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The effect of Acetobacter sp. and a sulfate-reducing bacterial consortiumfrom ethanol fuel environments on fatigue crack propagation in pipelineand storage tank steels q

J.W. Sowards a,⇑, C.H.D. Williamson b, T.S. Weeks a, J.D. McColskey a, J.R. Spear b

a National Institute of Standards and Technology, Applied Chemicals and Materials Division, Boulder, CO 80305, USAb Dept. of Civil and Environmental Engineering, Colorado School of Mines, Golden, CO 80401, USA

a r t i c l e i n f o

Article history:Received 24 January 2013Accepted 31 October 2013Available online 9 November 2013

Keywords:C. Corrosion fatigueC. Hydrogen embrittlementC. Intergranular corrosionC. Microbiological corrosionB. SEMA. Steel

a b s t r a c t

This paper evaluates the effects of microbiologically influenced corrosion (MIC) on fatigue-crack growthof candidate materials useful in expanding bio-ethanol usage, including a storage-tank steel (ASTM A36)and two pipeline steels (API 5L X52 and X70). The microbiological species sampled and cultivated from anethanol fuel production stream are responsible for both acetic acid and hydrogen sulfide production thatlead to significant increases in fatigue-crack growth rate across a wide range of stress-intensity-factoramplitudes (DK). The mechanism for increased fatigue damage is hydrogen uptake through adsorptioninto the steel, which embrittles material ahead of the growing fatigue crack.

Published by Elsevier Ltd.

1. Introduction

Understanding the corrosive effects of alternative fuel environ-ments on fuel storage and transport infrastructure is important forpredicting mechanical integrity of various components of the fueldelivery infrastructure. At a minimum, these components includepipelines, valves, storage tanks, ships, trucks and railcars. Theexisting infrastructure in the U.S., and throughout many othercountries, was designed to handle fossil fuels but could be repur-posed to handle emerging alternative fuels. A major concern is thatthe corrosive effects of alternative fuels are not well known forexisting transport and storage scenarios.

Fuel-grade ethanol (FGE) is one particular alternative that isseeing significant increases in consumption rates throughoutmuch of the world where biomass that is conducive for ethanolfuel production is available. FGE is a desirable alternative to fossilfuels since it can be blended with gasoline fuels already in use, aswell as serve as an oxygenator to reduce particulate emissionsduring the combustion process [1]. However, the sources ofbiomass for FGE are typically located in rural areas where the fuelis produced. The fuel then needs to be transported long distances

into existing infrastructure by railroad, truck, and barge tankersto primarily coastal regions where consumption rates are veryhigh [2]. Pipelines offer significant safety advantages and im-proved cost-effectiveness compared to other shipping methodsif the corrosive effects are predictable, especially with respect totheir mechanical integrity. However, degradation of the mechan-ical integrity of ethanol piping and steel tanks is established dueto the well-known phenomenon of ethanol stress corrosion crack-ing [3–6].

Microbiologically influenced corrosion (MIC) can be asignificant problem in the material systems that handle, store,transport, and distribute fuels [7–15]. MIC of oil and gaspipelines has been reported in multiple instances [14,16–26].The onset of MIC in pipeline systems is a result of many causes,from inadequate water handling practices during hydrostatictesting of pipelines to lack of water chemistry control, lack ofbiocide usage and oxygen scavenging, all of which can lead tothe premature degradation of alloys [27]. Aside from pipelines,fuel storage tanks are also susceptible to MIC, which can takethe form of general corrosion and pitting, when exposed to avariety of fuel systems [11–13,18,20]. Biocides are frequently ap-plied to storage tank systems as part of an anti-microbial strat-egy, but both the presence of microbial species and microbialbiocide resistance may vary with the fuel source. Sulfate-reducing bacteria (SRB) and acid-producing bacteria (APB) inparticular have both been known to contribute to the MIC ofengineered alloys including steels [7].

0010-938X/$ - see front matter Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.corsci.2013.10.036

q Contribution of NIST, an agency of the US government. Not subject to copyright.⇑ Corresponding author. Address: 325 Broadway, Boulder, CO 80305, USA. Tel.: +1

303 497 7960; fax: +1 303 497 5030.E-mail address: [email protected] (J.W. Sowards).

Corrosion Science 79 (2014) 128–138

Contents lists available at ScienceDirect

Corrosion Science

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SRB species have been the focus of many corrosion studies andare known to cause corrosion in pipeline systems associated withfossil fuels and be particularly problematic due to the productionof corrosive hydrogen sulfide [16,19,23,26,28,29]. The presence ofmolecular hydrogen (H2), and SRB-produced hydrogen sulfides en-hance anodic dissolution, and have an embrittling effect on steels.The hydrogen sulfides in particular slow the recombination ofmolecular hydrogen so that its uptake into the steel has more timeto proceed. In contrast, APB induce corrosion by metabolism of anorganic substance, such as a carbon source and H2 as an electrondonor resulting in secretion of corrosive organic acids [7]. Studieshave shown that APB influence corrosion of pipeline steels [19]and that microbial production of acetic acid enhances corrosion[30]. Pope et al. show that the observed corrosion damage byAPB was distinct from corrosion produced by protic acid of thesame pH, including that produced by SRB [30]. Though SRB andAPB have been implicated in corrosion of steels, often, differenttypes of microbes with various metabolic capabilities inhabit steelsurfaces as biofilms and impact corrosion processes. Microbial bio-films, which are communities of microbes immobilized by anorganically produced extracellular polymeric substance (EPS), caninfluence corrosion processes because they produce environmentsat the metal/biofilm interface that can have low values of pH anddissolved oxygen, and since they produce electrochemical reac-tions that significantly differ from those in the bulk solution [7].

