Bacterial adhesion and corrosion behavior of different pure metals induced by sulfate reducing bacteria

Abstract The corrosion behaviors of four pure metals (Fe, Ni, Mo and Cr) in the presence of sulfate reducing bacteria (SRB) were investigated in enriched artificial seawater (EASW) after 14-day incubation. Metal Fe and metal Ni experienced weight losses of 1.96 mg cm−2 and 1.26 mg cm−2, respectively. In contrast, metal Mo and metal Cr exhibited minimal weight losses, with values of only 0.05 mg cm−2 and 0.03 mg cm−2, respectively. In comparison to Mo (2.2 × 106 cells cm−2) or Cr (1.4 × 106 cells cm−2) surface, the sessile cell counts on Fe (4.0 × 107 cells cm−2) or Ni (3.1 × 107 cells cm−2) surface was higher.


Introduction
Microbiologically influenced corrosion (MIC) occurs when the metabolic activity of microorganisms accelerates the corrosion of metals either directly or indirectly (Thompson et al. 2022).Sulfate reducing bacteria (SRB) can speed up metal corrosion, primarily in anaerobic environments such as marine and oilfield conditions.The severity and consequences of MIC are exemplified by incidents such as the rupture of the Alaskan pipeline caused by SRB, resulting in the spillage of 750,000 liters of crude oil (Jacobson 2007).MIC not only leads to industrial accidents but also causes significant environmental damage and economic losses, amounting to billions of dollars annually.MIC can account for 20% of metallic material failures; in the marine, oil and gas industries, microorganisms account for almost 40% of material damage (Chen et al. 2022).
Stainless steel, characterized by its higher content of corrosion-resistant alloying elements such as nitrogen (N), nickel (Ni), molybdenum (Mo), and chromium (Cr), exhibits superior corrosion resistance compared to carbon steel.By facilitating the formation of a compact passive film on the surface of stainless steel, corrosion-resistant alloying elements can reduce the susceptibility of the metal matrix to corrosion.The primary constituents of the passive film are hydroxides and the oxide of Cr.In the case of Nicontaining stainless steel, a Ni metal layer can be formed between the metal matrix and the passive film (Olsson and Landolt 2003).The addition of Mo can promote re-passivation of the passive film and significantly increase the resistance of stainless steel to pitting (Montemor et al. 1999).N can also contribute to the improved pitting resistance of stainless steel by facilitating the generation of NH 4 þ , which mitigates the damage of H þ to the passive film (Jargelius-Pettersson 1999).As a result, Ni, Mo and Cr are often used as components in the composition of highentropy alloys and stainless steel.Both the number of sessile cells and the compactness of the passive layer can affect the MIC of stainless steel.It is believed that a denser passive film and a lower sessile cell count contribute to the higher resistance of stainless steels against MIC (Wan et al. 2023a(Wan et al. , 2023b)).
The composition of alloying elements in stainless steel affects not only the compactness of the passive film but also the sessile cell counts (Wan et al. 2023a).It is widely acknowledged that a higher content of the Ni alloying element in stainless steel promotes bacterial adhesion (Telegdi 2018;Tran et al. 2019).Conversely, higher contents of Mo and Cr alloying elements can inhibit bacterial adhesion (Telegdi 2018;Xu et al. 2023).Ni 2þ is known to be toxic to both bacteria and humans (Harasim et al. 2015); therefore, further research is necessary to comprehend the mechanism by which metal elements impact bacterial adhesion.However, the complexity of the alloying components in stainless steel presents challenges in investigating the individual influence of each alloying element on the count of sessile cells.Consequently, studying the corrosion behavior of pure metals such as Fe, Ni, Mo and Cr in the presence of SRB can provide a more straightforward understanding of how metal elements affect MIC.
The extracellular electron transfer mechanism (EET-MIC) and the metabolite mechanism (M-MIC) are the two primary mechanisms of corrosion of metals by SRB.EET-MIC is responsible for the corrosion of carbon steel (Fe) by SRB, which oxidizes Fe to produce energy for its own metabolism (Xu and Gu 2014;Anguita et al. 2022).Consequently, the weight loss of carbon steel due to corrosion by SRB increases with the number of sessile cells.M-MIC refers to the process in which metabolites of SRB, such as organic acids and H 2 S, accelerate the corrosion of metals.The corrosion of Cu by SRB is primarily driven by M-MIC, and the HS -produced by SRB will accelerate the corrosion of Cu (Dou et al. 2018(Dou et al. , 2020)).
This study focuses on investigating the corrosion mechanisms and sessile cell counts of four metals (Fe, Ni, Mo and Cr).It is apparent that these pure metals may exhibit distinct corrosion mechanisms in the presence of SRB.By comparing the sessile cell counts and corrosion mechanisms of Fe, Ni, Mo and Cr, this study aims to acquire a comprehensive understanding of their corrosion behavior in the presence of SRB.

