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06-18-2007 Journal Article1

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Diagnosing Microbiologically Influenced Corrosion: A State-of-the-ArtReview 5b. GRANT NUMBER

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PE0601153N

6. AUTHOR(S) 5d. PROJECT NUMBERB.J. Little, J.S. Lee, R.I. Ray

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73-5052-17-5

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Naval Research Laboratory REPORT NUMBER

Oceanography Division NRL/MR/7330-07-8999

Stennis Space Center, MS 39529-5004

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13. SUPPLEMENTARY NOTES

14. ABSTRACTDiagnosing microbiologically influenced corrosion (MIC) after it has occurred requires a combination of microbiological, metallurgical, and chemical analyses. MIC

investigations have typically attempted to 1 ) identify causative microorganisms in the bulk medium or associated with the corrosion products. 2) identify a pitmorphology consistent with an MIC mechanism, and 3) identify a corrosion product chemistry that is consistent with the causative organisms. The following

sections provide a discussion of available techniques, their advantages and disadvantages, and most importantly, their limitations.

15. SUBJECT TERMS

detection, methods, microbiologically influenced corrosion

16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON

a. REPORT b. ABSTRACT c. THIS PAGE ABSTRACT OF Brenda LittlePAGES

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Standard Form 298 (Rev. 8/98)Prescribed by ANSI Std. Z39.18

CORROSION SCIENCE SECTION

Diagnosing Microbiologically Influenced Corrosion:A State-of-the-Art Review

B.J. Little,* J.S. Lee,' * and R.I. Ray*

DISTI 1BUTW O STATE117NT AApproved for Public Release

Distribution Unlimited

ABSTRACT Culture TechniquesThe method most often used for detecting and

Diagnosing microbioloqtcally Iriluenced corrosion (MIC) after enumerating groups of bacteria Is the serial dilutionit has occurred requires a combination of microbiologlcal. met- to extinction method using selective culture media. Toallur•ical. and chemical analyses. MIC investigations have culture microorganisms, a small amount of liquid ortypically attempted to I) identifyj causative microorganisms inthe bulk medium or associated with the corrosion products. 2) solution or solid that contains nutrients (culture me-Identyiff a pit morphology consistent with an MIC mechantsm.and 3) ideni./f a corrosion product chemistry that is consistent dium). There are three considerations when growing

with the causative organisms. The following sections provide a microorganisms: type of culture medium, incubationdiscussion of available techniques. their advantages and dis- temperature, and length of incubation. The presentadvantages, and. most imporlantly, their limitations, trend in culture techniques is to attempt to culture

several physiological groups Including aerobic, het-KEY WORDS: detection, methods. mtcroblologlcally ir!Jluenced erotrophic bacteria; facultative anaerobic bacteria:corrosion sulfate-reducing bacteria (SRB). and acid-producing

bacteria (APB). Growth is detected as turbidity or aIDENTIFICATION OF CAUSATIVE ORGANISMS chemical reaction within the culture medium. Tradi-

For many years the first step In Identifying corrosion tional SRB media contain sodium lactate as the car-

as microbiologicaUy Influenced corrosion (MIC) was to bon source.- When SRB are present in the sample.

determine the presence of specific groups of bacteria sulfate is reduced to sulfide, which reacts with Iron

in the bulk medium (planktonic cells) or associated (either In solution or solid) to produce black ferrous

with corrosion products (sessile cells). There are four sulfide. Culture media are typically Incubated over

approaches: several days (30 days may be required for the growth

---culture the organisms on solid or in liquid media of SRB). There have been several attempts to improve

-- extract and quantify a particular cell constituent culture media and to grow higher numbers or bacteriaor to shorten the time required for some indication of

-demonstrate a spatial relationship between growth. A complex SRB medium was developed con-

microbial cells and corrosion products using taining multiple carbon sources that can be degradedmicroscopy to both acetate and lactate. In comparison tests, the

complex medium produced higher counts of SRB from

Submuitted for publication March 2006; in revised fonn. July waters and surface deposits among five commercially2006. available media. Jhobalia, et al., developed an agar-

•Corresponding author. E- mail: jlee~nrls.,.c.navy.mljl.

Naval Restarch Laboratory,. Oceanography Code 7303 and 7332. based culture medium for accelerating the growthBldg. 1009, Roonm B131. Stennis Space Center. MS 39529-5004. of SRB.4 The authors noted that over the range from

0010-9312/06/000187/$5.00+$0.50/01006 © 2006, NACE International CORROSION-NOVEMBER 2006


1.93 g/L to 6.50 g/L SRB grew best at the lowest MIC. Unlike culturing techniques, biochemical assayssulfate concentration. Cowan developed a rapid cul- for detecting and quantifying bacteria do not requireture technique for SRB based on rehydration of dried growth of the bacteria. Instead, biochemical assaysnutrients with water from the system under investi- measure constitutive properties including adenosinegation." The author claimed that using system water triphosphate (ATP)."4 phospholipid fatty acids (PIFA).reduced the acclimation period for microorganisms by cell-bound antibodies,'6 and DNA."7 Adenosine-5'-ensuring that the culture medium had the same sa- phosphosulfate (APS) reductase'" and hydrogenase'linity as the system water used to prepare the inocu- have been used to estimate SRB populations.lum. The author reported quantification of SRB within Since ATP is a compound found in all living mat -one to seven days. ter. ATP assays estimate the total number of viable

