Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2

Corrosion Science xxx (2014) xxx–xxx

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Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2 Qing Qu a,⇑, Yue He a, Lei Wang a, Hangtian Xu a, Lei Li b, Yajun Chen a, Zhongtao Ding a a b

School of Chemical Science and Technology, Yunnan University, Kunming 650091, China Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming 650091, China

a r t i c l e

i n f o

Article history: Received 4 July 2014 Accepted 18 November 2014 Available online xxxx Keywords: A. Steel B. Polarization B. EIS C. Microbiological corrosion

a b s t r a c t Effect of Bacillus subtilis C2 (BS) on the corrosion behavior of cold rolled steel (CRS) in artificial seawater has been studied. A visible decrease in pH value and a noticeable decrease in open circuit potential were observed in solutions containing BS compared to the sterile solutions. Biofilm was evidently observed on CRS surface after immersion in solution containing BS for some time, the biofilm increased and became more and more compact with increasing immersion time. A significant reduction in the latter corrosion rate was observed although the initial corrosion was clearly accelerated in the presence of BS. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Microbiologically Influenced Corrosion (MIC) is a major problem in many industries such as oil and gas, as well as water utilities [1]. The term MIC is usually interpreted as to indicate an increase in corrosion rates due to the presence of bacteria that accelerate the rates of the anodic and/or cathodic corrosion reaction, while leaving the corrosion mechanism more or less unchanged [1]. However, an inhibiting effect of biofilm in aqueous environments (artificial seawater, for instance) has also been observed [2–4]. Thus, microorganisms may cause either microbially influenced corrosion acceleration (MICA) or inhibition (MICI). One of the first studies of MIC involved sulfate-reducing bacteria (SRB) that thrive only under anaerobic conditions and are found widespread in many waters and soils [1]. SRBs easily reduce inorganic sulfates to sulfides in the presence of hydrogen or organic matter and are aided in the process by the presence of an iron surface. Kuehr [1] in 1923 proposed the so-called cathodic depolarization mechanism which assumes that the SRBs remove atomic hydrogen from the iron surface which causes accelerated corrosion of iron. Then, SBR became the most extensively studied microorganisms in relation to biocorrosion [5–9]. However, it is interesting that recent studies suggest that SRB need not be present in abundance in all microbial communities responsible for microbially influenced corrosion [10,11]. By contrast, Bacillus species were usually found in the corrosive surfaces of metals in many environments ⇑ Corresponding author. Tel.: +86 871 65035798; fax: +86 871 65036538. E-mail address: [email protected] (Q. Qu).

and some of them were identified as the dominant bacterial species [12–16]. Macdonald and Brözel [12] determined the community structure in the studied open recirculating cooling-water system and noted that they did not observe any sulfate-reducing bacteria or Aeromonas. Rajasekar et al. [13] described bacterial enumeration and identification in diesel and naphtha pipelines located in the northwest and southwest region in India, but sulfate-reducing bacteria were not detected in samples from both pipelines, the samples obtained from the diesel and naphtha-transporting pipelines showed the occurrence of 11 bacterial species in these fields, and 7 of these species are belong to Bacillus species. Giacobonea et al. [14] identified eighteen microorganisms and affirmed that Bacillus cereus was the predominant organism isolated from a spent nuclear fuel pool in Argentina. Bolton et al. [15] confirmed that Bacillus pumilus was one of the predominant bacteria in a corroding galvanized steel pipes conveying water for specialist applications. Marques et al. [16] pointed out that Bacillus aquimaris and Bacillus licheniformis were the predominant strains on the corrosion surface of carbon steel coupons using reactors containing produced water from a Brazilian oil platform. In fact, as one of the most broad-spectrum species, Bacillus species can be readily isolated from soil and plant-associated environments, but are also found in other ecological niches such as deep-sea sediments, injection brine, fermented food, and the human gastrointestinal tract [17,18]. Of course, there will be existence of Bacillus species in the corrosion surfaces of metals in many environments. Therefore, the effects of Bacillus species on the corrosion of metals have attracted serious concern in the past decades [12–16,19–23]. However, only a few publications have dealt with MIC about Bacillus

http://dx.doi.org/10.1016/j.corsci.2014.11.032 0010-938X/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Q. Qu et al., Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2, Corros. Sci. (2014), http://dx.doi.org/10.1016/j.corsci.2014.11.032

