Cent. Eur. J. Eng. • 2(1) • 2012 • 113-122DOI: 10.2478/s13531-011-0056-z

Central European Journal of Engineering

Microbial Influenced Corrosion by Thermophilicbacteria

Review article

Suman Lata, Chhaya Sharma, Ajay K. Singh∗,

Department of Paper Technology, IIT-Roorkee, Saharanpur Campus, Saharanpur-247001, India

Received 15 July 2011; accepted 05 September 2011

Abstract: The present study was undertaken to investigate microbial influenced corrosion (MIC) on stainless steels dueto thermophilic bacteria Desulfotomaculum nigrificans. The objective of the study was to measure the extent ofcorrosion and correlate it with the growth of the biofilm by monitoring the composition of its extracellular polymericsubstances (EPS). The toxic effect of heavy metals on MIC was also observed. For this purpose, stainless steels304L, 316L and 2205 were subjected to electrochemical polarization and immersion tests in the modified Baar’smedia, control and inoculated, in anaerobic conditions at room temperature. Scanning electron microscopy (SEM)/energy dispersive spectroscopy (EDS) were used to identify the chemicals present in/outside the pit. The resultsshow maximum corrosive conditions when bacterial activity is highest, which in turn minimizes the amount ofcarbohydrate and protein along with the increase in the fraction of uronic acid in carbohydrate in EPS of the biofilm.However, although bacterial activity and corrosion rate decreases, the amount of biofilm components continue toincrease. It is also observed that the toxicity of metals ions affect the bacterial activity and EPS production. It wasobserved that Desulfotomaculum sp. has the ability to biodegrade its own EPS.

Keywords: Desulfotomaculum nigrificans • Stainless steel • Electrochemical polarization • Weight loss • Extracellular polymericsubstances© Versita sp. z o.o.

1. Introduction

Carbon steel is widely employed in most industries forits low cost, strength, and ability to fabricate in variousshapes and availability. However, their applications areoften affected by corrosion which necessitates the use ofstainless steels. Apart from chemical corrosion, the steelsmay also be affected by microbial influenced corrosion(MIC) which is caused by microbial colonization of metalsurfaces and formation of biofilms affecting the electro-chemical nature of the metal-environment system [1, 2].

∗E-mail: ajaycorr@rediffmail.com

Most microbial corrosion studies reported in literature arerelated to sulphate-reducing bacteria (SRBs), especiallymesophilic type (Desulfovibrio) [3–5] due to their preva-lence in various industrial media and their reputation asthe principal causative organism responsible for localizedcorrosion on stainless steels causing unexpected failure[6–10]. However, fewer studies have been done on thecorrosion aspects of thermophilic SRB (Desulfotomaculum),although these bacteria have been identified frequentlyin industrial media having moderately high temperaturese.g. condenser in electric power unit, oil wells etc. wherecorrosion of the metals has been detected. MIC due tothermophilic bacteria was first demonstrated by Torres-Sanchez and Magana-Vazquez [11] in a condenser of a

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Microbial Influenced Corrosion by Thermophilic bacteria

Table 1. Composition of steel samples.

Sample C Si Mn P S Cr Ni Mo N Cu Ti Co

304L 0.036 0.44 1.84 0.024 0.001 18.11 8.01 0.26 0.058 0.46 0.002 –316L 0.020 0.69 1.69 0.03 0.03 17.44 10.8 2.16 0.04 0.31 – 0.182205 0.02 0.52 1.45 0.02 0.002 22.25 5.48 3.08 0.15 - – –