The distinct advantage of culturing techniques to organisms in a sample. ATP assays are based on thedetect specific microorganisms is that low numbers of luciferin-luciferase reaction where ATI) provides thecells grow to easily detectable higher numbers in the energy for the oxidation of luciferin by the enzyme lu-proper culture medium. However, there are numerous ciferase. The procedure requires that a water samplelimitations for the detection and enumeration of cells be filtered to remove solids and salts that might inter-by culturing techniques. Several investigators have fere. A reagent is added to the filtered sample to lysefollowed the changes in microflora as a function of the cells and release the ATP. The reaction is sensitivewater storage. Zobell and Anderson' and Lloyd7 dem- to sulfide, some metals, and some types of biocides.onstrated that when water is stored in glass bottles. Emitted light is measured with a photometer and thethe bacterial numbers fall within the first few hours amount of light released during the reaction is directlyfollowed by an increase in the total bacterial popu- related to the amount of ATP in the sample.lation with a reduction in the number of species. If Biofilm community structure can be analyzed us-results from culturing techniques are to be related ing cluster analysis of PLFA profiles.'' Phospholipidsto the natural populations, culture media should be are found in the membranes of all cells. Under theinoculated within hours of collection and the sample conditions in natural communities, bacteria contain ashould be chilled during the interim. Under all cir- relatively constant proportion of their biomass ascumstances culture techniques underestimate the or- phospholipids. Phospholipids are not found in storageganisms in a natural population-" Kaeberlein. et al.. lipids and have a relatively rapid turnover so thatsuggest that 991% of microorganisms from the environ- their assay gives a measure of the viable cellular bio-ment resist cultivation in the laboratory."' One major mass. The phosphate of the phospholipids or theproblem in assessing microorganisms in natural en- glycerol-phosphate and acid-labile glycerol from phos-vironments is that viable microorganisms can enter phatidyl glycerol-like lipids can be assayed to increaseinto a nonculturable state." Another problem is that the specificity and sensitivity of the phospholipids as-culture media cannot approximate the complexity of a say. The ester-linked fatty acids in the phospholipidsnatural environment. Growth media tend to be strain- are both the most sensitive and useful chemical mea-specific. For example. lactate-based media sustain the sures of microbial biomass and community structure.growth of lactate-oxidizers, but not acetate-oxidizing PLFA profiles for natural biofilms have been shown tobacteria. Incubating at one temperature is further be more complex than profiles for laboratory biotlims.selective. The type of medium used to culture micro- None of the laboratory profiles clustered closely withorganisms determines, to a large extent, the numbers profiles from natural biofilms. Also, the PLFA profilesand types of microorganisms that grow. Zhu. et al.. for attached bacteria clustered separately from pro-demonstrated dramatic differences in the microbial files of the same bacteria in the bulk phase, suggest-population from a gas pipeline depending on the enu- ing that either the community or the physiology ofmeration techniques.'" For example, using culture attached bacteria differs from that of bulk phase bac-techniques SRB dominated the microfiora in most teria. Despite the fact the PLFA analysis cannot pro-pipeline samples. However, using culture-independent vide an exact description of each species in a givengenetic techniques they found that methanogens were environment, the analysis does provide a quantitativemore abundant in most pipeline samples than denitri- description of the microbiota in a particular environ-lying bacteria and that SRB were the least abundant ment. Analysis of other components of the phospho-bacteria. Similarly. Romero. et al., used genetic moni- lipids fraction give insight into community structure.toring to identify bacterial populations in a seawater For example. SRB contain lipids that can be used toinjection system. 3 They found that some bacteria identify at least a portion of the class. Dowling, et al.,present in small amounts in the original waters were Identilied unusual fatty acids as biomarkers for twoenriched in the culture process. SRB: iso 17: lw7c and branched monoenoics for a hy-

drogen-oxidizing Desuilovibrio sp. and 10 methyl 16:0Biochemical Assays for an acetate oxidizing Desulffobacter sp.2"

Biochemical assays have been developed for the Both Al-S reductase, an intracellular enzymedetection of specific microorganisms associated with found in all SRB. and hydrogenase. an enzyme pres-

CORROSION-Vol. 62, No. 11 1007


ent in some SRB (hydrogenase-positive), can be ex- Increasing shear force increased the rate of total lipidtracted from liquids or solids, including corrosion biosynthesis, but decreased per cell biosynthesis. In-products and sludge. In a procedure to quantify creasing fluid shear also increased cellular biomassAPS reductase. cells are lysed to release the enzyme, and greatly increased the ratio of extracellular poly-added to an antibody reagent and exposed to a color- mer to cellular protein. Maxwell developed a radiore-developing solution. In the presence of APS reductase spirometric technique for measuring SRB activity ona blue color appears whose intensity and develop- metal surfaces that involved two distinct steps: incu-ment rate is proportional to the amount of enzyme bation of the sample with "S sulfate and trapping theand roughly to the number of cells from which the released sulfide."Uenzyme was extracted. Similarly. hydrogenase activity Techniques for analyzing microbial metabolic ac-may be measured in a procedure where the enzyme tivity at localized sites have been developed. Franklin,is extracted from cells and exposed to hydrogen an- et al.. incubated microbial biofilms with "4C-meta-erobically. ' The rationale for relating hydrogenase to bolic precursors and autoradiographed the biolilmsMIC is that during corrosion in anaerobic environ- to localize biosynthetic activity on corroding metalments. molecular hydrogen is produced at the cath- surfaces./7 The localized uptake of labeled compoundsode. Some, but not all SRB, are hydrogenase positive, was related to localized electrochemical activities as-meaning that they possess the enzyme required to ox- sociated with corrosion reactions.idize molecular hydrogen. In the assay, hydrogenase Reporter genes can signal when the activity of areacts with hydrogen and reduces an indicator dye in specific metabolic pathway is induced. King, et al..solution. The activity of hydrogenase is established engineered the incorporation of a promotorless cas-by the development and intensification of a blue color sette of lux genes into specific operons of Pseudomo-proportional to the rate of hydrogen uptake by the nas to induce bioluminescence during degradation ofenzyme. The technique does not attempt to estimate naphthalene.' Using reporter genes. Marshall demon-specific numbers of SRB. Bryant, et al., suggested strated that bacteria immobilized at surfaces exhibitthat hydrogenase levels were better indicators of MIC physiological properties not found in the same organ-than numbers of SRB.2` Mara and Williams reported isms in the aqueous phase." Some genes are turnedthat hydrogenase was more important when the en- on at solid surfaces despite not being expressed In Iiq-vironment contained low concentrations of ferrous uid or on solid media. It is also likely that other genesions. but was less important in the presence of suf- are turned off at surfaces. They further demonstratedlicient ferrous ions to precipitate the sulfide produced gene transfers within blofilms even in the absence ofby SRB.22 Other investigators found no relationship imposed selection pressure.between levels of hydrogenase enzyme and the rate orextent of corrosion. Genetic Techniques

