microbial corrosion in linepipe steel under the influence of a

Microbial Corrosion in Linepipe Steel Under the Influenceof a Sulfate-Reducing Consortium Isolated

from an Oil FieldFaisal M. AlAbbas, Charles Williamson, Shaily M. Bhola, John R. Spear, David L Olson, Brajendra Mishra, and Anthony E. Kakpovbia

(Submitted January 13, 2013; in revised form June 4, 2013)

This work investigates microbiologically influenced corrosion of API 5L X52 linepipe steel by a sulfate-reducing bacteria (SRB) consortium. The SRB consortium used in this study was cultivated from a sour oilwell in Louisiana, USA. 16S rRNA gene sequence analysis indicated that the mixed bacterial consortiumcontained three phylotypes: members of Proteobacteria (Desulfomicrobium sp.), Firmicutes (Clostridiumsp.), and Bacteroidetes (Anaerophaga sp.). The biofilm and the pits that developed with time were char-acterized using field emission scanning electron microscopy (FE-SEM). In addition, electrochemicalimpedance spectroscopy (EIS), linear polarization resistance (LPR) and open circuit potential (OCP) wereused to analyze the corrosion behavior. Through circuit modeling, EIS results were used to interpret thephysicoelectric interactions between the electrode, biofilm and solution interfaces. The results confirmedthat extensive localized corrosion activity of SRB is due to a formed biofilm in conjunction with a porousiron sulfide layer on the metal surface. X-ray diffraction (XRD) revealed semiconductive corrosion productspredominantly composed of a mixture of siderite (FeCO3), iron sulfide (FexSy), and iron (III) oxide-hydroxide (FeOOH) constituents in the corrosion products for the system exposed to the SRB consortium.

Keywords biofilm, carbon steel API 5L X52, microbiologicallyinfluenced corrosion, pipeline, sulfate-reducingbacteria

1. Introduction

Microbiologically influenced corrosion (MIC), or bio-corrosion, is of considerable concern to the oil and gasindustry. MIC is induced by indigenous microorganisms thatnaturally reside in hydrocarbon and secondary water injectionsystems (Ref 1-3). Microbial metabolic products, for example,sulfide and organic acids such as acetic acid, may alter interfacechemistry, resulting in increased corrosion rates. Furthermore,biofilms consisting of complex communities of microbes andextracellular polymeric substances (EPS) that have developedon the metal surface create gradients of pH and dissolvedoxygen, leading to localized forms of corrosion, such as pittingand crevice formation (Ref 1-4).

Since the advent of modern oil and gas production, scientistsand engineers have faced problems caused by microorganisms(Ref 5). Hydrocarbons are an excellent carbon (food) source fora wide variety of microbes in all three domains of life—the

Bacteria, Archaea and Eucarya, and microbial representatives ofall the three domains likely play roles in MIC (Ref 2). Bacterialactivity (e.g., sulfate reduction) is believed to be responsible forgreater than 75% of the corrosion in production oil and gas wells,and it is likely that greater than 50% of failures of buriedpipelines and cables are due to the metabolic activities ofmicrobes (Ref 5). The main types of bacteria associated withmetals in pipeline systems are sulfate-reducing bacteria, ironreducing bacteria, iron and manganese oxidizing bacteria, andacid producing bacteria (Ref 1, 3, 5). For several reasons, SRBhave been recognized as the major contributor to MIC in pipelinesystems primarily due to their predominantly anaerobic lifestyleand continuous production of corrosive hydrogen sulfide (Ref 3, 5).However, in practical situations, MIC results from synergisticinteractions of different microbial consortia, which coexist in theenvironment and are able to affect the electrochemical processesthrough co-operative metabolisms (Ref 4).

As early as 1926, traditional microbiologists detected thepresence of SRB in oil environments (Ref 5). Studies have shownthat most kinds of cultivated SRB are responsible for theproduction of hydrogen sulfide,which is a toxic and corrosive gasin both aerial and aqueous form (Ref 4, 5). At a minimum,hydrogen sulfide is thought to be responsible for a variety ofenvironmental effects which include reservoir souring, contam-ination of natural gas and oil with H2S, corrosion of metalsurfaces, the ‘‘plugging’’ of reservoirs due to the formation ofextensive pore-space microbial biofilms and the precipitation ofmetal sulfides (Ref 5). SRB has received much attention in the oil& gas industry andMIC investigations, as these microorganismshave several detrimental metabolic activities including the abilityto: (1) oxidize hydrogen, (2) use O2 and Fe

3+, (3) utilize aliphaticand aromatic hydrocarbons, (4) couple sulfate reduction to theintracellular production of magnetite and (5) compete with

Faisal M. AlAbbas, Shaily M. Bhola, David L Olson, and BrajendraMishra, Department of Metallurgical and Materials Engineering,Colorado School of Mines, Golden, CO 80401; Charles Williamsonand John R. Spear, Department of Civil and EnvironmentalEngineering, Colorado School of Mines, Golden, CO 80401;and Anthony E. Kakpovbia, Inspection Department, Saudi Aramco,Dhahran 31311, Saudi Arabia. Contact e-mail: [email protected].

JMEPEG !ASM InternationalDOI: 10.1007/s11665-013-0627-7 1059-9495/$19.00

Journal of Materials Engineering and Performance

nitrate-reducing/sulfur-oxidizing bacteria (NRB-SOB) (sincethey may have a nitrite reducing activity) (Ref 5, 6). In total, allof these activities can significantly degrade the quality of oil andgas in a reservoir to be economically extractable. Moreover, SRBadversely impacts the integrity of oil and gas installations.