Mechanical degradation in biologically active environments hasbeen considered in several studies [26,31–38]. Pipelines and evenfuel storage tanks (e.g., in railcars, barges, and tanker ships) under-go cyclic loading due to e.g., fluctuations of internal pressure andwave loading, respectively. The combination of pitting and cyclicloading can readily have the undesired effect of fatigue crack initi-ation [39]. Environmentally assisted fatigue-crack propagation andstress-corrosion cracking (SCC) become a concern since both leadto eventual mechanical degradation, reduced component life, orworse, failure and the associated environmental consequences. Inregard to SRB in particular, fatigue-crack propagation rates inhigh-strength steels have increased by a factor of 50–1000 whenfreely corroding or when placed under applied cathodic potential[31,33,38]. These studies, which have focused on several offshoreenvironments, have shown that the extensive damage accumu-lated in test materials was related to sulfide-enhanced hydrogenuptake. Mechanical testing in inoculated media can be problem-atic, since transient crack propagation behavior has been causedby increases in metabolically produced species, which can compro-mise structural life prediction with the fatigue data produced inthe test [31].

Given that cyclic mechanical loading occurs in fuel transportsystems and that microbial species sampled from a FGE productionstream (including Desulfosporosinus sp. and Acetobacter sp.) havereportedly influenced corrosion of structural and pipeline steels[40,41], further investigation is needed on the effect of microbialcontamination of ethanol fuel environments on fatigue crack prop-agation. Specifically, the effect of the SRB Desulfosporosinus sp. [42]and the APB Acetobacter sp. [43] on corrosion has been demon-strated in other systems and warrants further study. There is nodescription available of the effects that SRB and APB have on thecracking of steels used for ethanol pipelines and storage tank alloys

under fatigue loading. This study evaluates the effect of these twotypes of microbes on the fatigue crack propagation in three steelsthat would be encountered in the storage and transport of alterna-tive fuels such as fuel-grade ethanol. Our results may have morebroad implications because similar species are found throughoutthe oil and gas industry.

2. Experimental procedures

2.1. Materials

Two grades of pipeline steel (API 5L X52 and X70) and a platesteel (ASTM A36) used for storage tank fabrication were obtainedfor this study from an oil & gas transmission company and a steeltank fabricator, respectively. The X52 pipe was 324 mm diameterwith 9.53 mm wall thickness, and the X70 pipe was 508 mm diam-eter with 6.60 mm wall thickness. The A36 plate was 6.35 mm inthickness. Chemical compositions are shown in Table 1. Represen-tative microstructures of the two pipeline materials are shown inFig. 1. Metallographic specimens were sectioned from the pipelinematerials and prepared with standard polishing methods for opti-cal microscopy (1 lm final polish and 2% nital etch). The A36 andX52 steel alloys have similar microstructure and contain a mixtureof polygonal ferrite and pearlite. The X70 material contains a finer-grained polygonal ferrite and bainitic structure. Note that the finergrain size in the X70 material, relative to A36 and X52, is a result ofthe higher microalloying content (Ti and Nb), which aids in grainrefinement during thermo-mechanical controlled processing.Tensile tests of the three steels were performed in air accordingto ASTM E8/E8M Standard Test Methods for Tension Testing ofMetallic Materials [44] by use of a 250 kN servohydraulic testframe. The average mechanical properties from three tests, andthe corresponding standard deviations of the measured propertiesare shown in Table 2.

2.2. Fatigue crack propagation measurements

Baseline tests performed in air and simulated fuel-gradeethanol with the test procedure summarized here are reported ingreater detail elsewhere [45]. Compact tension C(T) specimens(Fig. 2) were machined from the three steels (in the LT orientation)in accordance with ASTM E647 Standard Test Method for Measure-ment of Fatigue Crack Growth Rates [46]. The pipeline steel speci-mens were machined from curved pipe sections that were notflattened prior to specimen sectioning. The X70 pipe wall section(6.60 mm) limited the ‘‘B’’ dimension (specimen thickness) to5.715 mm due to pipe curvature. All specimens were made to thisthickness so that the stress state of the crack would be consistentamong the tests. Fatigue precracking and crack propagation mea-surements were performed with the compliance technique. Crackmouth opening displacements (CMOD) were used for compliancemeasurements. Displacement was measured in the load line witha coated 6.35 mm gauge length crack-opening displacement gaugeattached to integral knife edges machined at the crack mouth. AnRTV coating was applied to prevent degradation of the clip gaugein the corrosive test environments. Calibration of the gauge wasperformed after application of the coating and verified between

Table 1Chemical compositions (wt.%) of steel alloys.

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

A36 0.22 0.04 0.08 0.08 0.9 0.23 0.02 <0.01 <0.01 0.03 <0.01 0.01 0.01X52 0.070 0.195 0.030 0.020 1.050 0.050 0.004 0.021 0.001 0.029 0.003 0.008 0.008X70 0.050 0.185 0.043 0.017 1.505 0.030 0.01 0.084 0.015 0.032 0.01 0.006 0.012

J.W. Sowards et al. / Corrosion Science 79 (2014) 128–138 129


tests. Precracking was performed in air at 20 Hz, and loads wereincrementally shed so that initial loads during crack propagationstudies were greater than final loads during fatigue precracking.Tests were performed in a 100 kN closed loop servohydraulic testframe with a sinusoidal waveform and constant force (P) ratioR = Pmin/Pmax = 0.1. Environmental testing was performed with aloading frequency (f) of 0.1 Hz. Liquid pipelines likely experiencehigher R and lower loading frequency than those reported here;however, we selected a particular mechanical condition to evaluatethe effect of bacteria under accelerated conditions. Stress-intensityamplitude was controlled by software and continually increasedduring testing at a rate, C = 0.15 mm�1, which is the normalizedK-gradient.