Bacteria and materials
Table 1 presents the primary compositions of various metals used.For the electrochemical testing, cylindrical coupons measuring 1.0 cm in diameter and 1.0 cm in height were employed.Prior to testing, each coupon was equipped with a copper wire at the back, covered in epoxy, and left with its top portion (0.785 cm 2 ) exposed.Square coupons, measuring 0.3 cm in height and 1.0 cm in length and breadth, were employed for the remaining tests.All coupons were polished from 180# to 1,200# finish using silicon carbide papers (Wan et al. 2023a).After being washed with acetone and ethanol, the coupons and electrodes were sterilized using a UV lamp (with a wavelength of 253.7 nm) for 30 min prior to incubation.The bacteria utilized in this study were isolated from the South China Sea (Zhanjiang city, Guangdong province, China) and identified as Desulfovibrio sp.(Wan et al. 2023a).The incubation medium employed was enriched artificial seawater (EASW), as previously reported (Wan et al. 2023a).Prior to inoculation, the EASW was adjusted to a neutral pH (7.0) and autoclaved (121 � C) for 20 min to ensure sterilization.To remove oxygen from the bottle, the medium was sparged with N 2 (99.999%) for 60 min.Three parallel coupons were placed in an anaerobic vial.Subsequently, a three-day-old inoculum was introduced to the incubation medium with 100 mg l −1 Lcysteine.Each anaerobic experiment was conducted in a N 2 gas-filled anaerobic chamber.Each anaerobic vial was placed in a 37 � C incubator for 14 days.The only difference in the experimental setup under abiotic conditions was the absence of inoculated bacteria in the culture medium.

Electrochemical tests
For the electrochemical experiments, 250 ml of growth medium was introduced into 330 ml glass cells.In the electrochemical cells, the saturated calomel electrode (SCE) and platinum sheet served as the reference electrode (RE) and counter electrode (CE), respectively, and different pure metals served as the working electrodes (WE).An electrochemical workstation (Model CS350, Corrtest, Wuhan, China) was utilized to monitor the open circuit potential (OCP), linear polarization resistance (LPR), and electrochemical impedance spectroscopy (EIS) on a daily basis.Upon achieving a stable OCP, LPR measurements were performed between -10 mV and 10 mV (vs.OCP) at a positive scanning rate of 0.167 mV s −1 .Subsequently, EIS was conducted over a frequency range of 10 4 -10 −2 Hz, with an amplitude of 10 mV.After 14-day SRB incubation, potentiodynamic polarization (PDP) experiments were carried out between −200 mV and 200 mV (vs.OCP) at a positive scanning rate of 0.5 mV s −1 .Additional cyclic PDP was employed to evaluate the pitting characteristics of the coupons.The EIS results were analyzed using Zview (Version 3.0a).The LPR and PDP results were analyzed using Cview (Version 2.6) software (Scribner, North Carolina, USA).

