Microbiologically influenced corrosion of 304 stainless steel by halophilic archaea Natronorubrum tibetense

Journal Pre-proof Microbiologically influenced corrosion of 304 stainless steel by halophilic archaea Natronorubrum tibetense Hongchang Qian, Lingwei Ma, Dawei Zhang, Ziyu Li, Luyao Huang, Yuntian Lou, Cuiwei Du

PII:

S1005-0302(20)30045-1

DOI:

https://doi.org/10.1016/j.jmst.2019.04.047

Reference:

JMST 1912

To appear in:

Journal of Materials Science & Technology

Received Date:

19 December 2018

Revised Date:

11 March 2019

Accepted Date:

11 April 2019

Please cite this article as: Qian H, Ma L, Zhang D, Li Z, Huang L, Lou Y, Du C, Microbiologically influenced corrosion of 304 stainless steel by halophilic archaea Natronorubrum tibetense, Journal of Materials Science and amp; Technology (2020), doi: https://doi.org/10.1016/j.jmst.2019.04.047

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Research Article Microbiologically influenced corrosion of 304 stainless steel by halophilic archaea Natronorubrum tibetense

Hongchang Qian, Lingwei Ma, Dawei Zhang*, Ziyu Li, Luyao Huang, Yuntian Lou, Cuiwei Du*

Beijing Advanced Innovation Center for Materials Genome Engineering, Institute for Advanced

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Materials and Technology, University of Science and Technology Beijing, Beijing 100083, China

*Corresponding authors.

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E-mail addresses: [email protected] (D. Zhang); [email protected] (C. Du).

Abstract

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The corrosion behavior of 304 stainless steel (SS) in the presence of aerobic halophilic archaea Natronorubrum tibetense was investigated. After 14 days of immersion, no obvious pitting pit was

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observed on the SS surface in the sterile medium. By contrast, the SS exhibited serious pitting corrosion with the largest pit depth of 5.0 μm in the inoculated medium after 14 days. The results

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of electrochemical tests showed that the barrier property of the passive film decreased faster in the inoculated medium. The X-ray photoelectron spectroscopy results indicated that the detrimental Fe2+ and Cr6+ increased in the passive film under the influence of archaea N. tibetense, which

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resulted in the accelerated deterioration of passive film and promoted the pitting corrosion. Combined with the energy starvation tests, the microbiologically influenced corrosion mechanism

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of 304 SS caused by halophilic archaea N. tibetense was discussed finally.

Keywords: Stainless steel; Archaea; Microbiological influenced corrosion

1. Introduction The adhesion of microbial cells on metal surface and subsequent biofilm formation may accelerate or inhibit the metal corrosion, which is well known as microbiologically influenced corrosion (MIC).

MIC accounts for approximately 20% of the total corrosion damage [1]. In the past few decades, MIC has attracted extensive research interests. Except for the commonly mentioned sulfatereducing bacteria, the influence of other bacteria, such as acid-producing bacteria and iron-oxidizing bacteria, on the corrosion of various metallic materials has also received attentions [2-8]. A number of MIC mechanisms have been proposed [9-12]. Up to now, almost all MIC studies focused on the field of bacteria. In recent years, some researchers began to explore the MIC caused by eukaryotic fungi in atmospheric and aqueous environments [13, 14]. As another important part of the microbial community, archaea microorganisms also have important

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impact on social life and industrial production, which have been widely applied in biogas preparation, environmental management and biological metallurgy because of its unique cell microstructure and metabolism process [15, 16]. Archaea microorganisms mainly live in extreme natural environments, such as alkaline soil, polar zone and deep sea, which can be classified into

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the following types: methanogenic archaea, thermophilic archaea, halophilic archaea, acidophilic

archaea and ammonia-oxidizing archaea [17, 18]. As early as the 1990s, the oil and gas industry has

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realized the influence of archaea on the metal corrosion [19]. Davidova et al. [20] investigated the MIC of a thermophilic archaea, Thermococcales sp. against pipeline steel, and this archaea was

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isolated from the inner wall of a hot oil pipeline. The results revealed that this thermophilic archaea could promote the dissolution of metallic iron under anaerobic condition. In addition, the corrosion

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promoting effect of methanogenic archaea isolated from marine environment or oil pipeline on carbon steel has also been evaluated [21, 22]. Although the MIC of methanogenic archaea and thermophilic archaea began to draw attention, relevant studies were very limited. As more oil and

