Surface Changes in Mild Steel Coupons from the Action of Corrosion-Causing Bacteria
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1981, p. 766-774 Vol. 41, No. 3
0099-2240/81/030766-09$02.00/0
Surface Changes in Mild Steel Coupons from the Action of
Corrosion-Causing Bacteria
CHRISTIAN 0. OBUEKWE,2 DONALD W. S. WESTLAKE,' FRED D. COOK,2 AND J. WILLIAM
COSTERTON3
Departments of Microbiology,1 and Soil Science,2 University ofAlberta, Edmonton, Alberta T6G 2E9; and
Department of Biology, University of Calgary, Calgary, Alberta T2N 1N4,3 Canada
Changes which occur on the surface of mild steel coupons submerged in cultures
Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
of an Fe(III)-reducing bacterium, isolated from corroded pipe systems carrying
crude oil, were studied microscopically to investigate the interaction between the
corrosion-causing bacterium and the corroding mild steel coupon. Under micro-
aerobic conditions and in the absence of the bacteria, a dense, crystalline,
amorphous coat formed on the surface of the steel coupons. In the presence of
bacteria the surface coat was extensively removed, exposing the bare metal to the
environment. After about 2 weeks of exposure, the removal of the surface coating
was followed by colonization of the metal surface by the bacteria. Colonization
was mediated by fibrous, exopolysaccharidic material formed by the bacteria.
Extension of studies to other bacteria isolated from crude oil and corroded pipes
reveals that the formation of exopolysaccharide fibers and possession of adherent
properties are common characteristics of bacteria from crude oil systems.
Microorganisms, in particular the anaerobic, mented (1, 11, 17, 21, 25) and possible mecha-
sulfate-reducing bacteria, have been implicated nisms by which it occurs have been proposed (3,
in the corrosion of metals used in industry (1, 4, 10, 23, 25, 26), no systematic study of the
11, 21). Interest in this phenomenon has cen- physical interaction between a corrosion-related
tered in the petroleum industry, as economic bacterium and a metal surface has been re-
losses are more clearly defined in this industry. ported. It would be interesting to see whether
Corrosion becomes a major problem when oil the initiation and progression of the corrosion
fields have to be subjected to secondary recovery process, as in bacterial animal infections (9, 18),
procedures, such as the use of injection waters, are associated with adherent properties of bac-
to maintain oil flow. Such waters are often taken teria.
from nearby sources such as streams and sloughs This paper reports the results of microscopic
and used without sterilization so that the oil- studies of the interaction under laboratory con-
bearing formations are inoculated with aquatic ditions between some bacterial isolates from
bacteria. An example of an oil field in this stage Pembina crude oil, produced water from the oil
of maturation is the Pembina field of north- field, and corrosion products of failed pipes and
central Alberta, Canada, where a variety of aero- mild steel coupons submerged in cultures of the
bic, facultative aerobic, and anaerobic bacteria organisms.
are found. Representatives of these groups can
be readily isolated from oil, produced water, and MATERIALS AND METHODS
internal pipeline encrustations or "tubercles" Mild steel coupons. The corrosion test specimens
(Obuekwe, Ph.D. thesis, University of Alberta, (coupons) were AISI 10-18 mild steel of dimensions
Edmonton, 1980). Among such bacteria were a 5.0 by 1.2 by 0.1 cm obtained from Caproco Corrosion
group of facultative aerobes which were capable Prevention Ltd., Edmonton. Each coupon was
under anaerobic conditions of reducing ferric to punched out on a die from a sheared, cold-roller sheet
ferrous iron and reducing sulfite, thiosulfate, and of the metal. A uniform bright finish was achieved by
elemental sulfur (but not sulfate) to sulfide along blasting with powdered glass. Before submersion in
with the classical anaerobic sulfate reducers. cultures of bacteria, each coupon was sterilized by
The result of the synergistic interaction of such immersion in 70% ethanol for 10 min and degreased in
bacteria would be an increase in the concentra- 95% ethanol for 15 min and quickly dried under ultra-
tion of ferrous ions together with an enhanced violet light in a stream of warm, sterile air.
Bacterial cultures. The bacteria were isolated
level of production of sulfide which result in the from crude oil samples, produced water from oil wells,
sustained production of the potentially corrosive and corrosion products of failed pipes. These orga-
ferrous sulfide (17). Whereas the role of bacteria nisms were chosen for this work because of their
in the corrosion process has been well docu- corrosive activities (Obuekwe et al., Abstr. Annu.
