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A comparative study of corrosion of 316L stainless steel in biotic and abiotic sulfide environments
International Biodeterioration & Biodegradation 120 (2017) 91e96
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
A comparative study of corrosion of 316L stainless steel in biotic and abiotic sulfide environments Shiqiang Chen a, Y. Frank Cheng a, *, Gerrit Voordouw b a b
Department of Mechanical & Manufacturing Engineering, University of Calgary, Calgary, Alberta, T2N 1N4, Canada Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 September 2016 Received in revised form 18 January 2017 Accepted 10 February 2017
Electrochemical corrosion of 316L stainless steel in a biotic sulfate-reducing bacteria (SRB) culturing medium and an abiotic sulfide sterile solution was investigated by biotesting, cyclic voltammetry and surface characterization techniques. It was attempted to determine if the abiotic sulfide solution is capable of reproducing the corrosive environment that is comparable to that in the biotic SRB medium. Results demonstrated that sulfides generated from SRB metabolism result in a heterogeneous iron sulfide film on the steel surface. Corrosion occurs at locations where sulfides accumulate, increasing the surface roughness of the steel. Like those generated in the biotic medium, sulfides formed in the sterile medium can increase the steel corrosion. However, the generated sulfide film is more uniform, compact and stable than that formed in the SRB medium. Moreover, the steel possesses different cyclic voltammetry behavior. The prepared abiotic sulfide solution is not representative of the SRB medium for corrosion studies. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Corrosion 316L stainless steel Sulfate reducing bacteria Biotic bacterium culturing medium Abiotic sulfide solution
1. Introduction Microbiologically influenced corrosion (MIC) has been one of primary mechanisms resulting in pipeline failures in petroleum industry (AlAbbas et al., 2013; Maruthamuthu et al., 2011; Usher et al., 2014; Videla and Herrera, 2009). In particular, sulfate reducing bacteria (SRB) are identified as main microorganisms contained in highly corrosive environments to generate metabolites (e.g., sulfides, extracellular polymeric substances (EPS), etc.), contributing to metallic MIC (Little et al., 2006; Enning and Garrelfs, 2014; Zapata-Penasco et al., 2016) and even stress corrosion cracking (Javaherdashti et al., 2006). Stainless steels have been used to make oilfield devices due to their good machinability and excellent corrosion resistance. Generally, the resistance of stainless steels, such as 316L stainless steel, to corrosion is due to the formation of a layer of passive film, which is usually composed of an inner chromium oxide layer and an outer mixed iron oxide and hydroxide layer, on the steel surface (Macdonald, 1992). However, the steel is prone to suffer from severe corrosion in the presence of SRB in the service environment
* Corresponding author. E-mail address: [email protected] (Y. Frank Cheng). http://dx.doi.org/10.1016/j.ibiod.2017.02.014 0964-8305/© 2017 Elsevier Ltd. All rights reserved.
(Bachmann and Edyvean, 2006; Sheng et al., 2007; Unsal et al., 2016). There has been much work conducted to investigate corrosion of 316L stainless steel in SRB-containing environments. It was found that the metabolic activity of SRB is able to accelerate corrosion of the steel (Unsal et al., 2016). The SRB result in generation of iron sulfide film on the steel surface. The sulfides are the metabolites of SRB, and are highly corrosive. In addition to cause corrosion of steels that are in active dissolution state in environments, they can result in breakdown of the passive film formed on passivated steels, affecting the further corrosion and localized corrosion (Duan et al., 2006; He et al., 2009). As the presence of sulfides in the environment is critical to steel corrosion, extensive work has been performed to investigate MIC using artificially prepared, abiotic sulfide solutions (Cord-Ruwisch, 1985; Mendili et al., 2013; Sherar et al., 2011). However, it has been questionable whether an abiotic sulfide solution is representative of the corrosive environment associated with the biotic SRB medium. As a result, the essential difference of the steel corrosion in biotic and abiotic sulfide environments is yet understood mechanistically. In this work, a comparative study of electrochemical corrosion of 316L stainless steel was conducted in a biotic SRB culturing medium and a sulfide-containing sterile solution, respectively, by cyclic voltammetry and various surface analysis techniques,
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including confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), X-ray energy dispersive spectrometer (EDS) and atomic force microscopy (AFM). Both electrochemical behavior and corrosion product film formed during the steel corrosion were analyzed in both media. It is anticipated that this work provides an essential insight into the corrosivity of the biotic SRB culturing medium and the sulfide-containing sterile solution, and the mechanism of steel corrosion in both media. 2. Experimental 2.1. Material Specimens were cut from a 316L stainless steel plate, with a chemical composition (wt.%): 0.019 C, 1.18 Mn, 0.48 Si, 16.72 Cr, 12.10 Ni, 2.05 Mo, 0.038 P, 0.0013 S, 0.10 N and balance Fe. The dimension of the specimen was 1 cm 1 cm 0.3 cm. The steel coupons were embedded in epoxy resin, leaving a working area of 1 cm2. The exposed face of the specimen was sequentially ground with silicon carbide emery papers up to #1200 grit, and was then rinsed with deionized water, degreased with absolute ethyl alcohol, and dried with pure nitrogen. 2.2. Bacterial culturing The SRB used in this work were extracted from oil sludge collected from an oil well in Alberta, Canada. The 16S rRNA genes were amplified by polymerase chain reaction (PCR), and sequenced using Illumina Miseq in order to identify the SRB extracted from the oil sludge sample (Sharma et al., 2016). The sequencing results showed that the dominant SRB were Desulfomicrobium. The modified coleville synthetic brine culture solution was used for SRB growth. The solution was prepared by the following procedure. Chemicals including 0.05 g KH2PO4, 0.32 g NH4Cl, 0.21 g CaCl2, 0.54 g MgSO4, 1.42 g Na2SO4 and 1.36 g sodium formate were added to 1 L of ultrapure water, and the sealed mixture was autoclaved at 121 C for 20 min. After cooling down in air to ambient temperature, the culture medium was purged with N2/CO2 (9:1) gas for 20 min to remove oxygen until the content of dissolved oxygen was below the detection limit of 0.001 mg/L of a dissolved oxygen meter (ExStik DO600). The 30 mL NaHCO3 (1 M) and 1 mL selenite were added to the medium using a 0.22 mm filter for sterilization. The pH of the prepared culture medium was adjusted to 7.2 ± 0.1 using 1 M HCl solution. The extracted SRB were then added in the medium for growth. The growth curve of SRB was measured using turbidimetry at 600 nm with an ultraviolet and visible light spectrophotometer (UV-1800, SHIMADZU). The concentration of sulfide, CS2-, was measured using the methylene blue method (Cord-Ruwisch, 1985). The artificial sulfide solution was prepared by adding 2 mM Na2S solution in the sterile culture solution mentioned above.