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gas resources were found in extreme environments, such as deep-sea and desert, the exploration of energy resources will inevitably move towards these extreme environments in the near future. As

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the dominant microorganisms in these extreme environments, other types of archaea besides methanogenic and thermophilic archaea may also have an important impact on the corrosion of metallic materials. However, as far as we know, there was no research about the MIC of other types of archaea until now. In the west of China, there are vast alkali soils and salt lakes with high salinity. Thousands of kilometers of steel pipelines and railways pass through these high-salinity environments [23], and abundant halophilic archaea communities have been discovered to inhabit in these environments

[24, 25]. Hence, it is of great significance to investigate the MIC of halophilic archaea on metallic materials. In our previous study, we have clarified the influence of one halophilic archaea on the corrosion of carbon steel for the first time [26]. The results showed that the halophilic archaea Natronorubrum tibetense promoted the corrosion of carbon steel by using metal irons as energy source. Electrons could transfer from the exposed iron surface into the attached archaea biofilms through extracellular electron transfer (EET) process. However, for the metal protected by oxide layer, such as stainless steel (SS) with passive film, it remains unclear whether this halophilic archaea can also promote the corrosion process. Furthermore, no article has focused on the influence

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of any archaea on the corrosion of SS materials. The purpose of this study is to get a throughout understanding on the influence of halophilic archaea N. tibetense on the corrosion behavior of 304 SS. Firstly, the morphologies and coverage conditions of the biofilms on 304 SS surfaces were observed. Then the sizes of the pitting pits in the sterile and

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inoculated media after different immersion days were counted to compare the corrosion degrees. Electrochemical tests were also utilized to further character the effect of this halophilic archaea on

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the corrosion process of the SS. Subsequently, the compositions of the passive films in different media after 14-day immersion were analyzed by X-ray photoelectron spectroscopy (XPS) to see the

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influence of archaea N. tibetense on the passive film. Finally, combined with the energy starvation

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tests, the MIC mechanism of 304 SS caused by N. tibetense was proposed and discussed.

2. Experimental methods

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2.1 Metal material, archaea strain and culture medium The 304 SS samples were cut into 10 mm × 10 mm × 2 mm. The SS samples were sealed with epoxy

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resin and only left with 1 cm2 of exposed area. The exposed areas were polished by 240, 400, 800, 1500 grit abrasive papers subsequently, followed by ultrasonic cleaning with deionized water and ethanol. All samples were then sterilized under UV light for 20 min. The pure archaea N. tibetense strain was purchased from China General Microbiological Culture Collection Center (CGMCC). The culture medium was also provided by CGMCC and contained following compositions in 100 mL deionized water: 1.5 g casamino acid, 0.3 g sodium citrate, 0.25 g glutamic acid, 0.25 g MgSO4·7H2O, 0.2 g of KCl, and 25.0 g NaCl. Before autoclaved treatment,

the pH value of the culture medium was adjusted to 8.5 by sterile Na2CO3 solution. After autoclaved treatment for 30 min at 115 °C, the archaea strain was inoculated into the culture medium and cultivated at 37 °C. The detailed process of immersion test was described in previous study [26], and the SS samples were immersed into the sterile and inoculated media for 3, 7 and 14 days. 2.2 Surface analysis The N. tibetense biofilm morphologies were observed by scanning electron microscopy (SEM, FEI Quanta 250). Before SEM observation, in order to immobilize the biofilm, the samples with biofilms were immersed into 2.5% (v/v) glutaraldehyde solution for 8 h. The biofilms were subsequently

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dehydrated with ethanol solutions (50%, 60%, 70%, 80%, 90% and 100%). The adhesion conditions of archaea on the sample surfaces were also observed by confocal laser scanning microscopy (CLSM, Nikon Model C2 Plus). The thicknesses of N. tibetense biofilms were measured by CLSM,

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and the coverage rates of N. tibetense biofilms were calculated using Image J software (National

Institutes of Health, Bethesda, MD, USA). Before the observation, the biofilms were stained in dark

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with SYTO-9 and propidium iodide (PI) dyes in phosphate buffer saline (PBS) for 20 min [27, 28]. The sizes of the pitting pits were observed and counted by confocal laser scanning microscopy

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(CLSM, KEYENCE VK-X). The chemical compositions of passive films after immersion in different media were analyzed by XPS (Thermo escalab 250Xi). The release amounts of the main metal elements from the SS surface after immersion were measured by the atomic absorption