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species, and the strains in these studies are rather extensive. 7 strains of Bacillus species including Bacillus subtilis, Bacillus megaterium, B. pumilus, B. licheniformis, Bacillus brevis, B. cereus, Bacillus mycoides have been reported in the literature available to date. Mansfeld et al. [21] showed that B. subtilis and B. licheniformis can inhibit the corrosion of cartridge brass, aluminum 2024 and carbon steel in artificial seawater; but Jack et al. [22] found that pure culture of Bacillus sp. induced greater corrosion of mild steel initially by 2- to 6-fold, and the rate of this corrosion decreased to that of a  nas et al. [23] compared the corsterile control after 17 days; Juzeliu rosion activity of abiotic samples with that of the samples colonized with B. mycoides indicated microbially influenced corrosion acceleration for zinc, inhibition for aluminum, and indifference for mild steel. Bolton et al. [15] showed that B. pumilus accelerated the corrosion of galvanized coupons but did not increase the corrosion of steel. The results of Giacobonea et al. [14] showed that major pits covered with deposits were found on AA 6061 samples exposed to B. cereus but not on 99.999% Al. It easy to draw a conclusion from literatures that the results are in variety endless due to different strains of Bacillus species and different metals being used in different studies. And the results are likely to be different even when the same strain and metal are used in different studies. Just like MIC of other microorganisms, the mechanisms involved in MIC of Bacillus species are very complicated, as the process is affected by many factors, many mechanisms, such as formation of differential aeration cells caused by oxygen respiration, production of corrosive agents and organic and/or inorganic acids, metal-deposition, hydrogen embrittlement, and metal-binding effect of extracellular polymeric substance, inactivation of corrosion inhibitors and cathodic and anodic depolarization, were suggested, with no single mechanism identified as playing a major role in MIC of Bacillus species [1]. In recent years, popular viewpoint involved is that biofilm has a great impact on MIC, and some researchers also suggested that biofilm formed by some strains of Bacillus species e.g. B. subtilis may be beneficial to reduce MIC rates of metals [1,3,24–27]. For example, Du et al. [27] suggested that the compact biofilm formed by one strain of Bacillus species isolated from oil storage tank could effectively protect A3 steel from being corroded by solution, unfortunately, they did not showed the specific strain. Zuo et al. [3] demonstrated that B. subtilis retarded the corrosion of Al 2024 in AS by formation of a live biofilm, but they also deemed that the mechanism by which BS and other bacteria protect metallic surfaces from corrosion remained unclear. Furthermore, it is well known that many strains including B. subtilis can produce organic acid such as lactic acid in their physiological activities [28,29], and organic acid decreases pH values, but there are no studies involved in the effect of pH values in the presence of B. subtilis. Furthermore, microorganisms can colonize and form biofilm on metal surface during several days [5–7,19,20,27], the early corrosion information may provide helpful insights in establishing an accurate theoretical background on the microbiological influence on corrosion, but publications dealt with the corrosion in the early stage are rather scare. The objective of this investigation is to determine the effect of B. subtilis C2 (BS) on the corrosion of cold rolled steel (CRS) in artificial seawater (AS) at the early stages. Meanwhile, a possible mechanism is presented to explain the experimental observation.

2. Experimental methods 2.1. Material The experiments were performed with cold rolled steel (CRS) specimens with the following chemical composition (wt.%): C 0.050, Si 0.02, Mn 0.28, Cu 0.25, Ni 0.25, S 0.023, Cr 0.15, P 0.019, Fe remainder.