2.4. Morphological and compositional characterization Bacterium attachment and biofilm formation on the 316L stainless steel surface were observed by a CLSM. After immersion in SRB medium and sulfide solution for 7 days, the steel specimens were washed with ultrapure water, and were then stained with a fluorescent dye (Molecular Probes™ FilmTracer™ LIVE/DEAD® Biofilm Viability Kit) in the darkness according to the manufacturers' procedure. All these procedures were conducted in an anaerobic glove box. Similarly, after immersion in the SRB medium and the sulfide solution for 7 days, the steel specimens were washed with ultrapure water, and were then cleaned with absolute ethyl alcohol to remove bacteria and biofilm. The morphology of the steel electrode was characterized by a field emission SEM. Elemental maps were obtained through an EDS coupled with the SEM. An AFM (Keysight 5500) was used for topographic characterization of the steel specimen. A scanner carrying a long rectangular cantilever with a spring constant of 0.2 N/m (apex radius <10 nm) was placed above the specimen. The scanning mode was configured as contact, with a scanning rate of 1 Hz and a resolution of 512 512 pixel. A region of 5 mm 5 mm was selected for AFM characterization. The obtained images were processed to remove background signals, and the surface roughness was extracted. 3. Results and discussion 3.1. SRB growth curve Fig. 1 shows the growth curve of SRB in the biotic culturing medium, where the growth of SRB is composed of three stages. In stage one, i.e., in the first two days, the number of SRB keeps at a low level, which is called the adaptive stage. In this stage, SRB have a low metabolic activity, producing few metabolites. From day two to the eighth day, the number of SRB increase quickly with time. This stage is called the exponential phase, where the speed of the bacterium's reproduction is much greater than the rate they die. The SRB are featured with a vigorous metabolic activity, producing extensive metabolites accumulating in the culture medium. After the eighth day, the speed the bacterium multiplication is smaller than the death rate, and the number of SRB decreases with time. This stage is called the death phase, where the rate of bacterium death is greater than the production rate. To maintain a stable metabolic activity of SRB in this work, the maximum growth of bacteria at the exponential phase, i.e., the seventh to the eighth day,
2.3. Measurements of cyclic voltammograms CV measurements were conducted on a three-electrode cell using a Solartron 1280C electrochemical testing system. The 316L stainless steel electrode, a graphite sheet, and a saturated calomel electrode (SCE) were used as the working, counter and reference electrodes, respectively. After immersion of the steel electrode in the test solution for 1 h, the open-circuit potential of the steel reached a relatively stable value. The CV was measured with a potential range from 0 V (SCE) to 1.1 V (SCE) at a potential scanning rate of 2 mV/s. All measurements were performed at 25 ± 2 C. To ensure reproducibility of testing data, all tests were performed under controlled conditions at least three times.
Fig. 1. The growth curve of SRB in the prepared culturing medium.
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is chosen to study the SRB induced corrosion of 316L stainless steel. Fig. 2 shows the concentration of sulfides in the biotic SRB culture medium and the abiotic sulfide solution, respectively, as a function of time. It is seen that the sulfide concentration in both media maintain approximately stable at 2 mM during the testing period. 3.2. Cyclic voltammogram (CV) measurements Fig. 3 shows the CV curves measured on 316L stainless steel electrode in the sterile, sulfide-free culture medium as a function of time. One anodic current peak is observed at about 0.5 V(SCE) ~ 0.3 V(SCE), which is associated with the oxidation of Fe2þ to Fe3þ (Kocijan et al., 2007). The peak current density is almost constant at about 6 mA/cm2 after 1 day of testing. Other than the hydrogen evolution reaction occurring more negative than 0.9 V(SCE), there is no obvious cathodic current peak in the negative potential sweep direction. Fig. 4 shows the CV curves measured on the steel electrode in biotic SRB culture medium as a function of time. The weak oxidative current shoulder at about 0.5 V(SCE) after 2 h of immersion, as marked in the figure, is attributed to the oxidation of Fe2þ to Fe3þ,
Fig. 2. Concentrations of sulfides in the SRB culture medium and the sterile sulfide solution, respectively, as a function of time.
