Effects of Ag and Cu ions on the microbial corrosion of 316L stainless steel in the presence of Desulfovibrio sp.

Bioelectrochemistry 110 (2016) 91–99

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Effects of Ag and Cu ions on the microbial corrosion of 316L stainless steel in the presence of Desulfovibrio sp. Tuba Unsal a, Esra Ilhan-Sungur a,⁎, Simge Arkan a, Nurhan Cansever b a b

Istanbul University, Faculty of Science, Department of Biology, 34134 Vezneciler, Istanbul, Turkey Yıldız Technical University, Faculty of Chemistry–Metallurgy, Metallurgical and Materials Engineering Department, 34210 Esenler, Istanbul, Turkey

a r t i c l e

i n f o

Article history: Received 14 December 2015 Received in revised form 22 March 2016 Accepted 26 March 2016 Available online 11 April 2016 Keywords: 316L stainless steel Microbiologically influenced corrosion Desulfovibrio sp. Biofilms Electrochemical tests Ag and Cu ions

a b s t r a c t The utilization of Ag and Cu ions to prevent both microbial corrosion and biofilm formation has recently increased. The emphasis of this study lies on the effects of Ag and Cu ions on the microbial corrosion of 316L stainless steel (SS) induced by Desulfovibrio sp. Electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization were used to analyze the corrosion behavior. The biofilm formation, corrosion products and Ag and Cu ions on the surfaces were investigated using scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS) and elemental mapping. Through circuit modeling, EIS results were used to interpret the physicoelectric interactions between the electrode, biofilm and culture interfaces. EIS results indicated that the metabolic activity of Desulfovibrio sp. accelerated the corrosion rate of SS in both conditions with and without ions. However, due to the retardation in the growth of Desulfovibrio sp. in the presence of Ag and Cu ions, significant decrease in corrosion rate was observed in the culture with the ions. In addition, SEM and EIS analyses revealed that the presence of the ions leads to the formation on the SS of a biofilm with different structure and morphology. Elemental analysis with EDS detected mainly sulfide- and phosphorous-based corrosion products on the surfaces. © 2016 Elsevier B.V. All rights reserved.

1. Introduction A cooling tower is a heat exchanger that provides water stream at lower temperatures to industrial systems through the release of waste heat into the atmosphere. It is an essential part of an industrial system; especially, open-circuit cooling towers provide ideal conditions with the recirculation of water, different pH and temperature zones, and a suitable nutrient balance for the existence and growth of microorganisms [1]. The bacteria, fungi, and algae that enter into the cooling systems through the water and the air in contact with water lead to biofilm formation on the internal surfaces of cooling towers. Once adhered to the surface, bacteria begin to grow and produce extracellular polymeric substances (EPS), promoting the structure of biofilm [2,3]. The developing biofilm allows for the formation of different oxygen gradients which promote the growth of anaerobic sulfate-reducing bacteria (SRB) in fully aerated systems such as cooling towers [1,4]. SRB are considered the major bacterial group involved in microbiologically induced corrosion (MIC) and are frequently isolated from cooling towers [5–7]. Desulfovibrio sp. is particularly one of the most abundant and dominant genera of SRB in cooling tower water [8]. MIC is a corrosion process influenced by the metabolic activity of microorganisms. The main factors that affect corrosion are the variety of ⁎ Corresponding author. E-mail address: [email protected] (E. Ilhan-Sungur).

http://dx.doi.org/10.1016/j.bioelechem.2016.03.008 1567-5394/© 2016 Elsevier B.V. All rights reserved.