This study investigates the impact of environmental SRB(cultivated from oil field samples rather than obtained from aculture collection) on the corrosion behavior of API 5L X52linepipe steel. The SRB consortium used in this study wascultivated from an oil well in Louisiana, USA (Ref 7). 16SrRNA gene sequence analysis was performed to identify theSRB species. The nature and kinetics of chemical andelectrochemical reactions introduced by SRB activities onAPI 5L X52 carbon steel coupons were characterized usingelectrochemical impedance spectroscopy, linear polarizationresistance (LPR) and open circuit potential (OCP). The biofilmand corrosion morphology were investigated using fieldemission scanning electron microscopy coupled with energydispersive spectroscopy (EDS). The composition of corrosionproducts was evaluated using x-ray diffraction analysis.

2. Materials and Methods

2.1 Microorganisms and Testing Medium

The SRB consortium used in this study was cultivated fromwater samples obtained from an oil well located in Louisiana,USA. The water samples were collected and bottled at the wellhead from an approximate depth of 2200 ft. as described underthe NACE Standard TM0194 (2004) (Ref 8). The SRB werecultivated in a modified Baar!s medium (ATCC medium 1250).Baar!s medium is reported to be suitable when studying theinfluence of mixed bacterial communities on steel corrosion,though many different media recipes exist (e.g., the Postgateseries of media) and any number of them likely work (Ref 9).This growth medium was composed of magnesium sulfate (2.0 g),sodium citrate (5.0 g), calcium sulfate di-hydrate (1.0 g),ammonium chloride (1.0 g), sodium chloride (25.0 g),di-potassium hydrogen orthophosphate (0.5 g), sodium lactate60% syrup (3.5 g), and yeast extract (1.0 g). All componentswere per liter of distilled water. The pH of the medium wasadjusted to 7.5 using 5 M sodium hydroxide and sterilized in anautoclave at 121 "C for 20 min. The SRB species were culturedin the growth medium with filter-sterilized 5% w/w ferrousammonium sulfate added to the medium at a ratio of 0.1-5.0mL respectively. The bacteria were incubated for 72 h at 37 "Cunder an oxygen-free nitrogen headspace.

2.2 Sulfate-Reducing Consortium Identifications

Genomic DNA was extracted from the bacterial consortiumusing the MoBio Powersoil DNA extraction kit (MoBio,Carlsbad, CA) with the 10-min vortexing step replaced by1 min of bead beating. Primers 515F (5¢-GTGCCAGCMGCCGCGGTAA-3¢) and 1391R (5¢-GACGGGCGGTGWGTRCA-3¢) (Ref 7, 10) were used to amplify 16S rRNA genes.Polymerase chain reaction (PCR), cloning and transformationwere conducted as described by Sahl et al. (Ref 11). Uniquerestriction fragment length polymorphisms (RFLP) weresequenced on an ABI 3730 DNA sequencer at Davis Sequenc-ing, Inc. (Davis, CA), and Sanger sequence reads were calledwith PHRED (Ref 12, 13) via Xplorseq (Ref 14). Sequences

were aligned with the SINA aligner (Ref 15) and added(parsimony insertion) to the Silva SSURef111_NR guide tree(Ref 16) with the ARB software package (Ref 17). Sequenceswere also compared to the Genbank database (Ref 18) via theBasic Local Alignment Search Tool (BLAST) (Ref 19).Phylogenetic trees were created with RAxML (Ref 20).Relevant sequences were obtained from the Genbank databaseand aligned and masked with ssu-align (Ref 21). Phylogenetictrees were created using the GTR substitution model and thegamma distribution of rate heterogeneity. The number ofbootstrap replicates was determined using the RAxML fre-quency-based criterion (Ref 22). 16S rRNA gene sequencesproduced during this research have been deposited in theGenbank with accession numbers KC756849-KC756851.

2.3 Sample Preparation

Pipeline steel (API 5L X52) coupons, provided by a localenergy company (Saudi ARAMCO), were used for this study,and their chemical compositions are shown in Table 1.Metallographic specimens from the received materials wereprepared with standard methods for optical microscopy (1 lmfinal polish and 2% nital etch). Representative microstructurecontains a mixture of polygonal ferrite and pearlite structures.

For corrosion evaluations, the coupons were machined to asize of 10 mm9 10 mm9 5 mm and embedded in a mold ofnon-conducting epoxy resin, leaving an exposed surface area of100 mm2. For electrical connection, a copper wire was solderedat the rear of the coupons. The coupons were polished with aprogressively finer sand grinding paper until a final grit size of600 lm was obtained. After polishing, the coupons were rinsedwith distilled water, ultrasonically degreased in acetone andsterilized by exposing to pure ethanol for 24 h.

