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F7 IION PAG21Form Approved Public re(cid:127)"(cid:127) If II IiI~lIlIlI 111)1fl~1 11111111 1111 ilI_I_r per rusponse. including the Tiimfore reviewing instructions. searOchiBngM e xNisotig. 0da7ta0 4so-0uroc1es8, 8gatheiir.tw-d maintain Send comrnents regarding this burden or any other aspect oft his collection of information, including suggestions for reduc ation Operations and Reports. 1215 Jefferson Davis Highway, Suite 1204. Arlington. VA 22202-4302. and to the Offic isthIngton. DC 20503. 1. Agency use Only (Leave blank). 2I.1 R9e9po4r t Date. 3. Report Type and Dates Covered. Final - Conference Proceedings 4. Title and Subtitle. 5. Funding Numbers. Advances in MIC Testing Program Element No. 0601153N Project No. 03103 6. Author(s). TaskNo. 320 . Little, B., and P. Wagner . .. Accession No. DN094463 Work Unit No. 13331A 7. Performing Organization Name(s) and Address(es)., 8. Perforrnlng Organization Naval Research Laboratory . Report Number. Oceanography Division Std. Tech. Publication 1232- Stennis Space Center, MS 39529-5004 1994, Amer. Soc. for Testing & Matls. 9. Sponsorlng/Monitoring Agency Name(s) and Address(es). 10. Sponsoring/Monitoring Agency Naval Research Laboratory Report Number. Requirements Division NRL/PP/92:077:333 Stennis Space Center, MS 39529-5004 11. Supplementary Notes. 12a. Distribution/Availability Statement. 12b. Distribution Code. Approved for public release; distribution is unlimited. 13. Abstract (Maximum 200 words). The study of microbiologically influenced corrosion (MIC) has progressed from phenomenological case histories to a mature interdisciplinary science including electrochemical, metallurgical, surface analytical, microbiological, biotechnological, and biophysical techniques. With microelectrodes and gene probes it is now possible to measure interfacial dissolved oxygen, dissolved sulfide and pH, and to determine microbial species responsible for localized chemistry. Biofilms can be tailored to contain consortia of specific microorganisms and naturally-occurring biofilms can be dissected into cellular and extracellular constitutents. Scanning vibrating electrodes can be used to map the distribution of anodic electrochemical activity. Electrochemi- cal impedance spectroscopy and electrochemical noise analysis techniques have been developed to non-destructively evaluate localized corrosion due to MIC. The development of environmental scanning electron, atomic force, and laser confocal microscopy makes it possible to image cells on surfaces and to accurately determine the spatial relationship between microorganisms and localized phenomena. Transport of nutrients through biofilms can be modeled using techniques including optical density measurements to precisely locate the water/biofilm interface and nuclear magnetic resonance imaging to visualize flow characteristics near surfaces colonized with microorganisms. The ways in which new techniques can be used to understand fundamental mechanisms and to discriminate MIC will be discussed in this paper. 14. Subject Terms. 15. Number of Pages. Microbiologically influenced corrosion (MIC), culture techniques, electrochemistry, surface 11 analyses 16. Price Code. 17. Security Classification 18. Security Classification 19. Security Classification 20. Limitation of Abstract. of Report. of This Page. of Abstract. Unclassified Unclassified Unclassified SAR NSN 7540-01-280-5500 Standard Form 298 (Rev. 2.89) Prescribed by ANSI Std Z19 18 298-102 Authorized reprint from Standard Technical Publication 1232- 1994 Copyright American Society for Testing and Materials. 1916 Race Street, Philadelphia, PA 19103 Keynote Address Brenda J. Little' and PatriciaA . Wagner' Advances in MIC Testing REFERENCE: Little. B. and Wagner. P.. "Advances in MIC Testing," Wicrobiological In- fluenced Corrosion Testing, ASTM STP 1232. Jeffery R. Kearns and Brenda J. Little. Eds.. American Society for Testing and Materials, Philadelphia, 1994. pp. 1-11. ABSTRACT: The study of microbiologically influenced corrosion (MIC) has progressed from phenomenological case histories to a mature interdisciplinary science including electrochemical. metallurgical, surface analytical. microbiological. biotcchnological, and biophysical techniques. With microelectrodes and gene probes it is now possible to measure interfacial dissolved oxygen, dissolved sulfide and pH. and to determine microbial species responsible for localized chemistry. Biofilms can be tailored