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The influence of cathodic protection potential on the biofilm formation and corrosion behaviour of an X70 steel pipeline in sulfate reducing bacteria media
Journal of Alloys and Compounds 729 (2017) 180e188
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Journal of Alloys and Compounds journal homepage: http://www.elsevier.com/locate/jalcom
The influence of cathodic protection potential on the biofilm formation and corrosion behaviour of an X70 steel pipeline in sulfate reducing bacteria media Tao Liu a, b, Y. Frank Cheng b, * a b
College of Ocean Science and Engineering, Shanghai Maritime University, Shanghai, China Department of Mechanical Engineering, 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 15 June 2017 Received in revised form 9 September 2017 Accepted 16 September 2017 Available online 19 September 2017
This work investigated the biofilm formation and microbiologically influenced corrosion (MIC) of a pipeline steel under cathodic protection (CP) in an extracted soil in solution with addition of sulfate reducing bacteria (SRB), Desulfovibrio desulfuricans. It was found that the application of CP did not affect the planktonic bacterial growth in the solution. The CP facilitated the bacterial attachment to the steel in this system, and a layer of biofilm formed on the cathodically protected steel, decreasing the CP effectiveness for corrosion protection. The CP potential of 850 mV vs. copper sulfate electrode (CSE) was not sufficient to protect the steel from corrosion in SRB containing media. Despite a further negative shift of the CP potential to 1000 mV vs. CSE was effective to control uniform corrosion of the steel, pitting corrosion still occurred under biofilm on the steel. © 2017 Elsevier B.V. All rights reserved.
Keywords: Cathodic protection Pipelines Biofilm Microbiologically influenced corrosion Pitting corrosion
1. Introduction The presence of microorganism in environments is able to accelerate coating degradation and cause the so-called microbiologically influenced corrosion (MIC) even in right CP conditions [1e3], in which would not normally be seen corrosion without microorganisms. In aqueous solutions containing sulfate reducing bacteria (SRB), it was suggested that the applied CP potential should be shifted to 950 mV vs. CSE or even more negative values in order to fully protect steels from corrosion [4]. There have been some investigations on the mutual effect of CP and microbial activity. Guezennec paid attention to this problem 20 years ago [5]. In order to determine whether there was a relationship between cathodic polarization and the biofilm formation on steels, experiments were conducted on clean metal surfaces and biofilmed surfaces in natural or synthetic seawater. It was found that cathodically produced hydrogen facilitated growth of hydrogenase-containing SRB. Nekoksa and Gutherman's work also showed that the CP application enhanced the incubation and growth of microbes, with more bacteria settling on a cathodically
* Corresponding author. E-mail address: [email protected] (Y.F. Cheng). http://dx.doi.org/10.1016/j.jallcom.2017.09.181 0925-8388/© 2017 Elsevier B.V. All rights reserved.
protected stainless steel than on unpolarized ones [6]. Similar results were obtained by Zavala et al. [7], who found that the SRB population on CP-applied (i.e., 850 mV vs. CSE) pipeline steel was twice of the microorganisms on the coupons without CP application. Edyvean et al.’s work also investigated the interaction of CP and the biofilm formation on steels [8]. However, their finding was opposite to those listed above. On both stainless and non-stainless steels, CP retarded the development of bacterial fouling. Increasing the CP decreased the number of bacteria. Little and Wagner confirmed that, when the CP current was intermittent, the corrosion attack due to the microorganisms became more aggressive [9]. Regarding the mechanistic aspect of the effect of CP on MIC, Li et al. believed that there existed a synergistic effect of coating disbondment and partial shielding of CP current from reaching the disbonded crevice, where an environment favorable for active growth of SRB was generated [10]. A quick review of relevant literature on this topic shows that some of reported results are controversial each other [6e8]. The understanding of the mutual interaction of CP and MIC is vague. Moreover, uniform corrosion of metals in the presence of microorganisms is the focused topic in most work. The essential influence of cathodic polarization on MIC induced pitting corrosion has been lacking. The present work attempts to address these
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unknowns, and advances our understanding to this important problem. In this work, the biofilm formation and MIC of an X70 pipeline steel under various CP potentials in an extracted soil solution containing SRB, i.e., Desulfovibrio desulfuricans, were studied by biological testing and materials characterization techniques, including scanning electron microscopy (SEM), energy-dispersive X-ray spectrum (EDS), confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). The effect of CP application on bacterial growth was measured. The formation of biofilm on the steel under varied CP potentials was characterized. Both uniform corrosion and pitting corrosion of the steel in the SRB-containing soil solution were considered. The interactions between CP and the SRB induced MIC, particularly, pitting corrosion, were analyzed.
2. Experimental
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2.3. Surface characterization After 7 days of cathodic polarization in the test solution, the steel electrodes were removed from the solution, washed with a phosphate-buffered saline solution (pH 7.4), and then dipped in 2.5% glutaraldehyde fixative at 4 C for 12 h. The electrodes were dehydrated with various concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 10 min successively, and fully dried in N2 environment. A field emission SEM (Field Electron and Ion Co. Quanta 250 FEG) was used to characterize the surface morphology of the steel electrode. The elemental composition was obtained through the coupled EDS (Bruker Quantax EDS). It was confirmed with the EDS manufacturer that the penetration depth is 2e5 mm for the steel specimen under the testing condition in this work, where the operating voltage was 15 kV. Bacterial attachment and the biofilm formed on the steel electrode were characterized using a confocal laser scanning microscope (Olympus, FV1000). Prior to observation, the electrodes were
2.1. Material and specimen Specimens used in this work were cut from an X70 steel pipe, with the chemical composition (wt.%): 0.06 C, 1.44 Mn, 0.31 Si, 0.004 S, 0.01 P, 0.034 Ni, 0.16 Cr, 0.25 Mo, 0.005 V, 0.015 Cu, 0.01 Ti, 0.002 B, 0.029 Al and Fe balance. The outer and inner diameters of the pipe were 812.8 mm and 796.9 mm, respectively. The microstructure of the steel contained ferrite and pearlite. The specimens were machined into a circular shape, and sealed by epoxy resin, leaving an exposed area of 10 mm in diameter. The work face of the specimen was ground to a final 1200 grit SiC paper. The specimen was then degreased with acetone, washed with distilled water, and dried naturally in a high-purity nitrogen (99.999%) environment.