geothermal electric power unit, which operated in the tem-perature range of 40–150°C. They exposed 304L stainlesssteel tubes for several months to the condenser environment.The tubes developed pitting where Desulfotomaculum ni-grificans (DN) and Desulfotomaculum acetoxidans colonieswere observed. Almeida et al. [12] observed corrosion oncarbon steel coupons covered by biofilm, after exposure forseveral weeks at the outlet of a heat exchanger. The biofilmwas analyzed to have predominantly thermophilic speciesof SRB’s in planktonic and sessile phases. In anotherstudy, mild steel was exposed to four different cultures ofSRB including DN. Green rust 2 (GR2), ferrous sulfides,γ-FeOOH and super paramagnetic α-FeOOH in differentproportions were identified as corrosion products usingMössbauer spectroscopy. The formation of GR2 seemsto be the first step for the SRB induced corrosion [13].Anaerobic corrosion tests were carried out, at 40°C and50°C, on 316 stainless steel and carbon steel in the twostrains of SRB obtained from the condensate fluid of ageothermal electric power station. The microbial activitywas observed to influence the overall corrosion process,whereas, pitting and localized attack was found [14]. Effectof iron concentration on corrosion behavior was studiedby Çetin et al. [15] on low alloy steel in the presenceof Desulfotomaculum isolated from an oil production well.Çetin and Donmez [16], studied corrosion behavior of lowalloy steel, in the presence of anaerobic sulfate-reducingDesulfotomaculum sp. isolated from an oil production well.They found the corrosion activity depends upon bacte-rial metabolites, ferrous sulfide, hydrogen sulfide, ironphosphide, and cathodic depolarization effects. They alsostudied the influence of two biocides (formaldehyde andglutaraldehyde) on corrosion behavior. The same group[17] also studied the effect of D. nigrificans with electro-chemical impedance spectroscopy and scanning electronmicroscopy (SEM). The incubation of the SRB in culturemedium accelerates the cathodic depolarization process oflow-alloy steel, but slows down the anodic process. It wasobserved that the biofilm formation initiates after lapse ofa certain incubation period and that the corrosion products(iron sulfides) start affecting the biofilm after a certainincubation period. Anandkumar et al. [18]; Anandkumarand Rajeshkar, [19], investigated the role of Desulfotomac-ulum geothermicum and Desulfotomaculum kuznetsovii inmild steel corrosion. The presence of bacteria enhances

corrosion by accelerating cathodic reaction and suppress-ing anodic reaction. Pitting was also observed, probablydue to cathodic depolarization. The study implicates theimportance of D. geothermicum in the corrosion of coolingtowers of the petroleum refinery. In another study, cor-rosion was found to be enhanced under biotic conditionsin steel containers meant for nuclear waste disposal ina repository, thereby indicating the possibility of SRBgrowth (including Desulfotomaculum) and faster corrosionunder the disposal condition, if water is available [20].The studies performed until now are mostly on carbonsteel and low alloy steel and relate to (i) MIC underin-plant test conditions, (ii) characterization of corrosionproducts, (iii) effect of biocides on MIC, and (iv) influenceof change in anodic and cathodic tafel slopes on corro-sion reactions. Work done previously on other ferrousmetal-microbe systems have shown that MIC is expectedto depend upon the nature of the biofilm since the extracel-lular polymeric substances (EPS) formed by the bacteriafavor attachment of cells to ferrous materials [1, 21–23]so that the existing macromolecules e.g. carbohydratesand proteins in the biofilm may influence considerably theelectrochemical reactions at the metal-biofilm interface[1]. Therefore, to study the dependence of MIC on thenature of the biofilm, stainless steels in the presence ofDesulfotomaculum nigrificans, were subjected to immersiontest, electrochemical polarization test, and SEM/energydispersive spectroscopy (EDS). It was observed that mostcorrosive conditions prevail when bacterial activity is high-est, together with minimum amount of carbohydrate andprotein, and increased fraction of uronic acid in carbohy-drates of EPS in the biofilm. The toxicity of metals ionswas observed to affect the bacterial activity and EPS pro-duction whereas Desulfotomaculum sp. showed the abilityto biodegrade its own EPS.

2. Experimental DetailsSamples of austenitic 304L, 316L and duplex stainlesssteel 2205 (Table 1) were tested in the present study. Thecoupons of these samples (size 5× 2× 0.25 cm) were pol-ished progressively from coarse to fine (up to 1000 grit)emery paper and then subjected to 4/0 (equivalent to 2000grit) (‘3M’ and ‘Premier’ make) for final finish. The polished