Surface analysis
Prior to the examination of the biofilm using an environmental scanning electron microscope (ESEM) (Quanta 200, FEI, Eindhoven, Netherlands), the coupons after 14-day SRB incubation were subjected to a series of preparation steps.Firstly, the coupons were washed with phosphate buffered saline (PBS) to remove the planktonic cells.Subsequently, the coupons were immersed in a 2.5% (v v -1 ) glutaraldehyde solution for 2 h at 4 � C to fix the SRB cells.
Then, the coupons were dehydrated using a graded series of ethanol concentrations, starting from 25% and progressing to 50%, 75%, 90% and finally 100% (v v -1 ) anhydrous ethanol, with each step lasting for 10 min.After dehydration, the coupon surfaces were air-dried, coated with a thin layer of gold via sputtering, and observed by ESEM.The elemental compositions were analyzed by the energy dispersive spectrum (EDS) accessory.After 14-day SRB incubation, a soft brush was employed to remove the biofilm in the N 2 gas-filled anaerobic chamber.Subsequently, X-ray photoelectron spectroscopy (XPS) (AXIS-ULTRA DLD-600W, Kratos, UK) was utilized to analyze the compositions of the passive films formed on different pure metals.The XPS data were analyzed with CASAXPS software (Version 2.3.13,Casa Software Ltd, Abbotskerswell, UK).

Cell counts and biofilm analysis
The planktonic and sessile cell counts were determined using hemocytometers under an optical microscope at a magnification of 400�.The motility of the SRB cells facilitated their identification during the counting process.After a 14-day incubation, coupon surfaces were washed with PBS to remove planktonic cells, then sessile cells were scraped off from the coupons and suspended in a PBS solution for further analysis.
After the 14-day incubation, the metal specimens were gently washed with 1 M sterile PBS to remove loosely attached bacteria and fixed with 2.5% (v v -1 ) glutaraldehyde for 1 h.Then the samples were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, 50 ml stock solution in 1 ml PBS) and kept in the dark for 15 min, and gently washed with PBS to remove the residual DAPI solution.Subsequently, a confocal laser scanning microscope (CLSM) (Olympus, FV1200, Tokyo, Japan) was employed to observe the sessile cells.

Headspace hydrogen concentration and pH
The pH of the media and headspace hydrogen concentrations in the vials were monitored after the 14day incubation.pH measurements were conducted using a pH tester (Shanghai Yidian Group Co., Shanghai, China) with an accuracy of 0.01.Hydrogen concentration analysis was performed using a H 2 gas detector (BH-90, Bosean Electronic, Henan, China) capable of detecting concentrations ranging from 1 to 1,000 ppm, with an accuracy of 1 ppm.To collect the headspace gas, a 20 ml syringe was employed to extract the gas in the headspace (25 ml) of the 125 ml anaerobic vials until a negative pressure was achieved in the vials.The extracted gas was then introduced into a 200-ml gas collection bag for dilution.Subsequently, a 10-ml sample was withdrawn from the gas collection bag for testing purposes.

Weight loss and corrosion morphology
After the 14-day SRB incubation, fresh Clarke's solution was used to remove the surface layers (corrosion products and biofilm) of the coupons.Subsequently, the coupons were rinsed with ethyl alcohol and dried using N 2 .The masses of three replicate coupons were measured both before and after incubation to calculate the weight loss (the readability of the balance was 0.01 mg).A 3D stereoscopic microscope (VHX-1000E, Keyence, Tokyo, Japan) was employed to detect the corrosion pits.

Statistical analysis
All experiments were done in three replicates, and the results were expressed as the mean ± standard deviation (± SD).A t-test and one-way analysis of variance (ANOVA) were carried out using GraphPad Prism software (version 9.0.0 (121), GraphPad Software Inc., SanDiego, USA) to determine the variations.Differences were considered statistically significant at a p-value < 0.01.