2.2. Bacterium B. subtilis (BS) was obtained from Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resource of the Ministry of Education, Yunnan University (Kunming, China). The bacterial strain was cultured in Luria Bertani (LB) agar at 37 ± 2 °C. 2.3. Medium All tests were conducted using a nutrient-rich simulated seawater-based medium (AS). CRS was exposed to AS prepared as Vätäänen nine salts solution [30] (VNSS: NaCl: 17.6 g/l, NaHCO3: 0.08 g/l, KBr: 0.04 g/l, CaCl22H2O: 0.41 g/l, SrCl26H2O: 0.008 g/l, Na2SO4: 1.47 g/l, KCl: 0.25 g/l, MgCl26H2O: 1.87 g/l, H3BO3: 0.008 g/l, FeSO47H2O: 0.01 g/l, Na2HPO4: 0.01 g/l, peptone: 1.0 g/l, starch: 0.5 g/l, glucose: 0.5 g/l, yeast extract: 0.5 g/l). The pH of the medium was adjusted to 7.5 ± 0.1 using a 1 M NaOH solution and sterilized by autoclaving for 20 min at 121 °C and at 100 kPa. 2.4. Growth phase experiments Growth phase experiments were performed to determine the growth kinetics of BS in AS medium. A loop of BS cells from a slant culture of fresh nutrient agar was used to inoculate a 250 ml Erlenmeyer flask containing 100 ml Luria Bertani (LB) broth (pH 7.0). The flask was incubated on a rotary shaker at 150 rpm at 36 °C for 20 h until the BS was grown to an OD550 of 1.1, then 2 ml bacterial culture was taken out by sterile pipette and inoculated in AS to obtain BS inoculated medium with a volume of 150 ml in 250 ml flask. The flask was incubated with a stirring speed of 150 rpm using a polytetrafluoroethylene magnetic stirring at 25 °C by a thermostatically controlled water tank. In this process, three parallel flasks were performed. During incubation, 2 ml culture from each of three parallel flasks was collected and pooled every 4 h, the combined samples were then subjected to cell density measurement. The optical density of the culture at 550 nm (OD550) was measured over time using a photometer. The experiment was terminated until bacterial cells reached decline phase. All the experiments were performed in triplicate. 2.5. Fluorescent microscopy (FM) The CRS tablets (10  10  1 mm) were abraded with emery paper from 100 to 2000, then rinsed with distilled water, degreased with acetone (CH3COCH3), and dried with a warm air stream, sterilized with 2% glutaraldehyde solution for 1 h and rinsed with sterile distilled water. The treated CRS tablets were exposed to the BS inoculated medium without renewing the fresh medium under the conditions as described in growth phase experiments for 10, 24, 32, 72, 96 and 240 h, respectively. At the predetermined period of bacterial incubation, the specimens were retrieved and washed twice with a sterile phosphate buffered saline (PBS) solution to remove the dead and loosely attached bacteria. The specimens were washed thrice with the sterile PBS solution and deionization water, followed by staining with 2-(4Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) solution for 15 min. The specimens with immobilized bacterial cells were imaged under 100  magnifications using a Nikon E800 fluorescence microscope, equipped for epifluorescence with a mercury lamp. 2.6. pH tests pH tests were also performed in AS solutions containing working electrode with and without BS before electrochemical

Please cite this article in press as: Q. Qu et al., Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2, Corros. Sci. (2014), http://dx.doi.org/10.1016/j.corsci.2014.11.032

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experiments at 25 °C, pH tests was surveyed by PHS-25 and performed in pH-meter.

1.5

2.7. Electrochemical experiments

2.8. Surface topography analysis with SEM After immersion in AS with and without BS for 96 h, the samples were taken out. Prior to characterizing the morphologies of corrosion, the samples were carefully washed with distilled water, then fixed by 15 min immersion in 2.5% glutaraldehyde, dehydrated in a series of aqueous ethanol solutions (15%, 30%, 50%, 70%, 95% and 100%) for 15 min, coated with gold and examined by using scanning electron microscope (Holland yielding XL30 ESEM-TMP). To further evaluate possibility of pitting, biofilm on samples was cleaned by ultrasonication in 95% ethanol for 30 min, washed with distilled water and acetone, then the samples were taken out and subsequently immersed in ASTMG1-90 standard solution (Clark solution: 100 mL HCl + 2% Sb2O3 + 5% SnCl2) to remove the probable corrosion products.

3. Results and discussion 3.1. Growth phase Fig. 1 showed the growth phase of BS in sterile AS medium. Four different phases for BS were observed in this figure. In the lag phase, the OD value increases slowly from 2 to 12 h, showing that the bacteria grow slowly; In the log phase, OD value was in logarithmic phase during 12 and 32 h, suggesting that the bacteria grow quickly in this stage; In the stationary phase, OD value appears a plateau from 32 to 48 h corresponding to the maximum growth of the bacteria; In the decline phase, the OD value decrease quickly, which corresponds to bacterial lysis. After obtaining this growth phase curve, it was decided to evaluate corrosion resistance at different stages (2, 10, 16, 24, 48, 72, 96, 240 h), and 240 h was chosen as the maximum time.