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spectrometer (Thermo, IRIS Intrepid II). All surface characterizations were performed in triplicate. 2.3 Electrochemical tests

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The electrochemical tests for all samples were carried out using the electrochemical station (Gamry, Reference 600 Plus). The measurements contained a three electrode system, in which the sealed SS

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sample was used as working electrode; the platinum electrode and the saturated calomel electrode (SCE) were used as counter electrode and reference electrode, respectively. Firstly, the open circuit potentials were measured for 30 min to ensure the system stability. The electrochemical impedance spectroscopy (EIS) measurements were carried out with the frequency ranging from 100 kHz to 10 mHz, and the voltage amplitude of the sinusoidal perturbation was 10 mV. The potentiodynamic polarization curves were measured at the end of the 14 days with a scanning potential ranging from −200 mV vs. OCP, and the scanning rate was 0.166 mV/s. The Mott-Schottky measurements were

performed at a frequency of 1000 Hz with a potential step of 20 mV. Triplicate samples were prepared for each electrochemical test. 2.4 Energy starvation tests In order to create energy starvation condition, organic matters including casamino acid, sodium citrate and glutamic acid, were removed from the culture medium. In this part, the SS samples were immersed into different culture media, which are presented in Table 1. After 14 days of immersion at 37 °C, the growth conditions of halophilic archaea in different starvation groups were characterized by optical density at 600 nm (OD600) values using an ultraviolet spectrophotometer

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(Thermo Fisher, Bio Mate3S). The cell numbers were counted using a hemocytometer under a light

microscope at 400× magnification (Zeiss, Lab A1). The sizes of the pitting pits in different groups were also measured and compared by CLSM. Each measurement was conducted in triplicate.

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3. Results 3.1 Biofilm characterization

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The surface morphologies of the SS samples after 14-day immersion in the sterile and inoculated media are shown in Fig. 1. Compared with the clean sample surface in the sterile medium (Fig. 1(a)),

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most areas of the sample surface in the inoculated medium were covered by N. tibetense biofilms (Fig. 1(b)). From the enlarged image (Fig. 1(c)), it could be observed that the biofilms were composed of large amounts irregular shaped sessile cells with the length between 1 and 5 μm, which

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was consistent with the description in previous report [29]. The growth and coverage conditions of the biofilms were further quantitatively analyzed by CLSM

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[30]. After the staining process, living and dead microorganism cells were dyed bright green and bright red, respectively. The thickness and coverage of the biofilm were also measured and

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calculated. As shown in Fig. 2(a1), the living N. tibetense cells began to gather on the sample surface after 3 days. At this time, the average biofilm thickness was only ~ 5 μm and the average coverage rate of the biofilm was just ~ 20% (Fig. 3). With the increase of immersion time, the aggregation of living cells became more apparent. After 14 days, the average biofilm thickness gradually increased to ~ 15 μm, and the coverage rate increased to ~ 45%. It is worth mentioning that a low density of dead cells appeared on the sample surfaces during 14-day immersion period. Dead cells were mainly related to the insufficient supply of nutrients. This phenomenon suggested that N. tibetense cells

near the sample surface might be able to obtain a richer energy to sustain their growth. 3.2 Corrosion morphologies In order to investigate the influence of N. tibetense biofilm on the pitting corrosion behavior of 304 SS, the pit diameter and pit depth in sterile and inoculated culture media after different days were counted and showed in Fig. 4. No obvious pitting pit was found on the sample surfaces during 14 days of exposure in the sterile medium, indicating that the SS still maintained good corrosion resistance. As for SS immersion in the inoculated medium, there was no pitting pit on the sample surface in the first 3 days. However, pitting pits appeared on the sample surface after 7 days, and

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the average diameter and average depth of the pitting pits were ~ 17 μm and ~ 2.5 μm, respectively.

As time increased to 14 days, the pitting corrosion became more serious, and the average diameter

and average depth of the pitting pits further increased to ~ 19 μm and ~ 4.8 μm, respectively. The

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largest pit depth increased from 2.6 μm to 5.0 μm after 14 days of immersion (Fig. 5). These results indicated that the presence of N. tibetense biofilm accelerated the pitting corrosion of 304 SS.

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3.3 Electrochemical test results

Fig. 6 shows the EIS plots of the samples after 3, 7 and 14 days of exposure in the sterile and N.