Weight loss and sessile cell count of different metals
Figure 1 illustrates the weight losses of different metals after the 14-day incubation in abiotic and SRB medium.The weight losses of the metals Fe and Ni were greater in the SRB medium as compared to those in the abiotic medium (p < 0.0001), suggesting that SRB significantly accelerated the corrosion of Fe and Ni.The weight loss of Fe or Ni was much greater than that of Mo or Cr after the 14-day SRB incubation (p < 0.0001).The weight loss sequence was in line with both the dissolution rate of metal ions from pure metals and the sulfidation rate of different metals, as reported in previous studies (Herting et al. 2005;2008;Young 2008).The corrosion of Cr in the presence of SRB was influenced by the compactness of the passive film.The passive film on Cr could be readily formed with a trace amount of O 2 .Therefore, similar to stainless steel, the compact passive film could grant Cr higher corrosion resistance.
Figure 2a presents the pH values of the SRB medium containing different metal specimens after the 14-day incubation.The pH values of all five media were close to neutral and approximate (p > 0.05), indicating that acid corrosion contributed to only a small proportion of the MIC on the pure metals (Wang et al. 2024).Figure 2b illustrates the variation in H 2 concentration in the headspace of bottles containing different metals.The H 2 concentrations in five bottles all initially increased and then decreased over time.During the first three days, the nutrient availability was sufficient, resulting in the production of H 2 through the metabolism of SRB.The subsequent decrease in H 2 concentration can be attributed to the gradual depletion of nutrients from the third to the 14th day.As the nutrients became consumed, SRB would utilize H 2 as an energy source, leading to a continuous decrease in H 2 concentration (Telegdi et al. 2020;Wang et al. 2020).The headspace H 2 concentrations in the bottles containing different metal specimens were found to be similar to that of the control one, indicating that none of the four metals experienced significant H þ or H 2 S-induced corrosion.
Figure 3a presents the planktonic cell counts in the culture medium without and with different metals after the 14-day SRB incubation.The planktonic cell counts of the medium containing different metals were approximate (p > 0.05).Figure 3b displays the sessile cell counts on different metals after the 14-day SRB incubation.The sessile cell counts on Fe or Ni surface were approximately one order of magnitude higher than those of Mo or Cr (p < 0.0001), which was in accordance with the CLSM images in Figure 3.The densities of green spots on the surface of Fe and  Ni was significantly higher than those of Mo and Cr, as observed in the CLSM images.
Figure 4a shows the minimum inhibitory concentrations of different metal ions on SRB.It was observed that Fe 2þ was beneficial to the growth of SRB, and the growth of SRB remained uninhibited even with the addition of 1,000 mg l −1 Fe 2þ .Consequently, the minimum inhibitory concentrations of the other three metal ions (Ni 2þ , Cr 3þ and MoO 4 2-) were compared (Jia et al. 2019).The sequence of the minimum inhibitory concentrations for the metal ions was Ni 2þ < Cr 3þ < MoO 4 2-, indicating that Ni 2þ , Cr 3þ and MoO 4 2-exhibited toxicity towards SRB.To verify the precipitation rate of different metal ions by S 2-, 1 mg l −1 of different metal ions was added separately to a 2 mM aqueous Na 2 S solution (Chen et al. 2017), as shown in Figure 4b.Notably, distinct black precipitates appeared upon the  addition of 1 mg l −1 Ni 2þ , indicating the precipitation of nickel sulfide due to its high pK sp value (25.7 at 25 � C) (Blais et al. 2008).Consequently, 1 mg l −1 Ni 2þ could be rapidly precipitated by SRB-produced S 2-.However, precipitation of 1 mg l −1 of MoO 4 2-and Cr 3þ by 2 mM S 2-proved to be more challenging.