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coupons were ultrasonically degreased in acetone and ster-ilized by exposing to 70% ethanol for 4 hours [24] followedby drying under ultraviolet light in a stream of warm air[25]. Immediately after sterilization, the coupons were sub-merged in cultures of bacteria. For electrochemical studies,coupons of 1 cm2 were embedded in a mould of epoxy resinwith their electrical connection established via a copperwire. The test material was polished, degreased and ster-ilized as above. All the chemicals used were ‘Merck’ and‘Fisher scientific’ make of analytical reagent grade.The anaerobic SRB species, Desulfotomaculum nigrificans(DSM) were obtained from NCIM in India. The solutionused for cultivating this species and the test solution wasnutrient rich modified Baar’s medium [26]. The pH of themedium was adjusted to 7.5 and anaerobic conditions weremaintained using nitrogen gas. The SRB’s were cultivatedat 55°C. The bacterial concentration was estimated by mostprobable number (MPN) method [27]. For this purpose,biofilm was removed from the surface of exposed steelcoupon by swabbing with sterile cotton. The swab wassuspended in 10 ml modified Baar’s medium followed byhomogenization before undertaking the MPN procedure.The stainless steel coupons were immersed up to 144 hoursto characterize the biofilm and to study the life cycle ofmicrobes and pH of media with the aim of correlating withthe corrosion rate of the tested stainless steels. The biofilmformed on the coupons exposed for 72 hrs was analyzedfor SRB’s. For this purpose, the biofilm was immersed for1 hr in a 2% glutaraldehyde solution, to fix the biofilm, andthen was dehydrated using four ethanol solutions (15 min-utes each): 25, 50, 75 and 100%, successively [28]. Thetreated biofilm was rinsed in sterile distilled water, driedand subjected to examination under SEM (Quanta 200FCG, Netherland). For estimating the amount of EPS, thebiofilm was removed from the surface of exposed coupon bya sterile cotton swab and was suspended in 10 ml distilledwater for isolation [27]. This isolated EPS was analyzedfor carbohydrate, protein (29), uronic acid (30) and lipid(31, 32). For estimating the first three, the spectropoho-tometer was calibrated using standard solution of glucosefor carbohydrates, bovine serum albumin for proteins andD-glucuronic acid for uronic acid. Absorption of 620 nmwas measured for estimating carbohydrate, 660 nm for pro-tein and 520 nm for uronic acid. Lipid extraction was doneby Bligh and Dyer method [31]. Lipid quantification wasdone according to Rouser et al. [32]. Standard solution ofKH2PO4 was used for lipids estimation utilizing absorp-tion of 800 nm wavelength. KDO assay was also done todetermine cells lyses.Immersion tests were conducted from 7 to 90 days in con-trol and inoculated media. Since bacterial concentrationreaches maximum after about 96 hours (Table 2), 75% of thetest media was replaced by fresh media on every 5th day

Fig. 1 Scanning Electron Micrograph of Desulfotomaculum nigrificans bacteria on Stainless steel 304L after 72 hrs

Fig. 2 Corelation between corrosion rate (mpy) and EPS component (Carbohydrate and Protein)

of stainless steels with bacteria during immersion tests without media replenishing

Time (hrs)

0 20 40 60 80 100 120 140 160

Cor

rosi

on ra

te (m

py)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35 Carbohydrate and Protein conc. (m

g/cm2)6

8

10

12

14

16

18

20

22

24

26

Corr.rate 304LCorr.rate 316LCorr.rate 2205Carb. conc 304LCarb. conc. 316LCarb. conc. 2205Prot. conc. 304LProt. conc. 316LProt. conc. 2205

Figure 1. Scanning Electron Micrograph of Desulfotomaculum nigrif-icans bacteria on Stainless steel 304L after 72 hrs.

so as to maintain bacterial and chemical concentration.Carbohydrate and protein are the major components ofbiofilm in case of this bacterium so they were estimatedafter the exposure, as described above. The clean anddried coupons were weighed for estimation of corrosionrate as per standard procedure (ASTM G1-72) [33]. Extentof pitting was estimated by measuring maximum pit depthon the surface of cleaned coupons by using stereo micro-scope (Olympus) and optical microscope (Leica Q500MC).SEM/EDS analysis was done on corroded coupons forfinding the chemical composition outside/inside pits. Forestimating corrosion rate and its variation with bacterialgrowth and EPS, Tafel plots were measured. For thispurpose, the steel samples were incubated in Barr’s mediafrom 12 to 144 hours before they were subjected to the testunder anaerobic conditions. The measurements were doneusing a Radiometer ‘Voltalab’ Electrochemical LaboratoryModel PGZ301. Saturated calomel electrode (SCE) wasused as reference electrode, graphite rods as auxiliary andtest specimen as working electrode. Open circuit potential(OCP) and potentiodynamic polarization measurementswere also done as a part of electrochemical tests. Forthese measurements, the steel samples were put in blankand inoculated Baar’s medium for 0, 5 and 10 days underanaerobic conditions (represented in result as 0, 5, 10 DIPfor days’ incubation period) before they were subjected toelectrochemical tests.