2.4 Electrochemical Tests

The electrochemical measurements were made in a conven-tional three electrode ASTM cell coupled with a potentiostat and ahigh frequency impedance analyser (Gamry-600). The electro-chemical cells were composed of a test coupon as a workingelectrode (WE), a graphite electrode as an auxiliary electrode and asaturated calomel electrode (SCE) as a reference electrode. Theglasswareswere autoclaved at 121 "C for 20 min at 20 psi pressureand then air dried. Graphite electrodes, purging tubes, rubberstoppers and needles were sterilized by immersing in 70 vol.%ethanol for 24 h followed by exposure to a UV lamp for 20 min.Two solutions were used in this experiment. Under a sterilizedcondition (in a sterilized laminar flow hood), the first cell wasprepared with 600 mL of sterilized modified Baar!s growth media(described above) and the second cell was prepared with 600 mLsterilized Baar!s media inoculated with 5 mL of the SRBconsortium at 106 cell/mL. The electrochemical cells were purgedfor 1 h with pure nitrogen gas to establish the anaerobicenvironment. Electrochemical tests were performed at a flow rateof 0.9 m/s (100 rpm) under atmospheric pressure conditions at30 "C, to simulate the flow conditions of Saudi Aramco oilproduction pipeline.

Open circuit potential values of the systems were monitoredwith time during the immersion period followed by periodicreadings up to 336 h. Impedance measurements were performedon the system at the OCP for various time intervals fromimmersion up to 288 h. The frequency sweep was applied from105 to 10!2 Hz with an AC amplitude of 10 mV.


During the LPR technique, the polarization resistance (Rp)was measured on the system at a scanning amplitude of±10 mV with reference to the OCP for various time intervalsfrom immersion up to 336 h. The LPR plots were then fittedusing the Gamry-600 electrochemical software to obtain thegoodness of fit for corrosion rate values. The values were thencompared using the mathematical calculation as determined bythe equation given below (Ref 23);

icor "1

Rp

babc

2:3#ba $ bc%

! "#Eq 1%

where ba and bc is the anodic and cathodic slope, respec-tively.

2.5 Surface Analysis and Corrosion Product Compositionsof the Coupons Exposed to SRB

At the conclusion of each test, the WEs were carefullyremoved from the system for fixation. To fix the grown biofilmto the steel surface, the coupons were immersed for 1 h in a 2%glutaraldehyde solution, dehydrated with 4 ethanol solutions(15 min each): 25, 50, 75, and 100% successively, air driedovernight and then gold sputtered (Ref 24). After fixation, thecoupons were examined using field emission scanning electronmicroscopy coupled with EDS to evaluate the morphology andchemical composition of the biofilm. Corrosion productcomposition was obtained using the x-ray diffraction methodwith a Philips PW 3040/60 spectrometer using Cu Ka radiationsource. The coupons were then cleaned using a standardprotocol described under the ASTM-GI-03 (Ref 25), and the pitmorphology and density on the exposed coupons wereexamined using FE-SEM.

3. Results and Discussion

3.1 Identification of the Sulfate-Reducing Consortium

16S rRNA gene sequence analysis indicated that the mixedbacterial culture consortium contained three phylotypes that areclose to members of the Proteobacteria (Desulfomicrobiumsp.), Firmicutes (Clostridium sp.), and Bacteroidetes (Anaer-ophaga sp.) (Ref 7). The phylogenetic tree representative of thebacterial consortium has been shown in Fig. 1. Desulfomicr-obium, including mesophiles and thermophiles, are known tobe commonly associated with oil reservoirs. They have beenisolated previously from different oil fields in the North Sea(Ref 26) and in 5 of 6 different Alberta oil fields in Canada asobserved by Voordouw and colleagues (Ref 27). Leu et al.(1999) reported the presence of different strains of Desulfo-microbium obtained from various samples in distant oil fieldssuch as formation water and drilling mud (Ref 26). Desulfo-microbium sp. are anaerobic, Gram-negative, rod-shaped,sulfate-reducing bacteria that grow on different carbon sourcesubstrates that include lactate, pyruvate, glycerol, and ethanolwith optimal growth temperatures between 25 and 35 "C

(Ref 27). They are capable of using sulfate, thiosulfate or sulfiteas a terminal electron acceptor (Ref 6, 28). Several researchershave further reported that in the presence of 0.5% NaCl,Desulfomicrobium metabolize lactate and sulfate to produceacetate, CO2 and H2S as major end products (Ref 26-28). Thesemicroorganisms can play a significant role in oil field reservoirsouring by the reductive generation of hydrogen sulfide fromthese sulfur containing electron acceptors (Ref 28). Clostridiumsp. are anaerobic microbes that are Gram-positive, spore-forming bacteria. This type of bacteria is capable of survivinghigh temperatures due to the formation of heat-resistantendospores (Ref 1, 6, 28). Anaerophaga sp. have also beenpreviously identified in samples from a produced waterobtained from the high-temperature Troll oil formation in theNorth Sea (Ref 28). The cultivation of this consortium waspreviously reported (Ref 7).

3.2 Surface Morphology and Element Analysis

At the conclusion of the tests, the visual inspection of thecoupon exposed to the biotic system revealed dense, thick andblack products covered on the surface. There is a significantdifference in the appearance, structure, and morphology of thecorrosion products developed on the steel coupons exposed tothe biotic system compared to that exposed to the abioticsystem as shown in Fig. 2 and 3. Both the morphologicalobservations and EDS elemental analysis of corrosion productsof API X52 steel immersed in the biotic system are shown inFig. 2(a)-(c). As shown in Fig. 2(a), there are two distinctiveareas: A1 and A2. Quantitative EDS analysis shows A2 iscomposed of a higher amounts of sulfide, sodium salt,phosphate and carbon (Fig. 2(b) and Table 2). The light region(A2) is considered the outer layer where the sulfur is thepredominant constituent, Fig. 2(b). On the other hand, the darkregion, A1, is considered to be the inner layer in which the iron,oxygen, and carbon, in addition to sulfur and phosphorous, arepredominant, as shown in Fig. 2(c) and Table 2. The outer andinner areas were chosen based on our previous researchexperience, and no cross section view was performed on thesample (Ref 7). The spherical characteristic structures shown inFig. 2(a) might represent siderite (FeCO3) surrounded by amixture of iron sulfide (FeS) and iron oxides.