Electrochemical measurements
Figure 5 illustrates the variations in OCP and LPR of different metals during the 14-day SRB incubation.The OCP of Ni experienced a negative shift at the 10th hour, while the OCP of Mo and Cr exhibited a negative shift at the 24th hour (Figure 5a1).The compactness of passive film played a pivotal role in the variations of OCP.In the abiotic medium, the OCP of Ni displayed a negative shift, and the R p of Ni decreased on the fourth day (Supplementary material, Figure S1), indicating rupture of the passive film.The OCP of Mo and Cr were significantly higher than those of Fe and Ni in the SRB medium (Figure 5a2).The elevated OCP of Cr was ascribed to the presence of a compact and uniform Cr 2 O 3 /Cr(OH) 3 passive film, which readily formed on the surface of Cr (Shu et al. 2000;Tsuchiya et al. 2002).Metal Cr exhibited the highest R p under both abiotic (Supplementary material, Figure S1b) and SRB (Figure 5b) conditions, which was consistent with the lowest weight loss of Cr (Figure 1).In both abiotic (Supplementary material, Figure S1b) and biotic media (Figure 5b), metal Fe displayed the lowest R p, which could be attributed to the highest sessile cell count and the least dense passive film.
Figure 6 presents the Nyquist plots and Bode plots of different metals during the 14-day SRB incubation, and Table S3 (Supplementary material) displays the corresponding fitted data.During the 14-day incubation, metal Fe exhibited the smallest semi-circle diameters, which was consistent with the highest weight loss of Fe in Figure 1.The maximum phase angle shifted to lower frequencies for all four metals on day 2, indicating the adhesion of biofilm to the surfaces of metals.In the SRB medium, the sums of the film resistance (R f ) and charge transfer resistance (R ct ) of Ni were highest among the four metals (Supplementary material, Figure S4), which might be ascribed to the hindering effect of the nickel sulfide layer formed on the surface of Ni (Cheng et al. 2000).
Figure 7 depicts the cyclic PDP curves of different metals after the 14-day abiotic and SRB incubation, and the corresponding Tafel fitting data are presented in Table 2.The corrosion current density (i corr ) is a kinetic parameter that characterizes corrosion, and a lower i corr represents a higher resistance to general corrosion (Wang et al. 2022a;Yin et al. 2021).The potential at which a sharp increase in current density occurs during an anodic polarization scan is the pitting potential (E pit ).A higher E pit indicates higher resistance to localized corrosion.After both abiotic and SRB incubation, metal Cr exhibited the lowest i corr and the highest E pit , indicating superior resistance to both general and localized corrosion.Conversely, metal Fe exhibited the highest i corr and the lowest E pit , indicating the lowest resistance to general and localized corrosion.The E pit of metal Ni under abiotic conditions was −0.4 V vs SCE.However, the E pit in the presence of SRB increased to 0.4 V vs SCE and distinct passivated zones were observed.The higher E pit of Ni in the SRB medium might be attributed to the presence of the corrosion products Ni x S y (NiS, Ni 3 S 2 , and Ni 6 S 5 ), which could promote passivation and provide protection to the metal substrate (Young 2008;Magana-Zavala et al. 2009).