3. Results and Discussion

3.1. Bacteria and biofilm characterizationOn immersing stainless steel coupons in inoculated Baar’smedia, biofilm was observed after 12 hours. Fig. 1 shows

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Microbial Influenced Corrosion by Thermophilic bacteria

Table 2. Bacterial counting of Desulfotomaculum nigrificans (DN) by MPN Method.

Time (hrs) pH Planktonic Cells Sessile cells (per cm2)(per ml) 304L 316L 2205

0 7.23 2× 103 – – –12 7.32 21× 102 24× 102 22× 102 2× 103

24 7.48 7× 103 84× 102 78× 102 75× 102

36 7.59 54× 103 75× 103 69× 103 60× 103

48 7.69 73× 104 98× 104 82× 104 75× 104

60 7.73 32× 104 49× 104 42× 104 36× 104

72 7.79 23× 106 45× 106 36× 106 30× 106

84 7.8 28× 107 43× 107 35× 107 32× 107

96 7.79 15× 108 35× 108 29× 108 22× 108

120 7.76 1× 108 32× 108 25× 108 19× 108

144 7.74 67× 106 87× 107 7× 108 6× 108

Fig. 1 Scanning Electron Micrograph of Desulfotomaculum nigrificans bacteria on Stainless steel 304L after 72 hrs

Fig. 2 Corelation between corrosion rate (mpy) and EPS component (Carbohydrate and Protein)

of stainless steels with bacteria during immersion tests without media replenishing

Time (hrs)

0 20 40 60 80 100 120 140 160

Cor

rosi

on ra

te (m

py)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35 Carbohydrate and Protein conc. (m

g/cm2)6

8

10

12

14

16

18

20

22

24

26

Corr.rate 304LCorr.rate 316LCorr.rate 2205Carb. conc 304LCarb. conc. 316LCarb. conc. 2205Prot. conc. 304LProt. conc. 316LProt. conc. 2205

Figure 2. Corelation between corrosion rate (mpy) and EPS compo-nent (Carbohydrate and Protein) of stainless steels withbacteria during immersion tests without media replenish-ing.

Fig. 4 Corelation between corrosion rate (mpy) and EPS components (carb., protein and U.acid) of 304L with bacteria during immersion tests without media replenishing

Time (hrs)

0 20 40 60 80 100 120 140 160

Cor

rosi

on ra

te (m

py)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

EPS components (m

g/cm2)

0

5

10

15

20

25

Corr. rate CarbohydrateProteinU. acid

Fig. 3 Corelation in conc. of uronic acid and lipids of EPS with time on stainless steelswith bacteria during immersion tests without media replenishing

Time (hrs)

0 20 40 60 80 100 120 140 160

Uro

nic

Aci

d (m

g/cm

2 )

0

1

2

3

4

5

6

7 Lipids (µg/cm2)

8

10

12

14

16

18

20

U.A. 304LU.A. 316LU.A. 2205Lipids 304LLipids 316LLipids 2205

Figure 3. Corelation in conc. of uronic acid and lipids of EPS withtime on stainless steels with bacteria during immersiontests without media replenishing.

the SEM photograph of bacteria, rod shaped, anaerobicSRB’s with the typical morphology of the genus used(Desulfotomaculum, size between 0.7 µm–2.0 µm). Vari-ation of planktonic and sessile bacterial population andpH with time are shown in Table 2. Thus in differentcases, the bacterial concentration increased from ∼103 to∼108 cells/ml of media in case of planktonic and ∼103

to 109 cells/cm2 of biofilm (formed over coupons) in case

of sessile bacteria at 96 hrs of incubation. Afterwards itdecreases. The growth rate of sessile cells was greaterthan that of the planktonic cells between 12 to 96 hrsafter which cell concentration of both planktonic and ses-sile starts decreasing (Table 2) as also found elsewhere[27, 34, 35]. Comparison of Fig. 2 and Table 2 shows maxi-mum bacterial concentration when the carbohydrate andprotein concentration in the biofilm formed on all the threetypes of metals is observed to be the lowest. Comparisonof Fig. 3 and Table 2 shows minimum amount of uronicacid at highest bacterial concentration whereas the lipidsconcentration continues to increase until the exponentialphase of bacteria (96 hrs) after which it increases slightlybefore tapering off. Increase in lipids’ concentration maybe attributed to bacterial cells lyses. The decrease inthe amount of carbohydrate, protein and uronic acid isan indication of the biodegradation of EPS produced bythe bacteria, which was corroborated by performing thetest in the media without lactate and yeast. In this test,the coupons with biofilm were transferred into inoculatedcarbon free medium (without lactate and yeast) and theamount of carbohydrate and protein were measured atregular intervals. In pure cultures it has usually beenassumed that the bacteria do not degrade their own EPS,but present results showed otherwise. This type of prop-erty was shown by other bacteria also [27]. Some researchshows that mixed cultures or even pure culture degradedtheir own EPS material when they were in a starved state[36, 37]. The observance of decrease in the amount ofcarbohydrate, protein and uronic acid when the bacteria isin the most active stage indicates that nutrients media arenot enough and the bacteria is consuming sugar (carbohy-drate and uronic acid) and protein by degrading the EPSproduced by them. Afterwards, the bacterial concentrationstarts decreasing while amount of sugar and proteins in-crease which may be attributed to the death of bacterialcells. Fig. 2 and 3 shows higher amount of proteins thancarbohydrate and other components in the EPS produced