Fig. 3. CV curves measured on 316L stainless steel electrode in the sterile, sulfide-free medium, as a function of time.
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but this peak is not observed in the following days. In addition to the oxidative current peak for Fe2þ to Fe3þ and the hydrogen evolution reaction occurring more negative than 1.0 V(SCE), there are other two current peaks at about 0.2 ~ 0.1 V(SCE) and 0.9 V(SCE), respectively. The peak current density is much higher than that at about 0.5 V(SCE). Fig. 5 shows the CV curves measured on 316L stainless steel electrode in the sulfide-containing sterile medium as a function of time. There is also a pair of oxidative and reductive current peaks at 0.2 ~ 0.1 V(SCE) and 0.8 V(SCE), respectively, after 2 h of immersion. The current peak at 0.2 ~ 0.1 V(SCE) disappears in the following measurements. It was reported that the current peaks at these two potential ranges are attributed to oxidation and reduction of iron sulfide (Perini et al., 2013). The results in Figs. 4 and 5 indicate that, in the presence of sulfides in the solution, a layer of iron sulfide film can be formed on the steel surface. Moreover, the iron sulfide film can also be formed on the steel electrode in the biotic SRB culture medium because sulfides are generated from the SRB metabolism. Sulfides can attack passive films formed on metals such as stainless steels. It has been accepted (Ge et al., 2003; Seyeux et al.,
Fig. 4. CV curves measured on the steel electrode in biotic SRB culture medium as a function of time.
Fig. 5. CV curves measured on 316L stainless steel electrode in sulfide-containing sterile medium as a function of time.
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2015) that the sulfides change the composition and structure of passive films formed on stainless steel surface, decreasing the corrosion resistance of the steel. The passive film can be reduced by sulfides to produce iron sulfides. Thus, the oxidative current peaks of Fe2þ to Fe3þ in both abiotic sulfide-containing and biotic SRB media disappear after 1 day of immersion, as the formed iron sulfide film would inhibit the oxidation of Fe2þ to Fe3þ. Fig. 6 shows the time dependence of oxidative and reductive peak current densities (iO and iR) of iron sulfide in the SRB culture medium and the sulfide-containing sterile medium, respectively. After 2 h of immersion of the steel electrode in individual media, the absolute values of iO and iR for iron sulfide are almost identical, but the current densities are larger than 40 mA/cm2 in the SRB medium and are less than 20 mA/cm2 in the sulfide-containing sterile solution. Generally, the value of i can be influenced by either the concentration of sulfides or the micro-morphology of the iron sulfide film. Since the concentration of sulfides is identical in both media, as seen in Fig. 2, the micro-morphology of the iron sulfide film formed on the steel surface plays a key role in the different values of iO and iR obtained in the two media. It has been accepted (Veleva et al., 2002; Zarasvand and Rai, 2014) that a heterogeneous dissolution of the iron sulfide film can generate a higher anodic current, and a lower oxidative current density indicates the formation of a more stable film. The higher values of iO and iR obtained in the SRB culture medium show that the SRB are able to increase dissolution of the film, and thus,
Fig. 6. Oxidative and reductive peak current densities (iO and iR) of iron sulfides in both the SRB culture medium and sulfide-containing sterile medium, respectively, as a function of time.
increase the steel corrosion. The absolute values of both iO and iR decrease with the immersion time in both media, indicating that the iron sulfide film becomes more stable with time in the environment. After 7 days of immersion, the iO and iR decrease to 2.41 and 1.54 mA/cm2, respectively, in the sulfide-containing sterile medium. This shows that the iron sulfide film experiences a quite low dissolution in the environment, protecting the steel from corrosion attack (Homborg et al., 2014; Seth and Edyvean, 2006). The iO and iR decrease to 9.49 and 14.37 mA/cm2, respectively, in the SRB medium, which are larger than those obtained in the sulfide sterile solution. Thus, the iron sulfide film formed in the SRB medium is less stable than that formed in the sulfide sterile medium. 3.3. Surface characterization Fig. 7 shows the CLSM views of the 316L stainless steel electrode after 7 days of immersion in sulfide-containing sterile and SRB culture media, respectively. It is seen in Fig. 7B that bacteria are present on the steel surface, and some bacteria accumulate to form local clusters. This is a typical characteristic of SRB adhering to metals. In the sulfide sterile medium (Fig. 7A), there is no sign of bacterium adsorption. The SRB adsorption on 316L stainless steel can result in formation of a layer of inhomogeneous biofilm, which makes the local chemical environment different from the bulk solution. For example, the local sulfide concentration can be elevated greatly compared to that in the bulk solution (Little et al., 2008). Fig. 8 shows the SEM views and EDS elemental analysis of the 316L stainless steel electrode after 7 days of immersion in the SRB culture and sulfide-containing sterile media, respectively. In the sulfide sterile medium, the corrosion product film is dense and homogeneous (Fig. 8A). The distribution of sulfur (S) element is also homogeneous on the electrode surface (Fig. 8B). The results indicate that the formed iron sulfide film is present homogeneously on the steel surface, protecting the steel from further corrosion. However, in the SRB culture medium, corrosion products accumulate locally on the steel surface (Fig. 8C). The EDS results show that they are S-enriched corrosion products (Fig. 8D). Thus, the presence of SRB in the medium would result in a heterogeneous iron sulfide film on 316L stainless steel surface. It is noted that the steel electrode is under the identical surface preparation for testing. While the scratches are visible on the steel electrode in the SRB culture medium (Fig. 8C), there is no scratch observed on the electrode in the sulfide sterile medium (Fig. 8A). This shows the compactness and homogeneousness of the surface films formed in the two media. Figs. 9 and 10 show the AFM topographic images and the
Fig. 7. CLSM views of the 316L stainless steel electrode after 7 days of immersion in (A) sulfide-containing sterile medium and (B) biotic SRB culture medium, respectively.