Similar features have been reported by Hendrik Venzlaff et al.for steel surfaces exposed to a SRB strain, Desulfopilacorrodens. The fact that Desulfomicrobium have the capacityto convert the carbon source (lactate) through pyruvate to acetatealong with the production of carbonate supports the formation ofFeCO3 (Ref 6, 29). Furthermore, the presence of di-potassiumhydrogen orthophosphate and sodium chloride in the growthmedia might lead to the precipitation of phosphorous-basedcompounds and sodium chloride on the surface as suggested bythe EDS spectra (Ref 30).

The morphological observations and elemental analysis ofsurface deposits and corrosion products on API X52 carbonsteel coupons exposed to the abiotic system are shown inFig. 3(a) and (b). There is one coherent homogenous layer ofcorrosion product with salt crystal deposits on the surface. As

Table 1 The chemical composition of API-5L X52 carbon steel coupons, wt.%

Fe C Si Cr Ni Mn Cu Mo Nb Ti Al V S P

Bal. 0.070 0.195 0.03 0.02 1.05 0.05 0.004 0.021 0.001 0.029 0.003 0.008 0.008


Fig. 1 The phylogenetic characterization of SRB consortium

Fig. 2 FESEM and EDS analysis for the system under biotic conditions. (a) FESEM Image of carbon steel exposed to Barr!s medium inocu-lated with SRB, at 1009. Arrow points to a magnified image (2009) of iron carbonate layer. EDS analysis corresponding to the FESEM outerand inner region shown in Fig. 2(b) and (c), respectively


shown in Fig. 3(b) and Table 2, quantitative EDS analysisshows that this layer might be composed of iron oxides mixedwith sodium chlorides, calcium and carbon-based compoundsthat accumulated from the growth medium (Ref 30).

As shown in Table 2 and Fig. 2 and 3, the biotic and abioticsystems displayed significant differences in distribution andcomposition of corrosion products. In the presence of the SRBconsortium, there is significant accumulation of sulfur-basedcompounds and FeCO3, and a substantial amount of corrosion

products growing upward is observed, and the interface doesnot have a rigid appearance, most likely due to the biofilmmatrix, nature of which is polysaccharidic and viscoelastic(Fig. 2 and 4), while the corrosion products for the abioticsystem exhibits a completely different thin-flat layer with a hardtexture (Fig. 3) (Ref 1-4).

The biofilm developed in the presence of the SRBconsortium together with the produced corrosion products havea heterogeneous morphology and thickness (Fig. 4). The

Fig. 3 FESEM and EDS analysis on the API X52 exposed to sterilized Baar!s medium

Table 2 Comparative of EDS analysis corresponding to the abiotic and the biotic systems, respectively

Element, wt.% C O Na Si Fe S Cl Mn Ca P Total

Abiotic systemWhole region 0.01 18.73 4.54 … 68.38 1.54 4.61 2.11 0.08 0.00 100

Biotic systemOuter region (A2) 14.99 4.20 8.66 1.69 10.06 55.09 0.00 0.00 0.00 6.84 100Inner region (A1) 10.35 23.58 6.96 0.89 26.98 22.43 0.00 0.00 0.00 8.72 100

Fig. 4 FESEM image for the biofilm developed on the API X52 exposed to the SRB-containing medium 10009 and 20009


FESEM micrograph (Fig. 4) reveals the presence of thecorrosion products, cells, spores, and EPS fibers distributedover the coupon. At the conclusion of the experiment, the steelsubstrate was hardly visible. It was covered with a porous blacklayer. A jelly-like substance could be observed among thecorrosion products, which was speculated to be biofilmproduced EPS (Ref 30, 31). Rod-shaped and round shapedbacteria occupied a significant volume fraction qualitatively,(no quantitative measurements were performed); however, EPSand corrosion products have been reported to occupy 75-95%of biofilm volume, while 5-25% is occupied by the metabo-lizing cells (Ref 1, 30). Higher concentrations of hydrogensulfide, phosphate-based compounds, and other potentiallybiotically-generated, corrosion-influencing compounds arelikely to be promoted by SRB metabolism and biofilmformation (Ref 1, 3, 30, 31). Conversely, the nature ofcorrosion film for the abiotic system exhibits a completelydifferent thin-flat layer with a hard texture (Fig. 3).

3.3 XRD Results

The XRD spectra of API X52 steel coupons exposed to thebiotic system are displayed in Fig. 5. The XRD patternconfirmed the formation of a significant amount of iron sulfidecompounds that include mackinawite (Fe1+xS), other biogeneticiron sulfide (FeS), siderite (FeCO3), and iron (III) oxide-hydroxide (FeOOH) (Ref 29, 32, 33). The formation of pyriteand mackinawite films in the presence of SRB is expected as aconsequence of their production of sulfide. Due to the very highreactivity of H2S with iron, the mackinawite layer has beenshown to form, which is much faster than what would havebeen expected from the typical kinetics for a precipitationprocess. The formed mackinawite is not stable and maydissolve depending on the solution saturation level. For the pHranging between 4 to 7, which is a typical SRB-containingenvironment, the solution is often supersaturated with respect toiron sulfide, and the layer does not dissolve (Ref 34). The ironsulfide films could protect or deteriorate the surface. Thin

protective films are associated with low ferrous ion concentra-tion rates while active films are believed to form in the presenceof high ferrous ion concentrations. These protective films havebeen proposed to fail due to disruption by microbial action,bulky growth, and oxidation (Ref 34). It has been proposed thatthe corrosion rate for steel can be defined by the rates of ironsulfide layer formation and breakdown (Ref 7).

