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Eect of Sulfate-reducing Bacteria on Corrosion Behavior of Mild Steel in Sea Mud

Xiaodong ZHAO1,2) , Jizhou DUAN1) , Baorong HOU1) and Suru WU3)
1) Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2) Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 3) College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266003, China
[Manuscript received June 12, 2006, in revised form July 31, 2006]

Microbiologically inuenced corrosion (MIC) is very severe corrosion for constructions buried under sea mud environment. Therefore it is of great importance to carry out the investigation of the corrosion behavior of marine steel in sea mud. In this paper, the eect of sulfate-reducing bacteria (SRB) on corrosion behavior of mild steel in sea mud was studied by weight loss, dual-compartment cell, electronic probe microanalysis (EPMA), transmission electron microscopy (TEM) combined with energy dispersive X-ray analysis (EDX) and electrochemical impedance spectroscopy (EIS). The results showed that corrosion rate and galvanic current were inuenced by the metabolic activity of SRB. In the environment of sea mud containing SRB, the original corrosion products, ferric (oxyhydr) oxide, transformed to iron sulde. With the excess of the dissolved H2 S, the composition of the protective layer formed of FeS transformed to FeS2 or other non-stoichiometric polysulphide, which changed the state of the former layer and accelerated the corrosion process. KEY WORDS: Microbiologically inuenced corrosion (MIC); Sulfate-reducing bacteria (SRB);
Mild steel; Sea mud

1. Introduction Along with the development of marine exploitation more and more metal constructions are connected with sea mud environment such as pipelines for transportation of oil, gas, and water, seabed cable, and protective material of sacricial anode[17] . The corrosion of metal in sea mud is of great importance[8] . In the middle of 1930s, Khr et al.[9] armed that u sulfate-reducing bacteria (SRB) were responsible for the serious corrosion of steel immersed in wet anaerobic mud. About 50%70% corrosion of underground pipeline is due to microbiologically inuenced corrosion (MIC). Although extensive research has been carried out on corrosion induced by SRB in various natural environment and culture medium, corrosion directly involved with environment of sea mud was not investigated deeply. Investigation of corrosion in inorganic medium and microbiological environment shows that the formation of iron sulde has great inuence on the proceeding of the corrosion reaction. Iron sulde has characteristics of semiconductor, which acts as medium for the transportation of electron in galvanic couple with the steel substance, enhancing the anodic and cathodic reaction[10] . Jack[11] studied the inuence of FeSx as a layer of corrosion product on the substrate of steel in soil containing SRB, which indicated that the content of FeSx had positive pertinence with the corrosion rate, and the presence of dissolved oxygen would promote the corrosion. It is sometimes neglected that ferric oxide emerges with the formation of ferrous sulde or before it in the initial stage of the corrosion process. Ferric oxide should be responsible for the emergence and development of microbiologically inuenced corrosion
Prof., Ph.D., to whom correspondence should be addressed, E-mail: duanjz@ms.qdio.ac.cn.

(MIC), which haven t been well investigated yet. In this paper, the corrosion behavior of mild steel has been investigated in sea mud containing active SRB. Considering the corrosion rate, galvanic corrosion, formation of corrosion product, as well as the emergence and transformation of oxide and sulde, we discussed the corrosion process inuenced by SRB. 2. Experimental 2.1 Organisms and culture Sulfate-reducing bacteria were enriched and cultivated from the sea mud in the Huanghai Sea, China. A modied Postgate s C medium was used for enrichment culture, which contained 0.5 g KH2 PO4 , 1 g NH4 Cl, 0.06 g CaCl2 6H2 O, 0.06 g MgSO4 7H2 O, 6 ml 70% sodium lactate, 1 g yeast extract, 0.3 g sodium citrate in 1 L aged seawater from Qingdao oshore area. The medium was deaerated by purging with high-purity nitrogen for 20 min and autoclaved at 121 C, then was added sterile 0.004 g/L FeSO4 7H2 O. The sea mud used in the experiment was taken from Jiaozhou Bay of Qingdao, rich in SRB. Parts of the sea mud were autoclaved at 121 C and infused with sterile seawater, serving as sea mud without SRB; others were autoclaved and infused modied medium inoculated SRB, stirred adequately with a sterile glass stick and deposited for 24 h, as sea mud with SRB, so that the SRB number in the parallel sea mud sample could be approximately the same. 2.2 Material Mild steel (C 0.17, Si 0.073, Mn 0.70, S 0.008, P 0.012) was used for this study. The specimens for weight loss test were 50 mm20 mm7 mm in size. The specimens for microscopy observation were 20 mm10 mm2 mm in size, with a 2 mm hole drilled on the margin. 10 mm column specimens were used as working electrodes for electrochemical