to contain consortia of specific microorganisms and naturally-occurnng biofilms can be dissected into cellular and extracellular constituents. Scanning vibrating electrodes can be used to map the distribution of anodic electrochemical activity. Electrochemical impedance spectroscopy and electrochemical noise analysis techniques have been developed to non-destructively evaluate localized corrosion due to MIC. The development of environmental scanning electron, atomic force, and laser confocal microscopy makes it possible to image cells on surfaces and to accurately determine the spatial relationship between microorganisms and localized phenomena. Transport of nutrients through biofilms can he modeled using techniques including optical density measurements to precisely locate the water/ biofilm interface and nuclear magnetic resonance imaging to visualize flow characteristics near surfaces colonized with microorganisms. The ways in which new techniques can be used to understand fundamental mechanisms and to discriminate MIC will be discussed in this paper. KEYWORDS: microbiologically influenced corrosion (MIC). culture techniques, electrochemistry, surface analyses Introduction Corrosion associated with microorganisms has been recognized for over 50 years. vet the study of MIC is a relatively new. multidisciplinary field. In 1985 an International Conference on Biologically Influenced Corrosion was sponsored by the National Association of Cor- rosion Engineers (NACE). Of the thirty-six papers in the proceedings volume, roughly half the papers are descriptive case studies and ten titles contain the words sulfate-reducing bacteria (SRB) [11. In 1985 many of the techniques for evaluating MIC depended on char- acterizing shape, color, and smell of surface deposits in addition to the presence of specific types of bacteria, usually SRB. In contrast, today there is general recognition that SRB contribute and control many cases of MIC, but that it is the total community of bacteria within the biofilm that is responsible for MIC and there is no correlation between numbers and types of cells and localized corrosion [21. Electrochemical. surface analytical. and microbiological techniques are now routinely combined to9 elu4cid-ate t1he 9com7pl3exi ti1es of microbial interactions with metal substrata. Research chemist and oceanographer. resp Center. MS 39529-5004. 4 . . 1J (cid:127).- - 2 MICROBIOLOGICALLY INFLUENCED CORROSION TESTING Techniques for Determining Cellular Constituents Within Biofilms (ulture [t'chntques For many ,ears, the standard for eialuatinn MIC has been the enumeration of SRB either in bulk liquids or in surface deposits using a liquid or solid 131 medium with sodium lactate as the carbon source 14.5j. When SRB are present in the sample. sulfate is reduced to sulfide which reacts with iron in solution to produce black ferrous sulfide. Blackening ot the medium oer a 28-dav period signals the presence ol SRB. Usually. I mL samples are injected bv ,,,rine into media bottles for Il-fold dilutions. It is assumed that only a single living bacterium is required to blacken a bottle, The simplest interpretation ot test results is to consider that if one bottle is blackened, the sample contained at least I organism, if two bottles are blackened. the sample contained I10 organisms; three bottles. I(X) organisms and so) on. Agar slants can be inoculated by dipping a pipe cleaner into a liquid sample and insertinga it into a single vial of ,olid or semi-solid agar. Mineral oil and a CO-generating tablet are usually added to exclude oxgen, and the vial is capped. incubated for 5 days. and checked daily for blackening. The distinct advantage of culturing techniques is that they are extremely sensitive. Low numbers of SRB grow to easilv detectable higher numbers in the proper culture medium. However, growth media tend to be strain-specific For example. lactate-based media sustain the grow-th of lactate oxidizers but not acetate-oxidizing bacteria. Incubating at one temperature is further selecti(cid:2)e Culturing methods using agar media cannot distinguish between a single SRB cell and a clump o( SRB cells 131 The present trend in culture techniques is to attempt to culture several ph,,iolog.cal groups including aerobic. heterotrophic bacteria; facultative anaerobic bacteria, and acid-producing bacteria in addition to sulfate-reducing bacteria 161. A complex SRB medium containing multiple carbon sources that can be de- graded to both acelate and lactate has