Genetic techniques using ribosomal RNA (rRNA)Cell Activity or their genes (rDNA) have been used to identify and

Roszak and Colwell reviewed techniques com- quantify microbial populations in natural environ-monly used to detect microbial activities in natural ments.' 32 These techniques involve amplification ofenvironments, Including transformations of radio- 16S rRNA gene sequences by polymerase chain re-labeled metabolic precursors." Phelps. et al.,"4 and action (PCR) amplification of extracted and purifiedMittelman. et al.,25 used uptake or transformation of nucleic acids. The PRC products can be evaluated"4C-labeled metabolic precursors to examine activi- using community fingerprinting techniques such asties of sessile bacteria in natural environments and denaturing gradient gel electrophorests (DGGE). EachIn laboratory models. Phelps. et al.. used a variety DGGE band is representative of a specific bacterialof "t C-labeled compounds to quantify catabolic and population and the number of distinctive bands is in-anabolic bacterial activities associated with corrosion dicative of microbial diversity. The PCR products cantubercles in steel natural gas transmission pipelines. 24 also be sequenced, and the sequences are comparedThey demonstrated that organic acid was produced to the sequences in the Genbank database."' whichfrom hydrogen and carbon dioxide in natural gas by allows the identity of the species within an environ-acetogenic bacteria, and that acidification could lead mental sample. Horn, et al.. identified the constitu-to enhanced corrosion of the steel. Mittelman, et al.. ents of the microbial community within a proposedused measurement of lipid biosynthesis from '4C-ac- nuclear waste repository using the following twoetate, In conjunction with measurements of microbial techniques:3

biomass and extracellular polymer, to study effects of -isolation of DNA from growth culture and sub-differential fluid shear on physiology and metabolism sequent Identification by 1 6S rDNA genesof Alteromonas (formerly Pseudomonas) atlantica.2 " -isolation of DNA directly from environmental

Genfank. National Center for I .National samples followed by subsequent identificationLibrary of Medicine. 38A. h8N05. 8600 Rockvflle Pike. fr theatai , of the amplified 16S rDNA genes (Table I andMD 20894. Figure 11

1008 CORROSION-NOVEMBER 2006


TABLE 1 MpiY= 4Organisms Isolated after Growth in Various Yucca Mountain

Simulated Groundwaters and 16S rDNA Sequence Aru

Divergence from Reference Organisms-7 AkM1h. ~Py

Growth Medium from Ynoa A' 14'

Which Organism Isolated Zo, 1~7

lxJl3 lxJl3 y 13-A 5Synthetic Synthetic YR3

with without , R i-2Glucose Glucose t k~ aglmrn

% D iv e rg e n c e - Y- -P B- C 3 co s i M R - 1Organism"'1 from Database1 a'~ YMRBA Cynobate3

Raistonia picketfil YkR ,4Raitona etrohus4.5/MS 6.9/MS "4vo~ r.~~ t ~

Burkholderia cepacia :t oBloST~.l1

Blastobacter natatorius lo~ba ~kcier spSphingomonas paucimobilis 2.0/GB YR-lMethylobacterium meSOphilicurfl YMAB-1315Caulobacter sub vibroidies YtlM e C20h artoaeuUncultured bacterium oxSCC-6(cý 4.0/GB YI tM d lpa roacwi

Pseudomonas (Janth) mephitica 6.06/MS Upedaha protebactetur

Microbactenumr barked 4.55/MS Yfymi'R'cl , "hMicrobactenum, keratanolyicum 4.15/IMS 4.1 5/MS KR-2Microbacterium chocolatum 5.16/MS YMRB-Dna3loasapArthrobacter sp. SMCC G964101 0.0/GB Y.-R&A7Pseudomonas stutzed 3.93/MS 1.0/GB YR-IAtipia genosp. 14 4.0/GB Bailj Amin-o13

R~~a_,Ius sapr hytlcus Gram()Closest relative in 16S rONA sequence comparisons to three Clostri um aminovalaricom positivesseparate databases (i.e., MS, GB, ROP, below). Cl~trdum Moulinrn~ei

(B) MS, MicroSeq database (Applied Biosystems, Foster City, Cali- YMAB.D31

fornia); GB, GenBank database (National Center for Biotechnolo- AnnornycealQs

gy Information, Bethesda, Maryland); RDP, Ribosomal Database eniharopoalluaesp

Project (Michigan State University, East Lansing, Michigan). 6atumcfnsic( H. L~demann, 1. Arth, W. Liesack, AppI. Environ. Microbiol. 66 YMAis.CIe

(2000): p. 754-762. LYi8A" L.G. VanWaasbergen, D.L. Balkwill, F.H. Crocker, B.N. Blorns- MýFtad, and RNV. Miller, Appl. Environ. Microbiol. 66 (2000): p. 3,454- YMR A18

3,463. YMR iA 14YMRB-C25

YMRB.EAYMRS-F16High G + C

YMRB-QComparison of the data from the two techniques dem- Nigoacteriurn keralano4lyfcum gram positivesonstrates that culture-dependent approaches under- M~crobaclerium esieraromaicurn

estimated the complexity of microbial communities. M oaddereiNocardoides p.seliZhu, et al., used genetic techniques to characterize erbcrap

the types and abundance of bacterial species in gas Atpipeline samples and made similar observations.' Arthrobasp