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test. The surfaces were treated according to GB 5776-86 of China, polished with a series of silicon carbide papers, cleaned with ethanol using an ultrasonic cleaner, followed by rinsing with sterile distilled water to remove the contamination. Then they were kept in a sterile and oxygen-isolated environment and sterilized by ultraviolet lamp for 30 min before test, insuring of no contamination by other bacteria. 2.3 Instrumentation and experimentation Three sets of parallel specimens for weight loss test were immersed in the sea mud with and without SRB for 60 d at room temperature (25 C). They were cleaned in an acid pickling bath (conc. HCl, Sb2 O3 20 g/L, SnCl2 20 g/L) for 1015 s to remove the corrosion products after taken out from the sea mud. Corrosion rate was calculated by data of weight loss according to GB 5776-86 of China. Galvanic corrosion test was carried out in selfmade dual-compartment cell lled with sea mud with and without SRB, respectively, which was conductive by luggin capillary. Test samples were two electrochemical working electrodes mentioned above. The galvanic current was tested at certain time by homemade ZRA-2 galvanic corrosion meter and the test period lasted for about a month. Specimens for observation were incubated in the sea mud containing SRB for 3 weeks and taken out at dierent incubation time. Corrosion morphology and element analysis were studied by using a JEOL JXA8800R electronic probe microanalyzer (EPMA) and selected area electronic diraction (SAED) analysis by HITACHI H-800 transmission electron microscopy (TEM) combined with PV9900 energy dispersive Xray analysis (EDX). EIS tests were carried out by using an EG&G Princeton Applied Research, Parstat 2273 electrochemical analyzer system. Ruthenium-titanium electrode with an area of 2 cm2 was used as a counter electrode and saturated calomel electrode (SCE) as reference electrode. Luggin capillary was used for decreasing IR drop. Working and counter electrodes were buried in the sea mud. EIS measurements were conducted at dierent exposure time of immersion. The potential amplitude was set to 10 mV in opencircuit and the frequency range was from 95 kHz to 10 MHz. 3. Results 3.1 Corrosion characteristics of mild steel in sea mud Corrosion rate of mild steel immersed in sea mud with and without SRB for 60 d were 0.0321 and 0.0114 mm/a, respectively. Inuenced by the metabolic activity of SRB the corrosion rate of mild steel in sea mud with SRB was increased by 182% compared with that in sterile sea mud. The it curve of galvanic current density changing with time is shown in Fig.1. At the beginning, the specimen immersed in sea mud with SRB acted as cathode and that in sterile sea mud acted as anode. After a period the current direction gradually reversed in the opposite direction, because of the change of the surface state of the specimen immersed in the sea mud inoculated SRB. The reversion occurred again after a week. In conclusion, the amplitude of galvanic current underwent an increase-decrease alter-

Fig.1 Galvanic current density dependence with time

native process. Galvanic potential presented a similar changing tendency to that of galvanic current. It may be deduced that the variation of the current was inuenced by the metabolic activity of SRB. In addition, many factors such as oxygen concentration dierence cell etc can also generate galvanic current. Under the current condition, the role of bacteria and subsequently change of electrode were the essential reason. 3.2 EIS analysis When mild steel was exposed to sea mud inoculated SRB, the EIS spectra varied signicantly with exposure time. The impedance spectra could be tted by two equivalent circuits as shown in Fig.2. Equivalent circuit (a) is tted for the impedance spectra before 180 h exposure when the diusion is not very distinct and equivalent circuit (b) for the later time with an diusion impedance W Rs represents resistance of the electrolyte (sea mud). Rp is the polarization resistance of the substrate of steel; Q is the capacity of layer of corrosion products; W is the Warburg impedance due to the diusion. The tting parameters are listed in Table 1. Either the tting data or the simulated curves (not shown) conrm the reliability of the equivalent circuit.