different corrosion mechanisms. The mechanisms proposed to explain anaerobic MIC by SRB include: cathodic depolarization [9], precipitation of iron sulfide, which can induce cathodic depolarization [10–12], anodic depolarization resulting from local acidification at the anode [13], metal ion chelating by (EPS) [14], and galvanic coupling with EPS [15]. SRB cause serious corrosion of various metal structures in cooling towers, such as circulation pipes, the water basin, and heat exchangers [16–19]. Cooling towers are available in a wide variety of configurations and materials to suit the usage purpose of the industrial system. 316L stainless steel (SS) is the most preferred material in cooling tower systems, especially in areas where water accumulates, such as the cold-water basin, owing to its excellent corrosion resistance. The improved level of corrosion resistance in 316L SS is due to the formation of a chromium oxide film layer (passivation layer) on the surface of the metal as a result of the chromium and nickel content, and the presence of molybdenum [20,21]. However, cooling water conditions and the microbial activity of SRB may cause severe corrosion that result in structural failures of the SS systems. In recent years, the Ag and Cu ionization system has been used as an alternative to biocides to prevent both microbial corrosion and the formation of biofilm in order to improve water systems. Although it is a more effective and environmentally friendly method of improving water systems, the usage of Ag and Cu ionization technology is less preferred because of the simplicity of the application of chemicals.

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However, in contrast to Ag and Cu ionization technology, using chemicals can cause health, environmental, and corrosion problems. It has been reported that Ag and Cu ions deactivate the bacteria as a result of their synergistic effect [22,23]: while Cu ions weaken cell membranes, Ag ions bind the cellular protein of bacteria cells [24]. Studies featuring Ag and Cu ions have generally focused on the biocidal effect of ions on microorganisms. However, no data has been found concerning the effect of Ag and Cu ions on the microbial corrosion behavior of 316L SS in the presence of Desulfovibrio sp. The aims of the present study were to establish the effects of Ag and Cu ions on the MIC of 316L SS by using electrochemical techniques and also to determine the corrosion behavior of 316L SS in the presence of Ag and Cu ions. 2. Material and methods 2.1. Preparation of specimens The SS specimens, with dimensions of 25 × 25 × 1 mm, were cut from a 316L SS sheet; the nominal elemental composition (%, by mass) of the specimens was: C 0.022, Cr 16.02, Ni 11.44, Mo 1.95, Mn 0.984, Si 0.382, P 0.035, S 0.010. The coupons were abraded through 240, 320, 400, 600 and 800-grit silicon carbide sandpaper, polished with aluminum oxide, washed with sterile distilled water, dried, and kept in a desiccator until use. For electrochemical measurements, the coupons were protected with silicon to leave a 1-cm2 surface in contact with the medium, then immersed in ethanol, and dried with hot air [25]. 2.2. Medium and test conditions The study was performed using a pure culture of the strain Desulfovibrio sp., isolated from cooling tower water in our previous study [8]. All experiments were carried out in sterile Postgate's medium C (PC) and pure Desulfovibrio sp. culture with and without Ag and Cu ions (respectively 0.13 ppm and 0.3 ppm). The PC medium consisted of sodium lactate (6.0 g L−1), KH2PO4 (0.5 g L−1), NH4Cl (1.0 g L−1), Na2SO4 (4.5 g L− 1), CaCl2·6H2O (0.06 g L− 1), MgSO4·7H2O (0.06 g L−1), yeast extract (1.0 g L− 1), resazurin (0.001 g L− 1), FeSO4·7H2O (0.004 g L−1), C2H3O2SNa (0.1 g L−1), C6H5O7Na3·2H2O (0.3 g L−1) [26]. The pH was adjusted to 7.2 by 10% NaOH. C2H3O2SNa and FeSO4·7H2O were added after boiling the medium, passing it through 99.9% N2 gas. The medium was sterilized in an autoclave for 20 min at 121 °C and then cooled in a glove-box system. For preparation of the Desulfovibrio sp. culture before the experiments, the PC medium (800 ml) was inoculated with appropriate volumes (10%) of 1-day-old Desulfovibrio sp. culture resulting in an initial concentration of 25 × 106 per ml [27]. The final concentrations of Cu and Ag ions were adjusted to 0.3 ppm and 0.13 ppm in the sterile PC medium and the 1-day-old Desulfovibrio sp. culture, respectively. The experiments were carried out under anaerobic conditions in the glovebox system at 30 °C.