YMRS-D5B_Another example of' genetic techniques is the fluo- MBE

rsetin situ hybridization (FISH). which uses the YMRB- 8rescentYMRB-A16

specific, fluorescent (lye-labeled oligonucleotide probes YR-2YMRB.Dgto selectively identify and visualize SRB both in estab- 0.1

lished and developing multispecies biofllms.3 2 Change/sKequece postion

FIGURE 1. Phylogenetic tree of Yucca Mountain (YM) bacterialMicroscopy community as identified by 16S ODNA analysis of DNA extracted

Ugqht Microscopy - Using light microscopy and from YM rock.33proper staining. investigators have demonstrated arelationship between an unusual variety of coppercorrosion and gelatinous. polysaccharide-containing This phenomenon is distinct from other types of cop-biofllmiS.1 3 7 Blue water (also called copper by-product per corrosion in that it does not significantly corn-release or cuprosolvency) is observed in copper tub- promise the Integrity of the tube, but Instead leads toIng. primarily in soft waters after a stagnation period copper contamination and coloring of the water.of several hours to days and is typically associated Epifluorcscerice Microscopy - lmmunofluo-with copper concentrations of 2 mg/L to 20 mgIL.. rescence techniques have been developed for the



Identilication of specific bacteria in blofilms.:'•' Epi- have been derived from traditional scanning electronfluorescence cell surface antibody (ECSA) methods microscopy (SEM) and transmission electron micros-for detecting SRB are based on the binding between copy (TEM). SEM has been used to image SRB fromSRB-specific antibodies and the SRB cells, and subse- corrosion products on Type 904L (UNS N08904).42

.111

quent detection of SRB-specific antibodies with a sec- microorganisms in corroding gas pipelines.:" and iron-ondary antibody through two approaches. First, the oxidizing Gaillonella in water distribution systems."secondary antibodies can be linked to a fluorochrome TEM has been used to demonstrate that bacteria arethat enables bacterial cells marked with the second- intimately associated with sulfide minerals and thatary antibody to be viewed with an epifluorescence on copper-containing surfaces the bacteria were foundmicroscope. Second, the secondary antibodies can be between alternate layers of corrosion products and at-conjugated with an enzyme (alkaline phosphatase) tached to the base metal."that then can be reacted with a colorless substrate to In traditional SEM. nonconducting samples in-produce a visible color proportional to the quantity cluding biofilms associated with corrosion productsof SRB present. Detection limits for the field test are must be dehydrated and coated with a conductive film10.000 SRB mm-' filter area. The color reagent used of metal before the specimen can be viewed. Tradl-for field tests is unstable at room temperature and tional TEM methods for imaging biolilms require fixa-tends to bind nonspecifically with antibodies adsorbed tion of biological material, embedding In a resin anddirectly at active sites on the filter, creating a false thin-sectioning to achieve a section that can transmitpositive that may Interfere with the detection of SRB an electron beam. Environmental electron microscopyat levels below 10.000 cells mm-2. Antigenic struc- includes both scanning (ESEM) and transmissiontures of marine and terrestrial strains are distinctly (ETEM) techniques for the examination of biologi-different and therefore antibodies to either strain did cal materials with a minimum of manipulation. i.e..not react with the other. fixation and dehydration. Little. et al.. used ESEM to

CornJocal Laser Scanning Microscopy - Confocal study marine biofilms on stainless steel surfaces."'laser scanning microscopy (CLSM) permits one to They observed a gelatinous layer in which bacteriacreate three-dimensional images. determine surface and microalgae were embedded. Traditional SEM Im-contour in minute detail, and accurately measure ages of the same areas demonstrated a loss of cellularcritical dimensions by mechanically scanning the and extracellular material (Figure 2). Little and co-object with laser light. A sharply focused image of a workers used ESEM to demonstrate sulflide-encrustedsingle horizontal plane within a specimen is formed SRB in corrosion layers on copper alloys (Figure 3)while light from out-of-focus areas is repressed from and iron-depositing bacteria in tubercles on stainlessview. The process is repeated again and again at steels (Figure 4).4647 Little, et al., used ETEM to imageprecise intervals on horizontal planes and the visual P. putida on corroding iron filings and to demonstratedata from all images are compiled to create a single, that the organisms were not directly in contact withmultidimensional view of the subject. Geesey. et al.. the metal.4" Instead, the cells were attached to theused CLSM to produce three-dimensional images of substratum with extracellular material (Figure 5). De-bacteria within scratches, milling lines, and grain sign and operation of the ESEM and ETEM have beenboundaries.'( described elsewhere.

Atomic Force Microscopy - Atomic force micros- There are fundamental problems in attemptingcopy (AFM) uses a microprobe mounted on a flexible to diagnose MIC by establishing a spatial relation-cantilever to detect surface topography by scanning at ship between numbers and types of microorganismsa subnanometer scale. Repulsion by electrons over- in the bulk medium and those associated with corro-lapping at the tip of the microprobe cause deflections sion products using any of the techniques previouslyof the cantilever that can be detected with a laser described. Zintel. et al., established that there werebeam. The signal is read by a feedback loop to main- no relationships between the presence, type. or levelstain a constant tip displacement by varying voltage to of planktonic or sessile bacteria and the occurrencea piezoelectric control. The variations in the voltage of pits.'9 Because microorganisms are ubiquitous, themimic the topography of the sample and, together presence of bacteria or other microorganisms does notwith the movement of the microprobe in the horizon- necessarily indicate a causal relationship with cor-tal plane. are converted to an Image. Telegdi. et al., rosion. In fact, microorganisms can nearly always beimaged microorganisms associated with corrosion on cultured from natural environments. Little. et al.. "several substrata.4 ' reported that electrochemical polarization could In-

Electron Microscopy - Many of the conclusions fluence the number and types of bacteria associatedabout blofilm development, composition, distribution, with the surface. 0 Artificial crevices created In 'ypeand relationship to substratum/corrosion products 304 (UNS S30400) stainless steel in abiotic seawater

were associated with large numbers of bacteria after"12' UNS numbers are listed In Metals and Alloys in the Unified Num-

bering S!Istem. published by the Society of Automotive Engineers 5-day exposures to natural seawater. Bacteria did not(SAE International) and o•sponsored by ASTM International cause the crevice: instead, bacteria were attracted to