2.2. Microbial cell culturing The SRB (i.e., Desulfovibrio desulfuricans) used in this work were purchased from America Typical Culture Collection. The soil was taken from Regina, Canada, and the chemical composition of the extracted soil solution is shown in Table 1. The medium for SRB culturing contained 0.5 g K2HPO4, 0.5 g (NH4)2SO4, 5 g sodium citrate, 3.5 g sodium lactate and 1 L extracted soil solution as mentioned above. The culturing medium was sterilized by autoclaving at 121 C for 20 min. It was then cooled down in ice water for 30 min, while purging with N2/CO2 (9:1, v/v) gas to remove oxygen until the content of dissolved oxygen was below 0.4 mg/L, as measured by a dissolved oxygen meter (ExStik DO600). 0.1 g of as-received SRB powders, which were in frozen state, was added in 10 mL of the culturing medium and kept in an incubator at 35 C for 3 days. After that, the 10 mL SRBcontaining medium was mixed with 500 mL culturing medium to prepare the test solution in this work. The number of SRB in the test solution was measured using the most probable number (MPN) method [11]. The concentration of sulfide in the solution was measured by the methylene blue method [12]. To ensure the reproducibility of data, the measurements of the sulfide concentration and the number of bacteria in the solution were conducted three times.
Table 1 Chemical composition of the extracted soil solution (in 1 L distilled water, pH ¼ 7.5). Chemicals Amount (g)
NaHCO3 0.0760
NaNO3 0.0014
NaCl 0.0089
Na2SO4 0.0775
CaSO4 0.8823
K2SO4 0.0619
MgSO4 0.3326
Fig. 1. Time dependence of (a) the sulfide concentration and (b) the number of Desulfovibrio desulfuricans in the test solution while the steel electrode is under a CP potential of 850 mV vs. CSE and without CP.
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washed with milli Q water, and then stained with a fluorescent dye (Molecular Probes™ FilmTracer™ LIVE/DEAD® Biofilm Viability Kit) in the darkness following the manufacturers' procedure. After SEM/EDS characterizations, a modified NACE RP07752005 protocol was used to remove corrosion products including biofilm from the electrode surface.18 The electrode was washed in dibutylthiourea-HCl solution, sodium bicarbonate solution, deionized water and acetone, respectively, for 2 min. According to NACE RP0755-2005,18 the specimen, upon removal of corrosion products and biofilm, is dried in a stream of air. This could cause oxidation of the steel in air. In this work, the specimens were dried naturally in the high-purity nitrogen environment, avoiding the steel oxidization. An AFM (Keysight 5500 scanning probe microscope system) was used to image the topography of the steel electrode, where 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 20 mm 20 mm was selected for AFM imaging. The topographic profile of the electrode was derived from AFM images. To ensure the reproducibility of the characterization results, three parallel specimens were prepared for each testing condition.
3. Results and discussion 3.1. Measurements of sulfide concentration and bacterial number in the test solution The SRB play a key role in the sulfur cycle. They can use sulfate (SO2 4 ) as a terminal electron acceptor in degradation of organic matters, producing sulfide by Ref. [13]:
Fig. 2. SEM morphological views of the steel electrodes at (a, b) OCP, (c, d) 850 mV vs. CSE and (e, f) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing test solution, where photos with two magnifications are provided for each electrode.
T. Liu, Y.F. Cheng / Journal of Alloys and Compounds 729 (2017) 180e188 2 þ 2C3H5O 3 þ SO4 / 2C2H3O2 þ 2HCO3 þ HS þ H
(1)
Fig. 1 shows the time dependence of the sulfide concentration and the planktonic bacterial number in the test solution while the steel electrode is under a CP potential of 850 mV vs. CSE. For comparison, the measurements were also conducted in the absence of CP application on the steel. It was seen that, in both the absence and presence of CP, the sulfide concentration increased from 0.8 mmol/L to 5.8 mmol/L after 5 days, and then approximately maintained at this value (Fig. 1a). It was noted that the initial quantity of sulfide was due to the reduction of sulfate contained in the SRB-containing medium prior to testing and measurements, as stated in Experimental Procedure. The bacterial number in the solution under a CP potential of 850 mV vs. CSE increased from 11.7 106 CFU/mL to 93.4 106 CFU/mL, where the CFU refers to colony-forming unit, after 3 days, and then kept approximately unchanged. Meanwhile, the bacterial number increased from 11.3 106 CFU/mL to 95.2 106 CFU/mL in the solution without CP after 7 days, as shown in Fig. 1b. The results showed that the SRB were active to reduce sulfate into sulfide in the solution. There was no obvious effect of the applied CP on the growth of planktonic bacterial cells and the sulfate reduction in the solution.