Fig.2 Equivalent circuit used for tting the impedance spectra: (a) single-layer model and (b) single-layer model including warburg impedance

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Table 1 Fitting parameters from the equivalent circuit for tting spectra in Figs.35 t/h Rs / Q n Rp / W 12 11.30 0.0001535 0.7954 3.332E4 36 13.58 0.0001922 0.7921 5.426E4 60 14.33 0.0002257 0.8056 4.127E4 84 14.43 0.0002618 0.8106 3.641E4 108 15.63 0.0003461 0.8237 3.468E4 132 15.84 0.000503 0.8592 2.834E4 180 16.13 0.001158 0.8769 4.152E4 3.862E6 204 16.91 0.001405 0.8759 5.039E4 1.384E6 228 17.71 0.001666 0.8786 5.977E4 2.453E8

Fig.3 Nyquist (a) and bode spectra (b) for mild steel exposed to sea mud after SRB inoculation for 12 and 36 h

Fig.4 Nyquist (a) and bode spectra (b) for mild steel exposed to sea mud after SRB inoculation for 108 and 132 h

According to the EIS results the process can be divided into three stages. Nyquist and bode spectra for mild steel exposed to sea mud after SRB inoculation for 12 and 36 h are shown in Fig.3. At the rst stage, the original corrosion products are mainly ferric (oxyhydr) oxide. Sulfate-reducing bacteria attached on the substrate of the steel, assimilated nutrients and reduced sulfate to sulde ions. Biogenic H2 S and FeS are formed by the precipitation of ferrous ions with the sulde ions. A protective layer of FeS and iron oxide are formed on the substrate of the steel, which decreased the corrosion rate to some extent. Nyquist and bode spectra for mild steel exposed to sea mud after SRB inoculation for 108 and 132 h are shown in Fig.4. At the second stage, with the proliferation of SRB and the excess of dissolved H2 S, the composition of the corrosion products transformed from FeS to other polysulphide. Because of the changes of composition and crystal structure, this layer became loose, porous and easy to be desquamated. The substrate of the steel exposed to the corrosion medium

again, which accelerated the corrosion rate. At the third stage, the metabolic activity of the microorganisms declined gradually, and the ratio of S/Fe decreases with the reduction of H2 S. Protective layer of corrosion products is formed again; as a result, the corrosion rate decreases smoothly and tend to be constant. Nyquist and bode spectra for mild steel exposed to sea mud after SRB inoculation for 204 and 228 h are shown in Fig.5. Dependence of polarization resistance (Rp ) on time is shown in Fig.6. According to the gure, the value of Rp increases at the rst stage, decreases in the second stage with the variation of time, and increases again at the third stage. The metabolic activity of SRB and the change of state of the surface on the steel can be the explanation. 3.3 Surface morphology and element analysis by EPMA Morphology observation and element analysis of corrosion products of mild steel immersed in sea mud containing SRB after 7, 14 and 21 d by EPMA are

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Fig.5 Nyquist (a) and bode spectra (b) for mild steel exposed to sea mud after SRB inoculation for 204 and 228 h

Fig.6 Rp dependence with time

shown in Figs.79. First sulfate-reducing bacteria were supposed to attach on the substrate of the steel and biolm occurred for their metabolic activity. The biolm and induced products are inhomogeneous in distribution as well as thickness, as shown in Fig.7. Quantitative analysis shows that the main compositions are O 22.90, S 7.58, Fe 64.50 (wt pct) besides Si, Al element coming from the sand. It could be deduced that the corrosion products are mainly composed of iron oxide, sulde or sulfate after immersion for 7 d. The surface of the specimen immersed for 14 d was more inhomogeneous and the amount of corrosion products increased (Fig.8). Quantitative analysis shows that the main compositions are still S, O, and Fe, while the content of sulfur increases to 20.14 (wt pct). With further metabolic activity of SRB, corrosion products reveal the tendency of transformation from oxide to sulde, the conguration of the crystal needed further conrmation by TEM.