electrochemical impedance spectroscopy (EIS). EIS offers the advantage of providing enough insight on the formation and protection mechanisms of a given surface/biofilm layer. All electrochemical tests were carried out in a conventional electrochemical cell, with a carbon rod as the counter electrode, a saturated calomel electrode (SCE) as a reference electrode, and the prepared samples as the working electrode. The working solutions were mixed with a magnetic stirrer (150 rpm) and all of the measurements were performed at 30 °C. Square-shaped 316L SS coupons with a top surface area of 1 cm2 were exposed to the bacterial culture and sterile medium with and without Ag and Cu ions. The potentiodynamic polarization curves were obtained at a scanning rate of 1 mV/s between −800 mV and +1700 mV. The corrosion rate (Vcorr) values were derived from corrosion current density (icorr) over time. The EIS measurements were obtained from the instantaneous open circuit potential, scanning the frequency range from 105 Hz to 5 × 10−2 Hz, applying 5 mV of amplitude. Electrochemical measurements were carried out at certain time intervals (8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, 240 h, 360 h, 480 h, 600 h and 720 h). 2.5. Surface characterization Biofilm formation and corrosion products formed on the specimens were analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS) at the end of the experiment. The specimens were fixed with 2.5% glutaraldehyde, followed by dehydration in a graded series of ethanol and air drying [28]. The dried samples were coated with gold and imaged with a ZEISS LS10 SEM. Chemical analyses and distribution of the Ag and Cu ions on the surfaces were done by EDS and elemental mapping. 3. Results and discussion 3.1. Corrosion behavior of 316L SS in the Desulfovibrio sp. culture The 316L SS specimens were exposed to the sterile PC medium and Desulfovibrio sp. culture for 720 h. Potentiodynamic polarization curves of the specimens for 8 h, 144 h, and 720 h of exposure are shown in Fig. 1. It was observed that the corrosion behavior of 316L SS changed after the addition of Desulfovibrio sp. to the medium. While the Ecorr value of 316L SS in the sterile PC medium was − 0.651 V (vs. SCE) after 8 h, it shifted to noble values in the presence of Desulfovibrio sp. after 8 h of exposure and was detected as being − 0.333 V (vs. SCE) (Fig. 1). The shift in Ecorr to more positive values could be related to the growth of Desulfovibrio sp. and biofilm formation on the 316L SS specimens, as reported by Alabbas et al. [29]. Then variations in the Ecorr values were observed (Fig. 2), indicating the effect of the presence

2.3. Generation of Ag and Cu ions Ag and Cu ions were produced electrolytically by applying a voltage of 17 V via two electrodes in deionized water employing potentiostat equipment (LINK, AC-50). The electrodes were constructed from Ag and Cu plates (99.9% purity) 38 mm thick. The electrode areas were 1.133 mm2. The solutions of Ag and Cu ions were analyzed immediately after generation and used freshly. 2.4. Electrochemical measurements The electrochemical corrosion tests were performed with a computer-controlled testing device (Gamry-Interface1000, USA) by measuring the potentiodynamic polarization curves and

Fig. 1. Polarization curves of the 316L SS specimens exposed to the sterile PC medium for ) and Desulfovibrio sp. culture for 8 h ( ), 144 h ( ) and 720 h ( ). 8h(

T. Unsal et al. / Bioelectrochemistry 110 (2016) 91–99

Fig. 2. Ecorr values of the 316L SS coupons exposed to the sterile PC medium and Desulfovibrio sp. culture in the presence and absence of Ag and Cu ions over experiment: ) and with ( ) Ag and Cu ions; Desulfovibrio sp. Sterile PC medium without ( ) and with ( ) Ag and Cu ions. culture without (