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Fig. 8. SEM views (A, C) and EDS elemental analysis (B, D) of the 316L stainless steel electrode after 7 days of immersion in sulfide-containing sterile medium (A, B) and SRB culture medium (C, D), respectively.
Fig. 9. AFM topographic images of the 316L stainless steel electrode after 7 days of immersion in (A) sterile, sulfide-free medium, (B) sulfide sterile medium, and (C) SRB culture media, respectively.
derived surface roughness of the 316L stainless steel electrode after 7 days of immersion in sterile sulfide-free, sulfide sterile and SRB
culture media, respectively. In the sulfide-free sterile medium, surface irregularities such as scratches and grooves generated
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during electrode preparation are visible (Fig. 9A), and the average roughness of the electrode surface is 3.14 nm only (Fig. 10). A layer of thin and uniform passive film is formed on the steel to protect it from corrosion attack. In the sulfide sterile medium, some granular corrosion products are formed and scattered on the electrode surface, as seen in Fig. 9B. The surface roughness of the electrode increases to 5.99 nm (Fig. 10). According to the EDS results, these granular products are mainly iron sulfide. Thus, the formation of the iron sulfide film increases the roughness of the steel, but the corrosion is uniform. In the SRB culture medium, the size and number of the granular corrosion products increase remarkably (Fig. 9C), and the surface roughness of the electrode increases to 9.75 nm. Obviously, the SRB adhere to the steel surface and form a heterogeneous biofilm, as seen in Fig. 7. The chemical substances in the SRB medium, such as sulfides, distribute heterogeneously on the steel surface. Local corrosion occurring at a high rate causes deposit of corrosion products, increasing the surface roughness of the electrode. The presence of SRB leads to a heterogeneous iron sulfide film than that in sulfide sterile medium. The iron sulfide film, with a less protective ability and heterogeneous growth, causes localized corrosion of the steel. Therefore, the stainless steel is vulnerable to corrosion in SRB medium. The simulated sulfide solution is not able to reproduce the corrosive environment that is associated with the SRB medium.
4. Conclusions Sulfides generated from SRB Desulfomicrobium metabolism would result in formation and growth of a heterogeneous iron sulfide film on the stainless steel surface. Localized corrosion occurs preferentially at locations where sulfides accumulate, increasing the surface roughness of the steel. Sulfides contained in the sterile medium are able to increase the steel corrosion, but the formed iron sulfide film is more uniform, compact and stable than that formed in the SRB culturing medium. The corrosion of the steel in the sterile solution is smaller than that in the SRB culture medium. Although the primary products of Desulfomicrobium metabolism are sulfides, the simulated abiotic sulfide solution is not able to reproduce the corrosive environment associated with the biotic SRB culturing medium.
Fig. 10. Surface roughness of the 316L stainless steel electrode after 7 days of immersion in the sterile sulfide-free, sulfide sterile and biotic SRB culture media, respectively.
Acknowledgements This work was supported by the University of Calgary's Eyes High Postdoctoral Scholar Program.
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Passivation model of 316 stainless steel in simulated cooling water and the effect of sulfide on the passive film. Appl. Surf. Sci. 211, 321e334. He, W., Knudsen, O.O., Diplas, S., 2009. Corrosion of stainless steel 316L in simulated formation water environment with CO2-H2S-Cl. Corros. Sci. 51, 2811e2819. Homborg, A.M., Morales, C.F., Tinga, T., De Wit, J.H.W., Mol, J.M.C., 2014. Detection of microbiologically influenced corrosion by electrochemical noise transients. Electrochimica Acta 136, 223e232. Javaherdashti, R., Singh Raman, R.K., Panter, C., Pereloma, E.V., 2006. Microbiologically assisted stress corrosion cracking of carbon steel in mixed and pure cultures of sulfate reducing bacteria. Int. Biodeterior. Biodegrad. 58, 27e35. Kocijan, A., Donik, C., Jenko, M., 2007. Electrochemical and XPS studies of the passive film formed on stainless steels in borate buffer and chloride solutions. Corros. Sci. 49, 2083e2098. Little, B.J., Lee, J.S., Ray, R.I., 2006. Diagnosing microbiologically influenced corrosion: a state-of-the-art review. Corrosion 62, 1006e1017. Little, B.J., Lee, J.S., Ray, R.I., 2008. The influence of marine biofilms on corrosion: a concise review. Electrochimica Acta 54, 2e7. Macdonald, D.D., 1992. The point defect model for the passive state. J. Electrochem. Soc. 139, 3434e3449. Maruthamuthu, S., Kumar, B.D., Ramachandran, S., Anandkumar, B., Palanichamy, S., Chandrasekaran, M., Subramanian, P., Palaniswamy, N., 2011. Microbial corrosion in petroleum product transporting pipelines. Ind. Eng. Chem. Res. 50, 8006e8015. Mendili, Y.E., Abdelouas, A., Bardeaum, J.F., 2013. The corrosion behavior of carbon steel in sulfide aqueous media at 30C. J. Mater. Eng. Perform. 