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OCP, 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing test solution. At OCP and 850 mV vs. CSE, the distributions of elements C and O were extensive, accumulating on the electrode surface, as seen in Figs. 3 and 4. They were mainly from corrosion products and the metabolic substances of SRB, such as iron oxide and extracellular polymeric substance [14]. However, at 1000 mV vs. CSE, the distribution of elements C and O was dispersive (Fig. 5). They were mainly from the biofilm. For element Fe, at OCP and 850 mV vs. CSE, its nonuniform distribution and even elemental accumulation showed that the Fe was from corrosion products. The distribution of Fe at 1000 mV vs. CSE was quite uniform, indicating that the element was from the steel substrate. The element S was mainly from biofilm and corrosion products. The color indicative of this element faded with the negative shift of the potential, which was due to the reduced thickness of the biofilm, as characterized by CLSM below. Fig. 7 shows the CLSM imaging of the biofilms forming on the
3.2. Morphological and compositional characterization of the corroded steel electrodes Fig. 2 shows the SEM morphological views of the steel electrodes at open-circuit potential (OCP), 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing test solution, where photos with two magnifications are provided for each electrode. It is noted that the overlapping of bacterial cells with corrosion products makes it impossible to count the number of SRB cells on the steel specimen. Moreover, most cells are actually buried under the corrosion products. Thus, a qualitative description is given herein. It is seen from Fig. 2 that, when the steel was at OCP, extensive corrosion products along with SRB cells formed on the electrode surface, as marked in Fig. 2a (1000). From the enlarged view in Fig. 2b (10,000), a great number of SRB cells were overlapped with the corrosion products. When the CP potential of 850 mV vs. CSE was applied on the electrode, the content of corrosion products decreased (Fig. 2c) compared to that in Fig. 2a. From the enlarged view in Fig. 2d, the corrosion products were still overlapped with the SRB cells. At 1000 mV vs. CSE, the steel electrode contained a few corrosion products only, with extensive SRB cells present (Fig. 2e). Moreover, the SRB cells overlapped each other, with some corrosion products existing among the cells. It was found that the bacterial cells seemed “branched”, which was actually a misvisualization generated during specimen preparation for SEM observation. When the steel electrodes were not properly coated with gold, the SEM electron beam could damage the SRB cells. Moreover, when the cells with various orientations overlap each other, the observed “branching” phenomenon also appears in the SEM views. Thus, the “branched” cells was not due to the contaminated microbes. Instead, the testing condition was controlled very carefully to avoid any potential contamination. The characterization of corrosion morphology qualitatively confirmed that the CP potential of 850 mV vs. CSE was not effective to protect the steel from corrosion in the presence of SRB, as evidenced by the extensive corrosion products generated on the steel surface. When the CP potential was shifted to 1000 mV vs. CSE, the uniform corrosion of the steel could be prevented, with only SRB present on the electrode surface. Obviously, the CP would affect the growth of sessile bacteria on the steel specimens. Figs. 3e6 show the SEM and EDS spectra of the steel electrode at
Fig. 3. SEM and EDS spectra measured on the steel specimens after 7 days of testing at different potentials (a) OCP, (b) 850 mV vs. CSE, (c) 1000 mV vs. CSE.
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Fig. 4. EDS spectra of the steel electrode at OCP after 7 days of testing in the SRB-containing test solution.
Fig. 5. EDS spectra of the steel electrode at 850 mV vs. CSE after 7 days of testing in the SRB-containing test solution.
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Fig. 6. EDS spectra of the steel electrode at 1000 mV vs. CSE after 7 days of testing in the SRB-containing test solution.
electrodes at OCP, 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB culturing solution. When the electrode was at OCP, the formed biofilm was thick, with an average thickness of 65.0 mm (Fig. 7a). Moreover, the dead sessile cells, as colored in red, were much more than live cells colored in green. At the CP potential of 850 mV vs. CSE, the biofilm was not continuous, and its thickness was about 43.5 mm, as shown in Fig. 7b. When the applied CP potential was 1000 mV vs. CSE, the biofilm was thinner and denser, and the average thickness was approximately 34.2 mm. The number of live sessile cells (green color) increased obviously (Fig. 7c), compared to that obtained at OCP in Fig. 7a. The measurements of the SRB number and sulfide concentration in Fig. 1 showed that the CP did not affect the SRB activity in the culturing solution. The morphological observation, especially at 1000 mV vs. CSE, showed that SRB can adhere to the steel surface extensively to form a layer of biofilm. The CLSM imaging further demonstrated that the biofilm can form on the steel surface under CP application, even at a very negative potential of 1000 mV vs. CSE. Generally, the presence of corrosion products on the steel could improve the biofilm attachment. However, the CP application protected the steel from corrosion and reduced the generation of corrosion products, especially at the sufficiently negative potential of 1000 mV vs. CSE, as seen in Fig. 2. Thus, the biofilm formation on the CP-applied steel, as evidenced in Fig. 7, indicated that the CP could facilitate the bacterial attachment on the steel surface in this testing system. As the CP potential was shifted negatively, the measured biofilm thickness reduced. Combined with the morphological observation in Fig. 2, it can be concluded that the large thickness of the surface film obtained at OCP was associated with the mutual overlapping of the biofilm and corrosion products. The imaged biofilm information
by CLSM gave a porous, loose structure because the corrosion products were not captured in the image. When the CP potential was not sufficiently negative, such as at 850 mV vs. CSE, corrosion still occurred on the steel. Since the amount of corrosion products reduced compared to that at OCP, the overlapping of corrosion products with biofilm was not remarkable. Thus, the captured biofilm had a reduced thickness. As the CP potential was further shifted to 1000 mV vs. CSE, corrosion products almost disappeared at this negative cathodic potential. The formed film was almost purely the biofilm, which became more compact and thinner. 3.3. Characterization of corrosion pits on the steel surface After removal of corrosion products and biofilm from the electrodes, which were at OCP, 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing solution, corrosion pits were identified by AFM observation, as shown in Fig. 8. The derived topographic profiles of the electrodes, especially the identified corrosion pits, are shown in Fig. 9. It was noted that, in this work, isolated pits were formed randomly, rather than extensively, on the electrode surface, especially at sufficiently negative CP potentials such as 1000 mV vs. CSE. Actually, many AFM scans and imaging were conducted over the electrode surface, and the images showed a similar result. Thus, only single AFM scan was selected for presentation. Some big and deep pits were selected to show that, even at CP, pitting corrosion still occurred in the SRBcontaining medium in the present system. At OCP, the formed pit #1 was 15.8 mm in diameter and 3.2 mm in depth, with an approximately semispherical shape. The pit #2 was 7.2 mm in diameter and 2.3 mm in depth, which was formed on the electrode at 850 mV vs. CSE. Pit #3, which was formed at 1000 mV vs. CSE,
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Fig. 7. CLSM imaging of the biofilms forming on the electrodes at (a) OCP, (b) 850 mV vs. CSE and (c) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRBcontaining test solution.