of Desulfovibrio sp. on the corrosion process. This effect could be related to the biofilm formation, the activity and growth of Desulfovibrio sp., and to the production of some metabolites (fatty acids, H2S, etc.) affecting electrochemical behavior on the metal surface [30]. Fig. 1 also shows that the anodic current density increased significantly and the width of the passive range increased in the Desulfovibrio sp. culture with time. The widths of the passive ranges in the bacterial culture after 8 h, 144 h, and 720 h were 0.983 V (− 0.147 to 1.130 V (vs. SCE)), 1.053 V (− 0.171 to 1.223 V (vs. SCE)), and 1.306 V (− 0.134 to 1.44 V (vs. SCE)), respectively. However, the width of the passive range decreased according to the sterile PC medium. These results indicate that the passive film on the metal surface was changed by Desulfovibrio sp. over time. The performance of the passive film could be changed by the sulfide produced by SRB. Marcus [31] reported that the sulfide can attenuate the binding force of the metal-to-metal bonds on the surface, resulting in the increment of the anodic dissolution rate of metal. The corrosion rate plots obtained from icorr over time for the sterile PC medium and Desulfovibrio sp. culture are shown in Fig. 3. Vcorr values show that the corrosion rate of the specimens increased in the presence of Desulfovibrio sp. when compared to the sterile PC medium and a maximum value of 0.119 mm·y−1 after 144 h was detected (Fig. 3). Arkan et al. [32] reported in their study, which was conducted in a closed lab-scaled test system with the same experimental conditions, that the maximum corrosion rate of the coupons was detected after 144 h of exposure time and there was a significant relationship between the corrosion rate of the coupons and the protein amount of the EPS. This might be caused by the fact that the iron used by the hydrogenase enzymes of the SRB is supplied by the corrosion of the metal. The dissolved iron from the surface increases the synthesis of hydrogenase enzyme, and more synthesized hydrogenase enzyme

Fig. 3. Corrosion rates of the 316L SS coupons exposed to the sterile PC medium and Desulfovibrio sp. culture in the presence and absence of Ag and Cu ions over experiment: ) and with ( ) Ag and Cu ions; Desulfovibrio sp. Sterile PC medium without ( ) and with ( ) Ag and Cu ions. culture without (

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increases the metabolic activity of the bacteria, thereby encouraging the corrosion rate as reported by Lata et al. [33] and Da Silva et al. [34]. The Nyquist diagrams of the 316L SS specimens exposed to the sterile PC medium and Desulfovibrio sp. culture as a function of immersion time are given in Fig. 4. The Nyquist plot of specimens in the sterile PC medium indicates a capacitive behavior and thus high corrosion resistance of the 316L SS specimens. The maximum phase angle for the sterile PC medium was 76.9 in a frequency range of 1 to 100 Hz after 8 h. The maximum angle appearing in the frequency range 1 to 100 Hz was ascribed to the formation of a protective film [35]. As seen in the Nyquist plots, the shape of the diagram gets closer with time to a real semicircle, in particular for specimens exposed to Desulfovibrio sp. for 720 h. The corrosion behavior of a 316L SS/biofilm system is typical of SS in the passive state, as reported by Sheng et al. [36] and Xu et al. [37]. The Bode phase angle plots show marked changes after different exposure periods due to the effect of Desulfovibrio sp. on the passive film. While the total impedance magnitude decreased up to 144 h, indicating the breakdown of the passive film on the SS surface, an increase was observed in the Bode magnitude plot after 720 h (Fig. 4). The Vcorr and EIS results indicate that the colonization and metabolic

Fig. 4. Impedance spectra of the 316L SS coupons exposed to the sterile PC medium for 8 h ), 144 h ( ) and 720 h ( ). Electrical ( ) and Desulfovibrio sp. culture for 8 h ( circuit diagrams suggested for electrode/solution interfaces in the absence (a) and presence (b) of the Desulfovibrio sp. after 720 h.