23, 1350e1357. Perini, N., Corradini, P.G., Nascimento, V.P., Passamani, E.C., Freitas, M.B., 2013. Characterization of AISI 1005 corrosion films grown under cyclic voltammetry of low sulfide ion concentrations. Corros. Sci. 74, 214e222. Seth, A.D., Edyvean, R.G.J., 2006. The function of sulfate-reducing bacteria in corrosion of potable water mains. Int. Biodeterior. Biodegrad. 58, 108e111. Seyeux, A., Zanna, S., Allion, A., Marcus, P., 2015. The fate of the protective oxide film on stainless steel upon early stage growth of a biofilm. Corros. Sci. 91, 352e356. Sharma, M., An, D., Baxter, K., Edillon, M., Henderson, L., Voordouw, G., 2016. Understanding the Role of Microbes in Frequent Coiled Tubing Failures. Corrosion’2016, Paper No. 7815. NACE International, Houston, TX, USA, 2016. Sherar, B.W.A., Power, I.M., Keech, P.G., Mitlin, S., Southam, G., Shoesmith, D.W., 2011. Characterizing the effect of carbon steel exposure in sulfide containing solutions to microbially induced corrosion. Corros. Sci. 53, 955e960. Sheng, X., Ting, Y.P., Pehkonen, S.O., 2007. The influence of sulphate-reducing bacteria biofilm on the corrosion of stainless steel AISI 316. Corros. Sci. 49, 2159e2176. 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Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
A comparative study of corrosion of 316L stainless steel in biotic and abiotic sulfide environments Shiqiang Chen a, Y. Frank Cheng a, *, Gerrit Voordouw b a b
Department of Mechanical & Manufacturing Engineering, University of Calgary, Calgary, Alberta, T2N 1N4, Canada Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 September 2016 Received in revised form 18 January 2017 Accepted 10 February 2017
Electrochemical corrosion of 316L stainless steel in a biotic sulfate-reducing bacteria (SRB) culturing medium and an abiotic sulfide sterile solution was investigated by biotesting, cyclic voltammetry and surface characterization techniques. It was attempted to determine if the abiotic sulfide solution is capable of reproducing the corrosive environment that is comparable to that in the biotic SRB medium. Results demonstrated that sulfides generated from SRB metabolism result in a heterogeneous iron sulfide film on the steel surface. Corrosion occurs at locations where sulfides accumulate, increasing the surface roughness of the steel. Like those generated in the biotic medium, sulfides formed in the sterile medium can increase the steel corrosion. However, the generated sulfide film is more uniform, compact and stable than that formed in the SRB medium. Moreover, the steel possesses different cyclic voltammetry behavior. The prepared abiotic sulfide solution is not representative of the SRB medium for corrosion studies. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Corrosion 316L stainless steel Sulfate reducing bacteria Biotic bacterium culturing medium Abiotic sulfide solution
1. Introduction Microbiologically influenced corrosion (MIC) has been one of primary mechanisms resulting in pipeline failures in petroleum industry (AlAbbas et al., 2013; Maruthamuthu et al., 2011; Usher et al., 2014; Videla and Herrera, 2009). In particular, sulfate reducing bacteria (SRB) are identified as main microorganisms contained in highly corrosive environments to generate metabolites (e.g., sulfides, extracellular polymeric substances (EPS), etc.), contributing to metallic MIC (Little et al., 2006; Enning and Garrelfs, 2014; Zapata-Penasco et al., 2016) and even stress corrosion cracking (Javaherdashti et al., 2006). Stainless steels have been used to make oilfield devices due to their good machinability and excellent corrosion resistance. Generally, the resistance of stainless steels, such as 316L stainless steel, to corrosion is due to the formation of a layer of passive film, which is usually composed of an inner chromium oxide layer and an outer mixed iron oxide and hydroxide layer, on the steel surface (Macdonald, 1992). However, the steel is prone to suffer from severe corrosion in the presence of SRB in the service environment
* Corresponding author. E-mail address: [email protected] (Y. Frank Cheng). http://dx.doi.org/10.1016/j.ibiod.2017.02.014 0964-8305/© 2017 Elsevier Ltd. All rights reserved.