OD550

1.0

A three-electrode system including a working electrode, an auxiliary electrode and a reference electrode was used for electrochemical measurements. The working electrodes were made of the steel specimen in PVC holder using epoxy resin with an exposed area of 1.0 cm2, abraded with emery paper from 100 to 2000 grades on the test face, rinsed with distilled water, degreased with acetone (CH3COCH3), and dried with a warm air stream, sterilized with 2% glutaraldehyde solution for 1 h, rinsed with sterile distilled water, and then exposed in UV light for 15 min for sterilization. The auxiliary electrode is a platinum foil and the reference electrode is a saturated calomel electrode (SCE) with a Luggin capillary positioned close to the working electrode surface in order to minimize ohmic potential drop. EIS measurements were conducted at the end of open circuit potential (OCP) measurements to ensure it was in steady state and carried out in a frequency range of 0.1–105 Hz using a 10 mV peak-to-peak potential perturbation at the open circuit potential using PARSTAT 2263 Potentiostat/Galvanostat (Princeton Applied Research). The Tafel polarization curves were carried out by polarizing in the range between 250 and 250 mV with respect to Ecorr vs. SCE at a scan rate of 1 mV s1. All experiments were performed in AS solutions with a stirring speed of 150 rpm using a polytetrafluoroethylene magnetic stirring with or without BS. And the test temperature was kept at 25 °C in the whole test process by means of a thermostatically controlled water tank. Each experiment was repeated at least three times to ensure the reproducibility.

0.5

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Time (h) Fig. 1. Growth phase of BS in sterile AS medium at 25 °C.

3.2. The bacterial colonization study with FM Fig. 2 showed the fluorescence images of the aspect of BS biofilm, formed by microcolonies attached to the experimental CRS surface during constant agitation of the medium. Before 10 h, only sparse BS cells were distributed over the specimen surface, and no bacterial clusters appeared (Fig. 2A). In the subsequent period, the bacteria grew rapidly and cumulatively. After 10 h of exposure, some distinguishable bacterial cells were spotted over CRS surface (Fig. 2B and C). With the increase in exposure time, the BS cells became dense, and aggregated to form bacterial clusters or a patchy biofilm, the patchy biofilm increased in thickness and size, and became more compact, then, a thick and compact biofilm was formed on the surface of CRS (Fig. 2D–F), especially the biofilm after 240 h immersion became more uniform and denser. It is, therefore, easy to conclude that the coverage of the BS cells on CRS surface increased with time. This confirms that the BS can be seen adhering to the surface of the sample and form the biofilm. 3.3. pH results Fig. 3 gave the pH value of AS solution containing working electrode with and without BS at 25 °C for different times. It is easy to see from this figure that, all the solutions in the absence of BS showed weak basicity and the pH value slightly increased with increase in immersion time, the anodic dissolution of CRS in AS solution without BS was balanced by oxygen-consuming corrosion in the cathodic areas, which results in slight increase of pH. On the contrary, all the solutions in the presence of BS appeared weak acidity, pH clearly decreased with increase in immersion time from 0 to 16 h and then slightly increased from 16 to 96 h, that is, there was a minimal pH value in their rapid growth period at 16 h. This may have been a result of the following reasons: (a) BS can produce organic acid in their physiological activities which results in decrease of pH, organic acid secreted by BS is associated with many factors, but the metabolic capability is one of the main factor [28,29], the stronger the physiological activities, the more organic acid BS produces; (b) the anodic dissolution of CRS in AS solution with BS is balanced by hydrogen and oxygen consuming corrosion in the cathodic areas which results in increase of pH. 3.4. Open circuit potential (OCP) The OCP experiments of CRS in sterile AS or the same medium inoculated with BS at 25 °C were recorded after 2 h immersion and plotted in Fig. 4. The corresponding OCP remained stable in

Please cite this article in press as: Q. Qu et al., Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2, Corros. Sci. (2014), http://dx.doi.org/10.1016/j.corsci.2014.11.032

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Fig. 2. Fluorescence images of bacterial cells colonized on CRS specimens in the BS inoculated AS medium after various exposure times (A) 10 h in lag phase; (B) 24 h in logarithmic phase; (C) 32 h in stationary phase; (D) 72 h in decline phase; (E) 96 h in decline phase; (F) 240 h in decline phase. Scale bar: 20 lm.