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activity of Desulfovibrio sp. can cause the modification of the chemical composition of passive film and/or breakdown of the film, resulting in acceleration of the corrosion process. The maximum phase angles for 8 h and 144 h were 66 and 64.7 in the low-frequency range of 0.1 to 100 Hz, respectively, which was probably associated with the inhomogeneity of the surface (Fig. 4). At 720 h, the maximum phase angle appeared in the high-frequency range of 100 to 10,000 Hz, indicating the presence of the pores in the biofilm (Fig. 4). The SEM micrographs validated that the biofilm layer formed by the Desulfovibrio sp. was heterogeneous and porous covering all the surface of the 316L SS specimen (Fig. 5). Also, it was observed that the bacteria were embedded in their own EPS. Kramers–Kronig (KK) transformations were performed to validate the EIS experimental data. The values of goodness of fit were provided by software and determined by chi-squared value [38]. The chi-square method calculates the standard deviation between the original data and the calculated spectrum. The chi-square gives a good indication of the quality of the fit: if data without variances are used a value of 10−5 for chi-square indicates a reasonable to good fit [39]. The value of chi-square variance of the measurements was in the range of 10−3 to 10−4. The values of goodness of fit for Desulfovibrio sp. determined after 8 h, 144 h, and 720 h were around 10− 4, and the values fall by the middle of the determined range [40]. The EIS results were further analyzed by fitting them with appropriate equivalent electrical circuits. Fig. 4 shows the equivalent circuit representation for the sterile PC medium and Desulfovibrio sp. culture. Since the bulk solution/electrical double layer and the electrical double layer/ electrode interface does not behave as an ideal capacitor in the presence of the dispersing effect, in the present study a constant phase element (CPE) was used as a substitute for the capacitor in the equivalent circuit to fit the EIS data more accurately. The CPE defined in the usual impedance format is: ZCPE ¼ 1=Y 0 ðjωÞn :

ð1Þ

In this equation, j is the imaginary root, ω (= 2πf) is the angular frequency. The factor n is a dimensionless parameter that lies between 0 and 1, which is correlated with the surface inhomogeneity. The factor Y0 denotes a parameter related to the capacitance. In Fig. 4a, the circuit includes: (1) resistance Rs considered as the solution resistance, (2) the parallel combination of a charge transfer resistance (Rct) and CPE1 associated with 316L SS surface double layer capacitance, (3) the parallel combination of a film resistance (Rf) and CPE2 associated with 316L SS surface conditioning film. In Fig. 4b, the circuit includes: (1) Rs considered as the solution resistance, (2) the parallel combination of a pore resistance (Rpo) including Rct and CPE1, (3) the parallel combination of biofilm resistance (Rb)

together with an infinite Warburg element (W) and CPE2 associated with the biofilm layer including corrosion products on the 316L SS surface. According to the impedance results, different corrosion behavior was observed in different media. In the case of the sterile PC medium, Rf related to the resistance of the film formed on the surface had a high value (2666 Ω cm2) after 8 h of exposure, in contrast to the biotic conditions (Table 1), indicating a low corrosion rate, possibly due to the effect of phosphate salt in the medium, as reported by Duan et al. [41]. In the presence of Desulfovibrio sp., Rb and Rpo values increased over time, detected as being 173.2 Ω cm2 and 229.3 Ω cm2 after 720 h (Table 1). The increase in impedance after 720 h testifies for the protective effect of the biofilm and/or the corrosion products [42]. However, according to the Bode curves, there appears to be a layer of porous biofilm on the 316L SS surface for Desulfovibrio sp. (Fig. 4). It is indicative that the biofilm with pores can permit easy access of aggressive ions to the metal surface and accelerate corrosion as time progresses [36]. Fig. 6 shows the EDS analysis results of the corrosion products on the surface of the 316L SS. S content on the 316L SS exposed to the sterile PC medium was observed (Fig. 6a), assumed to have originated from the medium content. In the presence of Desulfovibrio sp., the corrosion products on the 316L SS surface were mainly composed of Fe, Cr, P, Ni, S, and O (Fig. 6b). The appearance of the S peak is due to the presence of FeS formed as a result of Desulfovibrio sp. metabolic activities. It is well known that higher concentrations of FeS in corroded products indicate the influence of SRB in the corrosion processes [43].