(a) (b)

FIGURE 2. (a) ESEM image of wet estuanne biofilm on Type 304 stainless steel surface. (b) ESEM image of estuarinebiofilm on Type 304 stainless steel surface after treatment with acetone/xylene.'s

(a) (b) (c)

FIGURE 3. ESEM images of bacteria within corrosion layers on copper foils (a, b markers = 5 pm; c marker I pm). Arrowsindicate suffide-encrusted cells in 3c.46

the anodic site. Several other Investigators have made -large craters from 5 cm to 8 cm or greater insimilar observations. For example. de Sjanchez and diameter surrounded by uncorroded metalSchiffrln demonstrated that Cu(ll) and titanium ions (Figure 6)were strong attractants for Pseudornonas." Detection -cup-type hemispherical pits on the pipe surfaceor demonstration of bacteria associated with corrosion or In the craters (Figure 7)is not diagnostic for MIC. -striations or contour lines in the pits or craters

running parallel to the longitudinal pipe axisPIT MORPHOLOGY (rolling direction) (Figure 8)

-tunnels at the ends of the craters also runningPope completed a study of gas pipelines to deter- parallel to the longitudinal axis of the pipe (Fig-

mine the relationship between the extent of MIC and ure 9)the levels/activities of SRB. 5 2 He concluded that there Pope reported that these metallurgical features

was no relationship. Instead he found large numbers were "fairly definitive for MIC."52 However. the authorof APB and organic acids, particularly lactic acid, did not advocate diagnosis of MIC based solely on pitand identified the following metallurgical features in morphology. Subsequent research has demonstratedcarbon steel: that these features can be produced by abiotic reac-



(b) FIGURE 5. Hydrated Pesudomonas putida after removal of excessmoisture by circulation of air through the environmental cell."

welds was "characteristic of MIC."r'1 Hoffman sug-gested that pit morphology was a "metallurgical tin-gerprint...definitive proof of the presence of MIC."'Chung and Thomas compared MIC pit morphologywith non-MIC chloride-induced pitting in Tyzpes304/304L (UNS S30400/UNS S30403) and Type 308(tiNS S30800) stainless steel base metals and welds."A faceted appearance was common to both typesof pits in Types 304 and 304L base metals (FiguresI 1 [a] and [b]). Facets were located In the dendritic

FIR skeletons in MIC and non-MIC cavities of the Type308 weld metal. They concluded that there were nounique morphological characteristics for MIC pits inthese materials. The problem that has resulted fromthe assumption that pits can be Independently inter-preted as MIC is that MIC is often misdiagnosed. Forexample. Welz and Tverberg reported leaks at weldsin a stainless steel (Type 3161. tUNS S316031) hotwater system in a brewery after six weeks in opera-tion were a result of MIC.`'4' The original diagnosiswas based on the circumstantial evidence of attackat welds and the pitting morphology-scalloped pitswithin pits. However, after a thorough investigation.MIC was dismissed. There were no bacteria associatedwith the corrosion sites: deposits were too uniformto have been produced by bacteria. The hemispheri-cal pits had been produced when carbon dioxide gastCo wave lbeepratued and lwpbublce.Tes nueaispedri-

FIGURE 4. (a) Tubercles associated with pitting in galvanized steel surface discontinuities.pipe from water distribution system (2X); (b) and (c) ESEM views of More recently, several Investigators have demon-Gallionella filaments observed in tubercles. Horizontal field width: (b) strated that the initial stages of pit formation due to57 pm and (c) 29 pm.4

7 certain types of bacteria (to have unique characteris-

tics. Geiser. ei al., found that puts in Type 316L stain-

tons5 3 and cannot be. used to Independently diagnose less steel due to the manganese -oxidizing bacteriumMIC. Leptothrix discophora had different morphologies than

Other investigators described ink bottle-shaped pits initiated by anodic polarization. " Pits Initiatedpits in 300 series stainless steel that were supposed by these organisms in a solution of sodium chlorideto be diagnostic of MIC (Figure 10). Borenstein and were approximately 10 times longer than they wereLindsay reported that dendritic corrosion attack at wide (Figures 12[a] and [bh).`N Pits produced by micro-



SP. SOTHS

FIGURE 6. Cup-type scooped out hemispherical pits on flat surfaces FIGURE 7. Close-up of sand-blasted surface showing MIC pattern £2

with craters in pits.5' Reproduced with permission from The Gas Reproduced with permission from The Gas Technology Institute.Technology Institute.

FIGURE 8. Corrosion pits with stnationsY Reproduced withpermission from The Gas Technology Institute.

FIGURE 9. Close-up view of tunnels (10OX). ý Reproduced withpermission from The Gas Technology Institute.

a possibility that the pits were initiated at the siteswhere the microbes were attached. Eckert used API51, steel to demonstrate micro-morphological char-acteristics that could be used to identify MIC initia-tionYw Coupons were installed at various points in apipeline system and were examined by SEM at 1 .OOOXand 2,OOOX. They demonstrated that pit initiationand bacterial colonization were correlated and thatpit locations physically matched the locations of cells.Telegdi. et al., used AFM to image biofilm formation,

FIGURE 10. An illustration of an ink bottle-type pit noted in many extracellular polymer production. and subsequentcases of MIC and commonly found in the Type 904L tubes from corrosion."' Pits produced by ltiobactllus interrnedtusfailed heat exchangers. had the same shape as the bacteria. None of these

investigators claim that these unique features ('an bedletectedI with the unaided eye or that the features will

organisms were much smaller than, and not nearly be preserved as pits grow, propagate, and merge.as deep as, pits produced in the same solution byelectrochemical means. Pits had almost Identical sizes CHEMICAL TESTINGand aspect ratios as the sizes and aspect ratios of themnanganese-oxidizing bacteria. The similarity between Analyses for corrosion product chemistry canthe dimensions of the bacterial cells attached to the range from simple field tests to mineralogy and iso-surface and the dimensions of corrosion pits indicate tope fractionation. Field tests for solids and corrosion