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Fig. 3. pH value of AS solution containing working electrode with and without BS at 25 °C for different times.

Fig. 4. Variation of the OCP of CRS in AS solution without and with BS at 25 °C.

the 0.67 to 0.71 V (SCE) potential range in sterile AS medium, whereas the OCP in the presence of BS were in the 0.74 to 0.77V (SCE) range. Only small variations were observed in both cases indicating there were no obvious pitting occurred. It also is

easy to see from Fig. 4 that there was a noticeable decrease in OCP in solutions containing BS compared to the sterile solutions. The decrease of OCP in presence of BS compared to the sterile solution can be attributed to biofilm formation; biofilm can slow down

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-0.4

times, respectively. From Fig. 5a it is easy to see that, in the absence of BS, the anodic current densities increased obviously with increase in immersion time from 2 to 48 h and then slightly decreased with increase in immersion time from 48 to 240 h, suggesting that the maximum anodic dissolution may appear at 48 h in the absence of BS. However, Fig. 5b shows that, in the presence of BS, the anodic current densities slightly increased with increasing immersion time from 2 to 16 h and then quickly decrease from 16 to 240 h, suggesting the anodic dissolution accelerate during 2– 16 h and decelerate during 16–240 h. Tafel polarization parameters including corrosion potential (Ecorr), cathodic Tafel slopes (bc), anodic Tafel slopes (ba) and corrosion current density (icorr) obtained by extrapolation of the Tafel curves were shown in Table 1. It can be observed from this table that the corrosion current density (icorr) gradually increased with increasing of the immersion duration in the sterile artificial seawater medium before 48 h, however, after 48 h, the value decreased, the maximum icorr was 22.1 lA/cm2 at 48 h, and then slightly decreased to 16.9 lA/cm2 at 240 h, it could be due to the gradual accumulation of corrosion products on CRS surface which slows down the further corrosion. In the BS inoculated medium, before 16 h icorr increases with increasing immersion time. In 16 h, icorr reached the maximum value of 21.3 lA/cm2. However, after 16 h, icorr decreased remarkably, in general, icorr decreased in the range from 21.3 lA/cm2 to 1.1 lA/cm2, indicative of the change in the corrosion rate of CRS with time owing to the cooperation of the formation of biofilm and decrease in pH which is attributed to the metabolisms of the microorganisms. Table 1 also showed that, the corrosion of CRS in AS without BS was greater than that in AS with BS before 16 h immersion, but the result was the opposite after 16 h immersion, suggesting that BS can accelerate the initial corrosion and then retard the corrosion clearly. Javed et al. [32] studied MIC of Escherichia coli in different media, they deemed that bacteria test media could determine the outcome of MIC and supposed that corrosion inhibition by bacteria in the nutrient broth (NB) might be linked to the alkaline by-products generated by the bacteria in the presence of peptone in aerobic conditions in addition to the bacterial respiration. But in our study, the metabolic by-products was weak acid which might accelerate the initial corrosion, but with immersion time increased, the nutrition was consumed by BS, metabolic by-products decreased with creasing the immersion time, thus, the formation of biofilm began to play the main role and might be responsible for inhibition of the latter corrosion.

(a)

E vs. SCE (V)

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log (i, A/cm ) Fig. 5. Potentiodynamic polarization curves of CRS exposed to AS medium without (a) and with BS (b) at 25 °C for different times.

the diffusion of the cathodic depolarization agents such as oxygen, hydrogen ions, which results in decrease of the cathodic reduction potential. Therefore, biofilm can decreases OCP in the presence of BS according to the mixed potential theory. Similar results were also fund observed by Jack et al. [22], they deemed that biofilm formation and electrode colonization were responsible for the decreases of OCP in the presence of Bacillus sp. for carbon steel. Cheng et al. [31] also suggested that biofilm formation result in more evident decrease of the OCP value in the presence of bacteria in their study.