2.3. Biological culturing and fatigue testing

Microbiological species including Acetobacter sp. and asulfate-reducing consortium that included Desulfosporosinus sp.and Clostridia sp. were cultivated from industrial ethanol contain-ment-tank samples [47]. These tanks contained fuel-grade ethanoland water. Acetobacter sp. were maintained in a medium contain-ing yeast extract (0.5 g L�1), peptone (0.3 g L�1), and sodium chlo-ride (1 g L�1) in distilled water (adapted from Lisdiyanti et al. [48]).

Ethanol (5% by volume) was added as a carbon source. Acetobactersp. are aerobic bacteria that metabolically oxidize ethanol into ace-tic acid. The sulfate-reducing consortium was maintained in amodified Postgate B medium that included potassium dihydrogenphosphate (0.5 g L�1), ammonium chloride (1 g L�1), calcium sul-fate (1 g L�1), magnesium sulfate 7-hydrate (2 g L�1), yeast extract(1 g L�1), ascorbic acid (0.1 g L�1), thioglycollic acid (0.1 g L�1), andiron sulfate 7-hydrate (0.5 g L�1) [49]. Ethanol (2% by volume) wasadded as a carbon source after 0.2 lm filtering. The SRB consor-tium was maintained under a nitrogen headspace. This consortiumincluded Desulfosporosinus sp. and Clostridia sp. Desulfosporosinussp. are anaerobic spore-forming bacteria capable of reducingsulfate to sulfide in an eight electron energy transfer, and these mi-crobes have been associated with fuel-contaminated environmentsas well as iron transformations [42,50]. Clostridia sp. are anaerobic,spore-forming bacteria capable of various metabolisms [51]. It isexpected that APB and SRB could be found together as a consor-tium in a biofilm on a steel surface since aerobic species (APB)could thrive in an oxic environment and consume oxygen, therebyproviding an anoxic environment suitable for anaerobic SRB.

In this study, the impact of APB and SRB metabolisms on crackgrowth were examined. Cultures were inoculated into test solu-tions in a controlled laboratory environment. Test solutions were

Fig. 1. Optical micrographs showing representative microstructures of (a) X52 and (b) X70 pipeline steels.

Table 2Mechanical properties of steel alloys.

Alloy 0.2% Offset yield strength (MPa) Ultimate tensile strength (MPa) Total elongation (%)

A36 292 ± 3.1 451 ± 0.5 37.8 ± 1.8X52 397 ± 4.2 491 ± 1.0 21.3 ± 0.2X70 561 ± 14.5 655 ± 9.1 17.2 ± 0.8

Ø12.70

30.48

30.48

5.715

13.97

13.971.52

1.52

10.16

63.5050.80

Chevron Notch

Clip GaugeOpeningAnd Notch

Fig. 2. Schematic drawing showing compact tension specimen configuration and nominal dimensions (mm).

130 J.W. Sowards et al. / Corrosion Science 79 (2014) 128–138


poured into a 6 L test vessel, and a mechanical reaction frame con-taining the C(T) specimen was lowered into the test environments.Care was taken when preparing the testing environment, becausepast work has demonstrated that metabolic transients can affectthe applicability of fatigue test results based on the concept ofsimilitude [31]. To avoid such transients in this study, microbialcultures were maintained in the stationary phase, i.e., the growthrate and death rate of bacterial cultures are equal. Cells werecounted with a Petroff-Hausser cell counting chamber and aphase-contrast microscope before and after fatigue-crack propaga-tion testing. Cell densities were maintained on the order of 107

cells per mL of media for inoculated tests. Solution acidity wasmeasured with a pH probe at the beginning and end of fatigue test-ing. Relevant conditions of the test solution, including pH and cellcounts, are reported in Table 3.

Fig. 3 shows a schematic of the mechanical reaction frame thatwas implemented to completely submerge C(T) specimens intotest environments. The reaction frame, which was made of high-strength stainless steel, was electrically isolated from the C(T)specimen with Teflon washers and ceramic loading pins to preventgalvanic interactions. The test chamber and seals were made ofpolymeric materials with good chemical resistance to ethanol.Air-tight seals were maintained throughout testing to maintainrelatively constant test conditions, namely to prevent solutionevaporation or contamination. Precracked C(T) specimens weresubmerged in the test environment under free-corrosion condi-tions. The precrack was grown for a period of �24 h before com-mencing data collection. APB test solutions were exposed to airin the headspace above the test solutions, but were not mechani-cally aerated. SRB test solutions were covered with approximately25 mm of vegetable oil and purged with high purity N2 gas tomaintain anaerobic conditions. Anaerobic conditions were con-firmed in the test chamber by the redox indicator resazurin. Thiscompound is an oxygen-reduction indicator that turns solutionsto a reddish hue if proper deaeration is not maintained. Fatiguetesting in the APB and SRB environments last �5 days perexperiment.