766VOL. 41, 1981 CORROSION OF MILD STEEL COUPONS 767
Meet. Can. Soc. Microbiol. 1979, p. 13). The organisms metal, albeit rough, was clearly exposed. When
included a Pseudomonas sp. (isolate no. 200) (Ob- immersed in either uninoculated Butlins or B10
uekwe, Ph.D. thesis) and other bacteria referred simn- medium, however, the coupons were covered by
ply as isolates no. 2, 42, 66, 230, and 218. a crystalline or amorphous coating (or both).
Short-term submersion (up to 2 weeks) of the mild Such crystals appear very densely packed (Fig.
steel coupons to pure cultures of the organisms was 1B).
carried out in static cultures grown in stoppered 500-
ml Erlenmeyer flasks containing 400 ml of medium. In the uninoculated B10 medium (control)
Cultures for long-term (over 2 weeks) submersion of after 6 days of immersion the coupon surfaces
coupons were cultivated in a continuous culture sys- were completely obliterated by the closely
tem. Coupons were suspended in 500-ml Erlenmeyer packed surface coat (Fig. 1C). This deposit was
flasks (fitted with an overflow arm) which were con- thought to be a corrosion product which accu-
Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
tinuously fed sterile medium from a 44-liter reservoir mulated on the metal surface. When the coupons
at a dilution rate of 0.012 h-', which would result in were exposed to cultures of Pseudomonas sp.
microaerobic conditions. The media employed were as isolate no. 200 in B10 medium, the surface coat-
follows. Modified Butlin medium contained (per liter): ing was extensively removed, exposing the bare
K2HPO4, 0.5 g; NH4Cl, 1.0 g; Na2SO4, 2.0 g; MgSO4-
7H20, 0.1 g; sodium lactate (60%), 1.5 ml; and yeast metal (Fig. 1D), with the surface coats or depos-
extract (Difco Laboratories, Detroit, Mich.), 1.5 g, at its occurring only as isolated patches or eroded
a final pH of 7.2. B10 medium contained (per liter): crystalline material surrounded by the exposed
K2HPO4, 0.8 g; KH2PO4, 0.2 g; Na2SO4, 0.4 g; MnSO4, metal surface. A similar observation was made
0.001 g; NaMoO4, 0.001 g; yeast extract (Difco), 5.0 g; in uninoculated Butlin medium (Fig. 2A, con-
peptone (Difco), 5.0 g; soluble FePO4 (City Chemical trol) and in Butlin medium inoculated with iso-
Corp., New York, N.Y.), 4.7 g; and CaSO4 (saturated late no. 200 (Fig. 2B). Thus, the presence of this
solution), 10 ml, at a final pH of 7.2. pseudomonad prevented the formation of a pro-
Epifluorescence microscopy. Previously im- tective surface coat on the mild steel coupons.
mersed coupons were rinsed in running distilled water The nature of the coupon surfaces immersed
(4 liters/min) and stained in acridine orange (10 mg/
100 ml of distilled water) for 3 min. The dye solution in cultures of this pseudomonad did not seem to
was initially filtered through membrane filters (pore differ markedly with time up to a period of 2
diameter, 0.32 ,um; Millipore Corp., Bedford, Mass.) to weeks. However, the degree of surface coating
remove particulate matter. Coupons were destained was less with extended exposure time. After 2
with three changes of isopropanol and then air-dried. weeks of exposure, bacteria-like structures ap-
The specimens (coupons) were then examined with a peared attached to the surfaces of the sub-
standard Zeiss light microscope fitted with epifluoresc- merged coupons. The demonstration by scan-
ence illumination system, including a halogen lamp. ning electron microscopy of attachment of this
Transmission electron microscopy. Bacterial pseudomonad after 2 weeks of submersion of the
cells recovered by low-speed centrifugation (3,000 x g
for 5 min) for ultrastructural studies were fixed with coupons was not unequivocal; it was difficult to
or without ruthenium red by the procedure of Patter- differentiate amorphous inorganic deposits from
son et al. (20), dehydrated and embedded in low-vis- bacteria. However, epifluorescence microscopic
cosity resin by the procedure of Spurr (24), and ex- examination of coupons exposed for 2 weeks
amined in an AE1 801 electron microscope. revealed the attachment or close association of
Scanning electron microscopy. Freshly with- bacteria with the coupon surface. Rinsing of the
drawn coupons were rinsed in distilled water or 0.1 M coupons in running distilled water (4 liters per
cacodylate buffer, fixed in 5% (vol/vol) glutaraldehyde min) did not dislodge the bacteria from the
in cacodylate buffer (pH 7.0) with or without 0.025 M metal surface. The quantitation of bacterial at-
ruthenium red, washed, and postfixed in 0.5% glutar-
aldehyde. The coupons were then dried in graded tachment (cells per unit area) was not possible
ethanol concentrations up to absolute ethanol, then because many of the cells were embedded in the
dried in graded Freon 113 concentrations made up in uneven surface coat and could only be seen at
absolute ethanol, and finally dried to the critical point varied planes of focus.
