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Microbial consortium influence upon steel corrosion rate, using polarisation resistance and electrochemical noise techniques
Electrochimica Acta 49 (2004) 4295–4301
Microbial consortium influence upon steel corrosion rate, using polarisation resistance and electrochemical noise techniques M.J. Hernández Gayosso a,1 , G. Zavala Olivares b,∗ , N. Ruiz Ordaz a , C. Juárez Ramirez a , R. Garc´ıa Esquivel b , A. Padilla Viveros b a
b
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. Prolongación de Carpio y Plan de Ayala, México, DF, C.P. 11340, Mexico Instituto Mexicano del Petróleo, Grupo de Corrosión. Eje Central Lázaro Cárdenas 152, Col. San Bartolo Atepehuacan, México, DF, C.P. 07730, Mexico Received 3 November 2003; received in revised form 8 March 2004; accepted 12 March 2004 Available online 10 June 2004
Abstract The microbiologically influenced corrosion (MIC) is a process, which affects the oil industry, particularly the hydrocarbons extraction, transport and storage. MIC evaluation has been normally based upon microbiological tests, and just a few references mention alternating methods, such as the electrochemical techniques, which can be used as criteria for their evaluation. In this work, two different electrochemical laboratory techniques, polarisation resistance and electrochemical noise were used, in order to determine the corrosion behaviour of a microbial consortium, obtained from a gas transporting pipeline, located in the southeast of Mexico. The bacteria population growth was found to be different for sessile and plancktonic microorganisms. Moreover, long incubation times were required to reach the maximum concentration of sessile bacteria. The electrochemical techniques used in this study exhibited a similar tendency on the corrosion rate behaviour with time, and values above 0.3 mm year−1 were observed at the end of the experiments. The experiments were complemented with surface analysis. Scanning electron microscope observation of APIXL52 steel coupons, exposed to the consortium action, revealed bacteria presence, as well as a damaged steel surface. A type of localized corrosion was observed on the metal surface, and it was associated to the bacteria effect. © 2004 Elsevier Ltd. All rights reserved. Keywords: Microbiologically influenced corrosion; Electrochemical techniques; Biofilm
1. Introduction The microbiologically influenced corrosion (MIC) is a very dangerous process, which affects the oil industry, particularly during the hydrocarbon extraction, transport and storage [1–3]. The activity and microorganisms’ growth at the pipelines steel may cause surface modifications, which induce a more complex corrosion process. In general, the study of the MIC has been based upon microbiological tests and gravimetric techniques, and just few references mention alternating methods, which can be used as criteria for their evaluation.
∗
Corresponding author. Tel.: +52-55-9175-6838; fax: +52-55-9175-6844. E-mail address: [email protected] (G. Zavala Olivares). 1 Tel.:+52-55-57296000. 0013-4686/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2004.03.038
The use of electrochemical techniques [4–6] for the evaluation of MIC, such as polarisation resistance (PR) and electrochemical noise (EN), has been considered during the corrosion rate determination and this is an interesting area which requires more research, in order to implement and standardize their laboratory experiments and field application. Most of the study involving electrochemical techniques and MIC processes has been done considering short experimental times. Under these conditions, mainly low corrosion rates can be observed. Usually, the experimental time is established considering several aspects, such as: • kinetics growth of microorganisms in the electrolyte (plancktonic microorganisms), • sulphate consumption • sulphuric acid production.
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However, a basic characteristic on the MIC process frequently is not considered: biofilm formation on the metal surface. Kinetics growth is different for plancktonic microorganisms and those established at the metal surface (sessile microorganisms). Once the biofilm is formed, the corrosion damage on the metal surface should be dependent mainly on the sessile microorganisms, as the plancktonic microorganisms are not in contact with the metal surface. In this study, the PR and EN techniques were used to evaluate the corrosion rate of carbon steel in presence of plancktonic and sessile microorganisms, in order to determine the influence of both: plancktonic and sessile microorganisms, on the MIC processes.
using poliacrylamide gel electrophoresis [10]. The bacterial group present on the surface was identified by means of the complete gene amplification (1502 bp). Using the 8FPL and 1492 RPL initiators [11], the obtained sequence was processed using the National Center for Biotechnology Information (NCBI) data base: http://www.ncbi.nlm.nih.gov. 2.3. Surface analysis of the coupons exposed to microorganisms The coupons were immersed for 1 h in a 2% glutaraldehyde solution, in order to fix the biofilm to the steel surface, and then become dehydrated using four ethanol solutions (15 min each): 25, 50, 75 and 100%, successively. After that, the samples were taken to the SEM for their analysis.