2.4. Characterization procedures

C(T) specimens were sonicated in 200 proof ethanol immedi-ately following fatigue propagation testing. Fracture surfaces wereliberated in liquid nitrogen after cleaning and then stored in a des-iccated container. A scanning electron microscope (SEM) coupledwith energy dispersive X-ray spectroscopy (EDS) was used toevaluate fracture surfaces and remnants of biological activity(e.g., mineral deposits). An accelerating voltage of 15 kV and alight-element detector window were used for chemical analyses.EDS spectra were not quantitative due to the unavailability ofappropriate spectrographic material standards used for chemicalanalysis of corrosion product and biological materials. Fracture

specimens were not coated (e.g., with carbon or gold sputter coat)prior to examination.

3. Results and discussion

This study builds on a previous study that reported effects ofsimulated fuel-grade ethanol (SFGE) on the fatigue crack propaga-tion in A36, X52, and X70 tank and pipeline steels [45]. That studyshowed that each of these steels may experience degradation dueto superimposed mechanical fatigue and ethanol stress-corrosioncracking by fatigue loading during transport of FGE. Jain [41] foundthrough electrochemical studies that MIC produced acceleratedcorrosion rates and localized corrosion on A36, X52, and X70 steelsin FGE environments using the same culture strains and materialheats studied here. That study also suggested that MIC may occursimultaneous to fatigue and stress-corrosion cracking and thesefindings formed the basis of the research questions addressedherein.

Due to the hygroscopic nature of ethanol, water-rich phasesthat could harbor microbiological life are generally not expected.However, upset conditions such as those experienced duringhydrostatic testing are conducive to microbiological growth [27].In addition, the presence of any significant or remnant water layer(i.e., the Helmholtz Layer) on a steel surface can provide an essen-tial need for microbial/biofilm life. The introduction of water (arti-ficially and/or otherwise) into a pipeline or tank that contain

Table 3Test solution cell counts and acidity at the beginning and end of fatigue tests.

Test number Initial cell count (#/mL) Final cell count (#/mL) Initial solution acidity (pH) Final solution acidity (pH)

X70–5 (Control Test Solution) 0 9 � 106 6.60 4.82X70–6 (APB + Test Solution) 2 � 107 3 � 107 3.60 3.58X70–7 (SRB + Test Solution) 8 � 107 6 � 107 6.5–7.0a 6.5–7.0a

X52-6 (Control Test Solution + 1 g L�1 glutaraldehyde) 0 – 6.0–6.5a 6.0–6.5a

X52-5 (APB + Test Solution) 2 � 107 3 � 107 3.35 3.36X52-7 (SRB + Test Solution) 7 � 107 6 � 107 6.5–7.0a 6.5–7.0a

A36-5 (APB + Test Solution) 6 � 107 5 � 107 3.15 3.12A36-4 (SRB + Test Solution) 4 � 107 5 � 107 6.5–7.0a 6.5–7.0a

APB Test Solution: 5% ethanol (vol), 1 g L�1 NaCl, 0.5 g L�1 yeast extract, 0.3 g L�1 peptone, balance H2O.SRB Test Solution: 2% ethanol (vol), balance Postgate B medium, Purged with N2.

a Values are approximate and were measured with pH paper. Other values were measured with pH probe.

CLIPGAUGE

FUEL LEVEL

MECHANICALACTUATOR

CONTAINMENTTANK

C(T)SPECIMEN

FUEL/GROWTHMEDIUM

Fig. 3. Fatigue crack propagation testing apparatus used for ethanol fuelenvironments.



remnants of FGE establishes an ideal situation for MIC since waterand a carbon source (ethanol) are necessary. Electron donors andacceptors are also needed for metabolic processes and can be pro-vided by trace contaminants and H2 found in the host steel and inthe fuel and water in the pipelines. Therefore, if biofilms are estab-lished during the hydrostatic testing, there is a possibility that theyremain intact after fuel transport recommences. The biofilms mayharbor localized environments significantly different from those inthe pipeline and result in surface pitting [7], for example. Giventhat pits are likely sites for fatigue crack initiation [39], and thatmicrobiological species living within biofilms can producechemical species that degrade the mechanical integrity of steel,the following results are applicable for describing upset conditionsin the presence of APB (e.g., Acetobacter sp.) and SRB (e.g., Desulfosp-orosinus sp.) in ethanol environments where water contents areinitially high enough to promote and sustain microbiological life.However, the chemical species produced by these bacteria arenot specific to ethanol, virtually any hydrocarbon makes for anideal carbon source for a diversity of microbiota, and thus these re-sults may have broader applicability to other biofuels and the oiland gas industry.

3.1. Crack growth in APB environment

A drastic increase in fatigue-crack propagation rates is inducedby APB (shown in Fig. 4) for all three of the steel alloys tested herewhen compared to air and simulated FGE environments [45]. Notethat at low levels of DK (the stress-intensity-factor amplitude)there is a strong dependence of crack growth rate on DK. At a DKlevel of 20 MPa m1/2 there is an approximate 25-fold increase inthe rate of crack propagation, relative to air, in the two pipelinesteels. Crack growth data of all three materials tested in the APBsolution essentially overlay one another below DK values ofapproximately 25 MPa m1/2. A significant transition in the slopeof da/dN vs. DK occurs near this level, indicating a wide variationin Paris Law exponents and DK-independent crack growth rates.The presence of this plateau region is explained as being relatedto mass transport limitations of chemical species at the crack tip[38]. The plateau limits crack growth at intermediate levels ofDK to approximately 2 � 10�6 m/cycle in the A36, and

1.5 � 10�6 m/cycle in the two pipeline steels. Note that the pHwas lowest in the A36 APB test (due to APB-produced organicacids), which may contribute to the higher observed crack growthrates in the plateau region, although the increase in crack growth,relative to air, is still lowest in A36. As DK values increase beyondthe plateau, the da/dN values resume the increasing trend in eachmaterial, where they converge towards the crack growth rate datameasured in air. This behavior can be explained as the purelymechanical damage component begins to dominate at the higherDK levels.