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tibetense inoculated media. The evolution of the EIS results with time in different media exhibited significant difference. For the samples in the sterile culture medium, the Nyquist curves changed little during the whole immersion process (Fig. 6(a)). In the corresponding Bode plots (Fig. 6(b)),

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the impedance values in the low-frequency region stayed almost constant. Only one time constant could be seen from the phase angle curves during 14 days, which was associated with the protective

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passive film on the sample surface. This result revealed that the passive film remained integrity and the corrosion reaction at the metal/passive film interface has not started, which supported the results

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of CLSM observation (Fig. 4). In contrast, when the biofilms colonized and expanded on the steel surfaces, the radius of the arcs in the low-frequency region of the Nyquist curves decreased more quickly (Fig. 6(c)), and the impedance values in the low-frequency region decreased sharply from ~ 1.0×105 Ω cm2 to ~ 3.2×104 Ω cm2 after 14 days (Fig. 6(d)). From the phase angle plots, a new time constant appeared in the low-frequency region after 7 days, which indicated the charge transfer process in the electric double layer at the metal/electrolyte interface [31]. This result revealed that the pitting corrosion occurred on the 7th day in the inoculated medium.

Two electrical equivalent circuits (Fig. 7) were adopted to fit the EIS results. The electrical equivalent circuit with two time constants (Fig. 7(b) was used to fit the EIS results of the 7th day and the 14th day in Fig. 6(c). Other EIS results were fitted using the electrical equivalent circuit with one time constant (Fig. 7(a)). Rs represents the solution resistance, Qp represents the constant phase element (CPE) of the passive film. The CPE was used to more precisely fit the non-ideal capacitive behavior of the passive film [32]. For the samples in the sterile medium, Rp represents the resistance of the passive film. For the samples in the inoculated medium, Rp represents the combined resistance of the passive film and the N. tibetense biofilm. Qdl and Rct are the CPE of

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double layer capacitance and the charge transfer resistance, respectively. The fitting results of Rp in different media are shown in Fig. 8. Compared with the Rp values in sterile condition, the one in inoculated medium decreased faster, which proved that the formation of N. tibetense biofilms was

harmful to the barrier property of passive film. It is worth noting that the Rp in the inoculated culture

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medium increased after 3 days, which may be caused by the physical barrier effect of the

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extracellular polymeric substances (EPS) adhering to the sample surface.

Potentiodynamic polarization curves were also conducted to compare the corrosion resistance of the

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samples in sterile and inoculated culture media after 14 days of immersion (Fig. 9(a)). Influenced by the N. tibetense biofilms, the corrosion potential value (Ecorr) shifted negatively from ~ -0.55 V to ~ -0.60 V after 14 days of immersion. For the samples in the sterile medium, a wide passive

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region with a pitting potential (Epit) of ~ 0.10 V could be observed. In comparison, the Epit value decreased to ~ -0.05 V and the width of the passive region became smaller in the presence of N.

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tibetense biofilms. Furthermore, the passive current density (Ipit) in the inoculated medium increased by nearly one order of magnitude compared with that in the sterile medium.

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Fig. 9(b) shows the Mott-Schottky plots for the passive films on 304 SS after 14 days of exposure in the sterile and inoculated media. From the Mott-Schottky plots, two linear regions with the turning potentials around - 0.3 V could be observed for both plots, suggesting that the N. tibetense biofilm did not change the semiconductor property of the passive film within 14 days. From -0.3 V to 0.6 V, the linear region with positive slope represents n-type semiconductive property, which is related to the iron oxide or hydroxide in the passive film [33]. From -0.6 V to -0.3 V, the linear region with negative slope behaves as p-type semiconductive property, which reflects the chromium

oxide in the passive film [34]. The donor density (Nd) and the accept density (Na) in the passive film could be calculated from the slopes of two linear regions [35], and are shown in Table 2. After 14 days of exposure, the Nd and Na in the inoculated medium were higher than those in the sterile medium, which resulted from the modification of iron oxide and chromium oxide by the N. tibetense biofilm [36, 37]. The increase in Nd and Na promoted the electron transfer in the passive film and accelerated the dissolution of passive film [38]. The results of potentiodynamic polarization and Mott-Schottky measurements further proved that the adhered N. tibetense biofilms could destroy the barrier function of the passive film and increased the possibility of pitting corrosion.