Possibly, the capacity of the bacterial consortium (Desul-fomicrobium & Clostridium) to convert the carbon source(lactate) through pyruvate to acetate and produce carbonateresulted in the formation of FeCO3 (Ref 29). Some bacteriaof the Clostridium genus (present in the consortium) arecapable of fermentative utilization of lactate depending onthe pH medium and produced acetate, bicarbonate orpropionic acid that may be relevant to our system; however,due to our limitations, such measurements have not beenmade (Ref 6). It might be possible that the bacterialconsortium work in a synergistic way and affect thecorrosion of the alloy steel.

EDS and XRD results suggested that the following reactionsoccurred as a result of metabolic activities of the SRBconsortium and corrosion behavior of the steel surface. Thegeneral chemical and electrochemical reactions can varyaccording to the surface content and EDS analyses, butgenerally include a cathodic reaction under natural deaeratedconditions (Ref 1, 3, 30, 31, 35):

2H2O $ 2e! ! H2 $ 2OH! #Eq 2%

The SRB Desulfomicrobium sp. may use cathodic hydrogento reduce sulfate to sulfide as follows (Ref 1-4, 31)

SO2!4 $ 9H$ $ 8e! ! HS! $ 4H2O #Eq 3%

Reaction [3] normally happens at a slow rate without bio-catalysis from bacteria, however; in systems that containmicrobes, the reaction can be rapid, being enzymaticallycatalyzed by the hydrogenase enzyme system of Desulfomicr-obium sp. Some hydrogen sulfide ions will convert to hydrogensulfide especially at acidic pH as described (Ref 3, 30, 33)

HS! $ H$ ! H2S #Eq 4%

Reaction [4] is rapidly facilitated by the presence ofDesulfomicrobium sp. at a pH between 1 and 7. The productionof hydrogen sulfide and the oxidation of iron (anodic reaction) leadto the formation of different types of iron sulfide as follows(Ref 30, 31):

Fe0 ! Fe2$ $ 2e! #Eq 5%

Fe2$ $ H2S ! FeySx $ 2H$ #Eq 6%

According to Liu et al. (Ref 36), the ferrous ions (Fe2+)produced by the dissolution process react with sulfidemetabolized by the bacteria with the subsequent productionof different forms of iron sulfide (FeSx). Therefore, it isexpected that the structure of the biofilm changes accordingto the different growth phases of the SRB (Ref 30, 31, 35,36).

The rate determining step (RDS) for the SRB metabolicactivities, Reaction [3], is the proton reduction to formhydrogen. The kinetics of this hydrogen reduction are expectedto be slow due to the fact that the concentration of protons isextremely low under natural de-aerated conditions. It has beenreported that in a de-aerated water environment, the formation

20 40 60 80 1000

2000

4000

6000

8000

10000

12000

14000

!!

""

+

"#

"In

tens

ity (

CP

S)

2$ (degree)

#

"•!

# •

Fig. 5 XRD spectra for the system under biotic conditions for 14days exposure with SEM image of the examined surface


of hydrogen on the surface is extremely low because of limitedprotons (Ref 37).

H$ $ 1e! ! Hads #Eq 7%

Therefore, when there is not enough hydrogen to drive themetabolic reaction (Reaction [3]) Desulfomicrobium sp. mayswitch to convert the carbon source (lactate) through pyruvateto acetate with the production of carbonate (Ref 6, 29, 30).When SRB utilize lactate as an electron donor, they produceextra ATPs by the proton motive force and the oxidation oflactate to acetate plus carbon dioxide (Ref 6). The producedhydrogen crosses the cytoplasmic membrane and is oxidized byperiplasmic hydrogenase to initiate a proton motive force,which in turn bio-catalyzes Reaction [3] (Ref 4, 6). The primereactions of this process are as follows:

2CH3CHOHCOO! $ 2SO2!

4

! 4CH3COO! $ HCO!3 $ H2S $ HS! $ H$ $ CO2

#Eq 8%

Carbonate ions will react with ferrous ions and forminsoluble siderite (FeCO3) in addition to iron sulfide (FeS). Thenet reaction will be as follows (Ref 29):

4Fe$ SO2!4 $ 3HCO!3 $ H2O

! FeS #s) $ 3FeCO3 #s)$ 5OH!; DG& " !86:1 kJ/mol Fe

#Eq 9%

Closer inspection of the API X52 carbon steel electrode inFig. 6 shows variations in surface roughness. Generalizedcorrosion was observed in an irregular pattern on the coupon asin Fig. 6(a). The images at higher magnifications in Fig. 6(b) alsoshow preferential etching of selected pearlite grains in a regular,rectangular, and repetitive manner. The attack appears to be in anun-preferred orientation. An inter-granular attack that producesmicrostructural relief and etching of pearlite microstructures canbe observed in Fig. 6(c) and (d).