Acidity values and results of bacterial cell counts of the APB testsolutions are included in Table 3, which represent test solutionconditions at the start and end of each fatigue test. Acetobactersp. oxidized ethanol into acetic acid and lowered the pH of the testsolutions from an initial ‘‘control’’ solution pH of approximately 6.6to pH levels of 3.1 to 3.6. Note that pH changed very little duringthe course of the tests in APB solutions and that bacterial cellcounts remained on the order of 107 cells per mL. Thus, significanttransients in crack growth behavior were not expected due to therelative constancy of the cell counts, and their subsequent meta-bolic reactions.

Crack-growth rates were measured in X70 in an ethanol testsolution ‘‘control’’ that was not inoculated with microbiologicalspecies as shown in Fig. 4(c). Differentiation between the effectof the high water content in the ethanol solutions, which promotesrapid microbiological growth, and the effect of the microbes them-selves is the basis for a control test. The control solution containedethanol, water, chloride, yeast extract, and peptone, just as the testsolution (essentially a minimum microbial growth medium). Aprevious report indicated that increases in the water content ofethanol increases fatigue crack growth rates in steel [52]. Whileit is known that aqueous chloride solutions can influence crackgrowth, an effect of yeast extract and peptone is not expected.However, contamination of the X70 control (test X70-5 in Table 3)became evident as microbes, including APB, are ubiquitousthroughout soils and any number of environments in nature. Thisnatural abundance made it difficult to run a true control solution.Cell counting in this control test showed a significant concentra-tion of putative APB cells in the solution (�106 cells per mL) albeitat concentration levels significantly lower than that of the inocu-

da/d

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15 20 30 40 50 607080 100 15 20 30 40 50 607080 100 15 20 30 40 50 6070 80 100

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AIR AIR AIR

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CONTROL +Glutaraldehyde

ΔK-ControlR = 0.1f = 0.1 Hz



ΔK (MPa·m1/2) ΔK (MPa·m1/2) ΔK (MPa·m1/2)

Fig. 4. Fatigue crack propagation rates of (a) A36, (b) X52, and (c) X70 in various test environments including air, simulated fuel-grade ethanol (SFGE), acid-producingbacteria (APB), and sulfate-reducing bacteria (SRB). Air and SFGE data are adapted from Ref. [45].



lated solutions maintained in the stationary phase. This concentra-tion was enough to lower pH from 6.60 (control condition) to 4.82after contamination. Still, the crack growth rates in this solutionare significantly lower than in the inoculated APB solution. Thissuggests that decreases in pH shift the plateau up and to the left,i.e., to promote the apparent effect of increasing crack growth ratesat significantly lower DK values.

A powerful protein cross-linker, glutaraldehyde is often used asan effective biocide in industrial operations in the oil and gasindustry. It was included in a control test of X52, at a concentrationof 1 g/L, to determine its effectiveness in preventing growth of theAPB; though, no APB were added to the test solution. Crack growthrates in this solution are shown in Fig. 4(b). The experiment thatcontained glutaraldehyde additions (test X52-6 in Table 3) showedthe lowest crack growth rates of all tests in the biological test med-ia. Acidity level did not change significantly during the control testcontaining the biocide glutaraldehyde even though microbes suchas APB are ubiquitous and readily contaminated the X70-5 controltest. Also, test solutions did not become cloudy as in other testsolutions. The effect of this biocide appears promising in controlof the APB species, but effects would have to be weighed uponthe pipeline and tank-stored fluids as well. It is also unclear howglutaraldehyde would affect other microbes. Note also that the pla-teau effect had significantly lower crack growth rates in this test.This test likely represents the combined effect of corrosion fatigueand possible environmental embrittlement of steel by the aqueous

ethanol solutions used to grow the bacteria. Increases in environ-mental crack growth beyond this must have been due to bacteriametabolic products.

3.2. Crack growth in SRB environment

In general, the highest increase in fatigue-crack propagationrates in the pipeline steels was induced by the SRB, as shown inFig. 4. Crack growth was accelerated by over 40-fold at the lowerstress intensities. However, the effect of SRB was not as potent inincreasing crack growth rates in the A36 steel, which may havelower hydrogen damage susceptibility due to its inherently lowerstrength compared to that of the two pipeline steels (Table 2).The SRB produced higher crack growth rates at lower stress inten-sity values than the APB environments in all three materials, whichindicates a significantly lower threshold DK level for the activedamage mechanism. The crack growth data of X52 and X70 overlayone another below 20 MPa m1/2. Note that in the A36 steel how-ever, the most distinct environmental cracking threshold is appar-ent where the SRB sharply increased crack growth at a DK value ofapproximately 15 MPa m1/2. Such a sharp increase in crack growthis not observed in any of the other tests. This distinct threshold ona plot of da/dN versus DK is normally associated with activation ofeither an SCC damage mechanism [53] or hydrogen-assisted dam-age mechanism [54]. In the present case, it would likely be a

Energy (kV)

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Low

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rmed

iate

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Fig. 5. Secondary electron SEM images of X52 fracture surface after testing in acid producing bacteria and sulfate reducing bacteria solutions showing intergranular fractureat lowDK and transgranular fracture at intermediateDK. Chemical analysis locations are indicated, including corresponding EDS spectra showing enrichment of organicelements in corrosion products.



hydrogen-damage mechanism related to biotic H2S production, asdiscussed below.