(Bachmann and Edyvean, 2006; Sheng et al., 2007; Unsal et al., 2016). There has been much work conducted to investigate corrosion of 316L stainless steel in SRB-containing environments. It was found that the metabolic activity of SRB is able to accelerate corrosion of the steel (Unsal et al., 2016). The SRB result in generation of iron sulfide film on the steel surface. The sulfides are the metabolites of SRB, and are highly corrosive. In addition to cause corrosion of steels that are in active dissolution state in environments, they can result in breakdown of the passive film formed on passivated steels, affecting the further corrosion and localized corrosion (Duan et al., 2006; He et al., 2009). As the presence of sulfides in the environment is critical to steel corrosion, extensive work has been performed to investigate MIC using artificially prepared, abiotic sulfide solutions (Cord-Ruwisch, 1985; Mendili et al., 2013; Sherar et al., 2011). However, it has been questionable whether an abiotic sulfide solution is representative of the corrosive environment associated with the biotic SRB medium. As a result, the essential difference of the steel corrosion in biotic and abiotic sulfide environments is yet understood mechanistically. In this work, a comparative study of electrochemical corrosion of 316L stainless steel was conducted in a biotic SRB culturing medium and a sulfide-containing sterile solution, respectively, by cyclic voltammetry and various surface analysis techniques,
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including confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), X-ray energy dispersive spectrometer (EDS) and atomic force microscopy (AFM). Both electrochemical behavior and corrosion product film formed during the steel corrosion were analyzed in both media. It is anticipated that this work provides an essential insight into the corrosivity of the biotic SRB culturing medium and the sulfide-containing sterile solution, and the mechanism of steel corrosion in both media. 2. Experimental 2.1. Material Specimens were cut from a 316L stainless steel plate, with a chemical composition (wt.%): 0.019 C, 1.18 Mn, 0.48 Si, 16.72 Cr, 12.10 Ni, 2.05 Mo, 0.038 P, 0.0013 S, 0.10 N and balance Fe. The dimension of the specimen was 1 cm 1 cm 0.3 cm. The steel coupons were embedded in epoxy resin, leaving a working area of 1 cm2. The exposed face of the specimen was sequentially ground with silicon carbide emery papers up to #1200 grit, and was then rinsed with deionized water, degreased with absolute ethyl alcohol, and dried with pure nitrogen. 2.2. Bacterial culturing The SRB used in this work were extracted from oil sludge collected from an oil well in Alberta, Canada. The 16S rRNA genes were amplified by polymerase chain reaction (PCR), and sequenced using Illumina Miseq in order to identify the SRB extracted from the oil sludge sample (Sharma et al., 2016). The sequencing results showed that the dominant SRB were Desulfomicrobium. The modified coleville synthetic brine culture solution was used for SRB growth. The solution was prepared by the following procedure. Chemicals including 0.05 g KH2PO4, 0.32 g NH4Cl, 0.21 g CaCl2, 0.54 g MgSO4, 1.42 g Na2SO4 and 1.36 g sodium formate were added to 1 L of ultrapure water, and the sealed mixture was autoclaved at 121 C for 20 min. After cooling down in air to ambient temperature, the culture medium was purged with N2/CO2 (9:1) gas for 20 min to remove oxygen until the content of dissolved oxygen was below the detection limit of 0.001 mg/L of a dissolved oxygen meter (ExStik DO600). The 30 mL NaHCO3 (1 M) and 1 mL selenite were added to the medium using a 0.22 mm filter for sterilization. The pH of the prepared culture medium was adjusted to 7.2 ± 0.1 using 1 M HCl solution. The extracted SRB were then added in the medium for growth. The growth curve of SRB was measured using turbidimetry at 600 nm with an ultraviolet and visible light spectrophotometer (UV-1800, SHIMADZU). The concentration of sulfide, CS2-, was measured using the methylene blue method (Cord-Ruwisch, 1985). The artificial sulfide solution was prepared by adding 2 mM Na2S solution in the sterile culture solution mentioned above.
2.4. Morphological and compositional characterization Bacterium attachment and biofilm formation on the 316L stainless steel surface were observed by a CLSM. After immersion in SRB medium and sulfide solution for 7 days, the steel specimens were washed with ultrapure water, and were then stained with a fluorescent dye (Molecular Probes™ FilmTracer™ LIVE/DEAD® Biofilm Viability Kit) in the darkness according to the manufacturers' procedure. All these procedures were conducted in an anaerobic glove box. Similarly, after immersion in the SRB medium and the sulfide solution for 7 days, the steel specimens were washed with ultrapure water, and were then cleaned with absolute ethyl alcohol to remove bacteria and biofilm. The morphology of the steel electrode was characterized by a field emission SEM. Elemental maps were obtained through an EDS coupled with the SEM. An AFM (Keysight 5500) was used for topographic characterization of the steel specimen. A scanner carrying a long rectangular cantilever with a spring constant of 0.2 N/m (apex radius <10 nm) was placed above the specimen. The scanning mode was configured as contact, with a scanning rate of 1 Hz and a resolution of 512 512 pixel. A region of 5 mm 5 mm was selected for AFM characterization. The obtained images were processed to remove background signals, and the surface roughness was extracted. 3. Results and discussion 3.1. SRB growth curve Fig. 1 shows the growth curve of SRB in the biotic culturing medium, where the growth of SRB is composed of three stages. In stage one, i.e., in the first two days, the number of SRB keeps at a low level, which is called the adaptive stage. In this stage, SRB have a low metabolic activity, producing few metabolites. From day two to the eighth day, the number of SRB increase quickly with time. This stage is called the exponential phase, where the speed of the bacterium's reproduction is much greater than the rate they die. The SRB are featured with a vigorous metabolic activity, producing extensive metabolites accumulating in the culture medium. After the eighth day, the speed the bacterium multiplication is smaller than the death rate, and the number of SRB decreases with time. This stage is called the death phase, where the rate of bacterium death is greater than the production rate. To maintain a stable metabolic activity of SRB in this work, the maximum growth of bacteria at the exponential phase, i.e., the seventh to the eighth day,
2.3. Measurements of cyclic voltammograms CV measurements were conducted on a three-electrode cell using a Solartron 1280C electrochemical testing system. The 316L stainless steel electrode, a graphite sheet, and a saturated calomel electrode (SCE) were used as the working, counter and reference electrodes, respectively. After immersion of the steel electrode in the test solution for 1 h, the open-circuit potential of the steel reached a relatively stable value. The CV was measured with a potential range from 0 V (SCE) to 1.1 V (SCE) at a potential scanning rate of 2 mV/s. All measurements were performed at 25 ± 2 C. To ensure reproducibility of testing data, all tests were performed under controlled conditions at least three times.