been developed and compared to five other com- merciallv available media using natural and produced waters and surface deposits 171 Rtochemical A 5sais, Biochemical assays have been developed for the detection of specific microorganisms associated with MIC. Unlike culturing techniques, biochemical assays for detecting and quantifying bacteria do not require growth of the bacteria. Instead. biochemical assays measure constitutive properties including adenosine triphosphate (ATP) 8I1. phospholipid fatty acids (PILFA) 121. cell-bound antibodies 19.10. and DNA (111. Adenosine-5'-phos- phosulfate CAPS) reductase 131. hydrogenase 1121. and radiorespirometric measurements have been used to estimate SRB populations and activity 113.141. ATP assavs estimate the total number of viable organisms by measurinet the amount of adenosine triphosphate in a sample. ATP is a compound found in all lihing matter. The procedure requires that a water sample be filtered to remove solids and salts which ma, interfere with the test. The filtered sample is added to a reagent that releases cell ATP An enzyme then reacts with the ATP to produce a photochemical reaction. Emitted light is measured with a photometer and the number of bacterial cells is estimated from the total light emitted. Biofilm community structure can be analyzed using cluster analysis of the PLFA profiles [21. PLFA profiles for natural biolilms have been shown to be more complex than profile', for laboratory hiofilms. None of the laboratory profiles clustered closely with profiles from natural biofilms. In addition, the PLFA profiles for attached bacteria clustered separately LITTLE AND WAGNER ON ADVANCES IN MIC TESTING 3 from profiles of the same bacteria in the bulk phase, suggesting that either the community or the physiology of attached bacteria differ from that of hulk phase bacteria. Immunofluorescence techniques have been developed for the identification of specific bacteria in hiofilms 115.11 I-p' ifluorescence cell surface antibody (ECSA) methods for detecting SRB are based on the use and subsequent detection of specific antibodies, produced in rabbits, that react with SRB cells [(V.I) A.\ secondarv antibody, produced in goats, is then reacted with the primarv rabbit antibodies bound to SRB cells, In some cases. goat antibodies are linked to a fluorochrome which enables bacterial cells marked with the secondarv antibody to be viewed with an epifluorescence microscope. In other cases. goat antibodies are conjugated with an enzyme (alkaline phosphatase) that can then be reacted with a colorless substrate to produce a visible color proportional to the quantity of SRB present. The detection limits for the field test are I) (MMS)tR B/mm: filter area. The color reagent used for the field tests is unstable at room temperature and tends to bind nonspe- cificallv with antibodies adsorbed directlv at active sites on the filter. creating a false positive that may interfere with the detection ot SRB at levels below It) (XX) cells,'mm-. Antigenic structures of marine and terrestrial strains are distinctly different and therefore antibodies to either strain did not react with the other. Furthermore. SRB antibodies did not react with non-SRB bacteria. The developers report a poor response of rabbit antibodies developed from pure SRB cultures to mixed populations [1/0 Rabbit SRB antibodies generated from fresh SRB strains from Prudhoe Bay. Alaska. as well as terrestrial and marine location%. were found to react better %%ih SRB from natural sources. It is possible to differentiate individual species within a biofilm bv reacting them with monoclonal antibodies specific to outer cell membrane antigens. Hogan 1/1I described a non-isotopic semi-quantitative procedure for the detection of Desulfobacterturn and I)e.ulftotnoaculum using DNA probes labeled with an acridinium ester that is sensitive to 10' organisms per min Direct molecular characterization of natural microbial populations can be accomplished with sequence analysis of 55 rRNAs 1/7.ISIJ. More rccentlv, fluorcscent dye-labeled oh- gonucleotide probes have been used for microscopic identification of sinle cells and characterization of mixed populations. Polvmerase chain reaction amplification. comparatioe sequencing and whole cell hybridization have been combined to ,clectivelv identit ,ind visualize SRB both in established and developing multispectes bitofilms (191. APS reductase is an intercellular enzyme found in all SRB. Briefly. cells are washed !o remove