3.2. Corrosion behavior of 316 L SS in the Desulfovibrio sp. culture with Ag and Cu ions Ag ions are poisonous to most microorganisms in trace amounts [44]. Cu has been used as an algaecide for many years and is reported to be one of the most toxic metals to heterotrophic bacteria in aquatic environments [45]. Further, Ag+ and Cu2+ show a synergistic disinfection effect [22]. In this study, the effects of electrolytically generated Ag and Cu ions on the MIC of 316L SS in the presence of Desulfovibrio sp. were investigated. The potentiodynamic polarization curves of the specimens in the presence of Ag and Cu ions for 8 h, 144 h and 720 h of exposure are shown in Fig. 7. The passive anodic current density in the culture with the ions increased and the Ecorr values shifted to more active values according to the sterile PC medium with the ions (Fig. 7). The addition of Ag and Cu ions to the Desulfovibrio sp. culture shifted the Ecorr value of the specimen to more active values after 8 h of exposure compared to the ion-free culture, and was detected as being − 0.510 V (vs. SCE),

Fig. 5. SEM micrographs of the biofilm formed on the 316L SS surfaces exposed to the Desulfovibrio sp. culture. a) bar = 50 μm, b) bar = 2 μm.

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Table 1 Electrochemical parameters obtained from the EIS results. Time (h)

8h

720 h

Solutions

Sterile PC PC+ Ag and Cu ions Dsv. Dsv.+ Ag and Cu ions Dsv. Dsv.+ Ag and Cu ions

Rs (Ω cm2)

33.35 24.40 14.29 39.36 58.26 22.11

Rf (Ω cm2)

2666 1003 – – – –

Rb (Ω cm2)

– – 99.13 8476 173.2 4516

Rct X102 (Ω cm2)

2081 569.2 – – – –

Rpo (Ω cm2)

– – 32.95 38.64 229.3 18.81

W X10−3 (Ω cm2)

– – 3.99 5.14 2.47 21.66

CPE1

CPE2

Y01 (μF cm−2)

n1

Y02 (μF cm−2)

n2

22.64 73.86 19.58 726 2.49 816.3

0.99 0.98 0.96 0.96 0.99 0.96

18.6 44.08 26.28 1.128 41.84 292.6

0.98 0.97 0.98 0.99 0.98 0.99

PC: Postgate C medium; Dsv.: Desulfovibrio sp.; Rs: resistance of the solution; Rf: resistance of the film; Rb: resistance of the biofilm; Rct: resistance of charge transfer; Rpo: resistance of pore; W: Warburg element; CPE1: constant phase element associated with 316 L SS surface double layer capacitance; CPE2: constant phase element associated with 316 L SS surface conditioning film; Y01,n1–Y02,n2: characteristic parameters of CPE1 and CPE2, respectively.

but then the Ecorr values were observed to be more noble values than the culture without the ions (Fig. 2). Similar passivation behavior was observed for the 316L SS specimens exposed to the culture and the sterile PC medium with the ions after 720 h. However, the addition of Ag and Cu ions to the Desulfovibrio sp. culture led to a decrease in the passive anodic current density of the 316L SS after 720 h (Fig. 7). Arkan et al. [32] reported that Ag and Cu ions encouraged the production of EPS by Desulfovibrio sp. on 316L SS surfaces. Thus, it is possible that the EPS could have supported the protective effect of the biofilm against corrosion by covering all the surface of the 316L SS. Another possibility is that the addition of Ag and Cu ions to

the Desulfovibrio sp. culture could have led to the formation of a biofilm with a different structure, which included corrosion products and the ions on the 316L SS, and could have affected the biofilm's protective feature. The corrosion rate of the specimens in the bacterial culture with the ions decreased according to the ions-free culture, and no major changes were observed during the exposure times. The maximum Vcorr values were detected as being 0.039 mm·y−1 after 72 h and 360 h (Fig. 3). Fig. 8 shows EIS results for 8 h, 144 h and 720 h of exposure. The values of goodness of fit for Desulfovibrio sp. with Ag and Cu ions determined after 8 h, 144 h, and 720 h were 1.236 × 10−4, 0.539 × 10−4, and

Fig. 6. SEM micrographs and EDS analysis of the corrosion products formed on the 316L SS surfaces. a) The control coupon exposed to the sterile PC medium, b) the test coupon exposed to the Desulfovibrio sp. culture.