2. Experimental 2.4. Corrosion rate determination using electrochemical techniques
2.1. Microorganisms cultivation The consortium used in this work was taken from a gas pipeline located in the Marine Region of Pemex, in México, during the inner cleaning procedure. The collected samples were inoculated in a selective medium, following the recommendations for field biological sampling, stated on API-RP 38 [7]. The microorganisms were maintained in the laboratory using the Posgate C [8] medium (Table 1), nitrogen atmosphere and at room temperature. The consortium was inoculated in vials with 100 ml Posgate C medium. API XL52 steel coupons (polished and degreased) were placed into the vials and incubated at room temperature. These vials were used as “sacrificial” to determine the consortium kinetics growth. This was done following the most probable number (MPN) method. 2.2. Microorganisms diversity The microorganisms diversity was determined using the thermal gradient gel electrophoresis (TGGE) technique with amplified fragments of 16S rDNA gene. The chain reaction (PCR) was carried out using the U968-GC and 1401 initiators [9]. The DNA fragments generated were observed Table 1 Composition of the Postgate C medium [8] Compound KH2 PO4 NH4 Cl Na2 SO4 MgSO4 ·7H2 O Sodium lactate CaCl2 ·2H2 O FeSO4 ·7H2 O Yeast extract Na citrate NaCl
These experiments were carried out in a 1 l standard cell, with a three electrode system: API XL52 steel as working electrode, a graphite rod as auxiliary and a saturated calomel electrode as reference. The electrolyte used was 800 ml Posgate C medium. Nitrogen gas was bubbled to remove all the oxygen and maintain anaerobic conditions. The electrochemical cell was connected to an ACM 772 potentiostat, and a PC was used for data recording. All experiments lasted approximately one month, and during this time, several PR and EN tests were carried out. All experiments were repeated twice. 2.5. Polarization resistance technique A potentiodynamic method was used to obtain the potential-current (E-I) ratio, applying a ± 10 mV overpotential, with respect to the free corrosion potential, Ecorr. The Sequencer® and Core running® software programs were used for data management, along with the V4 Analysis® software for data analysis. From this technique, resistance values are obtained (Rp ), which are used to calculate the corrosion current density, according to: icorr =
g/L 0.5 1.0 4.5 0.06 6.0 0.06 0.004 1.0 0.3 15
β Rp
(1)
where β is the Tafel slope, and a value of 0.026 V decade−1 is considered. Faraday’s law can be used to relate the corrosion current density to the corrosion rate, with the next equation: corrosion rate (CR) = K
icorr EW δ
(2)
where K is constant, EW is equivalent weight and δ is density.
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2.6. Electrochemical noise technique To perform these experiments, three identical steel electrodes were considered, using one of them as reference electrode. Measurements were taken with the Sequencer® and Core running® software programs, and the EN Analyse® program was used to analyse all data. Each experiment lasted approximately 2000 s, taking one point every second. The data obtained from this technique, are used to calculate, initially, the resistance noise, Rn, according to: Rn =
V I
(3)
where V is potential standard deviation and I is current standard deviation. The Rn value is equivalent to Rp value; therefore, the corrosion rate calculation is made according to Eqs. (1) and (2). Fig. 2. Pattern band for amplified fragments of 16S rDNA, obtained from TGGE technique.
3. Results and discusion 3.1. Kinetics growth
Log MPN
The kinetics growth for plancktonic and sessile microorganisms are shown in Fig. 1. The plancktonic microorganisms exhibit their fastest population growth around 150 h (approximately 107 bacteria ml−1 ); from this time, the bacteria population decreased. For the sessile microorganisms, the population growth is less accelerated than for the plancktonic and the maximum population is observed around 600 h. The plancktonic microorganisms, when in contact with an enriched medium, increase their population until the environmental conditions become adverse (nutrients decrease, pH, etc.); for the sessile microorganisms, the population growth is gradual and could be enhanced by a change on the conditions at the end of the kinetics [12]. There are some studies about MIC evaluation using electrochemical techniques, but only data about plancktonic microorganisms kinetics growth is provided [13,14]. In these
studies, only the time for the maximum plancktonic microorganisms growth is considered during the experiments. However, according to the kinetics growth observed in Fig. 1, the sessile microorganisms require more incubation time than the plancktonic, to get their maximum population and a direct relationship between the damage on the metal and the sessile population is expected. Therefore, the incubation time should be long enough to allow a complete sessile microorganisms growth. 3.2. Consortium diversity Using the TGGE technique, it was found that the consortium used in this work is constituted by five different bacteria species, as shown in Fig. 2. This five bacteria are observed
9 8 7 6 5 4 3 2 1 0 0
100
200
300
400
500
600
700
800
900
Time/hr Sessile/cm2
Plancktonic/ml
Fig. 1. Kinetics growth for plancktonic and sessile microorganisms in Postgate C medium. API XL 52 steel coupons.
Fig. 3. Biofilm formed on the metal surface, after 1000 h exposition time.
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(b) The light areas indicated, apart from the FeS, presence of nitrogen and carbon, due to the organic material (bacteria and exopolymer). Fig. 4 shows the biofilm formed on the metal surface, where typical Desulfovibrio vietnamensis bacteria, approximately 2 m size, are observed. 3.4. Electrochemical techniques
Fig. 4. Microorganisms in biofilm.
in the aqueous medium (plancktonic microorganisms), and only one is observed at the metal surface (sessile microorganism). The pattern band for the sessile microorganism is observed as well for plancktonic bacteria. The 16S rDNA gene of the sessile microorganism is formed of approximately 1500 bp and according to the gene amplification there is a similarity of 97% with Desulfovibrio vietnamensis. This specie has been isolated previously in the oil industry [15]. It is very important to point out that the Desulfovibrio vietnamensis is the specie at the metal surface and the main responsible for MIC processes, by means of the metabolic products formed at the interface. However, the plancktonic microorganisms should have some influence on the corrosion process. 3.3. Biofilm surface analysis The biofilm formed on the metal surface during the experiments is shown in Fig. 3, where the following can be observed: (a) The dark compact regions are formed by inorganic corrosion products and a microanalysis indicated high concentration of iron and sulfur (FeS).