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by SRB on respective stainless steel samples, as observedearlier [3] also. However, EPS harvested from SRBs grownin the presence of glass/plastic substrate shows no orlesser protein than other components [1]. This differencecould be attributed to the presence of metal centers (iron)in case of hydrogenase enzyme [39] of SRBs and that ironof these enzymes is being supplied due to the corrosionof the stainless steel coupons. This hypothesis is alsosupported by the comparison of present results with thoseof the corrosion experiments done in the presence of SRBsp. Desulfovibrio desulfuricans [40], which show highercorrosivity and higher amount of protein, in the harvestedEPS. The role of protein in influencing the compositionof the biofilm and hence the biocorrosion has also beensuspected [37]. One also observes that the decrease inthe concentration of carbohydrates starts occurring earlier(60 hours after start of the experiment) and its rate is alsohigher than that of the protein (Fig. 2). Possible use ofEPS as substrate by the bacteria and faster utilization ofcarbohydrate than protein [37] could be the reason for thisobservation.

3.2. Nature of media and CorrosionBaar’s media after inoculating with SRB shows an increaseof pH from 7.23 to 7.7/7.8 in around 4 days. This can beattributed to the metabolic reaction [41]:

8H+ = S0−4 + 8e− → S− + 4H20, (1)

which occurs in presence of hydrogenase in SRBs and toreactions

2H+ + S− → H2S. (2)

The e− s required for reaction (1) are available from (i) thepresence of ammonium ferrous sulphate and sodium lactatewhich act as electron donor and/or (ii) oxidation of Fe toFe2+. Thus the increase in pH can be assigned to thepresence and growth of SRB in the media. The evidenceof the presence of bacteria is indicated by the observationof corroded stainless steel coupon under SEM (Fig. 1), thesmell of H2S in the test cell and bacterial counting in thetest media (Table 2). The pH of the media is also observedto increase up to 7.7/7.8 in the electrochemical polarizationtests and the weight loss test whereas test in control mediashows change of pH up to only 7.4. This can be understoodsince due to anodic polarization of steel samples, more Feoxidizes, resulting in availability of larger number of e−’swhich in turn enhances the rate of the above indicatedmetabolic reactions.Fig. 2 shows corrosion rate, calculated from tafel plots,increasing up to 96 hours of exposure and then decreasing

Fig. 4 Corelation between corrosion rate (mpy) and EPS components (carb., protein and U.acid) of 304L with bacteria during immersion tests without media replenishing

Time (hrs)

0 20 40 60 80 100 120 140 160

Cor

rosi

on ra

te (m

py)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

EPS components (m

g/cm2)

0

5

10

15

20

25

Corr. rate CarbohydrateProteinU. acid

Fig. 3 Corelation in conc. of uronic acid and lipids of EPS with time on stainless steelswith bacteria during immersion tests without media replenishing

Time (hrs)

0 20 40 60 80 100 120 140 1608 Lipids 2205

Figure 4. Corelation between corrosion rate (mpy) and EPS com-ponents (carb., protein and U.acid) of 304L with bacteriaduring immersion tests without media replenishing.