interfacing chemicals, including hydrogen sulfide, and Ivsed to release APS reductase. The lysed sample is washed, added to an antibody reagent and exposed to a color- developing solution. In the presence of APS reductase a blue color appears within ( mmin The degree of color is proportional to the amount of enzyme and roughly to the number of cells from which the enzyme was extracted. Similarly, a procedure has been developed to quantify hydrogenase from hydrogenase-positive SRB requiring cells to be concentrated by filtration from water samples [121. Solids. including corrosion product and sludge. can be used without pretreatment. The sample is exposed to an enzyme extracting solution for 15 min and placed in an anaerobic chamber from which oxygen is removed by hydrogen The enzyme reacts with excess hydrogen and simultaneously reduces an indicator dye in solution. The activity of the hydrogenase is established by the development of a blue color in less than 4 h. The intensitv of the blue color is proportional to the rate of hydrogen uptake by the enzyme. The technique does not attempt to estimate specific numbers of SRB. Roszak and Colwell f201 reviewed techniques commonly used to detect microbial activities in natural environments, including transformations of radiolabelled metabolic precursors Phelps et al. [211 and Mittelman et al. 1221 used uptake or transformation of "C'-labelled metabolic precursors to examine activities of scssile bacteria in natural environments and 4 MICROBIOLOGICALLY INFLUENCED CORROSION TESTING in laboratory models. Phelps et al. 1211 used a variety ot ('-labelled compounds to quantfl(, catabolic and anabolic bacterial activities associated %kithc orrosion tubercles in stcel natural gas transmission pipelines. They demonstrated that organic acid was produced from Ii. and ('0. in n--ural gas bv acetogenic bacteria. and that acidification could lead to enhanced corrosion ot the steel. Nlittelman ct al. 1221 used measurement of lipid bios,,nthesis from '('-acetate. in conjunction with measurements of microbioal biomass and extracellular pol,- mer. to Ntudv effects of differential fluid shear on phvsiology and metabolism of .A.heromonas (formcrls Pteudorpionas) atlamca. Increasing shear force increased the rate of total lipid biosynthesis. but decreased per cell biosvnthesis,. Increasing fluid shear also increased cellular biomass and greatly increased the ratio of extracellular polymer to cellular protein. Techniques for analyzing microbial metabolic acti, itv at localizcd sites are also being developed, Franklin ct al. 123?1 incubated microbiail io(cid:127)ilnms with "('-metabolic precursors and autoradiographed the biotilms to locate biosynthetic activity on corroding metal surfaces. The uptake of the labelled compounds was related to localized electrochemical activities associated with corrosion reactions. A major breakthrough in determining bacterial activity within biotilms has been the use of '"reporter" genes that can signal the induction ot specific metabolic pathways. King et al. 1241 engineered the incorporation of a promotorless cassette of lux genes into specific operons of Pseudotnonas so that these operons induce bioluminescence during the degra- dation of naphthalene. Mittelman et al.' used the bioluminescent reporter gene to provide a quantitative measure ot attachment of microorganisms onto metal and glass surfaces in a laminar flow system. They found that biofilm light production was directly correlated with biofilm cell numbers in a range of 1t)'-10)' cells/cm-. Using reporter genes. Marshall et al. 1251 demonstrated that bacteria immobilized at surfaces exhibit physiological properties not found in the same organisms in the aqueous phase. Some genes are turned on at a solid surface despite not being expressed in liquid or on solid media. It is also likely that other genes are turned off at surfaces. They identified acid- and alkali-inducible genes in E. colt. Marshall et al. 1251 further demonstrated gene transfers within biofilms even in the absence of imposed selection pressure. Rosser and Hamilton 11.31. with subsequent modifications I141. developed a test tube technique for a "S sulfate radiorespirometric assay to measure SRB metabolic activity on the surface of metal coupons after exposure to corrosive environments- The coupon is placed into anaerobic filtered sterile seawater containing "S-sulfate. Oxygen-free zinc acetate is immediately injected onto an enclosed filter paper wick and the entire