was deeper (3.5 mm in depth) and narrower (6.2 mm in length). It has been established that, in the absence of SRB, the steel corrosion can be prevented by CP in the culturing solution [15]. This work shows that pitting corrosion still occurred on the cathodically polarized steel electrode under biofilm although the applied CP could reduce the steel corrosion, especially at the sufficiently negative potential of 1000 mV vs. CSE. When the steel was at OCP, the formed pit was wide. This is attributed to both anodic dissolution occurring on the steel surface and the SRB induced pitting corrosion. At CP potential such as 1000 mV vs. CSE, the general corrosion of the steel was controlled effectively. The microbial corrosion occurring under the biofilm resulted in pit growth and generated deep and narrow pits on the steel. Actually, the biofilm could entrap the microbial metabolites, and generated a local gradients of pH along the pit depth. The live cells inside the pit could also obtain electrons directly from the steel in the absence of organic carbon [16e18]. Thus, although the general corrosion was controlled under the CP potential of 1000 mV vs. CSE, the pit still grew to generate a deep, narrow geometry.
Fig. 8. AFM images of the pit morphology on the steel electrode which were at (a) OCP, (b) 850 mV vs. CSE and (c) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing solution.
3.4. Mechanism of the CP and MIC interaction Field experiences showed that microorganisms, typically SRB, contained in the soil participate in pipeline corrosion and stress corrosion cracking [19]. The SRB can reduce sulfate (SO2 4 ) to sulfides (e.g., H2S and HS), while utilizing natural organic compounds or molecular hydrogen (H2) as electron donors [20]: þ 4H2 þ SO2 4 þ 2 H / H2S þ 4H2O
(2)
T. Liu, Y.F. Cheng / Journal of Alloys and Compounds 729 (2017) 180e188 þ 8e þ SO2 4 þ 10H / H2S þ 4H2O
187
(3)
The ability of SRB to obtain electrons directly from Fe0 is not restricted to iron serving as the electron donor [22,23]. In this work, under the CP potential of 1000 mV vs. CSE, the iron oxidation would be prevented: Fe þ 2Hþ / Fe2þ þ H2
(4)
However, the number of sessile cells on the steel increases as the CP potential becomes more negative (as shown in Fig. 7). Since the anodic reaction is controlled at the potential of 1000 mV vs. CSE, Fe0 would not act as the electron donor. The increasing live cells adhering on the steel depend mainly on the excessive electrons supplied by the CP power source. The electrons that accumulated on the steel surface are used as the electron donor by SRB in their metabolism when other donors are not available, as schematically shown in Fig. 10a. Upon the adhesion of the cells, pitting corrosion would be initiated due to potential fluctuations caused by the shielding effect of the biofilm (Fig. 10b). Therefore, if the CP potential is sufficiently negative, the SRB use the electrons from the CP source. However, when the potential is not sufficient locally due to reasons such as the shielding effect of the surface film, the SRB use electrons from the steel directly. That is the reason that pitting corrosion would occur on the steel even the applied CP is as negative as 1000 mV vs. CSE.
4. Conclusions
Fig. 9. Derived topographic profiles by AFM imaging of the steel electrodes which were at (a) OCP, (b) 850 mV vs. CSE and (c) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing solution.
Dinh et al. [21] found that the SRB can also use Fe0 directly as the electron donor, which causes sulfate reduction much faster than the conventional H2-scavenging process:
Application of CP on X70 pipeline steel in the medium containing SRB, i.e., Desulfovibrio desulfuricans, does not affect the growth of planktonic bacterial cells in the solution, but affects the sessile bacteria on the steel. The presence of SRB in the environment decreases the CP effectiveness for corrosion protection, and the NACE recommended CP potential of 850 mV vs. CSE is not sufficient to protect the steel from corrosion. A further negative shift of the CP potential to 1000 mV vs. CSE is effective to control uniform corrosion of the steel. The CP facilitates the bacterial attachment to the steel in this system, and a layer of biofilm can be formed on the CP-applied steel in the solution containing SRB Desulfovibrio desulfuricans. At OCP, the biofilm is overlapped with corrosion products. With the negative shift the CP potential
Fig. 10. Schematic diagrams illustrating (a) SRB use electrons supplied by CP power source (at the first step), and (b) sessile SRB use electrons from the steel directly (at the second step).
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to 1000 mV vs. CSE, uniform corrosion is controlled, and the surface film is mainly composed of biofilm. However, pitting corrosion would still occur on the steel under the biofilm even when the steel is under the CP potential of 1000 mV vs. CSE. The number of live cells in the biofilm increases with the negative shift of the CP potential, and its role in pitting corrosion is probably related to the local shielding effect on CP permeation on the steel, and, as a result, the use of electrons by SRB directly from the steel. Acknowledgments This work was supported by National Basic Research Program of China (2014CB643306), Innovation Program of Shanghai Municipal Science and Technology (14DZ1205802, 14520501800) and Shanghai Natural Science Foundation (14ZR1419800). The biological testing and analysis were conducted in Dr. Gerrit Voordouw's laboratory in the Department of Biological Science at the University of Calgary.