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Fig. 7. Polarization curves of the 316L SS specimens exposed to the sterile PC medium for ) and Desulfovibrio sp. culture for 8 h ( ), 144 h ( ) and 720 h ( ) in 8h( the presence of Ag and Cu ions.

Fig. 8. Impedance spectra of the 316L SS coupons exposed to the sterile PC medium for 8 h ( ) and Desulfovibrio sp. culture for 8 h ( ), 144 h ( ) and 720 h ( ) in the presence of Ag and Cu ions. Electrical circuit diagrams suggested for electrode/solution interfaces in the absence (a) and presence (b) of the Desulfovibrio sp. in the presence of Ag and Cu ions after 720 h.

7.214 × 10−4, respectively, and these values fall by the middle of the determined range [40]. According to the Nyquist plots, the Ag and Cu ions led to a reduction in the resistance of the 316L SS in the sterile PC medium compared to the ion-free medium (Fig. 8). This is an indication of disrupting the stability of the layer formed on the 316L SS by Ag and Cu ions. The maximum phase angle for the sterile PC medium with the ions was 72.4 in the frequency of 10 to 100 Hz after 8 h of exposure. The smaller phase angle value is a sign of the deviation from the ideal semicircle and surface roughness [46]. Such non-uniformity could be related to the formation of the film layer on the surface, including ions accumulated from the medium and/or corrosion products. The shapes of the Nyquist curves in the culture with Ag and Cu ions indicate a capacitive behavior and thus a high resistance of the 316L SS specimens, based on the large-diameter semicircle, as reported by Moreno et al. [47] (Fig. 8). The Bode magnitude plot reveals that the modulus of the impedance was similar at 8 h and 144 h. However, an increase was established in the total impedance magnitude for 720 h. The changes in total impedance magnitude and spectra depending on time were different to those obtained from the ion-free culture (Figs. 4, 8). It was detected that the phase angles for the culture with Ag and Cu ions increased over time, indicative of the protective ability of the biofilm. The maximum phase angle was 64.7 in the low-frequency range of 0.1 to 1 Hz for 720 h, which was probably associated with the inhomogeneity of the biofilm (Fig. 8). SEM images show that the structures of the biofilms were heterogeneous, porous and fissured in the presence of Ag and Cu ions (Fig. 9). The cluster formation is a natural response of bacteria to avoid exposure to toxicity by reducing the total surface area in contact with the environment [48]. Similar observations have reported that aggregated bacteria are less sensitive to toxicants in solutions containing biocide than the same bacteria growing in dispersion [49]. Fig. 8 shows the equivalent circuits for the 316L SS in the sterile PC medium and culture with the ions. A different circuit was observed in the culture compared to the sterile medium. The fitting parameters of the EIS for 316L SS were given in Table 1. In Fig. 8a, the circuit includes: (1) Rs considered as the solution resistance, (2) the parallel combination of a charge transfer resistance (Rct) and CPE1 associated with 316L SS surface double layer capacitance, (3) the parallel combination of a film resistance (Rf) and CPE2 associated with the 316L SS surface conditioning film. In Fig. 8b, the circuit includes: (1) Rs considered as the solution resistance, (2) the parallel combination of Rpo and CPE1 associated with the formation of a heterogeneous layer composed of corrosion products and Ag and Cu ions along with other compounds deposited from the growth media, (3) the parallel combination of Rb together with a finite Warburg element (W) and CPE2 associated with the biofilm layer on the 316L SS surface. Although in the presence of Ag and Cu ions Rb decreased over time, it was observed that it reached a value (4516 Ω cm2 ) 26 times higher than that measured of that in the culture without the ions after 720 h (Table 1). This indicates that the layer formed in the presence of ions was more protective than that formed in the ion-free culture. Indeed, the corrosion rate of SS decreased in the Desulfovibrio sp. culture with ions when compared to the ion-free culture (Fig. 3). It was also detected that the addition of the Ag and Cu ions into the culture leads to decreased current density of the 316 SS after 720 h (Fig 7). This was possibly due to the negative effect of Ag and Cu ions on the growth of Desulfovibrio sp. During the experiment, it was observed that after the addition of the ions to the 1day-old Desulfovibrio sp. culture, the black color of the medium turned into a cloudy grayish color, indicative of the retardation of the growth of Desulfovibrio sp. caused by these ions. A high Warburg value in the culture with Ag and Cu ions indicates that the corrosion process is under diffusion control. Also, the small loop observed in the high-frequency region of the Nyquist curve validates that the protective ability of the biofilm decreases over time, as reported by Wang et al. [50].