system is incubated. Oxygen-free hydrochloric acid is then injected past the wick into the solution. Volatile acid sulfides. including any fi1"S formed, are trapped during an equilibration period. The wick is removed from the tube and the radioactivity measured using a liquid scintillation counter. after which the sulfate reduction rate is calculated. This technique has been used for both bulk and coupon samples. Techniques for Identification and Measurement of MIC Electrochemical Techniques Mansfeld and Little 1261 recently reviewed electrochemical techniques applied to MIC studies and no attempt will be made to discuss all the innovations in electrochemical techniques Three nondestructive electrochemical techniques, the scanning vibrating electrode technique (SVET). electrochemical impedance spectroscopy (EIS). and electrochemical M. W Mittelman. J M. H. King, 6. S. Savler. and D. C White. unpublished data, Univernii, of Tennessee. Knoxville. IN. 1992. LITTLE AND WAGNER ON ADVANCES IN MIC TESTING 5 noise analvsis [ NA) are currentl, heine used to provide unique insights into mechanism,, for MIC. SVET is used to determine the maenitude and si en of current densities oer freel] LOr- roding metals in solution 1271. Franklin et all 12S1 used SV[T to show a spatial relationship between localized corrosion and bacterial cells, on carbon steel surfaces. Pit propagaiton depended on the presence of bacteria. The authors proposed that biofilms inhibited migration of aggressive ions from pits or migration ot inhibiting ions from the hulk solution into pats. EIS techniques record impedance data as a function of the frequency of an applied signal at a fixed potential [291. A large frequency range (t)5 kliz to I mliz) must he inmestieated to obtain a complete impedance spectrum. l)owhng ct al. j301 and Franklin et aL 13-l demonstrated that the small signals required for -IS do not adversely affect the number. viabilitv, and activity of microorcanisms , ithin hioftilm. EIS data may be used to determine polarization resistance, the inverse of corrosion rate Sophisticated models have been developed for localized corrosion 132.331 that provide ad- ditional information from EIS data. Several reports have been published in which E[IS has been used to study the role of SRB in corrosion of buried pipes 134-361 and reinforced concrete 137-391. The formation of biofilms and calcareous deposits on three stainless steels and titanium during exposure to natural seawater was followed using EIS and surface analsis 140.411. Ferrante and Feron (421 used EIS data to conclude that the material composition of steels was more important for MW resistance than bacterial population. incubation time, sulfide content, and other products of bacterial growth. Jones et al. 1431 used EIS to determine the effects of several mixed microbiological communities on the protective properties of epoxy coatings on steel. A damage tunction was defined which allowed quahitatisc as- sessment of coating deterioration due to MIC. ENA follows fluctuations of potential or current as a function of time or experimental conditions. Analysis of the structure of the electrochemical noise using the frequency de- pendence of the power spectral densitv can provide information concerning the nature of corrosion processes and magnitude of corrosion rate. King ct al. 1.361 interpreted noise measurements for steel pipes in environments containing SRB as being indicative ot film formation and breakdown. iverson 144.451 used [NA to monitor corrosion of mild steel in a trypticase seawater culture of a marine SRB and concluded that breakdown of the iron sulfide film was accompanied by the generation of potential electrochemical noise. Surlace Analytical Techniques Nivens et al. 1461 demonstrated that attenuated total reflectance infrared spectroscopy (ATR-FT/IR) can be used to detect changes in sessile microbial biomass. The ATR-FT IR studies showed that changes in the physiological properties of attached bacteria were induced by changes in the bulk phase. They demonstrated that the number of attached ('aulohacter sp. was directly correlated with the intensity of the infrared amide 11a symmetrical stretch band at 1543 cm '. corresponding to bacterial protein. The technique was sensitive to lil bactena/cm'. and changes in the physiological status of the attached bacteria could be measured. For example, production of the intracellular storage lipid. poty-B hvdroxvalka- noate. and production of extracellular polymer, were