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Journal of Alloys and Compounds journal homepage: http://www.elsevier.com/locate/jalcom
The influence of cathodic protection potential on the biofilm formation and corrosion behaviour of an X70 steel pipeline in sulfate reducing bacteria media Tao Liu a, b, Y. Frank Cheng b, * a b
College of Ocean Science and Engineering, Shanghai Maritime University, Shanghai, China Department of Mechanical Engineering, 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 15 June 2017 Received in revised form 9 September 2017 Accepted 16 September 2017 Available online 19 September 2017
This work investigated the biofilm formation and microbiologically influenced corrosion (MIC) of a pipeline steel under cathodic protection (CP) in an extracted soil in solution with addition of sulfate reducing bacteria (SRB), Desulfovibrio desulfuricans. It was found that the application of CP did not affect the planktonic bacterial growth in the solution. The CP facilitated the bacterial attachment to the steel in this system, and a layer of biofilm formed on the cathodically protected steel, decreasing the CP effectiveness for corrosion protection. The CP potential of 850 mV vs. copper sulfate electrode (CSE) was not sufficient to protect the steel from corrosion in SRB containing media. Despite a further negative shift of the CP potential to 1000 mV vs. CSE was effective to control uniform corrosion of the steel, pitting corrosion still occurred under biofilm on the steel. © 2017 Elsevier B.V. All rights reserved.
Keywords: Cathodic protection Pipelines Biofilm Microbiologically influenced corrosion Pitting corrosion
1. Introduction The presence of microorganism in environments is able to accelerate coating degradation and cause the so-called microbiologically influenced corrosion (MIC) even in right CP conditions [1e3], in which would not normally be seen corrosion without microorganisms. In aqueous solutions containing sulfate reducing bacteria (SRB), it was suggested that the applied CP potential should be shifted to 950 mV vs. CSE or even more negative values in order to fully protect steels from corrosion [4]. There have been some investigations on the mutual effect of CP and microbial activity. Guezennec paid attention to this problem 20 years ago [5]. In order to determine whether there was a relationship between cathodic polarization and the biofilm formation on steels, experiments were conducted on clean metal surfaces and biofilmed surfaces in natural or synthetic seawater. It was found that cathodically produced hydrogen facilitated growth of hydrogenase-containing SRB. Nekoksa and Gutherman's work also showed that the CP application enhanced the incubation and growth of microbes, with more bacteria settling on a cathodically
* Corresponding author. E-mail address: [email protected] (Y.F. Cheng). http://dx.doi.org/10.1016/j.jallcom.2017.09.181 0925-8388/© 2017 Elsevier B.V. All rights reserved.
protected stainless steel than on unpolarized ones [6]. Similar results were obtained by Zavala et al. [7], who found that the SRB population on CP-applied (i.e., 850 mV vs. CSE) pipeline steel was twice of the microorganisms on the coupons without CP application. Edyvean et al.’s work also investigated the interaction of CP and the biofilm formation on steels [8]. However, their finding was opposite to those listed above. On both stainless and non-stainless steels, CP retarded the development of bacterial fouling. Increasing the CP decreased the number of bacteria. Little and Wagner confirmed that, when the CP current was intermittent, the corrosion attack due to the microorganisms became more aggressive [9]. Regarding the mechanistic aspect of the effect of CP on MIC, Li et al. believed that there existed a synergistic effect of coating disbondment and partial shielding of CP current from reaching the disbonded crevice, where an environment favorable for active growth of SRB was generated [10]. A quick review of relevant literature on this topic shows that some of reported results are controversial each other [6e8]. The understanding of the mutual interaction of CP and MIC is vague. Moreover, uniform corrosion of metals in the presence of microorganisms is the focused topic in most work. The essential influence of cathodic polarization on MIC induced pitting corrosion has been lacking. The present work attempts to address these
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unknowns, and advances our understanding to this important problem. In this work, the biofilm formation and MIC of an X70 pipeline steel under various CP potentials in an extracted soil solution containing SRB, i.e., Desulfovibrio desulfuricans, were studied by biological testing and materials characterization techniques, including scanning electron microscopy (SEM), energy-dispersive X-ray spectrum (EDS), confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). The effect of CP application on bacterial growth was measured. The formation of biofilm on the steel under varied CP potentials was characterized. Both uniform corrosion and pitting corrosion of the steel in the SRB-containing soil solution were considered. The interactions between CP and the SRB induced MIC, particularly, pitting corrosion, were analyzed.
2. Experimental
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2.3. Surface characterization After 7 days of cathodic polarization in the test solution, the steel electrodes were removed from the solution, washed with a phosphate-buffered saline solution (pH 7.4), and then dipped in 2.5% glutaraldehyde fixative at 4 C for 12 h. The electrodes were dehydrated with various concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 10 min successively, and fully dried in N2 environment. A field emission SEM (Field Electron and Ion Co. Quanta 250 FEG) was used to characterize the surface morphology of the steel electrode. The elemental composition was obtained through the coupled EDS (Bruker Quantax EDS). It was confirmed with the EDS manufacturer that the penetration depth is 2e5 mm for the steel specimen under the testing condition in this work, where the operating voltage was 15 kV. Bacterial attachment and the biofilm formed on the steel electrode were characterized using a confocal laser scanning microscope (Olympus, FV1000). Prior to observation, the electrodes were
2.1. Material and specimen Specimens used in this work were cut from an X70 steel pipe, with the chemical composition (wt.%): 0.06 C, 1.44 Mn, 0.31 Si, 0.004 S, 0.01 P, 0.034 Ni, 0.16 Cr, 0.25 Mo, 0.005 V, 0.015 Cu, 0.01 Ti, 0.002 B, 0.029 Al and Fe balance. The outer and inner diameters of the pipe were 812.8 mm and 796.9 mm, respectively. The microstructure of the steel contained ferrite and pearlite. The specimens were machined into a circular shape, and sealed by epoxy resin, leaving an exposed area of 10 mm in diameter. The work face of the specimen was ground to a final 1200 grit SiC paper. The specimen was then degreased with acetone, washed with distilled water, and dried naturally in a high-purity nitrogen (99.999%) environment.