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3.4 Surface analysis of passive film

In order to better understand how the N. tibetense biofilm modified the passive film of 304 SS, the chemical compositions of the SS surfaces exposed to different culture media for 14 days were

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analyzed by XPS. The high-resolution XPS spectra of Fe 2p from the passive films in the sterile and

inoculated media are shown in Fig. 10(a) and (b). Both spectra can be fitted with four peaks at

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binding energies of 706.9, 709.6, 710.8 and 712.8 eV, which were attributed to Fe, FeO, Fe2O3 and FeOOH, respectively. The relative amount of each component is listed in Table 3. For the samples

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in the sterile medium, the main forms of iron element in passive film were FeO, Fe2O3 and FeOOH, and a small amount of metallic Fe was detected. It has been reported that the passive film on the SS is very thin and even less than 2 nm in corrosive medium [39, 40], which may be smaller than the

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detection depth (1‒6 nm) of XPS [41]. Hence, the metallic iron matrix under the passive film could be detected by XPS. After immersion in the inoculated medium for 14 days, the relative amount of

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Fe increased to 10%, which indicated that the passive film became thinner under the influence of biofilm, and more iron matrix was detected by XPS. In addition, the Fe2+/Fe3+ ratio enhanced after

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14 days of immersion. It revealed that the N. tibetense biofilm promoted the reduction reaction from Fe3+ to Fe2+. It has been reported that the increase of Fe2+/Fe3+ ratio was not conducive to the stability of the passive film [42]. Fig. 10(c) and (d) shows the high-resolution Cr 2p spectra of the passive films after exposure in the sterile and inoculated media for 14 days. In these two media, the Cr 2p spectra can be curve-fitted with three peaks at 576.2, 577.3 and 579.0 eV, corresponding to Cr2O3, Cr(OH)3 and CrO3, respectively. In the presence of N. tibetense biofilm, the ratio of Cr(OH)3/Cr2O3 increased obviously

after 14 days. The Cr2O3 is the main component of passive film, so the reduction of Cr2O3 component will decrease the corrosion resistance of the passive film. It is worth noting that the relative amount of CrO3 enhanced after 14 days of immersion in the inoculated medium. With the participation of the N. tibetense biofilm, the oxidation process from Cr3+ to Cr6+ was accelerated. The formation of soluble and unstable Cr6+ will damage the passive film. The release amounts of some metallic elements from the steel surfaces after 14 days of immersion in different media were characterized by atomic absorption spectrometer and shown in Fig. 11. The concentrations of dissolved Fe, Cr and Ni elements in the inoculated medium were significant higher

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than those in the sterile medium. For the sterile medium, the dissolved Cr and Fe mainly came from the passive film. For the inoculated medium, the dissolved Cr and Fe not only came from Cr6+ and

Fe2+ in the passive film, but also came from the metal matrix dissolution during pitting corrosion.

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As the major components of the passive film, excessive consumption and release of Fe and Cr

elements will aggravate the degradation of the passive film. These results further verified that the

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N. tibetense biofilm could promote the destruction of passive film and accelerate the release of Fe and Cr elements. This may be the reason why the Nd and Na increased in the Mott-Schottky plots.

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3.5 Energy starvation test

In the energy starvation tests, the growth curves of planktonic N. tibetense in group A, group B, group C and group D are shown in Fig. 12. Although the organic matters were completely removed,

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archaea N. tibetense could still survive and sustain slow growth relying only on the SS (Group C). However, when organic matters and SS were all removed, archaea N. tibetense could not grow at

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all (Group D). This result indicated that the SS could be utilized by N. tibetense cells as an energy source. With the increase of the concentration of organic matters, the cell concentration at the

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stationary phase increased gradually. When the organic matters and SS were all added into the medium (group A), the cell concentration at the stationary phase reached 1.3×108 cells/mL, which was even higher than that in the regular medium without SS [26]. It revealed that the N. tibetense could simultaneously use organic matters and SS, leading to better growth. Fig. 13 exhibits the size variation of the pitting pits on the sample surfaces during 14 days of immersion in the 50% and 100% starvation culture media. In the 50% starvation culture medium (Group B), the pitting pits also appeared after 7 days, but the size of the pitting pits was larger than

that in the nutrient rich condition (Group A) during 14 days. When the organic matters were totally removed (Group C), the size of the pitting pits further increased during 14 days of immersion, and the pitting pits already appeared on the sample surface after only 3 days of immersion. It indicated that the pitting corrosion was the most serious in the 100% starvation culture medium. With the aggravation of starvation, the N. tibetense cells would be forced to uptake more energy from the metal surface, which accelerated the pitting corrosion of SS.