Corrosion rate values, obtained from the PR technique are shown in Fig. 5. From this Figure, an initial corrosion rate of around 0.15 mm year−1 was observed for the metal exposed to the Postgate C medium, and this value decreased during the next hours until negligible values were observed, around 0.005 mm year−1 . The Postgate C medium contains high concentration of aggressive anions, such as Cl− , SO4 2− and S2− , that could be responsible for a high initial corrosion rate. However, during the next hours, corrosion products are formed and the metallic surface is isolated from the Postgate C medium. On the other hand, the corrosion rate induced by the consortium exhibited an initial corrosion rate around 0.02 mm year−1 , which slightly increased with time until about 0.05 mm year−1 after 400 h. After that time, the corrosion rate was accelerated until values around 0.5 mm year−1 after 998 h. The corrosion rate observed for the metal exposed to the Postgate C medium, obtained from the EN technique, is shown in Fig. 6. From this figure, the initial corrosion rate value was around 0.2 mm year−1 for the sterile conditions, and this value decreased with time until about 0.01 mm year−1 at the end of the experiment. For the metal exposed to the bacterial consortium, an initial corrosion rate of about 0.025 mm year−1 was observed, remaining stable for about 500 h. After that time, an increment was observed, reaching values up to 0.35 mm year−1 at the end of the experiment. The corrosion rates obtained from both techniques, PR and EN, were very similar between them. Using these techniques, a sudden increase on the corrosion rate was observed after 400 h. It is clear that there is not any relationship 0.4
0.6
0.35 Vcorr/mm.y
Vcorr/mm.y
-1
-1
0.5 0.4 0.3 0.2
0.3 0.25 0.2 0.15 0.1 0.05
0.1
0
0 0
200
400
600
800
1000
0
200
Consortium
Fig. 5. Corrosion rates calculated from the PR technique. API XL 52 steel, exposed to sterile conditions and in presence of microorganisms.
600
800
1000
Time/hr
Time/hr Sterile conditions
400
Sterile conditions
Consortium
Fig. 6. Corrosion rates calculated from the EN technique. API XL 52 steel, exposed to sterile conditions and in presence of microorganisms.
M.J. Hern´andez Gayosso et al. / Electrochimica Acta 49 (2004) 4295–4301 1.00E- 07
0.4 0.35 0.3 0.25
6.00E- 08
0.2 4.00E- 08
0.15
Vcorr/mmy-1
Var I/V2
8.00E- 08
0.1
2.00E- 08
0.05 0.00E+00 0
Var
200
400
600
800
0 1000
Time/hr
Vcorr
Fig. 7. Current variance and corrosion rate values. API XL 52 steel exposed to the microbial consortium. 3
0.4 0.35
2.5
-1
0.25
1.5
0.2 0.15
1
Vcorr/mmy
Var E/A
2
0.3 2
0.1 0.5
0.05
0 0 Var
200 Vcorr
400
600
800
0 1000
Time/hr
Fig. 8. Potential variance and corrosion rate values. API XL 52 steel exposed to the microbial consortium.
between the steel corrosion kinetics (Figs. 5 and 6) and the plancktonic microorganisms kinetics (Fig. 1). When the plancktonic microorganisms reach their maximum population, the corrosion rate is relatively low. On the contrary, there is a direct relationship between the sessile microorganisms growth and the corrosion rate increment. It seemed to be a critical point where, after 400 h, the bacterial population at the surface reached values around 105 bacteria cm−2 , and the corrosion rate increased above 0.5 mm year−1 . This situation demonstrates that the corrosion damage at the surface is dependent upon the number of microorganisms
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at the metal surface, and independent of the plancktonic microorganisms. The RP technique has been used by other authors to relate the microorganism’s presence with a corrosion rate increment [16]. Keresztes et al. [17], indicated that there is an increment in the steel corrosion rate due to the presence of some bacteria, such as: Leptothix sp., Pseudomonas aeruginosa and Desulfovibrio sp. Galván et al. [14] found that when SRB are incorporated to an electrochemical system, there is an increment on the corrosion rate. However, these works do not mention anything about the kinetics corrosion and its relationship with the microorganisms kinetics growth at the metal surface. Additionally, the fact that the corrosion rate is increasing, even when the sessile microorganisms population is stable, could be due to metabolic products, which are produced and remained into the biofilm at the metal surface, increasing the corrosion process. Mora-Mendoza et al. [13] carried out a similar study, where the corrosion rate for API XL 52 steel was determined using the same Desulfovibrio sp., but as pure strain. According to their results, they observed very low corrosion rate values during their 350 h experiments. However, the corrosion rate values obtained in our study are higher than those reported by Mora-Mendoza et al. This situation could be due to two aspects: • The presence of different microorganisms groups in the medium, which could be acting in the corrosion process, even if they are not adhered at the metal surface. • The incubation time that, according to the results presented by Mora-Mendoza et al., could be long enough to reach a maximum plancktonic microorganisms population. But according to our results, could not be enough to get and appropriate microorganisms population at the metal surface (sessile microorganisms). On the other hand, the relationship between the microorganisms and the electrochemical noise technique has been considered in other works. Iverson and Heverly [18] performed some experiments to evaluate the iron corrosion rate induced by SRB. They found that the potential fluctuations
Fig. 9. Metal surface after 1000 h exposition time, under sterile conditions; (a) 100× and (b) 500×.