Fig. 1. The growth curve of SRB in the prepared culturing medium.
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is chosen to study the SRB induced corrosion of 316L stainless steel. Fig. 2 shows the concentration of sulfides in the biotic SRB culture medium and the abiotic sulfide solution, respectively, as a function of time. It is seen that the sulfide concentration in both media maintain approximately stable at 2 mM during the testing period. 3.2. Cyclic voltammogram (CV) measurements Fig. 3 shows the CV curves measured on 316L stainless steel electrode in the sterile, sulfide-free culture medium as a function of time. One anodic current peak is observed at about 0.5 V(SCE) ~ 0.3 V(SCE), which is associated with the oxidation of Fe2þ to Fe3þ (Kocijan et al., 2007). The peak current density is almost constant at about 6 mA/cm2 after 1 day of testing. Other than the hydrogen evolution reaction occurring more negative than 0.9 V(SCE), there is no obvious cathodic current peak in the negative potential sweep direction. Fig. 4 shows the CV curves measured on the steel electrode in biotic SRB culture medium as a function of time. The weak oxidative current shoulder at about 0.5 V(SCE) after 2 h of immersion, as marked in the figure, is attributed to the oxidation of Fe2þ to Fe3þ,
Fig. 2. Concentrations of sulfides in the SRB culture medium and the sterile sulfide solution, respectively, as a function of time.
Fig. 3. CV curves measured on 316L stainless steel electrode in the sterile, sulfide-free medium, as a function of time.
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but this peak is not observed in the following days. In addition to the oxidative current peak for Fe2þ to Fe3þ and the hydrogen evolution reaction occurring more negative than 1.0 V(SCE), there are other two current peaks at about 0.2 ~ 0.1 V(SCE) and 0.9 V(SCE), respectively. The peak current density is much higher than that at about 0.5 V(SCE). Fig. 5 shows the CV curves measured on 316L stainless steel electrode in the sulfide-containing sterile medium as a function of time. There is also a pair of oxidative and reductive current peaks at 0.2 ~ 0.1 V(SCE) and 0.8 V(SCE), respectively, after 2 h of immersion. The current peak at 0.2 ~ 0.1 V(SCE) disappears in the following measurements. It was reported that the current peaks at these two potential ranges are attributed to oxidation and reduction of iron sulfide (Perini et al., 2013). The results in Figs. 4 and 5 indicate that, in the presence of sulfides in the solution, a layer of iron sulfide film can be formed on the steel surface. Moreover, the iron sulfide film can also be formed on the steel electrode in the biotic SRB culture medium because sulfides are generated from the SRB metabolism. Sulfides can attack passive films formed on metals such as stainless steels. It has been accepted (Ge et al., 2003; Seyeux et al.,
Fig. 4. CV curves measured on the steel electrode in biotic SRB culture medium as a function of time.
Fig. 5. CV curves measured on 316L stainless steel electrode in sulfide-containing sterile medium as a function of time.
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2015) that the sulfides change the composition and structure of passive films formed on stainless steel surface, decreasing the corrosion resistance of the steel. The passive film can be reduced by sulfides to produce iron sulfides. Thus, the oxidative current peaks of Fe2þ to Fe3þ in both abiotic sulfide-containing and biotic SRB media disappear after 1 day of immersion, as the formed iron sulfide film would inhibit the oxidation of Fe2þ to Fe3þ. Fig. 6 shows the time dependence of oxidative and reductive peak current densities (iO and iR) of iron sulfide in the SRB culture medium and the sulfide-containing sterile medium, respectively. After 2 h of immersion of the steel electrode in individual media, the absolute values of iO and iR for iron sulfide are almost identical, but the current densities are larger than 40 mA/cm2 in the SRB medium and are less than 20 mA/cm2 in the sulfide-containing sterile solution. Generally, the value of i can be influenced by either the concentration of sulfides or the micro-morphology of the iron sulfide film. Since the concentration of sulfides is identical in both media, as seen in Fig. 2, the micro-morphology of the iron sulfide film formed on the steel surface plays a key role in the different values of iO and iR obtained in the two media. It has been accepted (Veleva et al., 2002; Zarasvand and Rai, 2014) that a heterogeneous dissolution of the iron sulfide film can generate a higher anodic current, and a lower oxidative current density indicates the formation of a more stable film. The higher values of iO and iR obtained in the SRB culture medium show that the SRB are able to increase dissolution of the film, and thus,
Fig. 6. Oxidative and reductive peak current densities (iO and iR) of iron sulfides in both the SRB culture medium and sulfide-containing sterile medium, respectively, as a function of time.