The increase of crack growth rate in SRB environments exam-ined here exhibit the plateau behavior described by Thomas et al.[38] at intermediate stress intensities much like the APB environ-ments. Crack growth rates peak in the region between DK levelsof approximately 30 MPa m1/2 to 35 MPa m1/2. Thomas et al. dem-onstrated that the plateau behavior was related to the H2S contentin high-strength microalloyed steels [38]. This plateau was shownto shift to higher crack growth rates and lower stress intensity lev-els as the H2S content increased, and eventually reached a behaviorwhere a steep threshold sharply transitioned into a linear variationof da/dN with DK as H2S became saturated. Based on that model,H2S saturation was not realized in the SRB environments here,which probably have concentrations well below 600 ppm (levelestablished in [38]), although measurements would be requiredto substantiate the actual concentrations. There is some consensusthat 50–200 ppm H2S is representative of SRB at the surface of me-tal [55], which would be well below the apparent values forsaturation.

3.3. Fracture behavior

The fracture behavior was evaluated by examining cracksurfaces with SEM. In a benign air environment these materialsexhibit the typical flat transgranular fracture where indications offatigue striations associated with each da/dN cycle are clearly evi-dent. Fig. 5 shows the fracture appearance of X52 tested in theAPB and SRB environments. The images are orientated so that thecrack growth direction is from right to left and the crack front fromtop to bottom. The plane of the crack is perpendicular to the image.Fracture-surface locations corresponding to low and intermediatelevels of DK were selected for analysis due to the differences in

crack-growth rate behavior observed in Fig. 4. Low DK values corre-spond to the behavior of rapid crack-growth rate increases withincreasing DK, i.e., less than approximately 20 MPa m1/2. Intermedi-ate DK values correspond to the plateau region.

In comparison to the fracture appearance in air, at the low lev-els of DK, the embrittling effect is quite clear in both APB and SRBenvironments. The intergranular nature of the environmental frac-ture is apparent. Qualitative estimates of the fracture in these lowDK regions suggest that approximately half of the fracture surfacearea is intergranular and half transgranular in an APB environment.Fractures appear to have more of an intergranular component inthe SRB environment as indicated qualitatively by a greater overallsurface ratio. Note that the intergranular facets have a corrodedappearance after exposure to the APB solution whereas they arerelatively undamaged after exposure in the SRB solution. This sug-gests that the anodic dissolution occurred along the fracture in theAPB solution, although it is not apparent at what point this oc-curred during the test, since the low DK portion of the crack wasexposed to test solution for several days after the crack propagatedthrough this region. Chemical analyses in the intergranular regionssuggest that Cl and S are enriched in the corrosion product on thefractures. Fractures have an entirely different appearance at theintermediate DK levels within the plateau region. The fractureappearance is exclusively transgranular. The features are againmottled on surfaces exposed to the APB environment, whereasthey are clearly serrated in the SRB environment. The fracture sur-faces of DK levels above the plateau are also transgranular in bothenvironments. In the SRB environment, the fatigue striations grewwith the level of DK, as expected by the increase in da/dN.

3.4. Biofilms associated with APB and SRB environments

Upon removal of the C(T) specimens from the testing environ-ments, biofilms were readily apparent on the specimen surfacesand were quite extensive. Corrosion product was also apparentand was associated with the biofilms. If the biofilms were not re-moved before drying out, they became firmly adhered to the spec-imen surfaces quite rapidly. Fig. 6 shows the typical appearance oftwo specimens immediately following removal from the test solu-tions. The APB biofilm had a light-colored opaque appearance, andthe steel beneath it had begun to oxidize rapidly upon removal,which is evident by the apparent rusty color under the biofilm.The SRB biofilm was black in color and appeared to be depositedin a layer much thicker than the APB film. This biofilm becamequite tenacious after drying, even to the extent that sonication inpure ethanol solution would not remove it from the specimen sur-face. The top half of the SRB test specimen shown in Fig. 6 waswiped with ethanol immediately after removal and shows exten-sive oxidation under the location of the biofilm. The tenacity ofthe SRB biofilm in pure ethanol is of concern. If such a biofilm wereto form under upset conditions where water may be present, theflow of ethanol fuel following the upset may not necessarily dis-solve the biofilm, based on the experience with sonication in pureethanol. Also given that the SRB species here are spore formers,they may go dormant until conditions are more conducive for themto begin growing again.

It is noteworthy that on the crack plane near the external sur-face of the C(T) specimen, biological debris resulting from theAPB biofilm was apparent, as shown in the SEM micrograph inFig. 7. Similar debris was apparent all along the fracture surface,as indicated in EDS analysis locations in Fig. 5. Survivability ofmicrobiological species is unclear in a crack that fully unloadsand closes, and should be a topic for prospective investigationson the relationship between MIC and growing tight fatigue cracks.Though given the micron size nature of most microbes, combinedwith possible formed pits in the steel, survivability is likely not an

Acid Producing Bacteria Sulfate Reducing Bacteria

20 mm

Fig. 6. Photographs showing biofilms on specimen surfaces following environmen-tal testing.