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Fig. 10. Metal surface after 1000 h exposition time, in presence of microbial consortium; Lower side view: (a) 10×, (b) 50×, (c) 200×, (d) 500X.
were according to breakdowns of the iron sulfide film, due to the SRB presence at the metal surface. Whitham and Huizinga [19] determined the localization index and the noise resistance during a microbiologically induced corrosion process; moreover, they presented a comparison between the noise resistance and polarization resistance values. Galvan [14] concluded that, according to the measurements obtained from his experiments, there is a direct relationship between the polarization resistance and noise resistance parameters. Two of the statistic parameters that can be obtained when processing the EN results are the current variance (VAR
I) and the potential variance (VAR E). Cottis and Turgoose [20] indicated that, when using the EN technique to evaluate the corrosion process, the VAR I increases when the corrosion rate increases and become localized. On the other hand, the VAR E decreases when the corrosion rate increases. For this system, the current variance is increased after 500 h (Fig. 7), indicating that the microbiologically induced corrosion is intensified after this time and the corrosion process become localized. At the same time, the potential variance decreased after 400 h (Fig. 8), indicating an increment on the corrosion rate.
Fig. 11. Metal surface after 1000 h exposition time under sterile conditions and in presence of microbial consortium.
M.J. Hern´andez Gayosso et al. / Electrochimica Acta 49 (2004) 4295–4301
3.5. Surface analysis of the corrosion damage at the metal surface The SEM analysis on the steel surface, carried out after the experiments were done, indicated that the metal exposed to sterile conditions exhibited uniform corrosion, as shown in Fig. 9. Likewise, once the biofilm was removed from the steel exposed to the consortium action, a localized corrosion process was observed at the metal surface, as shown in Fig. 10. This behaviours was expected, according to the results obtained from the electrochemical noise measurements. At the same time, the corrosion damage observed on the steel surface exposed to the consortium action was more severe than the damage observed under sterile conditions. To illustrate this situation, Fig. 11 shows images for both conditions, taken at the same magnification. From the above mentioned, when the metal is exposed to the consortium action, the plancktonic microorganisms reach their maximum population during the initial 400 h, and in this time low corrosion rate values are observed. After that, between 400 and 1000 h experimental time, the corrosion process become localized and an accelerated corrosion rate increment is observed. The localized corrosion process is supported with the surface analysis.
4. Conclusions The consortium isolated from the gas pipeline is constituted by five different bacteria species, but only one of them: Desulfovibrio vietnamensis, is the main microorganism at the metal surface. It is considered that the corrosion rate increment observed during the experiments is directly related to this bacteria specie. The results obtained from this study indicate that the damage observed on the metal surface depends upon the sessile microorganism’s population. Due to this situation, it is very important to determine the sessile kinetics growth when studying MIC processes. Determination of steel corrosion rate using electrochemical techniques (PR and EN), provide important information relating the process occurring at the metal surface. According to the results presented in this work, when using the electrochemical noise technique, it is possible to identify the moment when the corrosion process become localized. In this study the transition between uniform and localized corrosion was observed after approximately 500 h exposition time. There is a close relationship between the results observed with both electrochemical techniques. The corrosion rate values obtained are similar and they exhibited the same tendency.
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Therefore, it is considered that these techniques could be used as a tool for the study of the microbiologically influenced corrosion (MIC).
Acknowledgements The authors wish to thank the Instituto Mexicano del Petróleo (IMP), Escuela Nacional de Ciencias Biológicas (ENCB) and Consejo Nacional de Ciencia y Tecnolog´ıa (CONACyT), for their support in the production of this work.