Surface film characterization
The ESEM images of surface films on different metals after the two-day SRB incubation are presented in     significant alterations and the bacteria appeared ruptured and elongated.This observation suggests that metals Mo and Cr may possess toxicity towards SRB, leading to detrimental effects on the morphology of SRB. Figure 9 presents ESEM images, Fe element mapping, and EDS analysis of the corrosion products and biofilm formed on different metals after the 14-day SRB incubation.A significant accumulation of corrosion products and sessile cells was observed on the surfaces of metal Fe (Figure 9a) and metal Ni (Figure 9b).The EDS results (Table 3) indicated that the S contents on the surface of metal Fe and metal Ni were relatively high, reaching 17.75 and 24.88 wt%, respectively.Based on the elemental ratios in the EDS analysis, the corrosion products on the surface of metal Ni were primarily composed of Ni x S y .In contrast, only a small amount of solid precipitation adhered to the surfaces of metals Mo (Figure 9c) and Cr (Figure 9d), with the solid precipitations predominantly identified as Fe x S y based on the Fe element  mapping.The S element contents on the surface of metal Mo and metal Cr were lower (2.09 wt% for Mo and 2.99 wt% for Cr), as indicated by the EDS analysis (Table 3).After the 14-day abiotic incubation, the corrosion of metals Mo and Cr was negligible (Supplementary material, Figure S5).The cross-section images of different metals after the 14-day SRB incubation are displayed in Figure 10.The surface film on metal Fe exhibited the greatest thickness, measuring 45-55 lm, possibly as a result of higher adhesion of biofilm and corrosion products on the surface.In contrast, the surface film on metal Ni had a much thinner thickness, measuring only 2.8-3.3 lm, which might be due to the presence of a denser Ni x S y layer on the surface.The thickness of the surface film on metal Mo and metal Cr was measured to be 0.8-1.2lm and 0.4-0.6 lm, respectively.Both metal Mo and metal Cr exhibited relatively thin surface films, which could be attributed to the lower attachment of biofilm and corrosion products to the surfaces.
The corresponding XPS metal spectra of various metals before and after the 14-day SRB incubation are displayed in Figure 11.It is important to note that both the passive film and the depth of XPS detection are within the nanometer range.Consequently, the primary changes observed in the recorded XPS data following the biofilm removal process are attributed to the variations in the compositions of the passive film.The Fe 2p 3/2 spectra of metal Fe were composed of three peaks: Fe 3þ , Fe 2þ and Fe 0 (Figure 11a).The influence of SRB on the constituents of the passive film on metal Fe was minimal, as indicated by the slight variations in the Fe 2p 3/2 spectra before and after the incubation.The Ni 2p 3/2 spectra of the metal Ni are displayed in Figure 11b.The chemicals in the passive film on the metal Ni surface mostly transitioned from oxides and hydroxides to sulfides after 14-day SRB incubation.NiO constitutes the main component of the passive film on metal Ni, and Ni x S y can also serve as a protective layer (Magana-Zavala et al. 2009).The Mo 3d spectra of metal Mo before and after 14-day SRB incubation, shown in Figure 11c, demonstrated a decrease in the content of Mo (Ⅵ) (MoO 4 2-) after the incubation.The decrease might be due to the ability of SRB to reduce MoO 4 2-.Additionally, MoS 2 appeared on the surface of metal Mo after the 14-day SRB incubation.The Cr 2p 3/2 spectra of metal Cr before and after the 14-day SRB incubation are displayed in Figure 11d.After the 14-day SRB incubation, the contents of Cr 0 and Cr(OH) 3 increased, while the contents of Cr 2 O 3 decreased.This observation suggests that SRB may contribute to the destruction of Cr 2 O 3 .After the 14-day SRB incubation, the O 2-content in the passive films of all four metals decreased, as shown by the O 1s spectra (Supplementary material, Figure S7), which indicated that the presence of SRB could reduce the fraction of oxides.

Pit morphology
Figure 12 presents the 3D morphologies and pit depth profiles of different metals with corrosion products and biofilm removed after 14-day SRB incubation.As shown in Figure 12, the sequence of pit depths for the four metals was as follows: Fe (5.2 mm) > Ni (3.2 mm) > Mo (1.4 mm) � Cr (1.5 mm).The micrometer-level pit depths indicated that SRB-induced pitting corrosion of the four metals was in a stage of metastable pitting.This observation might be attributed to the relatively fewer defects and inclusions on the metal surfaces, leading to the dominance of general corrosion.

Corrosion mechanism of SRB to different metals
Thermodynamic analysis serves as a prerequisite for determining whether the corrosion of metals by SRB The anodic reactions and corresponding standard electrode potentials for the four metals are as follows (Stansbury and Buchanan 2000;Wang etal. 2023): where E o 0 in bioelectrochemistry is defined as the electrode potential (relative to standard hydrogen electrode, SHE) at 25 � C, a pH of 7, and 1 M solute (or 1 bar partial pressure of gas).Based on the anodic potentials of the four metals and the cathodic potential for sulfate reduction, metals such as Fe, Ni, and Cr were 'energetic' metals, as the electrons generated from the oxidation of these metals could be utilized in sulfate reduction by SRB (Krantz et al. 2019).