with Freon 113 as the desiccant. The dried coupons After long submersion periods (up to 9 weeks),
were coated with approximately 15.0 nm of gold film this pseudomonad formed thick fibrous exo-
sputtered in an Edmonds sputter-coater (Pirani Jen- polysaccharidic material in which the cells were
ning, Model 4) and examined with a Cambridge entrapped and attached to the metal surface
Stereoscan Model S4 or a Hitachi 450 scanning elec- (Fig. 3). Thin sections of the recovered, ruthe-
tron microscope at an accelerating voltate of 20 kV.
nium red-stained portions of the 9-week culture
RESULTS showed that the organism produced the exo-
The typical scanning electron microscopy of polysaccharidic material in the culture medium.
the surface of a fine glass-blasted mild steel Ruthenium red selectively stains acidic polysac-
coupon before submersion in bacterial cultures charides (13). The exopolysaccharide produced
is shown in Fig. 1A. The metal surface was free and surrounding and interconnecting the cells is
of any surface deposit or coating, and the clean shown in Fig. 4A. The exopolysaccharidic ma-768 OBUEKWE ET AL. APPL. ENVIRON. MICROBIOL.
Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
0
1
11111111 i;.: W
i 2ffka - ,1-11 d^b
I
I
FIG. 1. Scanning electron micrographs of (A) the surface of fine glass-blasted, unimmersed, mild steel
coupon, x2,485; (B) densely packed crystalline surface covering of mild steel coupon incubated in B1O medium
for 6 days, x2,275; (C) mild steel coupon incubated for 6 days in uninoculated (control) B10 medium, x455;
and (D) mild steel coupon incubated for 6 days in B10 culture of isolate no. 200, X455. D, Densely packed
crystals; ES, exposed metal surface.
terial occurred as a loose to dense mat of fibers. dense growth. Such adherence after 2 weeks of
Extension of these studies to other bacteria iso- incubation in batch cultures is shown in Fig. 5A
lated from Pembina crude oil samples revealed and B.
that these other bacteria readily formed exo-
polysaccharidic, fibrous material. These fibers DISCUSSION
occurred as a network of interconnecting fibers Electron and epifluorescence microscopic
filling the intercellular spaces (Fig. 4B) or were studies were undertaken to investigate the
condensed by dehydration and appeared as thick changes on the surfaces of mild steel coupons
strands (Fig. 4C and D). Among the isolates submerged in cultures of bacteria isolated from
obtained from Pembina crude oil were several the corrosive environment of Pembina crude oil.
unidentified gram-negative bacteria that pro- These surface changes arose from loss or depo-
duced extremely gummy colonies on solid agar sition of corrosion products and from the attach-
media. These bacteria readily adhered to the ment of the bacteria to the coupon surface.
surfaces of the mild steel coupons and formed Exposure of the coupons to uninoculated me-Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
FIG. 2. Scanning electron micrograph of (A) mild steel coupon incubated for 6 days in uninoculated
(control) Butlin medium, x4,550; and (B) mild steel coupon incubated for 6 days in Butlin medium culture of
isolate no. 200, x4,550. D, Densely packed crystals; AS, amorphous surface covering; ES, exposed metal
surface.
769Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
FIG. 3. Scanning electron micrograph of a mild steel coupon incubated for 9 weeks in B10 culture of isolate
no. 200; x9,200. EP, Exopolysaccharidic fibers; BC, bacterial cells.
770VOL. 41, 1981 CORROSION OF MILD STEEL COUPONS 771
Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
FIG. 4. Transmission electron micrographs of ruthenium red-stained, sectioned cells of (A) isolate no. 200
obtained from a 9-week B10 culture of the organism to which mild steel coupons were exposed, x40,000; (B)
18-h culture of isolate no. 2 and network of exopolysaccharide fibers, x37,500; (C) 18-h culture of isolate no.