The rectangular attack, found predominantly in the morecorroded areas on the surface, may be the result of the galvaniccoupling between the semiconductive iron sulfide, which actsas a cathode, and the steel surface, or anode. Galvanic corrosionis either a chemical or an electrochemical corrosion phenom-enon. The latter is due to a potential difference between twodifferent surfaces connected through a circuit that allows forcurrent flow to occur from more active metal (anode), to lessactive surface (cathode). In our case, the iron sulfide isconsidered the cathode where the calculated equilibrium

Fig. 6 FESEM analysis for the API X52 coupon surface after cleaning for the system under biotic conditions at 2009, 20009 and 40009


potential of sulfate reduction (SO42!/FeS) is !0.25 V, and thesteel surface is the anode with calculated equilibrium potentialof !0.44 V (Ref 29). Anodic corrosion of iron by galvaniccoupling with iron sulfide has been proposed previously(Ref 37, 38). Galvanic corrosion attacks the iron matrixadjacent to iron sulfide deposits.

Furthermore, the conductivity of the iron sulfide augmentsthis coupling between the solution and underlying surface that,in turn, catalyzes the corrosion process. The conductivity ofheterogeneous corrosion products composed of FeS andFeCO3 in SRB cultures has been reported to be around 50Sm!1, which is higher than that of many typical semiconduc-tors such as silicon. This conductivity is mainly due to thepresence of FeS as FeCO3, which is considered an insulatingmineral (Ref 29).

The preferred attacks shown in the pearlite microstructures(Fig. 6c) are attributed to the nature of heterogeneous structuresof pearlite. Pearlite consists of plates of cementite (Fe3C) in amatrix of ferrite (Ref 33). The structural and compositionalinhomogeneity within the pearlite structure invites the possi-bility of this preferred attack and enhances the localizedcorrosion (Ref 39). When pearlite is exposed to localizedcorrosion, as in our case, galvanic microcells between ferrite(cathode) and cementite (anode) are generated. Consequently,the surface will exhibit deeper and larger pits. However, in areal system where oxygen might exist, the corrosion mecha-nism becomes quite complicated, and not only the sulfate andsulfide but also several other ionic species (e.g. chlorides) maybe involved. On the other hand, minimal corrosion damage wasobserved in the abiotic system where the polishing marks arestill visible (Fig. 7).

3.4 Open Circuit Potential/Polarization Resistance

The OCP variations for the biotic and abiotic systems areshown in Fig. 8. The Ecorr as a function of time data revealedthat in the biotic system, a substantial shift of Ecorr towardsnoble values (-600 mV/SCE) occurred for the first 100 h andthen remained more or less stable throughout the period ofexposure. This potential shift was attributed to the growth ofthe SRB species, their metabolic activities, and subsequentaccumulation of iron sulfide on the surface. SRB attached to the

coupon surface, colonized and reproduced to form a biofilm,and the activity of microbes in this biofilm subsequently alteredthe electrochemical processes taking place at the steel surface.These alterations include pH changes, H2S production, ironsulfide formation and even EPS production. These factorscollectively enhance the reduction capacity of the system andaccelerate anodic dissolution (Ref 30, 31, 35, 36).

In stark contrast, the abiotic system had a notable increase ofthe Ecorr, which then remained more or less steady atapproximately !690 mV/SCE. This potential shift might beattributed to the accumulation of the growth medium constit-uents such as organic compounds, potassium, sodium chloride,and phosphorous on the coupon surface (Ref 31). There is adifference in noble direction of approximately 90 mV/SCEbetween the biotic and abiotic systems. This positive shift inEcorr is known as ennoblement. Ennoblement has been reportedfor different alloys exposed to microbes. It is probably the most

Fig. 7 FESEM analysis for the API X52 coupon surface after cleaning for the system under abiotic conditions exposure at 1009 and 5009

0 50 100 150 200 250 300 350-750

-700

-650

-600

OC

P (

mV

) /S

CE

Time (hrs)

Biotic System Abiotic System

Fig. 8 OCP variations under biotic and abiotic conditions


notable phenomenon in many MIC investigations (Ref 1-3).The ennoblement has been attributed to microbial colonization,biofilm formation and the deposition of sulfide, which collec-tively result in organometallic catalysis and acidification of theelectrode surface (Ref 1). The accumulation of corrosionproducts such as iron sulfide will result in galvanic couplingwith the steel surface and may contribute to this ennoblementphenomenon. Ennoblement promotes pitting corrosion, whichis more critical for passive alloys (Ref 1-3).

The polarization resistance variations for biotic and abioticsystems are shown in Fig. 9(a). The Rp as a function of timedata revealed that in the biotic system, a substantial decrease ofRp to 500 X cm2 was followed by another decrease toapproximately 250 X cm2 at 350 h. There was a noticeableincrease in the Rp to '3000 X cm2 at 100 h. This increase isattributed to the formation of a film of siderite (FeCO3) andmackinawite that provide short-term protection of the steelsurface. It has been reported that the mackinawite layer partiallyprotects the steel from corrosion (Ref 7). The substantial dropin the Rp is attributed to the formation of a stable biofilm andconductive iron sulfide mixed layers on the surface. SRBmetabolic activities produce hydrogen sulfide and form organiccompounds such as an EPS with acids at the metal/biofilminterface (Ref 1, 3, 30). These factors, along with galvaniccoupling between the iron sulfide and underlying surface, createan aggressive environment that leads to this substantialdecrease of polarization resistance.