CORROSION-Vol. 62, No.11 1013


(a) (b)FIGURE 11. (a) SEM of dendritic skeletons in MIC cavities in E308 stainless steel weld (1,000X). (b) Micrograph of the non-MIC chloride-induced corrosion pits in E308 weld root (300X).57

(a) (b)

FIGURE 12. (a) SEM image showing a heavy line on the left indicating square etching by iron milling. The indention in thecenter was detected after the coupon was microbially ennobled. It was not there before microbial colonization. (b) SEMimage of a typical pit initiated in Type 316L stainless steel using anodic polarization.57

products typically include pH and a qualitative analy- x-ray analysis (EDS) coupled with SEM can be usedsis for the presence of sulfides and carbonates. A drop to determine the elemental composition of corrosionof dilute hydrochloric acid placed on a small portion deposits. Because all living organisms contain ATl1. aof the corrosion product will indicate the presence of phosphorus peak in an EDS spectrum can be relatedcarbonates if noticeable bubbling occurs. If a rotten to cells associated with the corrosion products. Otheregg smell is present following acid treatment. sulfides potential sources of phosphorus. e.g.. phosphateare present in the corrosion product. Sulfides can be water treatments, must be eliminated. The activitiesverified by exposing a piece of lead acetate paper to of SRB and manganese-oxidizing bacteria producethe acidified corrosion product and watching for a surface-bound sulfur and manganese, respectively.color change from white to brown. Chloride is typically found in crevices and pits and

cannot be directly related to MIC. There are severalElemental Composition limitations for EDS surface chemical analyses. Sam-

Elements in corrosion deposits can provide infor- ples for EDS cannot be evaluated after heavy metalmation as to the cause of corrosion. Energy-dispersive coating: therefore. EDS spectra must be collected



prior to heavy metal coating. It is difficult or impos- TABLE 2sible to match spectra with exact locations on Images. Weight Percent of Elements Found on Commercially PureThis is not a problem with the ESEM because non- Copper Surfaces After Exposure to Estuarine Water

conducting samples can be imaged directly. meaning for 4 Months and Sequential Treatment

that EDS spectra can be collected of an area that is with Acetone and Xylene4

being imaged by ESEM. Little. et al., documented the Base After Exposure to After Afterchanges in surface chemistry as a result of solvent Element Metal Estuarine Water Acetone Xyleneextraction of water, a requirement for SEM (Table 2)." Al 9.49 1.22 0.74Other shortcomings of SEM/EDS include peak over- Si 21.38 1.89 1.27lap. Peaks for sulfur overlap peaks for molybdenum Cl 0.93 15.9 15.93and the characteristic peak for manganese coincides Cu 99.9 59.62 80.99 82.06

Mg 1.96 0 0with the secondary peak for chromium. Wavelength P 0.98 0 0dispersive spectroscopy can be used to resolve over- S 0.95 0 0lapping EDS peaks. Peak heights cannot be used to Ca 0.49 0 0determine the concentration of elements. It is also K 0.67 0 0

impossible to determine the form of an element with Fe 3.52 0 0

EDS. For example, a high-sulfur peak may indicatesullide, sulfate, or elemental sulfur.

Mineralogical Fingerprints Fe; Sn PyriteMcNeil. et al., used mineralogical data determined " -.

by x-ray crystallography, thermodynamic stability \"S -diagrams (Pourbaix). and the simplexity principle for .precipitation reactions to evaluate corrosion product Mackinawite -- Marcasitemineralogy.6" They concluded that many sulfides un- SO,-,der near-surface natural environmental conditions Greigitecould only be produced by microbiological action onspecifc precursor metals. They reported that djurlelte, Pyrrhotite

spinonkoptte, and the high-temperature polymorph FeCO S2 Smythiteof chalcocite were mineralogical fingerprints for the siderit-

SRB-induced corrosion of copper-nickel alloys. Theyalso reported that the stability or tenacity of sulfide FIGURE 13. Transformations of iron(ll) sulfides formed at pipelinecorrosion products determined their influence on corrosion sites (dashes, biological processes, solid lines, abiological

corrosion. processes).'

Jack. et al.. maintained that the mineralogy ofcorrosion products on pipelines could provide insightinto the conditions under which the corrosion took Fe(O)place."' For example, under anaerobic conditions in |the absence of SRB an iron(li) carbonate (siderite Initial corrosionIFeCO.]) was identified in water trapped under defec-tive coatings. Introduction of air caused a rapid dis-coloration of the white corrosion product to orange Fast oxidation Fast oxidationlron(Ilt) oxides. In the presence of SRB, indicator continuously wet dry conditions

minerals are siderite and iron(ll) sulfide in a ratio of3:1 or more (Figure 13). Mackinawlte (FeS), the first Magnetite, Fe 3 Maghemite, y-Fe2 O3formed crystalline sulfide, converts to gregite (Fe:S 4 })in a time- and pHi-dependent manner. Pyrrhotite Drying(Fe_,,S) may form after nine months. At aerobic corro- Slow oxidationslon sites, the minerals are iron(IIl) oxides: magnetite(Fe.,0j, hematite (Fe20j. lepidocrocite (y-FeO[OH]). Lepidocrocite, y-FeOOHand goethite (ot-FeOlOH]) (Figure 14). Nucleation + crystal growth

Isotope Fractionation IThe stable isotopes of sulfur (:"2S and :1S), natu- Geothite, a-FeOOH

rally present in any sulfate source, are selectively Hematite may form from magnetite as an intermediate.metabolized during sulfate reduction by SRB and theresulting sulfide is enriched in 32S.' The 'S accu- FIGURE 14. Transformation of iron(lloo oxides formed at pipeline

mulates in the starting sulfate as the :S is removed



and becomes concentrated in the sulfide. Little. et al.. 2. J.R. Postgate. The Sulphate-Reducing Bacteria (Cambridge, U.K,:demonstrated sulfur isotope fractionation in sulfide Cambridge University Press,. 1979). p. 26.