3.6. EIS EIS is a powerful, nondestructive electrochemical technique for the characterization of electrochemical reactions at the metal/biofilm interface and the formation of corrosion products and biofilm in MIC [33]. Nyquist Plots of CRS immersed for different times in the medium with and without BS at 25 °C were respectively presented in Figs. 6a and 7a. The corresponding Bode modulus

3.5. Polarization curves Fig. 5a and b presented the Tafel polarization curves of CRS exposed to AS medium with and without BS at 25 °C for different

Table 1 Tafel parameters of polarization curves for CRS in AS medium with and without BS at 25 °C for different exposure times. T (h)

2 10 16 24 48 72 96 240

AS

AS + BS

icorr (lA/cm2)

Ecorr (mV vs. SCE)

bc (mV dec1)

ba (mV dec1)

pH

icorr (lA/cm2)

Ecorr (mV vs. SCE)

bc (mV dec1)

ba (mV dec1)

pH

12.9 17.6 20.8 21.1 22.1 19.7 18.5 16.9

673 663 685 694 698 708 712 711

369 560 563 981 663 901 351 407

114 140 91 119 142 142 138 140

7.52 7.60 7.63 7.70 7.84 7.88 7.93 7.85

16.1 20.6 21.3 7.1 6.0 1.3 1.2 1.1

741 772 778 780 774 770 774 774

238 473 487 339 294 108 101 112

85 94 124 87 87 52 77 81

7.02 6.33 5.37 5.70 6.07 6.28 6.35 6.75

Please cite this article in press as: Q. Qu et al., Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2, Corros. Sci. (2014), http://dx.doi.org/10.1016/j.corsci.2014.11.032

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Frequency (Hz) Fig. 6. EIS of CRS immersed for different times in the sterile AS medium at 25 °C: (a) Nyquist plots, (b) Bode modulus diagrams and (c) Bode phase angle diagrams.

diagrams and Bode phase angle diagrams were also presented in Fig. 6b and c and 7b and c. From these figures it was clearly found that, all the diagrams were part of the imperfect semicircles and this was attributed to frequency dispersion [35]. The fact that impendence diagrams have a depressed semicircular appearance shows that the corrosion of steel is controlled by a charge transfer process and the presence of BS does not change the mechanism of dissolution of CRS [34]. In the sterile artificial seawater medium, the diameters of Nyquist plots decreased on increasing the immersion period from 2 to 48 h and then slightly increased from 48 to

Fig. 7. EIS of CRS immersed for different times in the sterile AS medium with BS at 25 °C: (a) Nyquist plots, (b) Bode modulus diagrams and (c) Bode phase angle diagrams.

240 h, indicating first increase and then decrease in the corrosion rate. In the BS inoculated medium, the diameters of Nyquist plots decreased firstly, but after 16 h, the diameters evidently increased, suggesting that the maximum corrosion rate appear at 16 h. The same phenomenon could be found in Bode modulus diagrams (Figs. 7b and 8b). Bode phase angle diagrams obtained for CRS electrodes (Figs. 7c and 8c) show two-time constant behavior [1]. Twotime constants relates to a two-layer structure developed during corrosion [1]. Considering that both the biofilm and corrosion

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Qfl

Qdl

Rf

Rct

pot-holed and cauliflower-like layer of deposits (Fig. 9A). In case of BS, a thicker, and more compact surface having bacterial deposition was noticed (Fig. 9B), magnified view of this film (Fig. 9C) further demonstrated that the compact biofilm was composed of numerous microorganisms (indicated by red arrows) with abundant extracellular polymeric substances, Fig. 9C also showed the biofilm was formed by multi-layer bacterial community, only small water-channel could be found in the same layer bacterial community. After the removal of the biofilm and products film (Fig. 9D), the metal surface was rather smooth, and the parallel features which could be associated with abrading scratches before immersion were still observed, the small particles in the abrading scratches might be the corrosion products which were not cleaned entirely by Clark solution. In addition, no evident pitting was observed after removing the films.

Rs

Fig. 8. Equivalent circuits used for fitting the impedance spectra based on a doublelayer model of surface film.