References [1] B.J. Little, P.A. Wagner, F. Mansfeld, Microbiologically Influenced Corrosión, NACE International, Houston, TX, 1997. [2] D.H. Pope, E.A. Morris, III. Mater. Perform. (1995) 23. [3] R. Towers, Protective Coatings Europe (2000) 30. [4] F. Mansfeld, B. Little, Corr. Sci. 32 (1991) 247. [5] S.C. Dexter, D.J. Duquette, O.W. Siebert, H.A. Videla, Corr. 47 (1991) 308. [6] H.A. Videla, Manual of Biocorrosion, CRC Lewis Publishers, USA, 1996. [7] American Petroleum Institute, API-RP 38, 1990. [8] J.R. Postgate, The Sulphate Reducing Bacteria, Cambridge University Press, Cambridge, 1984, p. 32. [9] A. Felske, B. Engelen, U. Nübel, H. Backhaus, Appl. Environ. Microbiol. 62 (1996) 4162. [10] C.A. Eichner, R.W. ERB, K. Timmis, I. Wagner-Dobler, Appl. Environ. Microbiol. (1999) 102. [11] A.D. Relman, Diagnostic Molecular Microbiology, Vol.490,American Society for Microbiology, Washington, DC 1993. [12] R.E. Tatnall, D.H. Pope, Identification of MIC. A Practical Manual on Microbiologically Influenced Corrosion. NACE International, Houston, 1993, p. 65. [13] J.L. Mora-Mendoza, A.A. Padilla-Viveros, G. Zavala-Olivares, M.A. Gonzalez-Nuñez, J.L. Moreno-Serrrano, M.J. Hernandez-Gayosso, R. Garc´ıa-Esquivel, J. Gal´ındez, Corrosion (2003) 03548. [14] M.R. Galván R. Mendoza-Flores, R. Duran-Romero, G. Garc´ıaCaloca, E. Ibarra-Núñez and R. Torres-Sánchez, Corrosion (2003) 03545. [15] N.D. Phuong, C.H.D. Thi, H.L. Thuy, H. Stand Lotter, Anaerobe 2 (1996) 385. [16] T.S. Rao, T.N. Sairam, B. Viswanathan, K.V.K. Nair, Corr. Sci. 32 (2000) 1417. [17] Zs. Keresztes J. Teledgi, J. Beczner, E. Kálmán, Electrochem. Acta 43 77. [18] W.P. Iverson, L.F. Heverly, Proceedings of the Symposium on Nondestructive Testing and Electrochemical Methods of Monitoring Corrosion in Industrial Plants, American Society for Testing and Materials, Philadelphia, PA, 1984. [19] T. Whitham, S. Huizinga, European Federation of Corrosion, Eurocor’96, 1997. [20] R.Cottis y S.Turgoose, Electrochemical Impedance and Noise, NACE International, USA, 1999.
Microbial consortium influence upon steel corrosion rate, using polarisation resistance and electrochemical noise techniques M.J. Hernández Gayosso a,1 , G. Zavala Olivares b,∗ , N. Ruiz Ordaz a , C. Juárez Ramirez a , R. Garc´ıa Esquivel b , A. Padilla Viveros b a
b
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. Prolongación de Carpio y Plan de Ayala, México, DF, C.P. 11340, Mexico Instituto Mexicano del Petróleo, Grupo de Corrosión. Eje Central Lázaro Cárdenas 152, Col. San Bartolo Atepehuacan, México, DF, C.P. 07730, Mexico Received 3 November 2003; received in revised form 8 March 2004; accepted 12 March 2004 Available online 10 June 2004
Abstract The microbiologically influenced corrosion (MIC) is a process, which affects the oil industry, particularly the hydrocarbons extraction, transport and storage. MIC evaluation has been normally based upon microbiological tests, and just a few references mention alternating methods, such as the electrochemical techniques, which can be used as criteria for their evaluation. In this work, two different electrochemical laboratory techniques, polarisation resistance and electrochemical noise were used, in order to determine the corrosion behaviour of a microbial consortium, obtained from a gas transporting pipeline, located in the southeast of Mexico. The bacteria population growth was found to be different for sessile and plancktonic microorganisms. Moreover, long incubation times were required to reach the maximum concentration of sessile bacteria. The electrochemical techniques used in this study exhibited a similar tendency on the corrosion rate behaviour with time, and values above 0.3 mm year−1 were observed at the end of the experiments. The experiments were complemented with surface analysis. Scanning electron microscope observation of APIXL52 steel coupons, exposed to the consortium action, revealed bacteria presence, as well as a damaged steel surface. A type of localized corrosion was observed on the metal surface, and it was associated to the bacteria effect. © 2004 Elsevier Ltd. All rights reserved. Keywords: Microbiologically influenced corrosion; Electrochemical techniques; Biofilm
1. Introduction The microbiologically influenced corrosion (MIC) is a very dangerous process, which affects the oil industry, particularly during the hydrocarbon extraction, transport and storage [1–3]. The activity and microorganisms’ growth at the pipelines steel may cause surface modifications, which induce a more complex corrosion process. In general, the study of the MIC has been based upon microbiological tests and gravimetric techniques, and just few references mention alternating methods, which can be used as criteria for their evaluation.
∗
Corresponding author. Tel.: +52-55-9175-6838; fax: +52-55-9175-6844. E-mail address: [email protected] (G. Zavala Olivares). 1 Tel.:+52-55-57296000. 0013-4686/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2004.03.038
The use of electrochemical techniques [4–6] for the evaluation of MIC, such as polarisation resistance (PR) and electrochemical noise (EN), has been considered during the corrosion rate determination and this is an interesting area which requires more research, in order to implement and standardize their laboratory experiments and field application. Most of the study involving electrochemical techniques and MIC processes has been done considering short experimental times. Under these conditions, mainly low corrosion rates can be observed. Usually, the experimental time is established considering several aspects, such as: • kinetics growth of microorganisms in the electrolyte (plancktonic microorganisms), • sulphate consumption • sulphuric acid production.