3P.J.H. Scott. M. D)avies, Mater. Perform. 31, 5 (1992): p. 64.corrosion deposits resulting from activities of SRB 4. C. Jhobalia. A. Hu. T. Gu, S. Ne•it, "Biochemical Engineering

within biofilms on copper surfaces.'4 32S accumulated Approaches to MIC." CORROSION/2005. paper no. 05500(Houston. TX: NACE International. 2005). p. 12.

in sulfide-rich corrosion products and ý'S was concen- 5. J.K. Cowan. "Rapid Enumeration of Sulfate-Reducing Bacteria,"trated in the residual sulfate in the culture medium. CORROSION/2005. paper no. 05485 (Houston. TX: NACE. 2005).

Accumulation of the lighter isotope was related to sur- p. 16.6. C. Zobell. D.Q. Anderson. B1ol. Bull. 71 (19361: p. 324.

face derivatization or corrosion as measured by weight 7. B. lloyd, J. Roy. Tech. Coll.. Glasgow 4 (1937): p. 173.

loss. Use of this technique to identify SRB-related cor- 8. S.J. GlovannonI. T.B. Britschgt, C.L. Mover. K.G. Field. Nature

rosion requires sophisticated laboratory procedures. 344 (1990): p. 60.9. D.M. Ward. M.J. Ferris. S.C. Nold. M.M. Batcson. Microblol. Mol.

Biol. Rev. 62. 4 (1998): p. 1.353.

CONCLUSIONS 10. T. Kaeberleln. K. lewis. S.S. Epstein, Science 226 (2002): p.13127.

11. D.B. Roszak. R.R. Colwell. Microbiol. Rev. 51.3 (19871: p. 365.4- The following are required for an accurate diagno- 12. X. Zhu. A. Ayala. H. ModIl. J.J. Kilbane. 11. "Application of

sis of MIC: a sample of the corrosion product or af- Quantitative. Real-Time PCR In Monitoring MleroblologlcallyInfluenced Corrosion (MIC) in Gas PIpelines." CORROSION/2005.

fected surface that has not been altered by collection paper no. 05493 (Houston. TX: NACE. 20051. p. 20.

or storage, identilication of a corrosion mechanism. 13. J.M. Romero, E. Velazquez. J.L. Garcla-Villalobos. M. Amaya. S.

identification of microorganisms capable of growth LIe Borgne. "Genetic Monitoring of Bacterial Populations In aSeawater Injection System, Identification of Bloelde Resistant

and maintenance of the corrosion mechanism in the Bacteria and Study of Their Corrosive Effect." CORROSION/2005.particular environment, and demonstration of an as- paper no. 05483 (llouston, TX: NACE, 20051. p. 9.

14. ASTM STP 641, "Use of ATP Extraction In Oil Field Waters" (Westsociation of the microorganisms with the observed Conshohocken. PA: ASTM International. 1977). p. 79.

corrosion. Three types of evidence are used to diagno- 15. M.J. Franklin, D.C. White, J. Blotechnol. 2 (1991): p. 450.

sis MIC: metallurgical, chemical, and biological. The 16. D.H. Pope. "Discussion of Methods for the Detection of Microorganisms Involved In Microbtologhcally Influenced Corrosion." In

objective is to have three independent types of mea- Biologically Induced Corrosion. Proc. Int. Conf. Biologically In-

surements that are consistent with a mechanism for duced Corrosion, ed. S.C. D)exter (Houston. TX: NACE. 1986),

MIC. p. 27 5.17. J.J. Hogan. "A Rapid. Non-Radioactive DNA Probe for Detectlon

* It Is essential In diagnosing MIC to demonstrate of SRBs." In Institute of Gas Technology Symp. on Gas. Oil. Coal.

a spatial relationship between the causative micro- and Environmental Blotechuologv (Chicago. IL: Institute of Gas

organisms and the corrosion phenomena. However, .Teclhology IIGTI. 1990).18. R.E. Talnall. K.M. Stanton. R.C. Ebersole. "Methods of Testing

that relationship cannot be independently Interpreted for the Presence of Sulfate-Reducing Bacteria." CORROSION/88.

as MIC. Pitting caused by MIC can Initiate as small paper no. 88 (Ifouston, TX: NACE. 1988). p. 34.and characteristics of 19. J. Bolvin. E.J. Laishlev. R.D. Bryant. J.W. Costerton. "The

pits that have the same size Influence of Enzyme Systems on MIC." CORROSION/90. paperthe causative organisms. These features are not obvi- no. 128 (Houston. TX: NACE. 1990). p. 8.

ous to the unaided eye and are most often observed 20. N.J.E. Dowling, P.D). Nichols. D.C. White. FEMS Microblol. F"eol.53 (1988): p. 325.

with an electron or atomic force microscope. MIC does 21. W. Bryant, W. Jansen, J. Bolvin. E.J. Laishley. J.W. Costerton.

not produce a macroscopic, unique. metallographic Appi. Environ. Microbiol. 57. 10 (1991): p. 2.804.

feature. Metallurgical features previously thought to 22. D.D. Mara. D.J.A. Williams. Br. Corros. J. 7. 5 (19721: p. 139.23. J. Jones-Meehan. J.W. Cofield. B. Little. R. Ray. IP. Wagner. M.

be unique to MIC. e.g., hemispherical pits in 300 se- McNeil. J. McKay. "Microbiologlcally Influenced Corrosion ofries stainless steel localized at weld or tunneling in Steels: Weight Los.s Measurements. ESEM/EDS. and XRD

Analyses." Int. Conf. MIC, paper no. 29 (Ilouston. TX: NACE.carbon steel. are consistent with some mechanisms 2003). p. 12.for MIC. but cannot be interpreted Independently. 24. T.J. Phelps. R.M. Schram. D. Rlngelberg. N.J. Dowling. D.C.