monitored by absorbance at 17311 cm (C=O stretch) and 1084 cm ' (C-0)s tretch). respectively Geesey and Bremer 1471 used ATR-FT/IR to evaluate non-destructivelv, in real time. interactions of bactena with thin films of copper dero)sited on germanium. ('hanges in the thickness of the copper films were measured as increased intensity of the infrared %iter absorption band at 1640 cm '. The authors compared copper loss in the presence of bacteria 6 MICROBIOLOGICALLY INFLUENCED CORROSION TESTING isolated from corroded copper sampl"es and were able to obsere difference,, between t1 n cultures. I sini this technique. Jollevc t al. 1.,I 1oh,,erýed copper oxidation hv three polmcrs,. including bacterial cxopolvmer. Niens el al. 1401 investigated the use of the quartz crystal microbalance (OCM). a %er% sensitite mass-sensing deilce, for detecting attached microbial films. rhe Q('M was more sensitive to changes in biomass than AVR-FT, IR. %ith a detection limit of 10' hacteria cm ,,nd a linear ranee ot at least two orders of magnitude. An interesting aspect of both A FR- FT IR and the .CMi s that substrata of both techniques can he used for electrochemical analyses so that corrosion information can he obtained while changes in microbial bhotilms are monitored. It is now generally recognized that biotilms alter hiofilm.,metal interfacial chemistries. Direct chemical measurement,, are restricted by biotilm thickness and the heterogeneous anisotropic nature of biofilms 1491. Ion-selective and gas sensing microprobes with tip di- aimeters less than 10 jaim have been developed for direct biohilm measurements. Lewandoski 1491 measured dissolved oxygen profiles in a continuous flow. open channel reactor with a mixed hiotilm on a metal surface. Van lloudt et al. 1501 developed a rugged iridium oxide p11 microelectrode with a 3 to 5 4m tip diameter to measure a p11 profile across a mixed population biofilm on a polycarbonate disc. An in-situ microtechnique has been developed for evaluating parameters of diffusion- controlled reactions in biofilms 1511. A microprobe 15 I-m in diameter was used to simultaneously measure dissolved oxygen and optical density at different depths in a submerged biofilm. The diffusion coefficient for dissolved oxygen, the dissolved oxygen flux. and the half vlcocitv coefficient were then calculated. Nuclear magnetic resonance imaging ( NNI RI). a non-invasive method, uses radiofrequency magnetic fields in the presence of a strong magnetic field to provide information about the concentration and physical state of specific atomic nuclei. Lcwandowski et al. 1521 demonstrated the use of NMRI to show distribution ot water, flow velocities, and biomass in a biofilm polycarbonate reactor system. Recent developments in image analysis systems and electron, atomic and laser micros- copies make it possible to image biologlical materials in the hydrated state. Mucllar ct al. 1531 were able to determine rate coefficients for early bacterial colonization on copper, silicon. stainless steel and glass using a chemostat. a flow cell. and a microscope equipped with an image analysis system. Substrata were monitored using reflective light from a microscope equipped with a Nomarski lens and video camera recorder. Fransmitted light aas used for transparent surfaces. rhev demonstrated that surface roughness and surface tree energv correlated positively with biological and abiological sorption processes. Little et al. 1541 used environmental scanning electron microscopy/energy-dispersive X- ray analysis (ESEMIEDS) to study biofilms on stainless steel surfaces, observing a gelatinous laver in which microalgae were embedded. Extracellular polymeric acidic polysaccharides hind and precipitate heavy metals. ESEM/EDS spectra indicated local concentrations ot Al. Ni. and Ti. Images of the same specimens made using traditional scanning electron milcrosopy I SEM) demonstrated a loss of cellular and cxtracellular material. Dclhvdration of the hiofilm with solvents, required for SEM. either extracted bound metals from the biofilm by ion exchange/solvent extraction or removed the metals with the extracellular polymeric material. Laser confocal microscopy permits one to create three-dimensional images. see surface contour in minute detail, and accurately measure critical dimensions by mechanically ,can- ning the object with laser light 1551. A sharply focused image of a single horizontal plane within a specimen is formed while light from out of focus areas is repressed from