products can be adsorbed onto the surface of matrix and their contributions are indistinguishable, the same equivalent circuit containing two-time constant equivalent circuit as shown in Fig. 8 was used in this study. The obtained fitting electrochemical parameters using the proposed equivalent circuits were listed in Table 2. In Fig. 8, Rs is the solution resistance, Rf is the resistance of film, Rct is the charge transfer resistance, Qfl and Qdl are the CPE parameters for film and double layer of film-electrolyte interface, respectively, the constant phase element (CPE) is usually used instead of a capacitance to account for the non-ideal capacitance response due to the almost complete absence of pure capacitance in the real electrochemical process [34–36]. n is a mathematical expression where 0 6 n 6 1. If n = 0 the impedance is entirely resistance, while it is capacitance if n = 1. And 0 < n < 1 represents deviation from the ideal capacitance, which is related to the surface roughness [34– 36]. As shown in Table 2, Rs, was quite low, due to the good conductivity in the presence of many salts in the medium with and without BS. In the absence of BS, the charge transfer resistance, Rct significantly decreased with increasing the immersion time from 2 to 48 h, then slightly increase from 48 to 240 h. In the presence of BS, before 16 h, Rct gradually decreased with time, indicating an increase in the corrosion rate of the bacteria-colonized specimens with time. But after 16 h, Rct increased. For the resistance of the film in the presence of BS, it did not have a clear trend due to its variation in magnitude with time for the formation and desorption of biofilm. The n2 values stayed above 0.8 in the presence of BS and were higher than that in absence of BS, indicative of the relatively homogeneity and compactness of the film in the presence of BS. From Table 2, it is clear that, Rct of CRS in AS with BS was smaller than that without BS at the beginning and subsequently became larger than that without BS, suggesting that BS can accelerate the initial corrosion and then retard the corrosion clearly. These results are in good agreement with the results obtained from Tafel polarization.

3.8. Mechanism In the sterile medium, in the early stages of the corrosion process, the anodic dissolution of CRS in AS is balanced by the cathodic reduction of oxygen:

Fe ! Fe2þ þ 2e

ð1Þ

H2 O þ 1=2O2 þ 2e ! 2OH

ð2Þ

Thus, countless electrochemical cells begin to form on the surface of CRS and accelerate the corrosion of CRS. Formation of OH in AS causes the slight increase in pH (Fig. 3). With the reaction going on, cations, e.g. Na+, Fe2+ migrate toward the cathodic areas while anions, e.g. Cl and OH move toward the CRS dissolution sites. The following reactions occur when the concentration of hydroxyl ions is increased to a certain level at anodic sites.

Fe2þ þ 2OH ! FeðOHÞ2

ð3Þ

4FeðOHÞ2 þ O2 þ 2H2 O ! 4FeðOHÞ3

ð4Þ

FeðOHÞ3 ! FeOOH þ H2 O

ð5Þ

Then FeOOH gradually transforms to more stable substance, Fe2O3

2FeOOH ! Fe2 O3 þ H2 O

ð6Þ

FeOOH and Fe2O3 are insoluble in weak basic solution. The formation of these insoluble substances forms a barrier film on CRS that may retard the anodic dissolution of CRS. Therefore the electrochemical corrosion rate slows down to some extent after concentration of OH is increased to a certain level (after 48 h immersion). In the presence of BS, it is well known that BS can produce organic acid (HOA) in their physiological activities which will decrease pH values; this phenomenon was also observed in our study (Fig. 3), significant reduction of pH in AS with BS compared

3.7. Surface topography analysis with SEM SEM micrographs of corrosion products formed on CRS immersed in the media with and without BS for 96 h were presented in Fig. 9A–D. As it can be seen from these figures, in the sterile artificial seawater medium, the corrosion surface of CRS looked rather uneven and CRS surface was covered with a fluffy,

Table 2 Analysis of EIS for CRS in AS medium with and without BS at 25 °C for different exposure times. AS

AS + BS 2

2

T (h)

Rs (X cm ) Cfl (lF cm

2 10 16 24 48 72 96 240

9.6 2.3 6.2 2.2 12.0 7.3 8.4 9.1

484 153 381 631 965 632 289 277

)

2

2

n1

Rf (X cm )

Cdl (lF cm

0.68 0.63 0.65 0.66 0.68 0.68 0.67 0.67

211 176 103 217 253 185 53 37

476 158 379 574 851 622 312 273

)

2

n2

Rct (X cm ) Rs (X cm2)

Cfl (lF cm2) n1

Rf (X cm2) Cdl (lF cm2) n2

Rct (X cm2)