In contrast, in the abiotic system, Rp decreased to about1000 X cm2 at the first 50 h followed by an increase to about1200 X cm2, which then remained more or less steadythroughout the experiment. The initial drop in the Rp was dueto the corrosion effects of the deposited nutrients (i.e., sulfideand sodium chloride) on the surface. However, when stabledeposits and a corrosion film were formed on the surface, itprovided protection as indicated by a steady resistanceafterwards (Ref 40).

The polarization resistance is inversely proportional to thecorrosion rate, implying a higher corrosion rate at lowerresistance. The corrosion rate plots over time for the biotic andabiotic systems are shown in Fig. 9(b).

The corrosion rate for the biotic system increased signifi-cantly after 100 h, which is in agreement with the shift of OCP.The corrosion rate reached a maximum value of 45 mpy after250 h, whereas the corrosion rate for the abiotic system for thesame interval is approximately 15 mpy. It should be noted thatthe utilization of the LPR method in this study should notreplace or underestimate the weight loss (WL) method indetermining the corrosion rate. The WL is absolutely morereliable as it provides direct proof of corrosion rather thanindirect electrochemical measurements (e.g., LPR).

Several general statements could be made to explain thehigh corrosion rates observed under the biotic conditions. Themicroenvironment at the metal-liquid layer could have beenaltered by biofilms via attachment of bacteria and deposition oforganic by-products such as: EPS, hydrogen sulfide, and ironsulfide (Reaction [3] and [8] and Fig. 4). These changesenhance the kinetics of the corrosion processes at the metalsurface, which was reflected in an increase in Ecorr accompa-nied by a decrease in Rp. Moreover, the accumulation of ironsulfide (Reaction [5] and [8]) on the carbon steel coupons formsa galvanic cell with the adjacent steel surface, resulting infurther enhancement of the corrosion process (Ref 1, 3). Theobserved rectangular pits and preferred pearlite microstructuresattacks (Fig. 6) are due to these galvanic couplings. However, itwas unclear what portion of an increase of corrosion resultedfrom the changes induced by the bacterial metabolic activitiesand what portion was due to other redox couples promoted bythe iron sulfide galvanic coupling.

3.5 Electrical Impedance Spectroscopy Results

Figure 10(a) displays the Nyquist plots for a carbon steelcoupon exposed to the abiotic system. At low frequencies (LF)(Fig. 10a), the magnitude of the capacitive loop represented bythe semicircle diameter increased with time. These LF magni-tudes represent the change in the charge transfer resistance (Rct)that describes the evolution of the anodic reaction controlled bycharge transfer processes (Ref 30, 31, 35, 41). The resistanceincreased to values around 4000 X cm2. The diagram obtainedat 192 and 288 h exhibited a bigger loop diameter, which

0 50 100 150 200 250 300 3500

500

1000

1500

2000

2500

3000 Biotic System Abiotic System

Rp (

%.c

m2 )

Time (hrs)

0 50 100 150 200 250 300 3500

10

20

30

40

50

60

70 Biotic System Abiotic System

Cor

rosi

on R

ate

(MP

Y)

Time (hrs)(b)(a)

Fig. 9 (a) Polarization resistance (Rp) and (b) corrosion rate variations under biotic and abiotic conditions


indicates an increase in charge transfer resistance. Under theseconditions, it was found that the corrosion process occurred inthe first 50 h, and a steady corrosion rate was observed as theexposure time increased (Fig. 9b). In the abiotic system, theanodic reaction is represented by reaction (5), and the cathodicreaction is shown by reaction (2). The steady corrosion rate ispossibly due to the protective effect of a mixed layer of depositsand corrosion products that are possibly composed of sodiumchloride, sulfide, potassium, and carbon-based compounds onthe electrode surface (Ref 40). The formation of a capacitivelayer on the steel surface is confirmed by phase angle spectra(Fig. 10b) that displayed one time constant or one peak at 10Hz. The equivalent electrical circuit based on the minimumdeviation between the measured and fitted data for the abioticcondition is shown in Fig. 11. The circuit includes:

• A resistance Rs considered as the solution resistance.• Parallel connection of a charge transfer resistance (Rct) for

the steel surface and constant phase element (CPE) associ-ated with a double layer capacitance due to the formationof a heterogeneous layer composed of corrosion productsalong with other compounds deposited from the growthmedia.

In general, a CPE is used instead of capacitor to compensate forthe deviation from ideal behavior. The impedance of CPE isdefined by the following equation (Ref 29, 41-43):

ZCPE " #CPE%!1#jx%!a #Eq 10%

When the carbon steel was exposed to the biotic system, theEIS spectra varied significantly with exposure time as shown inFig. 12(a). The LF magnitude, represented by the semicircle

diameter, significantly decreased with time, indicating adecrease in charge transfer resistance (Rct) and a subsequentincrease in the corrosion rate as supported by Fig. 9(b). The SRBconsortium impacts the corrosion rate by at least three mech-anisms, via biofilm formation, reduction of sulfates andproduction of hydrogen sulfide and subsequent formation ofiron sulfide (Ref 30, 31, 35). For the first 48 h, the mediumfrequency (MF) response presented in the phase diagram inFig. 12(c) shows one time constant that indicates an activationcontrol process, which is represented by the circuit diagram forthe abiotic system shown in Fig. 11. This behavior is attributedto the formation of an unstable conditioning layer based on amixture of inorganic / organic compounds; it is essentially thetime of biofilm formation (Ref 30, 31, 35). However, afterbiofilm formation in conjunction with the development of ironsulfide and siderite film and when a steady state is reached at 96h, another time constant shows up in the phase angle spectra inFig. 12(c). Figure 13(a) shows the circuit model used to fit theEIS data at 96 and 192 h, where the compact biofilm (bf) formedconstitutes an additional parallel combination of resistance andcapacitance. At 288 h, the compact biofilm layer and iron sulfidelayers start developing pores and the corresponding EIS data fitthe circuit model shown in Fig. 13(b). The enhancement of thedissolution kinetics of the metallic surface is evidenced by thedecrease of the magnitude of charge transfer resistance (Rct) withtime as shown in Fig. 12(a) and 13(b). EIS spectra suggest thatthe formation of an adherent biofilm along with a mixed layer ofiron sulfide and siderite established electrochemical cells on thesteel surface and subsequently enhanced the corrosion process.EIS results draw general statements about how the corrosionproceeds in the biotic system:

• Before 96 h, the MF response showed one time constant(Fig. 12c) that was attributed to the formation of an unsta-ble layer of a mixture of corrosion products, mainly ferrichydroxide and organic compounds. At this stage, the SRBbacteria attached to the surface, assimilated lactate andreduced sulfate to sulfide ions. Biogenic hydrogen sulfideand a subsequent mixed layer of iron sulfide and sideritealong with EPS are formed by the precipitation of ferrousions with sulfide and carbonate ions.

0 1500 3000 4500 60000

2000

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6000

-Z"

(%.c

m2 )

Z' (%.cm2)

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10-2 10-1 100 101 102 103 104 105

0

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-40

-60

-80

Pha

se A

ngle

(de

gree

)

Frequency (Hz)

0 h

96 h

192 h

288 h

(b)(a)

Fig. 10 EIS date abiotic system; (a) Nyquist plots and (b) phase angle plots

Fig. 11 Circuits model used to fit the EIS data for the abiotic sys-tem


• At 96 h, the mixed layers of EPS and semiconductive cor-rosion products were stable as evidenced by the phaseangle spectra that reveal two time constants (Fig. 12c). Atthis stage, the microenvironment changes induced by thebacterial metabolic activities in the biofilm and the gal-vanic coupling between the iron sulfide with the underly-ing surface increased the corrosion rate significantly, asshown in Fig. 9(b). The galvanic coupling attacks were

more pronounced with pearlite microstructures, as repre-sented in Fig. 6, due to their structure and compositionalheterogeneity that induced electrochemical potential gradi-ents.

• At the final stage, 288 h, with the proliferation of theSRB, production of excess hydrogen sulfide and accumu-lation of excess corrosion products, the biofilm and ironsulfide film decomposed, cracked and became loose andporous due to the production of polysulfide products andthe induced intrinsic physical growth stresses (Ref 7, 32,35, 44). Subsequently, the steel surface was exposed tothe aggressive medium again, which accelerated the corro-sion rate significantly ('45 mpy), as shown in Fig. 9(b).

4. Conclusions

In this study, MIC of API 5L X52 carbon steel coupons wasinvestigated by exposure of the coupons to a SRB consortiumcultivated from produced water from a production sour oil well.16S rRNA gene sequence analysis indicated that the mixed

0 1000 2000 30000

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-Z"

(%*c

m2 )

Z' (%*cm2)

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192 h

288 h

0 50 100 150 2000

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100

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288 hrs 366 hrs

-Z''

(%*c

m2 )

Z'(%*cm2)(a) (b)

10-2 10-1 100 101 102 103 104 105

0

-20

-40

-60

-80

Pha

se A

ngle

(de

gree

)

Frequency (Hz)

0 h 96 h 192 h 288 h

(c)

Fig. 12 EIS date for biotic system; (a), (b) Nyquist plots, and (c) phase angle plots

Fig. 13 Circuit models used to fit the EIS data for the biotic sys-tem (a) at 96 and 192 h (b) at 288 and 366 h


bacterial culture consortium contained three phylotypes that areclose to members of the Proteobacteria (Desulfomicrobiumsp.), Firmicutes (Clostridium sp.) and Bacteroidetes (Anaer-ophaga sp.); all described previously as species associated withMIC. In the presence of a SRB-biofilm, substantial levels ofsulfide were detected. XRD revealed the presence of differentphases, siderite (FeCO3), iron sulfide (FexSy), and iron oxide(FeOOH) constituents in the corrosion products for the systemexposed to the SRB consortium. The microenvironment at themetal-liquid layer became highly altered by the bacterialbiofilms via attachment of bacteria, deposition of organic by-products, (e.g., EPS and the production of hydrogen sulfide)and subsequent development of semiconductive iron sulfide.The metabolic activities resulted in accumulation of asubstantial amount of iron sulfide that promoted galvaniccoupling with the underlying surface and resulted in generalcorrosion. Thereafter, the corrosion rate increased dramatically.The corrosion of the steel coupons was significantly moresevere in the biotic conditions compared to the abiotic control.The corrosion rate was ' 45 mpy in the biotic system, while itwas '15 mpy for the abiotic system. The nature of thecorrosion was generalized with preferential etching of selectpearlite microstructures in a regular, rectangular, and repeatingmanner. The galvanic coupling attacks were more marked withpearlite microstructures due to high electrochemical potentialgradients. Moreover, the biofilm, as well as iron sulfide alteredthe kinetic behavior of the system by inducing an extra timeconstant in the circuit model.

Acknowledgments

The authors acknowledge and appreciate the Saudi Aramco andInspection Department Management for their continual support forthis project.

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microbial corrosion in linepipe steel under the influence of a

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