Bacteria do produce corrosion products that could White. Biofouling 3 (1991): p. 265.25. M.W. Mittelman. D.E. Nivens, C. Ltow, ID.C. White. Microbial Keol.

not be produced abiotically in near-surface environ- 19 (1990): p. 269.ments, resulting in isotope fractionatLion and miner- 26. S. Maxwell. SPE Prod. Eng. (1986): p. 363.

alogical fingerprints. The result is corrosion where 27. M.J. Franldin. D.C. White. H.S. Isaacs. Corros. Set. 33 (1992):p. 251.

none could be anUcipated based on the composition 28. J.M.II. King. P.M. DiGrazia. 13. Applegate. R. Buriage. J.

of the bulk medium, e.g., low-chloride waters, and Sanseverino. P. Dunbar. F. L.armer. G.S. Saylor. Science 249

corrosion rates that are exceptionally fast. Develop- (1990): p. 778.29. K.C. Marshall. "Analysis of Bacterial Behavior During Bholoullng

ment of sophisticated genetic and imaging techniques of Surfaces." in Blofouling and Blocorroston In Industrial Water

has made it possible to more accurately characterize Systems, eds. G.G. Geescy, Z. Lewandowski. H.-C. Flemming(Boca Raton. FL: Lewis Publishers Inc.. 1994). p. 15.

microorganisms and their spatial relationships to cor- 30 D.A. Stahl. D.J. Lane. G.J. Olsen. N.R. Pace. Science 224 (1984):rosion products and localized corrosion. However, the p. 409.requirements for diagnosing MIC have not changed. 31. D.A. Stahl. B. Flesher. II.R. Mansfield. L. Montgomery. Appl.

Environ. Microbiol. 54 (1988): p. 1.079.32. R.I. Amanm. J. Stromley. R. Devereux. R. Key. D.A. Stahl, Appl.

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34, X. Zhu. J. Lubeck, K. Lowe. A. Darami. J.J. Kilbane 11, "Improved 49. TI', Zintel. D.A. Kostuck. B.A. Cooklnghamu. 'Evaluation ofMethod for Monitoring Microbial Communities in Gas pipelines." Chemical T~reatmnents in Natural Gas Systems %,s. MIC and OtherCORROSION/2004. paper no. 04592 (Houston. TIX NACE. 2004). Forms of Internal Corrosion Using Carbo~n Steel Coupons."p. 13. CORROSION/2003. paper no. 03574 (Houston. TX: NACE. 2003).

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36. A.H.L. Chamberlain. W.R. Fischer. U. Hinze. Hit. Paradies. ships between Bacteria and Localized Corrosion." CORROSION/CA.C. Sequelra. HI. Siedlarek. M. Thies. D). Wagner. JXN Wardell. 96. paper no. 278 (Houston. TX: NACE. 1996). p. 8."An Interdisciplinary Approach for Microblologically Influenced 51. S.R. de Sainchez, D..J. Sehiffrln. J. Electroanal. Chem. 403 (1996):Corroison of Copper.- Microbial Corrosion. Proc. 3rd lot. EFC p. 39-45.Workshop, no. I5 (London. U1.K.: The Institute of Materials. 52. 1). Pope. GRI Field Guide: Microbiologically Intluenced Corrosion1995). p. 3. IMICI: Methods of D~etection in the Field (Chicago. II.: Gas Re-

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39. J.J. zambon. I'S. I luber. A.F. Meyer. J. Slots. M.S. Fornalik. 2003. paper no. 03544 (1llouston. TX: NACE. 2003). p. 13.R.E. Bater, AppI. Environ. Mierohiol. 48. 6 (1984): p. 1.214. 54. S.W. Dorenstein. PDB. Lindsay. Mater. Perorm. 33. 4(1994): p. 43.

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42. 1'.J.ll. Scott. M. Davies. Mater. Perform. 28, 5 (1989): p. 57. Non-MIC Chloride Induced Pits in ~ypes 304/3041,/P,30843. H.F. Ridgeway, .Il. Olson, App. Environ. Mierobiol. 41. 1 (1981): Stainless Steel Base Metal/Welds." CORROSION/99. paper no.

p. 274. 159 (Hlouston,'TX: NACE. 1999). p. 23.44. G. lBluiui. 'Biological Fouling of Copper and Copper Alloys." to 58. J. Welz. J. Tverberg. Mater. P'erfornm. 37 (1998): p. 66.

Biodeterluration VI (London. UK.: CAB International). p. 567. 59. M. Geiser. R. Avel. Z, Lewandowskl. "Pit Initiation on 3 16L45. B. Uitile. P. Wagner. R. Hay. R. Pope. R. Scheetz. J. Ind. Mierobiol. Stainless Steel in the Presence of Bacteria Leptothriix cliseophomn."

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47. B.J. Liitle, P.A. Wagner. Z. Lewandowskl. "The Role of Blominer- for Corrosion Processes Induced by Sulfate Reducing Bacteria."aIP.'aiion In Microblologically Influenced Corrosion." CORRO- CORROSION/91. paper no. 580 (Htouston. TX: NACE. 1991). p. 16.SION/98. paper no. 294 (Houston. TX: NACE, 19981. 62. T.R. Jack. M.J. Wilmoit. R.L. Sutberby. Mater. Perform. 34 (1995):

48. B.J. Little. R.K. Pope. T.L. Daulton. R-1. Ray. -Application of Envi- p. 19.romnoental Cell Transmission Electron Microscopy to Mlcrobio- 63. L.A. Chambers. P.A. Trudinger. Geomicroblo). J. 1 (1979): p. 249.logically Influenced Corrosion." CORROSION/2001. paper no. 64. B. Uitile. P. Wagner. R. Ray. M. McNeil. J. Jones-Meehan. Debalia01266 (Hlouston. TX: NACE. 2001). ?XIX Suppl. 11993): p. 287.

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