0.70 0.67 0.77 0.75 0.75 0.75 0.75 0.74

3271 2169 1716 1679 1583 1847 1991 2007

412 396 258 412 496 396 127 113

390 109 103 157 160 178 173 152

3110 2590 2399 2911 3153 3544 3799 3857

6.2 4.1 3.9 9.3 2.3 3.6 9.6 11.1

0.76 0.82 0.76 0.79 0.78 0.82 0.79 0.81

317 301 109 106 102 284 845 779

0.85 0.88 0.79 1.00 0.98 0.82 0.81 0.81

Please cite this article in press as: Q. Qu et al., Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2, Corros. Sci. (2014), http://dx.doi.org/10.1016/j.corsci.2014.11.032

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Q. Qu et al. / Corrosion Science xxx (2014) xxx–xxx

(B)

(A)

(A) 50 μm

50 μm

(C)

(D)

5 μm

50 μm

Fig. 9. SEM images of CRS in AS for 96 h. (A): without BS, (B) with BS, (C) magnified view of (B) (BS were indicated with red arrows) and (D) after the removal of the biofilm produced by BS and products film. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

with the sterile AS was observed from Fig. 3, and the solutions in the presence of BS presented as weak acid. Therefore, different from the sterile AS, in case of BS, the anodic dissolution of CRS in AS is balanced by HOA and oxygen consuming corrosion in the cathodic areas, that is, in the cathodic sites the following reaction takes place.

4HOA þ O2 þ 4e ! 4OA þ 2H2 O

ð7Þ

In the initial stages, few microcolonies are attached to CRS surface, the effect of pH on corrosion of CRS predominates in the corrosion process, and the corrosion of CRS is accelerated by lactic acid produced by BS. So the initial corrosion of CRS in AS with BS is larger than that in sterile AS. However, with time increased, the attached bacteria increase on CRS surface, simultaneously, some of them always occur detachment, finally, which reach to adsorption equilibrium and generate biofilm. That is, more and more BS colonize CRS surface by forming a more compact biofilm producing an environment at the metal/ biofilm interface which is different from the initial general medium [1,37,38]. Therefore, in the later stages, the corrosion of CRS in AS with BS may be up to the co-effect of pH and biofilm. The formation of biofilm can retard the corrosion of CRS which is due to a result of one or more of the following: (a) The influence of BS might be essentially due to the consumption of oxygen at the metal/electrolyte interface of the specimen, this can be clearly deduced from Eq. (7); (b) the presence of a biofilm acting as a barrier to the diffusion of corrosion products and thereby suppressing the process of metal dissolution; and/or (c) the bacteria producing a metabolic product that acts as corrosion inhibitor for CRS, adsorption and orientation of the metabolic product on CRS surface further retards the corrosion. This observation can further be supported by the SEM images obtained in this study, depicting corrosion product

build up and microbial adherence. Following this, the corrosion rate tends to decrease with further exposure. Actually, Mansfeld et al. [21] also suggested that the biofilm formed by BS which can produce polyaspartate may result in a significant reduction of corrosion rates of Al 2024. But the role of polyaspartate produced by BS in vivo in protection of metal should be further studied. As can be seen from Fig. 2, with time increased, the biofilm becomes more and more compact, and the effect of biofilm predominates gradually in the co-effect of pH and biofilm, thus, the corrosion can be clearly reduced by BS at the later stages.

4. Conclusion The microbially influenced corrosion of CRS in AS medium by B. subtilis C2 (BS) has been investigated using surface analysis and electrochemical techniques. The biofilm developed with time was analyzed using FM. Decrease in pH and OCP was clearly observed in the presence of BS comparing to the sterile AS. Only sparse BS cells were distributed over CRS surface in the initial colonization and then the BS cells on CRS surface became dense and aggregated to form a thick and compact biofilm. Corrosion of CRS was closely related to formation of biofilm. The later corrosion was obviously inhibited by BS when the compact biofilms were fully formed although the initial corrosion was accelerated because BS produced organic acid in their physiological activities.

Acknowledgement This work was financially supported by Chinese Natural Science Foundation under the Grant Nos. 51361028, 51161025, 21162036, and 20762014.

Please cite this article in press as: Q. Qu et al., Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2, Corros. Sci. (2014), http://dx.doi.org/10.1016/j.corsci.2014.11.032

Q. Qu et al. / Corrosion Science xxx (2014) xxx–xxx

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Please cite this article in press as: Q. Qu et al., Corrosion behavior of cold rolled steel in artificial seawater in the presence of Bacillus subtilis C2, Corros. Sci. (2014), http://dx.doi.org/10.1016/j.corsci.2014.11.032