2.2. Microbial cell culturing The SRB (i.e., Desulfovibrio desulfuricans) used in this work were purchased from America Typical Culture Collection. The soil was taken from Regina, Canada, and the chemical composition of the extracted soil solution is shown in Table 1. The medium for SRB culturing contained 0.5 g K2HPO4, 0.5 g (NH4)2SO4, 5 g sodium citrate, 3.5 g sodium lactate and 1 L extracted soil solution as mentioned above. The culturing medium was sterilized by autoclaving at 121 C for 20 min. It was then cooled down in ice water for 30 min, while purging with N2/CO2 (9:1, v/v) gas to remove oxygen until the content of dissolved oxygen was below 0.4 mg/L, as measured by a dissolved oxygen meter (ExStik DO600). 0.1 g of as-received SRB powders, which were in frozen state, was added in 10 mL of the culturing medium and kept in an incubator at 35 C for 3 days. After that, the 10 mL SRBcontaining medium was mixed with 500 mL culturing medium to prepare the test solution in this work. The number of SRB in the test solution was measured using the most probable number (MPN) method [11]. The concentration of sulfide in the solution was measured by the methylene blue method [12]. To ensure the reproducibility of data, the measurements of the sulfide concentration and the number of bacteria in the solution were conducted three times.
Table 1 Chemical composition of the extracted soil solution (in 1 L distilled water, pH ¼ 7.5). Chemicals Amount (g)
NaHCO3 0.0760
NaNO3 0.0014
NaCl 0.0089
Na2SO4 0.0775
CaSO4 0.8823
K2SO4 0.0619
MgSO4 0.3326
Fig. 1. Time dependence of (a) the sulfide concentration and (b) the number of Desulfovibrio desulfuricans in the test solution while the steel electrode is under a CP potential of 850 mV vs. CSE and without CP.
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washed with milli Q water, and then stained with a fluorescent dye (Molecular Probes™ FilmTracer™ LIVE/DEAD® Biofilm Viability Kit) in the darkness following the manufacturers' procedure. After SEM/EDS characterizations, a modified NACE RP07752005 protocol was used to remove corrosion products including biofilm from the electrode surface.18 The electrode was washed in dibutylthiourea-HCl solution, sodium bicarbonate solution, deionized water and acetone, respectively, for 2 min. According to NACE RP0755-2005,18 the specimen, upon removal of corrosion products and biofilm, is dried in a stream of air. This could cause oxidation of the steel in air. In this work, the specimens were dried naturally in the high-purity nitrogen environment, avoiding the steel oxidization. An AFM (Keysight 5500 scanning probe microscope system) was used to image the topography of the steel electrode, where 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 20 mm 20 mm was selected for AFM imaging. The topographic profile of the electrode was derived from AFM images. To ensure the reproducibility of the characterization results, three parallel specimens were prepared for each testing condition.
3. Results and discussion 3.1. Measurements of sulfide concentration and bacterial number in the test solution The SRB play a key role in the sulfur cycle. They can use sulfate (SO2 4 ) as a terminal electron acceptor in degradation of organic matters, producing sulfide by Ref. [13]:
Fig. 2. SEM morphological views of the steel electrodes at (a, b) OCP, (c, d) 850 mV vs. CSE and (e, f) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing test solution, where photos with two magnifications are provided for each electrode.
T. Liu, Y.F. Cheng / Journal of Alloys and Compounds 729 (2017) 180e188 2 þ 2C3H5O 3 þ SO4 / 2C2H3O2 þ 2HCO3 þ HS þ H
(1)
Fig. 1 shows the time dependence of the sulfide concentration and the planktonic bacterial number in the test solution while the steel electrode is under a CP potential of 850 mV vs. CSE. For comparison, the measurements were also conducted in the absence of CP application on the steel. It was seen that, in both the absence and presence of CP, the sulfide concentration increased from 0.8 mmol/L to 5.8 mmol/L after 5 days, and then approximately maintained at this value (Fig. 1a). It was noted that the initial quantity of sulfide was due to the reduction of sulfate contained in the SRB-containing medium prior to testing and measurements, as stated in Experimental Procedure. The bacterial number in the solution under a CP potential of 850 mV vs. CSE increased from 11.7 106 CFU/mL to 93.4 106 CFU/mL, where the CFU refers to colony-forming unit, after 3 days, and then kept approximately unchanged. Meanwhile, the bacterial number increased from 11.3 106 CFU/mL to 95.2 106 CFU/mL in the solution without CP after 7 days, as shown in Fig. 1b. The results showed that the SRB were active to reduce sulfate into sulfide in the solution. There was no obvious effect of the applied CP on the growth of planktonic bacterial cells and the sulfate reduction in the solution.