similar to the change observed for bacterial (planktonicand sessile) activity (Table 2). This is, however, the inverseof the trend observed for components of EPS, namelysugar and protein (Fig. 2). This observation suggestscorrelation between corrosion of steel and SRB activity,as proposed earlier [42]; i.e., that the dissolved metalconcentration can serve as an indicator of the bioactivityof the SRB. However, the impact of isoelectric potentialof the stainless steels on the adhesion of the bacteriaon the steel surface and on the metabolism of bacteriashould also be considered [43]. A comparison of variationof corrosion rate, carbohydrate and uronic acid contentwith time (Fig. 4), in case of all the three stainless steelsamples, shows increase in the fraction of uronic acid incarbohydrates as corrosion rate increases with time beyond60 hours of exposure. This fraction reaches a maximumafter the samples have been exposed for 96 hours, whencorrosion rate is also highest. This observation may beattributed to higher fraction of the acidic part of the sugar,i.e. uronic acid, which may result in overall decrease inpH of the solution. Work by Çetin et al. 2009 [17] showeddecrease in cathodic and increase in anodic tafel slopewith incubation time. However, no such dependence couldbe clearly observed in present work. To further observethe dependence of corrosion on the bacterial presencein media, immersion tests were done. Corrosion rates,calculated from these tests (Fig. 5), are observed to behigher in case of inoculated media as compared to control,an evidence of enhanced corrosion attack on steels due toSRBs. Pitting attack (Fig. 6) is observed to increase inthe presence of bacteria and with time. The SEM analysisof metal coupons exposed in sterile medium, exhibitedeither no or limited localized corrosion (Fig. 7), whereasthose exposed in inoculated medium showed significantpitting (Fig. 8). During immersion test, concentration ofcarbohydrates and proteins continued to increase (Fig. 9)which can be attributed to frequent addition of nutrientsin the media in these tests. Corrosion related parameters,

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Microbial Influenced Corrosion by Thermophilic bacteria

Fig. 5 Corrosion rate of stainless steels subjected to immersion tests with replenishment of media

Time (Days)0 20 40 60 80 100

Cor

rosi

on ra

te (m

py) i

n co

ntro

l med

ia

0.00

0.02

0.04

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0.08

0.10 Corrosion rate (m

py) in inoculated media

0.0

0.2

0.4

0.6

0.8

1.0

1.2

304L control2205 control304L inoculated2205 inoculated

Fig. 6 Maximum pit depth in case of stainless steels subjected to immersion tests with replenishment of media

Time (Days)

0 20 40 60 80 100

Max

imum

pit

dept

h (u

m)

0

20

40

60

80

100

120304L control316L control2205 control304L inoculated316L inoculated2205 inoculated

Figure 5. Corrosion rate of stainless steels subjected to immersiontests with replenishment of media.

Fig. 5 Corrosion rate of stainless steels subjected to immersion tests with replenishment of media

Time (Days)0 20 40 60 80 100

Cor

rosi

on ra

te (m

py) i

n co

ntro

l med

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py) in inoculated media

0.0

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304L control2205 control304L inoculated2205 inoculated

Fig. 6 Maximum pit depth in case of stainless steels subjected to immersion tests with replenishment of media

Time (Days)

0 20 40 60 80 100

Max

imum

pit

dept

h (u

m)

0

20

40

60

80

100

120304L control316L control2205 control304L inoculated316L inoculated2205 inoculated

Figure 6. Maximum pit depth in case of stainless steels subjected toimmersion tests with replenishment of media.

obtained from electrochemical tests are given in Fig. 10, 11and 12. It is observed that OCP in inoculated Baar’s mediashift towards more negative magnitudes as compared tothe respective values in control media. The OCP drop canbe attributed to the presence of sulfide in the inoculatedmedia due to the production of H2S by SRB activity. Oneobserves similar change in OCP with increase of incubationperiod in inoculated media (Fig. 10), which may be assignedto increased concentration of sulfides with time. Sulfidesolutions are known to be of reducing types hence theylower the OCP values. Anodic polarization curves (Fig. 13)show decrease in passivation range and pitting potential(Fig. 11 and 12) and increase in current density in caseof stainless steel samples exposed to inoculated vis-à-visthe control media. In case of measurements in inoculatedmedia, all the three parameters showed similar variationswith increase in incubation time (Fig. 13).The observance of a higher degree of corrosion attack asevidenced from increased corrosion rates, and deeper pits

Fig. 7 Scanning electron micrograph of stainless steel 304L after 50 days immersion test in Baar’s Media (Control)

Fig. 8 Scanning electron micrograph showing pits on stainless steel 304L exposed to inoculated Baar's media in 50 day's immersion test.

Figure 7. Scanning electron micrograph of stainless steel 304L after50 days immersion test in Baar’s Media (Control).