Fig.7 Surface morphology (a) and element analysis (b) by EPMA after 7 d

Fig.8 Surface morphology (a) and element analysis (b) by EPMA after 14 d

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Fig.9 Surface morphology (a) and element analysis (b) by EPMA after 21 d

Fig.10 Electronic diraction and composition analysis of corrosion products by TEM and EDX: (a) electron diraction pattern of Fe2 O3 , (b) electron diraction pattern of FeS, (c) electron diraction pattern of FeS2 , (d) composition analysis by EDX

A thick layer of corrosion products gradually formed on the surface of the specimen immersed for 21 d (Fig.9). The substrate of the steel could hardly be seen but the layer was loose and porous. Substances like jelly glue could be observed among the corrosion products, which was supposed to be extracellular polymer substances (EPS) resulting from metabolic activity. Element analysis showed that the content of Ca, Mg, Al, Cl and other negative and positive ions coming from the sea mud increased while content of S decreased comparatively. The loose and porous conguration formed by the precipitation of carbonates and polysulphide on the surface of steel could not protect the steel from the corrosion medium. On the contrary, attachment of bacteria was enhanced by the loose conguration and the corrosion of the

steel was aggravated to some extent. 3.4 Electronic diraction analysis of corrosion products by TEM Selected area electronic diraction (SAED) analysis of representative corrosion products is shown in Fig.10. SAED analysis of corrosion products after 7 d in sea mud is the pattern of Fe2 O3 along [101] crystal axis by calculation as shown in Fig.10(a). Figure 10(b) represents SAED of FeS along [211] crystal axis in corrosion products after 14 d and composition analysis by EDX shown in Fig.10(d). SAED of FeS2 along [157] crystal axis is also observed (Fig.10(c)). SAED and EDX analysis conrmed the presence of Fe2 O3 , FeS2 and FeS in the corrosion products after dierent time, as well as the transformation from

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Table 2 Properties of the iron suldes[1719] Mackinawite Fe(1x) S 0.0570.064 Tetragonal Pyrrhotite Fe(1x) S 0.000.14 NiAs type Compound Greigite Marcasite Fe(1x) S Fe(1x) S 0.25 0.5 Cubic Orthorhombic Pyrite Fe(1x) S 0.5 Cubic Smythit Fe(1x) S 0.000.18 Rhombohedral

Formula Value of x Crystal type

iron oxide to mackinawite, pyrite and other non-stoichiometric polysulphide inuenced by the metabolism of SRB. 4. Discussion In the environment of sea mud containing SRB the original corrosion products are mainly ferric (oxyhydr) oxide. In the presence of SO4 2 , (oxyhydr) oxide transforms to sulde with the reaction of biogenic hydrogen sulde, extracellular polymer substances (EPS) and organic acid. SRB metabolize as follows: 4H2 +SO4 2 +2H+ 4H2 O+ H2 S (role of hydrogenase) Or: 2Lactate+SO4 2 +H+ 2Acetate+2CO2 +2H2 O+HS (Lactate as the electron provider[12] ) Fe2+ can be acquired indirectly by the dissolution of ferric oxide and hydrogen sulde when ferrous salt is scarce in medium, the dissolution is as follows: 3H2 S + 2FeO(OH) 2FeS + S + 4H2 O Or the direct hydrogenase[13] : dissolution by SRB produces