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OCP, 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing test solution. At OCP and 850 mV vs. CSE, the distributions of elements C and O were extensive, accumulating on the electrode surface, as seen in Figs. 3 and 4. They were mainly from corrosion products and the metabolic substances of SRB, such as iron oxide and extracellular polymeric substance [14]. However, at 1000 mV vs. CSE, the distribution of elements C and O was dispersive (Fig. 5). They were mainly from the biofilm. For element Fe, at OCP and 850 mV vs. CSE, its nonuniform distribution and even elemental accumulation showed that the Fe was from corrosion products. The distribution of Fe at 1000 mV vs. CSE was quite uniform, indicating that the element was from the steel substrate. The element S was mainly from biofilm and corrosion products. The color indicative of this element faded with the negative shift of the potential, which was due to the reduced thickness of the biofilm, as characterized by CLSM below. Fig. 7 shows the CLSM imaging of the biofilms forming on the
3.2. Morphological and compositional characterization of the corroded steel electrodes Fig. 2 shows the SEM morphological views of the steel electrodes at open-circuit potential (OCP), 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing test solution, where photos with two magnifications are provided for each electrode. It is noted that the overlapping of bacterial cells with corrosion products makes it impossible to count the number of SRB cells on the steel specimen. Moreover, most cells are actually buried under the corrosion products. Thus, a qualitative description is given herein. It is seen from Fig. 2 that, when the steel was at OCP, extensive corrosion products along with SRB cells formed on the electrode surface, as marked in Fig. 2a (1000). From the enlarged view in Fig. 2b (10,000), a great number of SRB cells were overlapped with the corrosion products. When the CP potential of 850 mV vs. CSE was applied on the electrode, the content of corrosion products decreased (Fig. 2c) compared to that in Fig. 2a. From the enlarged view in Fig. 2d, the corrosion products were still overlapped with the SRB cells. At 1000 mV vs. CSE, the steel electrode contained a few corrosion products only, with extensive SRB cells present (Fig. 2e). Moreover, the SRB cells overlapped each other, with some corrosion products existing among the cells. It was found that the bacterial cells seemed “branched”, which was actually a misvisualization generated during specimen preparation for SEM observation. When the steel electrodes were not properly coated with gold, the SEM electron beam could damage the SRB cells. Moreover, when the cells with various orientations overlap each other, the observed “branching” phenomenon also appears in the SEM views. Thus, the “branched” cells was not due to the contaminated microbes. Instead, the testing condition was controlled very carefully to avoid any potential contamination. The characterization of corrosion morphology qualitatively confirmed that the CP potential of 850 mV vs. CSE was not effective to protect the steel from corrosion in the presence of SRB, as evidenced by the extensive corrosion products generated on the steel surface. When the CP potential was shifted to 1000 mV vs. CSE, the uniform corrosion of the steel could be prevented, with only SRB present on the electrode surface. Obviously, the CP would affect the growth of sessile bacteria on the steel specimens. Figs. 3e6 show the SEM and EDS spectra of the steel electrode at
Fig. 3. SEM and EDS spectra measured on the steel specimens after 7 days of testing at different potentials (a) OCP, (b) 850 mV vs. CSE, (c) 1000 mV vs. CSE.
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Fig. 4. EDS spectra of the steel electrode at OCP after 7 days of testing in the SRB-containing test solution.
Fig. 5. EDS spectra of the steel electrode at 850 mV vs. CSE after 7 days of testing in the SRB-containing test solution.
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Fig. 6. EDS spectra of the steel electrode at 1000 mV vs. CSE after 7 days of testing in the SRB-containing test solution.
electrodes at OCP, 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB culturing solution. When the electrode was at OCP, the formed biofilm was thick, with an average thickness of 65.0 mm (Fig. 7a). Moreover, the dead sessile cells, as colored in red, were much more than live cells colored in green. At the CP potential of 850 mV vs. CSE, the biofilm was not continuous, and its thickness was about 43.5 mm, as shown in Fig. 7b. When the applied CP potential was 1000 mV vs. CSE, the biofilm was thinner and denser, and the average thickness was approximately 34.2 mm. The number of live sessile cells (green color) increased obviously (Fig. 7c), compared to that obtained at OCP in Fig. 7a. The measurements of the SRB number and sulfide concentration in Fig. 1 showed that the CP did not affect the SRB activity in the culturing solution. The morphological observation, especially at 1000 mV vs. CSE, showed that SRB can adhere to the steel surface extensively to form a layer of biofilm. The CLSM imaging further demonstrated that the biofilm can form on the steel surface under CP application, even at a very negative potential of 1000 mV vs. CSE. Generally, the presence of corrosion products on the steel could improve the biofilm attachment. However, the CP application protected the steel from corrosion and reduced the generation of corrosion products, especially at the sufficiently negative potential of 1000 mV vs. CSE, as seen in Fig. 2. Thus, the biofilm formation on the CP-applied steel, as evidenced in Fig. 7, indicated that the CP could facilitate the bacterial attachment on the steel surface in this testing system. As the CP potential was shifted negatively, the measured biofilm thickness reduced. Combined with the morphological observation in Fig. 2, it can be concluded that the large thickness of the surface film obtained at OCP was associated with the mutual overlapping of the biofilm and corrosion products. The imaged biofilm information
by CLSM gave a porous, loose structure because the corrosion products were not captured in the image. When the CP potential was not sufficiently negative, such as at 850 mV vs. CSE, corrosion still occurred on the steel. Since the amount of corrosion products reduced compared to that at OCP, the overlapping of corrosion products with biofilm was not remarkable. Thus, the captured biofilm had a reduced thickness. As the CP potential was further shifted to 1000 mV vs. CSE, corrosion products almost disappeared at this negative cathodic potential. The formed film was almost purely the biofilm, which became more compact and thinner. 3.3. Characterization of corrosion pits on the steel surface After removal of corrosion products and biofilm from the electrodes, which were at OCP, 850 mV vs. CSE and 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing solution, corrosion pits were identified by AFM observation, as shown in Fig. 8. The derived topographic profiles of the electrodes, especially the identified corrosion pits, are shown in Fig. 9. It was noted that, in this work, isolated pits were formed randomly, rather than extensively, on the electrode surface, especially at sufficiently negative CP potentials such as 1000 mV vs. CSE. Actually, many AFM scans and imaging were conducted over the electrode surface, and the images showed a similar result. Thus, only single AFM scan was selected for presentation. Some big and deep pits were selected to show that, even at CP, pitting corrosion still occurred in the SRBcontaining medium in the present system. At OCP, the formed pit #1 was 15.8 mm in diameter and 3.2 mm in depth, with an approximately semispherical shape. The pit #2 was 7.2 mm in diameter and 2.3 mm in depth, which was formed on the electrode at 850 mV vs. CSE. Pit #3, which was formed at 1000 mV vs. CSE,
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Fig. 7. CLSM imaging of the biofilms forming on the electrodes at (a) OCP, (b) 850 mV vs. CSE and (c) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRBcontaining test solution.