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However, a basic characteristic on the MIC process frequently is not considered: biofilm formation on the metal surface. Kinetics growth is different for plancktonic microorganisms and those established at the metal surface (sessile microorganisms). Once the biofilm is formed, the corrosion damage on the metal surface should be dependent mainly on the sessile microorganisms, as the plancktonic microorganisms are not in contact with the metal surface. In this study, the PR and EN techniques were used to evaluate the corrosion rate of carbon steel in presence of plancktonic and sessile microorganisms, in order to determine the influence of both: plancktonic and sessile microorganisms, on the MIC processes.
using poliacrylamide gel electrophoresis [10]. The bacterial group present on the surface was identified by means of the complete gene amplification (1502 bp). Using the 8FPL and 1492 RPL initiators [11], the obtained sequence was processed using the National Center for Biotechnology Information (NCBI) data base: http://www.ncbi.nlm.nih.gov. 2.3. Surface analysis of the coupons exposed to microorganisms The coupons were immersed for 1 h in a 2% glutaraldehyde solution, in order to fix the biofilm to the steel surface, and then become dehydrated using four ethanol solutions (15 min each): 25, 50, 75 and 100%, successively. After that, the samples were taken to the SEM for their analysis.
2. Experimental 2.4. Corrosion rate determination using electrochemical techniques
2.1. Microorganisms cultivation The consortium used in this work was taken from a gas pipeline located in the Marine Region of Pemex, in México, during the inner cleaning procedure. The collected samples were inoculated in a selective medium, following the recommendations for field biological sampling, stated on API-RP 38 [7]. The microorganisms were maintained in the laboratory using the Posgate C [8] medium (Table 1), nitrogen atmosphere and at room temperature. The consortium was inoculated in vials with 100 ml Posgate C medium. API XL52 steel coupons (polished and degreased) were placed into the vials and incubated at room temperature. These vials were used as “sacrificial” to determine the consortium kinetics growth. This was done following the most probable number (MPN) method. 2.2. Microorganisms diversity The microorganisms diversity was determined using the thermal gradient gel electrophoresis (TGGE) technique with amplified fragments of 16S rDNA gene. The chain reaction (PCR) was carried out using the U968-GC and 1401 initiators [9]. The DNA fragments generated were observed Table 1 Composition of the Postgate C medium [8] Compound KH2 PO4 NH4 Cl Na2 SO4 MgSO4 ·7H2 O Sodium lactate CaCl2 ·2H2 O FeSO4 ·7H2 O Yeast extract Na citrate NaCl
These experiments were carried out in a 1 l standard cell, with a three electrode system: API XL52 steel as working electrode, a graphite rod as auxiliary and a saturated calomel electrode as reference. The electrolyte used was 800 ml Posgate C medium. Nitrogen gas was bubbled to remove all the oxygen and maintain anaerobic conditions. The electrochemical cell was connected to an ACM 772 potentiostat, and a PC was used for data recording. All experiments lasted approximately one month, and during this time, several PR and EN tests were carried out. All experiments were repeated twice. 2.5. Polarization resistance technique A potentiodynamic method was used to obtain the potential-current (E-I) ratio, applying a ± 10 mV overpotential, with respect to the free corrosion potential, Ecorr. The Sequencer® and Core running® software programs were used for data management, along with the V4 Analysis® software for data analysis. From this technique, resistance values are obtained (Rp ), which are used to calculate the corrosion current density, according to: icorr =
g/L 0.5 1.0 4.5 0.06 6.0 0.06 0.004 1.0 0.3 15
β Rp
(1)
where β is the Tafel slope, and a value of 0.026 V decade−1 is considered. Faraday’s law can be used to relate the corrosion current density to the corrosion rate, with the next equation: corrosion rate (CR) = K
icorr EW δ
(2)
where K is constant, EW is equivalent weight and δ is density.
M.J. Hern´andez Gayosso et al. / Electrochimica Acta 49 (2004) 4295–4301
4297
2.6. Electrochemical noise technique To perform these experiments, three identical steel electrodes were considered, using one of them as reference electrode. Measurements were taken with the Sequencer® and Core running® software programs, and the EN Analyse® program was used to analyse all data. Each experiment lasted approximately 2000 s, taking one point every second. The data obtained from this technique, are used to calculate, initially, the resistance noise, Rn, according to: Rn =
V I
(3)
where V is potential standard deviation and I is current standard deviation. The Rn value is equivalent to Rp value; therefore, the corrosion rate calculation is made according to Eqs. (1) and (2). Fig. 2. Pattern band for amplified fragments of 16S rDNA, obtained from TGGE technique.
3. Results and discusion 3.1. Kinetics growth
Log MPN
The kinetics growth for plancktonic and sessile microorganisms are shown in Fig. 1. The plancktonic microorganisms exhibit their fastest population growth around 150 h (approximately 107 bacteria ml−1 ); from this time, the bacteria population decreased. For the sessile microorganisms, the population growth is less accelerated than for the plancktonic and the maximum population is observed around 600 h. The plancktonic microorganisms, when in contact with an enriched medium, increase their population until the environmental conditions become adverse (nutrients decrease, pH, etc.); for the sessile microorganisms, the population growth is gradual and could be enhanced by a change on the conditions at the end of the kinetics [12]. There are some studies about MIC evaluation using electrochemical techniques, but only data about plancktonic microorganisms kinetics growth is provided [13,14]. In these
studies, only the time for the maximum plancktonic microorganisms growth is considered during the experiments. However, according to the kinetics growth observed in Fig. 1, the sessile microorganisms require more incubation time than the plancktonic, to get their maximum population and a direct relationship between the damage on the metal and the sessile population is expected. Therefore, the incubation time should be long enough to allow a complete sessile microorganisms growth. 3.2. Consortium diversity Using the TGGE technique, it was found that the consortium used in this work is constituted by five different bacteria species, as shown in Fig. 2. This five bacteria are observed
9 8 7 6 5 4 3 2 1 0 0
100
200
300
400
500
600
700
800
900
Time/hr Sessile/cm2
Plancktonic/ml
Fig. 1. Kinetics growth for plancktonic and sessile microorganisms in Postgate C medium. API XL 52 steel coupons.