view 'he process is repeated again and again at precise intervals on horizontal planes and the uýsual data from all images compiled to create a single. multidimensional view ot the subject. LITTLE AND WAGNER ON ADVANCES IN MIC TESTING 7 Geesev' used laser confocal microscopy to produce three-dimensional images of bacteria within scratches, milling lines and grain boundaries. The atomic force microscope (AFM) is related to the scanning tunneling microscope (STM). [he STM uses an atomically sharp conductive tip held angstroms from the surface to profile surface features with angstrom resolution. When the tip is electrically biased with respect to the sample. a current will flow between the surface atom closest to the tip and the nearest tip atom bv the quantum mechanical process of electron tunneling. While the STM requires the sample to be electrically conductive or coated with a conductive material. the AFM can be used to image non-conducting surfaces and does not rely on tunneling current. AFM provides exceptional detail and allows viewing of specimens in the hydrated state. AFM uses an extremely sharp scanning probe mounted on a flexible cantilever to record x.,v.: coordinates of a sample in fractions of a nanometer. Photodiode electrical outputs mimic sample topography and serve as the basis for the resulting image. AFM images of copper exposed to a bacterial culture medium for 7 days showed btofilms distrib- uted heterogeneously across the surface with regard to both cell numbers and depth 1561. Bacterial cells were associated with pits on the surface of the copper coupons. Conversion of metals to sulfides by SRB has been studied since the late 18(10s 1571. Baas- Becking and Moore identified mackinawite. gregrite and smvthite as indicators for SRB corrosion of ferrous metals in anaerobic environments 1581, McNeil et al. analyzed sulfide mineral deposits on copper alloys colonized by SRB in an attempt to identify specific mineralogies that were indicative of SRB activity 1591. They concluded that the formation of non-adherent layers of chalcocite (CuS) and the presence of hexagonal chalcocite were indicators of SRB-induced corrosion of copper. The compounds were not observed abiot- icallv and their presence in near-surface environments could not be explained thermods- namicallv. Sulfur isotope fractionation was demonstrated by Little et al. in sulfide corrosion deposits resulting from the activities of SRB within biofilms on copper surfaces 1601. ' S accumulated in sulfide-rich corrosion products, and "S was concentrated in the residual sulfate in the culture medium. Accumulation of the lighter isotope was related to surface derivatization or corrosion is measured by weight loss. Use of this and the preceeding mineralogicial technique to identify SRB-related corrosion requires sophisticated laboratory procedures Conclusions The combined testing approaches of microbiology. electrochemistry, and surface chemtltrx have been reviewed to provide insight into complex interactions between biofilms and metal surfaces. Multimedia microbiological cultures. biochemical assays and genetic probes are being used to demonstrate the presence of specific types of bacteria. ESEM. AFM and laser confocal microscopy have demonstrated the spatial relationship between bacteria and localized corrosion on hydrated surfaces. Dissolved oxygen, dissolved sulfides. pH nmd optical density profiles through biofilms have been made with microprobes. Electrochemical testink!. including EIS. SVET and ENA, has been used to demonstrate MIC for many all(-,,, in a large number of environments. Acknowledgments This work was supported by the Office of Naval Research. Program Element O) II1 5N through the Defense Research Sciences Program. NRL Contribution Number PR '92:077:333. (1( 6 (iceev.u npublished data. Montana State Ltnlvcr,,i,. liozcman. M I. I1112 8 MICROBIOLOGICALLY INFLUENCED CORROSION TESTING Reterences III Biologicait- inducedi ( orrosion, 1I (" 1)extcr. Ld .National Association ot Co~rrosion LniginccrN. Houston. Tecxas. 198h (21JF ranklin, Mi. J and Wkhit. 1) C . 13o corrosion. Ifiowi hnotoei, Vo 2. 'I991 pp 45f1-45t' I.?1 Tatnall. R. E1.. Stanton. K. MI.. and Fbersoke. R. Metlchods oft lcsrine tor the Presience of Sulfate- Reduciniz Bacteria. ( orrosion Nx. Piper No ,%S.N ational Association of Corrosion Ln- giflcer-. Houston. Icxas. 198SX 141 Postigate. J R . ii.upit-ei inHe ateria. ( nimbridte Ik in is rsitt I'ress. 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