Attachment of SRB on different metals
The chemical composition of steels could influence the initial bacterial attachment and subsequent MIC (Javed et al. 2016).Different metal elements could potentially exert diverse effects on bacterial attachment (Xia et al. 2015).The production of Fe-based enzymes was positively influenced by the concentration of Fe 2þ , and a higher Fe 2þ concentration could decrease the toxicity of S 2-to SRB.Therefore, the growth of SRB occurred at a higher extent on Fe 2þ (Reis et al. 1992;Jia et al. 2019).Additionally, the corrosion of metal Fe by SRB was in accordance with the EET-MIC mechanism, allowing sessile SRB to drive energy from the oxidation of metal Fe (Xu et al. 2016).
Metal Ni displayed sessile cell counts comparable to that of metal Fe (Figure 3b).It is reported that SRB has a higher affinity for metal Ni than for 304 stainless steel and polymethylmethacrylate (PMMA) because the Ni may encourage the adhesion of bacteria (Dec et al. 2017;Telegdi 2018;Tran et al. 2019).Depending on the concentration, Ni 2þ may have different effects on bacterial growth.For example, a low concentration (0.5 mg l −1 ) of Ni 2þ could promote the growth of SRB; conversely, high concentrations (� 5 mg l −1 ) of Ni 2þ might be toxic to SRB and cause a decrease in the bacterial population (Lopes et al. 2006).The promotion of SRB growth at low Ni 2þ concentrations could be attributed to the facilitation of nickel-based enzyme production (Mulrooney and Hausinger 2003).However, at high concentrations, Ni 2þ could displace other metal ions (such as Fe 2þ ) in the complexation of enzyme and nucleic acid, resulting in the inhibition of enzyme and protein activity and suppression of bacterial growth (Harasim et al. 2015).The quick precipitation of Ni 2þ by SRBproduced S 2-, which resulted in a low concentration of Ni 2þ near the surface of metal Ni (< 1 mg l −1 ), might be the cause of the high sessile cell counts on the Ni surface (Kikot et al. 2010;Pu et al. 2023a, Pu et al. 2023b).Additionally, the corrosion of metal Ni by SRB was consistent with the EET-MIC mechanism, further promoting the attachment of SRB (Pu et al. 2023a).
The sessile cell counts on metal Mo and metal Cr were one order of magnitude lower than that on metal Fe (Figure 3b), which was in accordance with the observed ruptured and elongated morphologies of SRB on Mo and Cr surfaces (Figure 8).The presence of molybdate (MoO 4 2-), which structurally resembles sulfate and possesses the capacity to inhibit the activity of adenosine 5 0 -triphosphate (ATP) sulfurylase and the reduction of sulfate by SRB, may be the cause of the decreased sessile cell counts on metal Mo (Rincon et al. 2008;Biswas et al. 2009).When the concentration of molybdate exceeded 2 mM, the generation of H 2 S by SRB was completely inhibited (Rincon et al. 2008).Therefore, the addition of Mo (1.0 wt%) in stainless steel could hinder the adhesion of SRB (Telegdi 2018).Because SRB and S 2-must work together to reduce MoO 4 2-to MoS 2 precipitates, S 2- had a decreased rate in precipitating MoO 4 2- (Biswas et al. 2009).Consequently, there might be a specific concentration of MoO 4 2-close to the surface of Mo, which would inhibit SRB attachment.Additionally, MoS 2 generated on the Mo surface also exhibited toxicity towards SRB and inhibited their adhesion (Wang et al. 2022b).Soluble metal ions produced by metal Cr include Cr(III) and Cr(Ⅵ); Cr(Ⅵ) is readily reduced to Cr(III) by S 2-produced by SRB.
Therefore, the main ion produced by the metal Cr in SRB medium is Cr(III) (Lai and McNeill 2006).Cr has the ability to inhibit the adhesion of SRB, resulting in a lower sessile cell counts on stainless steel surfaces compared to carbon steel surfaces (Telegdi 2018;Xu et al. 2023).This could be attributed to the detrimental effect of Cr 3þ on the ability of SRB to reduce sulfate ions (Fang et al. 2000;2002).Cr 2 S 3 is unstable in water, resulting in a lower precipitation rate of Cr 3þ compared to Ni 2þ by S 2-.The limited precipitation rate of Cr 3þ by S 2-allows a significant amount of Cr 3þ to remain near the surface of metal Cr (Lai and McNeill 2006), inhibiting the attachment of SRB due to the high sterilization efficiency of Cr 3þ on SRB (a minimum inhibitory concentration of 60 mg l −1 ).
Figure 13 illustrates the corrosion mechanism of Fe, Ni, Mo and Cr in the presence of SRB.Metals Fe and Ni were found to be vulnerable to MIC.The generated Fe 2þ and Ni 2þ resulting from the corrosion of Fe and Ni, respectively, were readily precipitated by S 2-.These precipitates, FeS and NiS, could attach to the surface of the metals (Fe and Ni), potentially affecting the corrosion process.The higher number of sessile cells on Fe and Ni surfaces could facilitate the formation of biofilm, and the metabolites produced by these sessile cells could further accelerate the corrosion of Fe and Ni.SRB could utilize electronic carriers such as riboflavin to promote electron transfer during the corrosion of Fe and Ni.In contrast, Mo and Cr exhibited the ability to produce substances that inhibited bacterial adhesion.Consequently, the number of sessile cells on the surfaces of Mo and Cr was low, resulting in a reduced corrosion rate.Furthermore, the presence of a compact passive film on Mo and Cr surfaces could effectively reduce corrosion in the presence of SRB.