4. Discussion

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The attachment and colonization of microbial cells on the metal surface and subsequent secretion

of EPS can lead to the formation of biofilms, which has been widely accepted to be responsible for the corrosion of the underlying metal substrates. The physical barrier function of biofilms often

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leads to the change of biofilm/metal interface environment, such as pH distribution and oxygen

concentration [43]. As shown in Fig. 2, only after 3 days, the N. tibetense biofilms began to form

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and expand on the 304 SS surface. In this N. tibetense culture medium, the special high salt content will reduce the concentration of dissolved oxygen. Moreover, the respiration and physical barrier

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function of aerobic biofilms could hinder the diffusion of oxygen to the metal surface [44]. Hence, the oxygen-poor environment will inevitably be formed at the bottom of the N. tibetense biofilm. Combined with the surrounding steel surface without biofilm coverage and with sufficient oxygen

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supply, the oxygen concentration cells were established on the SS surface, as described in Fig. 14(a). The passive films underneath biofilms became anode areas and the oxidation reaction happened

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[45]. The Cr3+ in the anode areas could be oxidized to harmful Cr6+, as shown in the XPS results (Fig. 10), which has been proved to be able to destroy the passive film [46, 47].

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The barrier effect of biofilms can also block the diffusion of some harmful metabolites into the bulk solution, such as H2O2 [48]. In order to avoid the toxic effect of H2O2, the N. tibetense cells will secrete catalase [29], which can catalyze H2O2 into water and oxygen easily: 2H2O2 → O2+2H2O

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which is also known as disproportionation [49]. The catalytic function of catalase is attributed to the protoporphyrin heme group, which contains a redox active centre of FeII/FeIII [50]. The report

from Bergel and Bergel [51] has indicated that this group enables the catalase to exchange electrons directly with the electrode surface. Hence, through the immobilized catalase at the interface of N. tibetense biofilm/passive film, the electrons lost from H2O2 (reaction 1) could be transfer into the passive film (Fig. 14(a)). This process could promote the reduction of Fe3+ to Fe2+ in passive film. The cooperation of Mott-Schottky and XPS results proved that the N. tibetense biofilms could modify the iron oxide and chromium oxide, which increased the concentration of Fe2+ and Cr6+ in the passive film after 14 days of immersion (Table 3). The increase of Fe2+ and Cr6+ could promote the dissolution of the passive film and reduce the corrosion resistance of the passive film.

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At the cathode area of oxygen concentration cell, oxygen molecules were reduced on the SS surface.

For the SS covered with biofilms, the oxygen reduction always occurs in two successive electronreduction steps [52]:

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O2 + 2H+ + 2e- → H2O2 H2O2 + 2H+ + 2e- → 2H2O

(2)

(3)

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Iken et al. [53] showed that the immobilized catalase on SS surface could also catalyze the cathode oxygen reduction process. Combined with the disproportionation of suspended H2O2, electrons lost

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from the cathode areas could be transferred into the bulk medium (Fig. 14(a)). The planktonic N. tibetense cells could uptake these electrons for survival and growth, which also promoted the

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cathode depolarization process and further promoted the anodic dissolution of the passive film under the biofilm. The results of energy starvation tests also showed that the N. tibetense could get energy from SS for survival (Fig. 12), which should be done through this way. From the above analysis, it

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is shown that the combination of catalytic behavior of catalase and oxygen concentration cell and

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energy uptake process of N. tibetense cells promoted the destruction process of passive film. Once the integrity of passive film was broken, the pitting corrosion began to take place under the biofilm. Thereafter, the anodic dissolution of exposed iron matrix occurred under the biofilm, and oxygen reduction reaction continued to take place on the cathodic areas (Fig. 14(b)). The corrosion cell promoted the pitting corrosion, and the size of the corrosion pit increased gradually, as shown in Fig. 4. Meanwhile, the planktonic N. tibetense cells continued to get electrons from the cathodic areas with the help of catalase, which promoted the anodic dissolution of the iron matrix under the

biofilm. The combination of oxygen concentration cell and energy uptake process of N. tibetense cells further aggravated pitting corrosion of 304 SS.