Fig. 7. Secondary electron SEM image showing (with arrows) biological debrisadjacent to crack mouth of X70 specimen tested in APB solution.



issue across the unload and closure regime. Mass-transport condi-tions at a submerged fatigue-crack mouth have been described innumerous studies, but the effect of flow on the ingress and egressof bacteria from the crack is not apparent.

This study did not consider the important aspect of corrosionfatigue-crack initiation. The likely fatigue-crack initiation sitescan be proposed based on evaluation of the biofilms on the exter-nal surfaces of the C(T) specimens, i.e., not the crack plane. Figs. 8and 9 characterize the regions in the immediate vicinity of thecrack on the external surface of the X52 C(T) specimens tested inAPB and SRB solutions, respectively. Pitting susceptibility of steelin simulated FGE decreases if water content becomes higher thanabout 10% [56]. Based on that finding, and the fact that APB solu-tions in this study had high water and low ethanol concentrations,the pitting behavior observed on C(T) specimens (shown in Fig. 8)can likely be attributed to the presence of acetic acid secreted bythe APB. Acetic acid produced by bacteria has been shown to attackthe matrix of pipeline steel at inclusion sites, resulting in adeep-pitting corrosion [30]. The secondary electron image inFig. 8 reveals significant corrosion pitting in the central locationof the image. The chemical analyses qualitatively show that theregions surrounding the pits have elevated carbon, oxygen, andchlorine content, whereas analysis at the center of the pits revealsmainly iron. The apparent organic debris also appears enriched inthose elements. The presence of differential aeration cells likelyresulted in some of the pitting, since the biofilms were not neces-sarily continuous on the surface. Deep pitting appeared to be

related to some of the APB biofilms. The apparently organic residueassociated with the SRB biofilm shown in Fig. 9 is enriched incarbon, oxygen, and chlorine and devoid of iron. Some sulfurenrichment is also notable, as would be expected with sulfatereducing species. The composition maps also show that Mn enrich-ment may coincide with the sulfur, indicating the presence of MnSinclusions. A pit was apparent at one location of elevated Mn and S.The organic residue along the bottom of the image tends to alignitself along the surface scratches. Areas of increased surface rough-ness, which are quite common, would be expected to be idealattachment points for the biofilms inside a pipe or tank, asreviewed elsewhere [7]. Jain has developed electrochemical-basedmodels that describe pit formation on these materials in the testedculture media [41]. That study was performed on bulk specimensand the electrochemical measurements would have less relevanceto the sharp fatigue crack tip tested here due to polarization,migration, and advection–diffusion behavior.

The possibility of both APB and SRB coexisting in a biofilmshould be considered. Both types of microbes were present inthe tanks from which our samples were acquired [47]. Also,other work has shown that an increase in metabolic productsthat lower pH, such as acetic acid, also increase the levels ofH2S and HS� by increasing Fe2+ availability for SRB [57]. Basedon that finding, we suggest that mixed cultures of APB andSRB in ethanol fuels such as those tested individually here,may in fact promote an even worse case for inducing fatiguedamage. Culturing a mixture of species was difficult in thepresent study and requires further development before fatiguetesting can be conducted.

3.5. Hydrogen damage mechanisms

The APB and SRB environments tested here resulted in a typicalcrack growth behavior that is characterized by: (1) a steep increasein crack growth rate at low DK followed by (2) a plateau region ofDK-independent crack growth rate across the intermediate valuesof DK. The behavior has been described for low-alloy steels sub-jected to fatigue loading in biotically produced H2S environments[38] and an X70 pipeline steel subjected to fatigue loading with im-pressed cathodic protection [58]. Fig. 10 shows the latter case, be-cause an X70 grade steel was also evaluated in the current work.The data set at �1.03 VSCE provides an environment where the testspecimen is cathodically charged with abiotic hydrogen. The APBdata here coincide very well with the abiotic source of hydrogen,as do the SRB data, although the crack growth rates are higher inthe latter case. Both the APB and SRB provide a source of hydrogen,which causes embrittlement of steel through hydrogen adsorption(Hads) followed by hydrogen entering into solution (Hsol), where itwill diffuse to the plastic zone ahead of the crack where stress tri-axiality is high. The slowest (rate-limiting) step in this process con-trols the rate of crack propagation. The adsorption and absorptionof hydrogen into the iron lattice are governed by Reactions (1) and(2):

H2ðadsÞ ! Hads þHads ð1Þ

Hads ! Hsol ð2Þ

The APB inoculated in the ethanol solutions in this work pro-duce acetic acid by nature. Acetic acid is a weak acid, and a poten-tial source of hydrogen to embrittle the crack tip upon adsorptionon the crack tip, dissociation, and finally absorption into the ironlattice, where it can migrate to the plastic zone. The cathodic reac-tions on pipeline steel in the presence of acetic acid have been re-viewed in Ref. [59], and are described by Reactions (3) and (4):

2Hþ þ 2e� ! H2 ð3Þ

0 1 2 3

Energy (kV)

46

0

0.1

0.2

0.3

0 1 2 3

Nor

mal

ized

C

ount

s

Energy (kV)

123

C

OFe Si S ClC

OFe Si S

Cl

C

FeCl

S

Mn

O

1

23

4

6

Fig. 8. Secondary electron SEM image showing APB biofilm residue and corrosionpits on external C(T) surface of X70 near the crack. Composition differences of pitregions are indicated by the spectra, and element maps show compositionvariations in the secondary electron image.