Conclusions
Metal Fe and Ni experienced significantly higher weight loss compared to Mo and Cr, which was attributed to the less protective passive film and the higher density of sessile cells on the surfaces of Fe and Ni.The highest sessile cell counts on the Fe surface was due to the favorable conditions provided by Fe 2þ for SRB growth.The elevated sessile cell count on Ni surface was attributed to the rapid precipitation of Ni 2þ by S 2-produced by SRB.On the other hand, the low number of sessile cells on Mo and Cr surfaces was attributed to their toxicity to SRB and the limited precipitation efficiency of MoO 4 2− and Cr 3þ by S 2-.In summary, this study provided a comprehensive investigation into the MIC of different metals and provided mechanistic insights to understand the influence of metal elements on MIC.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Weight loss of different metals after 14 days of incubation in abiotic and SRB medium.Error bars indicate standard deviation of three independent coupons in the same vial.���� p < 0.0001.

Figure 2 .
Figure 2. (a) pH of different SRB medium after 14 days of incubation; (b) H2 concentration in the headspace during the 14-day SRB incubation.Error bars indicate standard deviation of three independent samples in the same vial.ns p > 0.05.

Figure 3 .
Figure 3. (a) Planktonic cell counts of the culture medium without and with different metals after the 14-day SRB incubation; (b) sessile SRB cell counts after the 14-day incubation; CLSM images of biofilms on four metals after the 14-day SRB incubation: (c) Fe, (d) Ni, (e) Mo, (f) Cr.Error bars in (a) indicate standard deviation of three replicate samples in different anaerobic vials; error bars in (b) indicate standard deviation of three independent coupons in the same vial.ns p > 0.05, ���� p < 0.0001.

Figure 4 .
Figure 4. (a) Minimum inhibitory concentration of different ions; (b) appearance of 2 mM Na 2 S aqueous solution after the addition of 1 mg l −1 of different ions.

Figure 5 .
Figure 5. (a) OCP and (b) LPR variations of different metals during 14-day SRB incubation.Error bars indicate standard deviation from three independent electrochemical glass cells.

Figure 7 .
Figure 7. Cyclic potentiodynamic polarization curves of different metals after the 14-day (a) abiotic and (b) SRB incubation.

Figure 8 .
Figure 8. SEM images of surface films on different metals (a) Fe, (b) Ni, (c) Mo, (d) Cr after the two-day SRB incubation.

Figure 9 .
Figure 9. SEM images, Fe mapping, and EDS analysis of the biofilm and corrosion products on different metals (a) Fe, (b) Ni, (c) Mo, (d) Cr after the 14-day SRB incubation.

Figure 12 .
Figure 12. 3D morphologies and representative pit depth profiles of four metals with biofilms and corrosion products removed after the 14-day SRB incubation: (a) Fe, (b) Ni, (c) Mo, (d) Cr.

Figure 13 .
Figure 13.Mechanism of corrosion by SRB to Fe, Ni, Mo and Cr.