Fig. 3. Biofilm formed on the metal surface, after 1000 h exposition time.
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(b) The light areas indicated, apart from the FeS, presence of nitrogen and carbon, due to the organic material (bacteria and exopolymer). Fig. 4 shows the biofilm formed on the metal surface, where typical Desulfovibrio vietnamensis bacteria, approximately 2 m size, are observed. 3.4. Electrochemical techniques
Fig. 4. Microorganisms in biofilm.
in the aqueous medium (plancktonic microorganisms), and only one is observed at the metal surface (sessile microorganism). The pattern band for the sessile microorganism is observed as well for plancktonic bacteria. The 16S rDNA gene of the sessile microorganism is formed of approximately 1500 bp and according to the gene amplification there is a similarity of 97% with Desulfovibrio vietnamensis. This specie has been isolated previously in the oil industry [15]. It is very important to point out that the Desulfovibrio vietnamensis is the specie at the metal surface and the main responsible for MIC processes, by means of the metabolic products formed at the interface. However, the plancktonic microorganisms should have some influence on the corrosion process. 3.3. Biofilm surface analysis The biofilm formed on the metal surface during the experiments is shown in Fig. 3, where the following can be observed: (a) The dark compact regions are formed by inorganic corrosion products and a microanalysis indicated high concentration of iron and sulfur (FeS).
Corrosion rate values, obtained from the PR technique are shown in Fig. 5. From this Figure, an initial corrosion rate of around 0.15 mm year−1 was observed for the metal exposed to the Postgate C medium, and this value decreased during the next hours until negligible values were observed, around 0.005 mm year−1 . The Postgate C medium contains high concentration of aggressive anions, such as Cl− , SO4 2− and S2− , that could be responsible for a high initial corrosion rate. However, during the next hours, corrosion products are formed and the metallic surface is isolated from the Postgate C medium. On the other hand, the corrosion rate induced by the consortium exhibited an initial corrosion rate around 0.02 mm year−1 , which slightly increased with time until about 0.05 mm year−1 after 400 h. After that time, the corrosion rate was accelerated until values around 0.5 mm year−1 after 998 h. The corrosion rate observed for the metal exposed to the Postgate C medium, obtained from the EN technique, is shown in Fig. 6. From this figure, the initial corrosion rate value was around 0.2 mm year−1 for the sterile conditions, and this value decreased with time until about 0.01 mm year−1 at the end of the experiment. For the metal exposed to the bacterial consortium, an initial corrosion rate of about 0.025 mm year−1 was observed, remaining stable for about 500 h. After that time, an increment was observed, reaching values up to 0.35 mm year−1 at the end of the experiment. The corrosion rates obtained from both techniques, PR and EN, were very similar between them. Using these techniques, a sudden increase on the corrosion rate was observed after 400 h. It is clear that there is not any relationship 0.4
0.6
0.35 Vcorr/mm.y
Vcorr/mm.y
-1
-1
0.5 0.4 0.3 0.2
0.3 0.25 0.2 0.15 0.1 0.05
0.1
0
0 0
200
400
600
800
1000
0
200
Consortium
Fig. 5. Corrosion rates calculated from the PR technique. API XL 52 steel, exposed to sterile conditions and in presence of microorganisms.
600
800
1000
Time/hr
Time/hr Sterile conditions
400
Sterile conditions
Consortium
Fig. 6. Corrosion rates calculated from the EN technique. API XL 52 steel, exposed to sterile conditions and in presence of microorganisms.
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0.4 0.35 0.3 0.25
6.00E- 08
0.2 4.00E- 08
0.15
Vcorr/mmy-1
Var I/V2
8.00E- 08
0.1
2.00E- 08
0.05 0.00E+00 0
Var
200
400
600
800
0 1000
Time/hr
Vcorr
Fig. 7. Current variance and corrosion rate values. API XL 52 steel exposed to the microbial consortium. 3
0.4 0.35
2.5
-1
0.25
1.5
0.2 0.15
1
Vcorr/mmy
Var E/A
2
0.3 2
0.1 0.5
0.05
0 0 Var
200 Vcorr
400
600
800
0 1000
Time/hr
Fig. 8. Potential variance and corrosion rate values. API XL 52 steel exposed to the microbial consortium.