(2) In the environment of sea mud containing SRB, the original corrosion products were mainly ferric (oxyhydr) oxide. In the presence of SO4 2 , the original corrosion products, (oxyhydr) oxide, transformed to sulde with the reaction of biogenic hydrogen sulde, extracellular polymer substances (EPS) and organic acid. Biogenic H2 S and FeS were formed by the precipitation of ferrous ions with the sulde ions. A compact and protective layer of FeS was formed on the substrate of the steel in the initial stage of the corrosion process and the corrosion rate was decreased to some extent. (3) With the proliferation of SRB and the excess of dissolved H2 S, the composition of the corrosion products transformed from FeS to FeS2 and other nonstoichiometric polysulphide. Because of the changes of composition and crystal structure, the former protective layer became loose, porous and easy to be desquamated. The substrate of the steel exposed to the corrosion medium again, which accelerated the corrosion rate.
Acknowledgement The supports from the National Natural Science Foundation of China (No. 40376023 and 40406022) are gratefully acknowledged. REFERENCES [1 ] T.S.Rao and T.N.Sariram: Corros. Sci., 2000, 42(8), 1417. [2 ] J.R.Scully: Corrosion, 2000, 56(2), 199. [3 ] J.G.Kim and Y.W.Kim: Corros. Sci., 2001, 43(11), 2011. [4 ] J.Jan-Roblero and J.M.Romero: Appl. Microbiol. Biotechnol., 2004, 64, 862. [5 ] S.Glasauer and S.Langley: Science, 2002, 295, 117. [6 ] W.A.Hamilton: Biofouling, 2003, 19(1), 65. [7 ] Ravi K.Kukkadapu and John M.Zachara: Geochim. Cosmochim. Acta, 2004, 68, 2799. [8 ] Baorong HOU: Marine Corrosion and Protection, Science Press, Beijing, 1997, 5. (in Chinese) [9 ] Von Wolzogen Khr and G.A.H. van der Vlugt L.R.: u Water, 1934, 18, 147. [10] R.A.King and J.D.A.Miller: Nature, 1971, 233, 491. [11] T.R.Jack: Corrosion, 1998, 54(3), 246. [12] R.K.Thauer and K.Jungermann: Bacteriol. Rev., 1977, 41, 100. [13] M.Robert and J.Berthelin: Soil Science Society of America, Madison, Special Publication, No. 17, 453. [14] Jizhou DUAN and Baorong HOU: 13th AsianPacic Corrosion Control Conference, Osaka University, Japan, 2003, 16. [15] A.L.Neal and S.Techkarnjanaruk: Geochim. Cosmochim. Acta, 2001, 65(2), 223. [16] Robert Jerey and Robert E.Melchers: Corros. Sci., 2003, 45, 693. [17] J.S.Smith and J.D.A.Miller: Br. Corros J., 1975, 10, 136. [18] D.T.Richard: Stockholm Contr. Geol., 1969, 13, 49. [19] T.Hemmingsen and H.Lima: Electrochim. Acta, 1998, 43, 35.

Fe2 O3 + 6H+ + 2e 2Fe2+ (aq.) + 3H2 O Fe2 O3 is the dehydration product of FeO(OH). TEM analysis conrmed the presence of iron oxide and sulde, as well as the transformation process. These results were consistent with the former research[1416] . The sulfur/iron ratio can be increased by the excess of sulde: FeS + H2 S = FeS2 + 2H+ + 2e But most often non-stoichiometric compositions are obtained as shown in Table 2. A review on the nature of the products is given by Smith and Miller[17] , and a summary of the interrelationship of the major sulde products is given by Richard[18] . A compact and protective layer is formed on the substrate of the steel in the initial stage of the corrosion process and the corrosion rate is decreased to some extent. With the proliferation of SRB and the excess of dissolved H2 S, the composition and crystal type may be changed. When a crystal structure is converted into a denser structure, a cracking of the corrosion layer is expected. The substrate of the steel exposes to the corrosion medium again, which accelerates the corrosion process. 5. Conclusions (1) Inuenced by the metabolic activity of SRB the corrosion rate of mild steel in sea mud with SRB was increased by 182% compared with that in sterile sea mud. The variation of galvanic current between steel immersed in sea mud with and without SRB also revealed inuence of the metabolic activity of SRB.