plant extract. As a more compact surface layer was developed, the corrosion rate of 304 SS in the presence of A. annua dropped from 0.102 μg cm⁻² h⁻¹ to 0.026 μg cm⁻² h⁻¹ (Table 6). The inhibition efficiency (IE) estimated from ICP-OES reached 75%, aligning well with the values obtained from electrochemical measurements (Table 3 and Table 4).

3.4. Protection Action of A. annua Against MIC

The effect of A. annua aqueous extract on the formation of an oxide film on the 304 SS in simulated seawater in the presence of P. aeruginosa is illustrated in Scheme 1.

In the presence of P. aeruginosa (Scheme 1a), the self-passivating oxide layer on 304 SS was destabilized by chloride ions and biofilm formation, resulting in localized pitting corrosion (Figure 5b and Figure 6b). Porous Fe(II)/Fe(III) hydroxides and oxyhydroxides were detected on the 304 SS surface after incubation in biotic media (Figure 7, Table 3 and Table 5) [11,24,25]. These n-type semiconductor oxides (e.g., Fe₂O₃, FeOOH) facilitate electron conduction and support extracellular electron transfer (EET), thus promoting microbial corrosion [26,27,28,29]. The pH decrease observed in the biotic medium (Table 1) likely resulted from planktonic bacterial metabolism, enhancing Fe(III) solubility and enabling Fe(II)/Fe(III) redox cycling, in which iron undergoes continuous valence changes that support microbial electron transfer and contribute to corrosion of SS [30]. Shifts in pH also influence the redox potential of secreted mediators and the surface charge of the biofilm, which may reduce bacterial adhesion and compromise biofilm cohesion and structural integrity [31,32]. This is supported by impedance data, where time-dependent variation in the film resistance R₁ (from 9.65 to 20.49 kΩ cm², Table 2 and Table 3) and increasing charge transfer resistance R₂ (from 20.67 to 53.30 kΩ cm², Table 2 and Table 3) indicate biofilm restructuring and gradual formation of a more resistive surface layer.

In contrast, addition of A. annua extract (Scheme 1b) preserved the self-passivating Cr₂O₃-based film (Figure 6c, Table 5 and Table 6) and altered the corrosion product to Fe₃O₄ (Figure 7), a mixed-valence oxide associated with reduced corrosion severity. The identification of Fe₃O₄ as the dominant corrosion product in the inhibited system is supported not only by ATR-FTIR data (Figure 7), but also by complementary analyses, including Fe/O surface ratios from EDS (Table 5), corrosion rate data (Table 4 and Table 6), and film resistance values obtained from EIS measurements (Table 2 and Table 3). This convergence of results indicates the formation of a more compact, protective Fe₃O₄ layer compared to the porous Fe₂O₃-based films observed in the biotic medium. Although Fe₃O₄ can act as a redox mediator in microbial systems [33], its compact morphology likely contributed to surface stability and limited bacterial activity in the inhibited medium. ATR-FTIR analysis confirmed the presence of phenolic functional groups on the 304 SS surface after incubation in inhibited media (Figure 7), indicating effective adsorption of extract components. Additionally, EDS analysis revealed an increased surface carbon content, supporting the conclusion that extract-derived organic molecules were successfully adsorbed (Table 5). This reduced active sites for bacterial adhesion and metal dissolution, while also impairing biofilm development.

These findings emphasize the dual function of A. annua extract as both a surface-active inhibitor and antimicrobial agent. While the results are in agreement with prior literature and supported by surface and electrochemical analyses, additional studies are needed to quantify EET kinetics and characterize biofilm structure in the examined system more directly. Compared to conventional synthetic inhibitors, A. annua extracts are environmentally benign and biodegradable. However, potential ecological and economic impacts at large-scale applications, such as bioaccumulation, nutrient release, durability and actual production costs, require further assessment. One potential limitation is the variability in the composition of A. annua extracts due to differences in cultivation conditions, harvesting time, and extraction protocols. These variations may affect the reproducibility of the inhibition efficiency and require further standardization.

4. Conclusions

Electrochemical results showed that P. aeruginosa caused acceleration of corrosion rate of 304 SS in simulated seawater which was demonstrated by a decrease of R_p and increase of j_corr. Compared to negligible pitting in the abiotic medium, the pits formed in biotic media reached up to 3.75 μm. The synergistic effect of chlorides and bacterial activity led to the formation of porous FeOOH, which supported extracellular electron transfer and promoted localized corrosion. In contrast, in the presence of A. annua, self-passivating film stayed protected and a more compact oxide layer was formed, which decreased the diffusion rate of Fe at the electrode/electrolyte interface. The latter was associated with the adsorption of ionized chlorogenic acid on the 304 SS surface, which reduced the available sites for metal dissolution and adhesion of bacteria. The inhibition efficiency of A. annua against microbiological corrosion of 304 SS caused by P. aeruginosa was 74.4 ± 4.4%. The dual nature of A. annua extract, acting both as a surface-active inhibitor and as an antimicrobial agent, highlights its potential as a sustainable corrosion inhibitor in marine environments.