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Fig. 9. SEM micrographs of the biofilm formed on the 316L SS surfaces exposed to the Desulfovibrio sp. culture with Ag and Cu ions. a) bar = 100 μm, b) bar = 10 μm.

EDS and elemental mapping analyses of the coupons exposed to the sterile PC medium and culture with Ag and Cu ions were illustrated in Figs. 10 and 11. The corrosion products on the SS coupons exposed to the culture primarily contained Fe, O, Cr, P, S, and Ni. Other minor elements were present, namely Ca, Ag, and Cu. While in the sterile PC medium, the S peak was very small (Fig. 10b), a large S peak was noticed in the EDS spectrum of the culture. This significantly increased S peak probably indicates that the surface contains a microbial corrosion product FeS. The P peak could be related to Fe3P appeared as corrosion products in addition to FeS as reported

by Iverson and Olson [51] (Fig. 11b). Fe3P is formed by the reaction of iron with a highly active volatile phosphorus compound. This volatile phosphorus compound could be produced by Desulfovibrio sp. activity or by the reaction of hydrogen sulfide with inorganic phosphorus compounds in the environment. When a protective FeS layer does not break down, the volatile phosphorus compound acts on the steel surface and causes the corrosion of iron. Elemental mapping reveals low amounts of Ag and Cu on the corrosion products, and it was also observed that the distributions of Ag and Cu ions on the surfaces of the biofilms were similar (Fig. 11 c-d).

Fig. 10. Corrosion products deposited on the 316L SS surface in the Desulfovibrio sp. culture with Ag and Cu ions after 720 h of exposure, (a) SEM image, (b) EDS analysis of the corrosion products and (c–d) elemental mapping of Ag and Cu.

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Fig. 11. Corrosion products deposited on the 316L SS surface in the sterile PC medium with Ag and Cu ions after 720 h of exposure, (a) SEM image, (b) EDS analysis of the corrosion products and (c–d) elemental mapping of Ag and Cu ions.

4. Conclusions SEM, EDS and elemental mapping analysis were combined with potentiodynamic polarization and circuit modeling by EIS to clarify the differences in microbial corrosion of 316L SS induced by Desulfovibrio sp. in the presence and absence of Ag and Cu ions. The experiment results are summarized as follows: • The biofilms formed by Desulfovibrio sp. in the presence and absence of Ag and Cu ions show different morphology and polarization resistance. • The corrosion rate of 316L SS increase in the presence of Desulfovibrio sp. • Ag and Cu ions negatively affect the growth of Desulfovibrio sp. • Ag and Cu ions lead to a decrease in the corrosion rate of 316L SS coupons by Desulfovibrio sp. • The equivalent electrical circuit diagrams reveal that the morphology, structure and stratification of the biofilms on 316L SS are different in the presence and absence of Ag and Cu ions.

Acknowledgments Ag and Cu ionization system was provided by the LINK Technologies Ltd. This project was supported by the Research Fund of Istanbul University (Project Numbers: 41046 and UDP-44789).

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