Fig. 7 Scanning electron micrograph of stainless steel 304L after 50 days immersion test in Baar’s Media (Control)

Fig. 8 Scanning electron micrograph showing pits on stainless steel 304L exposed to inoculated Baar's media in 50 day's immersion test.

Figure 8. Scanning electron micrograph showing pits on stainlesssteel 304L exposed to inoculated Baar’s media in 50 day’simmersion test.

in immersion test and decreased values of pitting potentialand passivation range along with increased corrosion ratesin electrochemical tests in inoculated media as comparedto control, can be understood from the effect of the presenceof bacteria on the passivity of stainless steel. The passivityof stainless steel, in abiotic media, is attributed to the pres-ence of oxide and hydroxides of Cr on its surface. In thepresence of SRBs, biofilm forms on the metal surface [44]which affects the passive film through bacterially producedsulfides (Eq. 1) which results in formation of chromiumsulfides and iron/nickel sulfides due to presence of Fe, Ni

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Fig. 9 Relation between corrosion rate and EPS components in case of stainless steel 304L exposed to inoculated Baar’s media in immersion tests (with replenishment of media)

Fig. 10 OCP of stainless steels after exposure for 0, 5 and 10 days in inoculated and control media

Time (Days)

0 2 4 6 8 10

OC

P (m

V)

-500

-450

-400

-350

-300

-250

-200

304L control316L control2205 control304L inoculated316L inoculated2205 inoculated

Time (Days)

0 20 40 60 80 100

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rosi

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te (m

py)

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Carbohydrate and Protein conc.(m

g/cm2)

24

26

28

30

32

34

36

38

40

Corr.rate Carb.conc.Prot. conc.

Figure 9. Relation between corrosion rate and EPS components incase of stainless steel 304L exposed to inoculated Baar’smedia in immersion tests (with replenishment of media).

Fig. 9 Relation between corrosion rate and EPS components in case of stainless steel 304L exposed to inoculated Baar’s media in immersion tests (with replenishment of media)

Fig. 10 OCP of stainless steels after exposure for 0, 5 and 10 days in inoculated and control media

Time (Days)

0 2 4 6 8 10

OC

P (m

V)

-500

-450

-400

-350

-300

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304L control316L control2205 control304L inoculated316L inoculated2205 inoculated

Time (Days)

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py)

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Carbohydrate and Protein conc.(m

g/cm2)

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Corr.rate Carb.conc.Prot. conc.

Figure 10. 10 OCP of stainless steels after exposure for 0, 5 and10 days in inoculated and control media.

Fig. 11 Passivation range of stainless steels in media with/without bacteriaTime (Days)

0 2 4 6 8 10 12

Pass

ivat

ion

rang

e (m

V)

1100

1200

1300

1400

1500

304L control316L control2205 control304L inoculated316L inoculated2205 inoculated

Pitti

ng P

oten

tial (

mV

)

900

1000

1100

Figure 11. Passivation range of stainless steels in media with/withoutbacteria.

Fig. 11 Passivation range of stainless steels in media with/without bacteriaTime (Days)

0 2 4 6 8 10 121100

304L inoculated316L inoculated2205 inoculated

Fig. 12 Pitting potential of stainless steels in media with/without bacteriaTime (Days)

0 2 4 6 8 10

Pitti

ng P

oten

tial (

mV

)

500

600

700

800

900

1000

1100

304L control316L control2205 control304L inoculated316L inoculated2205 inoculated

Figure 12. Pitting potential of stainless steels in media with/withoutbacteria.

Fig. 13 Anodic polarization curves of stainless steel 304L in inoculated and control media

Fig. 14 Weight % of carbon, sulphur and phosphrous detected inside/outside pitformed on corroded stainless steel 304L

0 1 2 3 4

Wt %

0

10

20

30

40 Inside pitOutside pit

1- Carbon2 - Phosphrous3 - Sulphur

Figure 13. Anodic polarization curves of stainless steel 304L in in-oculated and control media.

and Cr in stainless steel (Table 1). These sulfides arebetter electron conductors, structurally more permeableand unstable; hence they make the passive film much lesseffective in protecting against corrosion attack. This resultsin enhanced corrosion rate and higher pitting attack incases where sulfide formation takes place in a localizedarea [45]. Thus pits observed on the corroded specimenscan be analyzed as due to SRB induced corrosion. Thisis supported by the observation of higher amount of C, Sand P inside pit as compared to their respective amountoutside the pit from the results of SEM/EDS (Fig. 8 and14). Higher amount of C inside pits can be assigned tothe amount of EPS which form as a result of metabolicactivities of bacteria while enhanced bacterial activity ofSRB’s inside pits leads to conversion of sulfates to sulfideions with higher rate. Iron phosphide is formed by the reac-tion of iron with phosphorus compound. When a protectiveFeS layer does not form or break down, the phosphoruscompound, formed by reaction of hydrogen sulfide withinorganic phosphorus compounds in the environment, acts