between the steel corrosion kinetics (Figs. 5 and 6) and the plancktonic microorganisms kinetics (Fig. 1). When the plancktonic microorganisms reach their maximum population, the corrosion rate is relatively low. On the contrary, there is a direct relationship between the sessile microorganisms growth and the corrosion rate increment. It seemed to be a critical point where, after 400 h, the bacterial population at the surface reached values around 105 bacteria cm−2 , and the corrosion rate increased above 0.5 mm year−1 . This situation demonstrates that the corrosion damage at the surface is dependent upon the number of microorganisms
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at the metal surface, and independent of the plancktonic microorganisms. The RP technique has been used by other authors to relate the microorganism’s presence with a corrosion rate increment [16]. Keresztes et al. [17], indicated that there is an increment in the steel corrosion rate due to the presence of some bacteria, such as: Leptothix sp., Pseudomonas aeruginosa and Desulfovibrio sp. Galván et al. [14] found that when SRB are incorporated to an electrochemical system, there is an increment on the corrosion rate. However, these works do not mention anything about the kinetics corrosion and its relationship with the microorganisms kinetics growth at the metal surface. Additionally, the fact that the corrosion rate is increasing, even when the sessile microorganisms population is stable, could be due to metabolic products, which are produced and remained into the biofilm at the metal surface, increasing the corrosion process. Mora-Mendoza et al. [13] carried out a similar study, where the corrosion rate for API XL 52 steel was determined using the same Desulfovibrio sp., but as pure strain. According to their results, they observed very low corrosion rate values during their 350 h experiments. However, the corrosion rate values obtained in our study are higher than those reported by Mora-Mendoza et al. This situation could be due to two aspects: • The presence of different microorganisms groups in the medium, which could be acting in the corrosion process, even if they are not adhered at the metal surface. • The incubation time that, according to the results presented by Mora-Mendoza et al., could be long enough to reach a maximum plancktonic microorganisms population. But according to our results, could not be enough to get and appropriate microorganisms population at the metal surface (sessile microorganisms). On the other hand, the relationship between the microorganisms and the electrochemical noise technique has been considered in other works. Iverson and Heverly [18] performed some experiments to evaluate the iron corrosion rate induced by SRB. They found that the potential fluctuations
Fig. 9. Metal surface after 1000 h exposition time, under sterile conditions; (a) 100× and (b) 500×.
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Fig. 10. Metal surface after 1000 h exposition time, in presence of microbial consortium; Lower side view: (a) 10×, (b) 50×, (c) 200×, (d) 500X.
were according to breakdowns of the iron sulfide film, due to the SRB presence at the metal surface. Whitham and Huizinga [19] determined the localization index and the noise resistance during a microbiologically induced corrosion process; moreover, they presented a comparison between the noise resistance and polarization resistance values. Galvan [14] concluded that, according to the measurements obtained from his experiments, there is a direct relationship between the polarization resistance and noise resistance parameters. Two of the statistic parameters that can be obtained when processing the EN results are the current variance (VAR
I) and the potential variance (VAR E). Cottis and Turgoose [20] indicated that, when using the EN technique to evaluate the corrosion process, the VAR I increases when the corrosion rate increases and become localized. On the other hand, the VAR E decreases when the corrosion rate increases. For this system, the current variance is increased after 500 h (Fig. 7), indicating that the microbiologically induced corrosion is intensified after this time and the corrosion process become localized. At the same time, the potential variance decreased after 400 h (Fig. 8), indicating an increment on the corrosion rate.
Fig. 11. Metal surface after 1000 h exposition time under sterile conditions and in presence of microbial consortium.
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3.5. Surface analysis of the corrosion damage at the metal surface The SEM analysis on the steel surface, carried out after the experiments were done, indicated that the metal exposed to sterile conditions exhibited uniform corrosion, as shown in Fig. 9. Likewise, once the biofilm was removed from the steel exposed to the consortium action, a localized corrosion process was observed at the metal surface, as shown in Fig. 10. This behaviours was expected, according to the results obtained from the electrochemical noise measurements. At the same time, the corrosion damage observed on the steel surface exposed to the consortium action was more severe than the damage observed under sterile conditions. To illustrate this situation, Fig. 11 shows images for both conditions, taken at the same magnification. From the above mentioned, when the metal is exposed to the consortium action, the plancktonic microorganisms reach their maximum population during the initial 400 h, and in this time low corrosion rate values are observed. After that, between 400 and 1000 h experimental time, the corrosion process become localized and an accelerated corrosion rate increment is observed. The localized corrosion process is supported with the surface analysis.
4. Conclusions The consortium isolated from the gas pipeline is constituted by five different bacteria species, but only one of them: Desulfovibrio vietnamensis, is the main microorganism at the metal surface. It is considered that the corrosion rate increment observed during the experiments is directly related to this bacteria specie. The results obtained from this study indicate that the damage observed on the metal surface depends upon the sessile microorganism’s population. Due to this situation, it is very important to determine the sessile kinetics growth when studying MIC processes. Determination of steel corrosion rate using electrochemical techniques (PR and EN), provide important information relating the process occurring at the metal surface. According to the results presented in this work, when using the electrochemical noise technique, it is possible to identify the moment when the corrosion process become localized. In this study the transition between uniform and localized corrosion was observed after approximately 500 h exposition time. There is a close relationship between the results observed with both electrochemical techniques. The corrosion rate values obtained are similar and they exhibited the same tendency.
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Therefore, it is considered that these techniques could be used as a tool for the study of the microbiologically influenced corrosion (MIC).
Acknowledgements The authors wish to thank the Instituto Mexicano del Petróleo (IMP), Escuela Nacional de Ciencias Biológicas (ENCB) and Consejo Nacional de Ciencia y Tecnolog´ıa (CONACyT), for their support in the production of this work.
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