2HAcþ 2e� ! H2 þ 2Ac� ð4Þ

The embrittling effect of the dissociation of molecular hydrogeninto adsorbed atomic hydrogen on steel is well known and isapparently readily available based on Reactions (3) and (4). How-ever, acetic acid in the presence of hydrated ethanol can have aninhibiting effect related to the surface adsorption of the acetateion [60]. The adsorption of acetate along the advancing fatiguecrack may play a role in the hydrogen uptake process and may alsodecrease contribution of the anodic dissolution to the overall rateof crack advance. Additions of sodium can accelerate the adsorp-tion on steel and increase the hydrogen evolution reaction [61].The test solutions used here contained 1 g L�1 NaCl, which mayhave participated in advancing the hydrogen evolution to a greaterextent than merely cathodic charging (abiotic H2 charging at �1.03VSCE in Fig. 10). This is supported by the observation of extensivechloride deposition on the fracture surfaces reported in Fig. 5.The kinetics of the acetate adsorption on newly exposed metal dur-ing fatigue loading would govern the contribution of dissolution tothe true corrosion fatigue. A pH-dependent behavior in APB envi-ronments is also shown in Fig. 10. Note that the lower pH conditionstill does not compare favorably with a case of true corrosion fati-gue (X70 in 3.5% NaCl at �0.7 VSCE potential), and the apparent

threshold (sharp increase in crack growth rate) and plateau effectare still noted. The line of true corrosion fatigue runs approxi-mately parallel to air data, indicating that dissolution is a relativelyconstant contribution to enhanced crack growth irrespective of DK.Therefore, it appears that decreasing pH increases available hydro-gen for adsorption, and that crack tip embrittlement is the mecha-nism, rather than increasing the crack advance by an anodicdissolution mechanism.

Based on the relative similarity in the observed plateau behav-ior in both the APB and SRB solutions, the effect of the SRB on ano-dic dissolution and its resulting contribution to the crack growth isminimal. The obvious explanation for the increased crack growthin SRB environments relative to APB environments is the presenceof sulfur in the cathodic reaction. The SRB, when present in neutralanaerobic environments, induces the cathodic reaction accordingto Reaction (5) [7] or more appropriately for the adsorption intosteel by Reaction (6) [55]:

H2Sþ e� ! HS� þ 1=2H2 ð5Þ

H2Sþ 2e� ! HS� þHads ð6Þ

H2S has greater thermodynamic stability than HS� at pH values be-low 7 [55] which corresponds to the SRB solutions here (Table 3).

Cl Fe

O SC

Mn

1

2 3

4

Energy (kV)

3

4

0

0.1

0.2

0.3

0 1 2 30 1 2 3N

orm

aliz

ed

Cou

nts

Energy (kV)

1

2

C

O Fe Si

SCl

COFe Si

SCl

Fig. 9. Secondary electron SEM image montage showing SRB biofilm residue and corrosion pits on external C(T) surface of X52 near the crack. Composition differences of pitregions are indicated by the spectra, and element maps show composition variations in the inset region.



The corrosion reaction becomes poisoned by the presence of sulfide,i.e., the rate of hydrogen recombination according to Reaction (3) isreduced. The presence of the hydrogen sulfide on the specimens isindicated by the overall higher sulfur intensity on the surfaces nearthe biofilms tested in the SRB solution (Fig. 8) relative to the sur-faces of those specimens tested in APB solution (Fig. 9).

4. Conclusions

(1) Microbiological species, including a sulfate-reducing bacte-rial consortium (Desulfosporosinus sp. and Clostridia sp.)and Acetobacter sp., were cultivated from a FGE environmentand inoculated into test media where fatigue testing showsmarked increases in the fatigue crack propagation rates inthe storage tank and pipeline steels evaluated here.

(2) The acid producing bacteria produced acetic acid, which dis-sociates, and provides a source of hydrogen for fatigue-cracktip embrittlement. Increases in solution acidity increase therate of crack growth and decreases the apparent thresholdstress intensity for environmental cracking. The hydrogencan also serve as a strong electron donor for microbiota pres-ent to further propagate both metabolism and MIC.

(3) Glutaraldehyde may inhibit growth of the Acetobacter sp.and other microbial species. The compound prevented APBcontamination of the control test; thus, eliminating theirembrittling effect during testing of X52 pipeline steel. Fur-ther testing is required to determine the efficacy of thiscompound.

(4) Sulfate reducing bacteria evaluated here produced the high-est observed crack growth rates as a result of sulfide-enhanced hydrogen embrittlement.

(5) The environmentally assisted fracture morphologies associ-ated with the acid producing bacteria and sulfate reducingbacteria vary, depending on stress-intensity amplitude.Low values of stress-intensity amplitude result in predomi-

nantly intergranular fracture, whereas intermediate andhigh values of stress-intensity amplitude result in a trans-granular fracture mode.

Acknowledgments

The authors acknowledge the assistance of Dr. Elisabeth Mans-field, Dr. Robert Amaro, Dr. Luke Jain, Mr. Ross Rentz, and Mr. KenTalley during experimental work. Financial support was providedin part by the U.S. Department of Transportation Pipeline and Haz-ardous Materials Safety Administration through the offices of JimMerritt and Robert Smith.

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ΔΔK (MPa m1/2)15 20 30 40 50 60 70 80 10010

da/d

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cyc

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)

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Author's personal copyjspear/pdf/publications/Sowards...Author's personal copy The effect ofAcetobactersp. and a sulfate-reducing bacterial consortium from ethanol fuel environments