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Microbial Influenced Corrosion by Thermophilic bacteria

Fig. 13 Anodic polarization curves of stainless steel 304L in inoculated and control media

Fig. 14 Weight % of carbon, sulphur and phosphrous detected inside/outside pitformed on corroded stainless steel 304L

0 1 2 3 4

Wt %

0

10

20

30

40 Inside pitOutside pit

1- Carbon2 - Phosphrous3 - Sulphur

Figure 14. Weight formed on corroded stainless steel 304L.

Fig. 15 Anodic polarization of stainless steels exposed to inoculated Baar’s media

after 10 days incubation period

Figure 15. Anodic polarization of stainless steels exposed to inocu-lated Baar’s media after 10 days incubation period.

on the steel surface and causes corrosion of iron [16]. Thepresence of higher phosphate concentration in EDS spec-tra indicates the role of phosphorus compounds in additionto sulfides on corrosion reactions. FeS decreases hydrogenover potential and cause cathodic depolarization [46, 47].This sulfide, in turn, reacts with iron (main constituentof steel) to form iron sulfide. When one puts the OCPvalues (Fig. 10) and pH of the inoculated test solutions(∼7.5) in E-pH diagram of Fe–S–H2O system [48] thissulfide appears to be mackinawite (FeS0.943). Mackinaw-ite is known to be black in appearance, dissolves easilyand is unprotective type. Also, mackinawite deposits arehypothesized to act as large surface area [49] and canact as a cathode in the galvanic couple with steel thusenhancing corrosion [39]. Inoculated test solutions in thepresent study are also observed to be dark in color afterthe end of the test indicating the formation of mackinawiteand their dissolution. Accordingly, iron inside pits corrodewith higher rate in the absence of any protective type ofcorrosion products leading to deeper pits.

Higher concentration of heavy metals (Ni, Cr and Mo)slows down the growth of bacteria and production of EPS[21, 42]. Accordingly, present results show maximum con-centration of sessile bacteria and of EPS components incase of 304L (minimum amount of Cr, Ni and Mo) andminimum in case of 2205 (maximum amount of Ni, Cr andMo) (Table 1 and Fig. 2 and 3). Results of immersion testshow extent of corrosion rate and pit depth to be maximumin case of stainless steel 304L followed by 316L and 2205(Fig. 5 and 6). Electrochemical test shows pitting potentialand passivation range; in general, to be highest in thecase of duplex stainless steel 2205 followed by austeniticstainless steels 316L and 304L (Fig. 11, 12 and 15). Thusboth the tests predict maximum corrosion resistance of2205 whereas least resistance of 304L in the studied me-dia. The relative resistance of the studied stainless steelsagainst corrosion may be correlated with their composi-tion through determination of pitting resistance equivalentnumber (PREN) as given below [50]:

PREN = %Cr + 3.3× %Mo + 16× %N.

Accordingly, the PREN of 2205 is maximum (34.8), that of316L is 25.2 and 304L is minimum (19.9).

4. ConclusionThe paper reports the results obtained from the electro-chemical polarization and immersion tests, SEM/EDS anal-yses conducted on stainless steels 304L, 316L, and 2205 inBaar’s media inoculated with Desulfotomaculum sp. Cor-rosivity of the solution is observed to increase with (i)increasing the activity of bacteria and (ii) the incubationtime. Maximum corrosive conditions are observed to prevailwhen bacterial activity is highest and EPS of the biofilmconsists of minimum amounts of carbohydrate and proteinalong with the increased fraction of uronic acid in carbohy-drate. It is also observed that Desulfotomaculum sp. hadthe ability to biodegrade its own EPS. Metal ions, presentin the corrosive media because of corrosion of stainlesssteel, show toxicity as they affect the bacterial activityand EPS production. Stainless steel 2205 shows maximumresistance against MIC followed by stainless steels 316Land 304L.

AcknowledgmentsOne of the authors (SL) acknowledges the Research fel-lowship received from MHRD, Govt. of India.

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S. Lata, C. Sharma, A.K. Singh

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