increase the steel corrosion. The absolute values of both iO and iR decrease with the immersion time in both media, indicating that the iron sulfide film becomes more stable with time in the environment. After 7 days of immersion, the iO and iR decrease to 2.41 and 1.54 mA/cm2, respectively, in the sulfide-containing sterile medium. This shows that the iron sulfide film experiences a quite low dissolution in the environment, protecting the steel from corrosion attack (Homborg et al., 2014; Seth and Edyvean, 2006). The iO and iR decrease to 9.49 and 14.37 mA/cm2, respectively, in the SRB medium, which are larger than those obtained in the sulfide sterile solution. Thus, the iron sulfide film formed in the SRB medium is less stable than that formed in the sulfide sterile medium. 3.3. Surface characterization Fig. 7 shows the CLSM views of the 316L stainless steel electrode after 7 days of immersion in sulfide-containing sterile and SRB culture media, respectively. It is seen in Fig. 7B that bacteria are present on the steel surface, and some bacteria accumulate to form local clusters. This is a typical characteristic of SRB adhering to metals. In the sulfide sterile medium (Fig. 7A), there is no sign of bacterium adsorption. The SRB adsorption on 316L stainless steel can result in formation of a layer of inhomogeneous biofilm, which makes the local chemical environment different from the bulk solution. For example, the local sulfide concentration can be elevated greatly compared to that in the bulk solution (Little et al., 2008). Fig. 8 shows the SEM views and EDS elemental analysis of the 316L stainless steel electrode after 7 days of immersion in the SRB culture and sulfide-containing sterile media, respectively. In the sulfide sterile medium, the corrosion product film is dense and homogeneous (Fig. 8A). The distribution of sulfur (S) element is also homogeneous on the electrode surface (Fig. 8B). The results indicate that the formed iron sulfide film is present homogeneously on the steel surface, protecting the steel from further corrosion. However, in the SRB culture medium, corrosion products accumulate locally on the steel surface (Fig. 8C). The EDS results show that they are S-enriched corrosion products (Fig. 8D). Thus, the presence of SRB in the medium would result in a heterogeneous iron sulfide film on 316L stainless steel surface. It is noted that the steel electrode is under the identical surface preparation for testing. While the scratches are visible on the steel electrode in the SRB culture medium (Fig. 8C), there is no scratch observed on the electrode in the sulfide sterile medium (Fig. 8A). This shows the compactness and homogeneousness of the surface films formed in the two media. Figs. 9 and 10 show the AFM topographic images and the
Fig. 7. CLSM views of the 316L stainless steel electrode after 7 days of immersion in (A) sulfide-containing sterile medium and (B) biotic SRB culture medium, respectively.
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Fig. 8. SEM views (A, C) and EDS elemental analysis (B, D) of the 316L stainless steel electrode after 7 days of immersion in sulfide-containing sterile medium (A, B) and SRB culture medium (C, D), respectively.
Fig. 9. AFM topographic images of the 316L stainless steel electrode after 7 days of immersion in (A) sterile, sulfide-free medium, (B) sulfide sterile medium, and (C) SRB culture media, respectively.
derived surface roughness of the 316L stainless steel electrode after 7 days of immersion in sterile sulfide-free, sulfide sterile and SRB
culture media, respectively. In the sulfide-free sterile medium, surface irregularities such as scratches and grooves generated
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during electrode preparation are visible (Fig. 9A), and the average roughness of the electrode surface is 3.14 nm only (Fig. 10). A layer of thin and uniform passive film is formed on the steel to protect it from corrosion attack. In the sulfide sterile medium, some granular corrosion products are formed and scattered on the electrode surface, as seen in Fig. 9B. The surface roughness of the electrode increases to 5.99 nm (Fig. 10). According to the EDS results, these granular products are mainly iron sulfide. Thus, the formation of the iron sulfide film increases the roughness of the steel, but the corrosion is uniform. In the SRB culture medium, the size and number of the granular corrosion products increase remarkably (Fig. 9C), and the surface roughness of the electrode increases to 9.75 nm. Obviously, the SRB adhere to the steel surface and form a heterogeneous biofilm, as seen in Fig. 7. The chemical substances in the SRB medium, such as sulfides, distribute heterogeneously on the steel surface. Local corrosion occurring at a high rate causes deposit of corrosion products, increasing the surface roughness of the electrode. The presence of SRB leads to a heterogeneous iron sulfide film than that in sulfide sterile medium. The iron sulfide film, with a less protective ability and heterogeneous growth, causes localized corrosion of the steel. Therefore, the stainless steel is vulnerable to corrosion in SRB medium. The simulated sulfide solution is not able to reproduce the corrosive environment that is associated with the SRB medium.
4. Conclusions Sulfides generated from SRB Desulfomicrobium metabolism would result in formation and growth of a heterogeneous iron sulfide film on the stainless steel surface. Localized corrosion occurs preferentially at locations where sulfides accumulate, increasing the surface roughness of the steel. Sulfides contained in the sterile medium are able to increase the steel corrosion, but the formed iron sulfide film is more uniform, compact and stable than that formed in the SRB culturing medium. The corrosion of the steel in the sterile solution is smaller than that in the SRB culture medium. Although the primary products of Desulfomicrobium metabolism are sulfides, the simulated abiotic sulfide solution is not able to reproduce the corrosive environment associated with the biotic SRB culturing medium.
Fig. 10. Surface roughness of the 316L stainless steel electrode after 7 days of immersion in the sterile sulfide-free, sulfide sterile and biotic SRB culture media, respectively.
Acknowledgements This work was supported by the University of Calgary's Eyes High Postdoctoral Scholar Program.
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