5. Conclusions The influence of a halophilic archaea N. tibetense on the corrosion of 304 SS in its culture medium was studied in this paper. The corrosion behaviors of the 304 SS after 3, 7 and 14 days of exposure in the sterile and N. tibetense-inoculated culture media were compared. Based on the results of XPS measurements and energy starvation tests, the MIC mechanism caused by N. tibetense was discussed.

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The following conclusions can be obtained:

(1) In the immersion tests, there was no obvious pitting pit on the SS surface after 14 days of exposure in the sterile medium. By contrast, the SS in the inoculated medium showed severe pitting

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corrosion after 14 days, which indicated an apparent accelerated corrosion process caused by archaea N. tibetense.

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(2) In the electrochemical tests, the Rp values of the passive films decreased more quickly in the inoculated media. The passive current density and doping density of the passive films increased

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under the influence of N. tibetense biofilms after 14 days.

(3) The N. tibetense biofilms promoted the formation of detrimental Fe2+ and Cr6+ in the passive

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films, and the N. tibetense could use SS as nutrient source. The cooperation of catalytic behavior of catalase and oxygen concentration cell and energy uptake from SS by N. tibetense promoted the

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destruction of passive film, and further accelerated the pitting corrosion.

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Acknowledgments

This work was supported by the Beijing Nova Program (No. Z171100001117076), the National Natural Science Foundation of China (Nos. 51871026, No. 51771029) and the National Environmental Corrosion Platform.

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Figure List

Fig. 1. SEM images of SS surface after 14 days of exposure in (a) the sterile medium and (b) the

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inoculated medium, and (c) the enlarged SEM image of biofilm.

Fig. 2. Fluorescence microscope images of the N. tibetense biofilms on the sample surfaces after (a1,

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a2) 3 days, (b1, b2) 7 days and (c1, c2) 14 days of exposure in inoculated media.

Fig. 3. (a) Biofilm coverage rate and (b) biofilm thickness of the N. tibetense biofilms on the sample

surfaces after different days of exposure in inoculated media.

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different days of exposure in sterile and inoculated media.

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Fig. 4. (a) Average diameter and (b) average depth of the pitting pits on the sample surfaces after

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Fig. 5. Maximum depth of the pitting pits on the sample surfaces after (a) 7 and (b) 14 days of

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exposure in inoculated media.

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media after different days.

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Fig. 6. EIS results of the samples in (a, b) the sterile media and (c, d) the N. tibetense inoculated

Fig. 7. Electrical equivalent circuits used for fitting EIS spectra.

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Fig. 8. Evolution of Rp values of the samples in different media.

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14 days of exposure in different media.

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Fig. 9. (a) Potentiodynamic polarization curves and (b) Mott-Schottky curves of the samples after

Fig. 10. High resolution XPS spectra of Fe 2p in (a) the sterile medium and (b) the inoculated medium after 14 days of exposure; High resolution XPS spectra of Cr 2p in (c) the sterile medium

and (d) the inoculated medium after 14 days of exposure.

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Fig. 11. Concentrations of the dissolved elements in different media after 14 days of exposure.

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Fig. 12. Growth conditions of N. tibetense cells in different groups.

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Fig. 13. (a) Average diameter and (b) average depth of the pitting pits on the sample surfaces after different days of exposure in 50% starvation and 100% starvation media.

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Fig. 14. Schematic figures of the MIC mechanism of the 304 SS (a) before and (b) after the passive

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film was destroyed.

Table List

Table 1 Comparison of four groups in the starvation tests. Group

A

B

C

D

N. tibetense

√

√

√

√

Organic energy sources

√

√(50%)

stainless steel

√

√

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√

Table 2 Doping density of the passive film on 304 SS after 14 days of exposure in the sterile and inoculated media. NA (cm-3)

Sterile medium

2.14×1021

3.64×1021

Inoculated medium

3.10×1021

4.45×1021

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ND (cm-3)

Table 3 Relative quantity of compounds in the Fe 2p and Cr 2p XPS spectra. Fe 2p

Cr 2p FeO

Fe2O3

FeOOH

Cr2O3

Cr(OH)3

CrO3

Sterile medium

0.05

0.17

0.41

0.37

0.70

0.27

0.03

Inoculated medium

0.10

0.43

0.27

0.20

0.54

0.36

0.10

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Fe