was deeper (3.5 mm in depth) and narrower (6.2 mm in length). It has been established that, in the absence of SRB, the steel corrosion can be prevented by CP in the culturing solution [15]. This work shows that pitting corrosion still occurred on the cathodically polarized steel electrode under biofilm although the applied CP could reduce the steel corrosion, especially at the sufficiently negative potential of 1000 mV vs. CSE. When the steel was at OCP, the formed pit was wide. This is attributed to both anodic dissolution occurring on the steel surface and the SRB induced pitting corrosion. At CP potential such as 1000 mV vs. CSE, the general corrosion of the steel was controlled effectively. The microbial corrosion occurring under the biofilm resulted in pit growth and generated deep and narrow pits on the steel. Actually, the biofilm could entrap the microbial metabolites, and generated a local gradients of pH along the pit depth. The live cells inside the pit could also obtain electrons directly from the steel in the absence of organic carbon [16e18]. Thus, although the general corrosion was controlled under the CP potential of 1000 mV vs. CSE, the pit still grew to generate a deep, narrow geometry.
Fig. 8. AFM images of the pit morphology on the steel electrode which were at (a) OCP, (b) 850 mV vs. CSE and (c) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing solution.
3.4. Mechanism of the CP and MIC interaction Field experiences showed that microorganisms, typically SRB, contained in the soil participate in pipeline corrosion and stress corrosion cracking [19]. The SRB can reduce sulfate (SO2 4 ) to sulfides (e.g., H2S and HS), while utilizing natural organic compounds or molecular hydrogen (H2) as electron donors [20]: þ 4H2 þ SO2 4 þ 2 H / H2S þ 4H2O
(2)
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(3)
The ability of SRB to obtain electrons directly from Fe0 is not restricted to iron serving as the electron donor [22,23]. In this work, under the CP potential of 1000 mV vs. CSE, the iron oxidation would be prevented: Fe þ 2Hþ / Fe2þ þ H2
(4)
However, the number of sessile cells on the steel increases as the CP potential becomes more negative (as shown in Fig. 7). Since the anodic reaction is controlled at the potential of 1000 mV vs. CSE, Fe0 would not act as the electron donor. The increasing live cells adhering on the steel depend mainly on the excessive electrons supplied by the CP power source. The electrons that accumulated on the steel surface are used as the electron donor by SRB in their metabolism when other donors are not available, as schematically shown in Fig. 10a. Upon the adhesion of the cells, pitting corrosion would be initiated due to potential fluctuations caused by the shielding effect of the biofilm (Fig. 10b). Therefore, if the CP potential is sufficiently negative, the SRB use the electrons from the CP source. However, when the potential is not sufficient locally due to reasons such as the shielding effect of the surface film, the SRB use electrons from the steel directly. That is the reason that pitting corrosion would occur on the steel even the applied CP is as negative as 1000 mV vs. CSE.
4. Conclusions
Fig. 9. Derived topographic profiles by AFM imaging of the steel electrodes which were at (a) OCP, (b) 850 mV vs. CSE and (c) 1000 mV vs. CSE, respectively, after 7 days of testing in the SRB-containing solution.
Dinh et al. [21] found that the SRB can also use Fe0 directly as the electron donor, which causes sulfate reduction much faster than the conventional H2-scavenging process:
Application of CP on X70 pipeline steel in the medium containing SRB, i.e., Desulfovibrio desulfuricans, does not affect the growth of planktonic bacterial cells in the solution, but affects the sessile bacteria on the steel. The presence of SRB in the environment decreases the CP effectiveness for corrosion protection, and the NACE recommended CP potential of 850 mV vs. CSE is not sufficient to protect the steel from corrosion. A further negative shift of the CP potential to 1000 mV vs. CSE is effective to control uniform corrosion of the steel. The CP facilitates the bacterial attachment to the steel in this system, and a layer of biofilm can be formed on the CP-applied steel in the solution containing SRB Desulfovibrio desulfuricans. At OCP, the biofilm is overlapped with corrosion products. With the negative shift the CP potential
Fig. 10. Schematic diagrams illustrating (a) SRB use electrons supplied by CP power source (at the first step), and (b) sessile SRB use electrons from the steel directly (at the second step).
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to 1000 mV vs. CSE, uniform corrosion is controlled, and the surface film is mainly composed of biofilm. However, pitting corrosion would still occur on the steel under the biofilm even when the steel is under the CP potential of 1000 mV vs. CSE. The number of live cells in the biofilm increases with the negative shift of the CP potential, and its role in pitting corrosion is probably related to the local shielding effect on CP permeation on the steel, and, as a result, the use of electrons by SRB directly from the steel. Acknowledgments This work was supported by National Basic Research Program of China (2014CB643306), Innovation Program of Shanghai Municipal Science and Technology (14DZ1205802, 14520501800) and Shanghai Natural Science Foundation (14ZR1419800). The biological testing and analysis were conducted in Dr. Gerrit Voordouw's laboratory in the Department of Biological Science at the University of Calgary.
[8]
[9] [10]
[11] [12]
[13]
[14]
[15] [16]
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