218, x36,000; and (D) culture of isolate no. 230, x37,500. EP, Exopolysaccharide fibers.Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
I _
S~ ~ ~ ~~ S
FI.5 cnigeeto mcorpssoigteataheto el fioaesn.6 A n 2()t
midselcuosatr2wesoficbto,X,1.M,M tasufc;B,bteilel.
772VOL. 41, 1981 CORROSION OF MILD STEEL COUPONS 773
dium or medium inoculated with the Pseudo- rides, whereas Jones et al. (12) described "poly-
monas sp. isolate no. 200 prevented or allowed, saccharide-like" material by which slime-form-
respectively, the formation of surface coats on ing bacteria attached to artificial surfaces in
the metal surfaces. The thick, densely packed flowing streams. Other workers (6, 16, 20) have
crystalline surface coating would probably cre- reported the mediation of exopolysaccharide
ate a barrier between the metal and its environ- (slime) in bacterial attachment to rumen wall
ment. In the presence of the Fe(III)-reducing and feed fibers, in freshwater and marine envi-
pseudomonad only minimal surface coating was ronments (7, 15,27,28), and in industrial systems
evident. Thus, the activities of this isolate pre- (5, 22). The true extent of the polysaccharide
vented the formation of the possibly protective bacterial glycocalyx is not always seen in elec-
surface coating. Since these studies were con- tron microscopy, because of the condensation of
Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
ducted under microaerobic conditions, the prod- this very hydrated structure during dehydration.
uct of coupon corrosion, Fe(o) -- Fe(II) + 2e&, The exopolysaccharide fibers are well main-
could be further oxidized by dissolved 02 to tained when they are attached at multiple points
Fe(III). A ferric oxide (y Fe2O3) film has been (Fig. 4A and B) or when they are stabilized by
previously implicated in the protection of metals reaction with lectins or specific antibodies (14).
against corrosion (19). Since the observed sur- Under natural conditions, this Pseudomonas
face coating was minimal in cultures of this isolate would exist together with other bacteria
pseudomonad, which reduces Fe(III) to Fe(II), in Pembina crude oil (Obuekwe, Ph.D. thesis).
it was inferred that these surface coats, which Among these bacteria were those that produced
were Fe(III) compounds which became solubi- very sticky colonies. Evidently, organisms which
lized to Fe(II), also occurred with the growth of do not readily produce slime or possess any
this organism. Presumably, in the absence of mechanism for adhesion would be easily entan-
isolate no. 200, the formation of a dense, crys- gled in the masses of the slime-producing bac-
talline, insoluble surface coating would reduce teria and thus bring them to close association
the contact between the metal and its environ- with the metal surface.
ment and in this manner prevent or reduce The relationship between bacterial attach-
corrosion. In contrast, the reduction of the gen- ment to metal and the corrosion of such struc-
erally insoluble Fe(III) compounds to the soluble tures is not known. However, the formation of a
Fe(II) forms by this pseudomonad [or any dense mass of attached bacterial cells may cause
Fe(III)-reducing organism] would expose the the development of concentration cells (electro-
metal to its harsh environment and lead to chemical) due to differential aeration. Further-
greater corrosion. This interpretation is sup- more, localization of bacterial activity to areas
ported by electrochemical data (Obuekwe, Ph.D. surrounding their colonies on metal surfaces will
thesis). cause discontinuity in the chemical environ-
Micrographs revealed that attachment of ment. Such differences on the metal surfaces
Pseudomonas sp. isolate no. 200 to the metal may give rise to galvanic couples (dissimilar
surface occurred after about 2 weeks of exposure. metal effect) and the attendant localized corro-
The inability of the running distilled water (used sion. Presumably, for an organism to play a
for rinsing the coupons) to dislodge the bacteria, significant role in the corrosion process, it should
as revealed by epifluorescence microscopy, led be closely associated with the corroding surface.
to the conclusion that the association between In the related phenomenon-ore leaching by
cells of isolate no. 200 and the coupon was strong bacteria-attachment of the bacteria to the ore
enough to be considered an attachment. particles appears to be common (2). However,
The production of exopolysaccharide fibers in attachment was not a prerequisite for leaching
which the cells were entrapped mediated the of ore material.
establishment and colonization of the metal sur- The interaction of an Fe(III)-reducing Pseu-
face. The formation of exopolysaccharidic ma- domonas bacterium with corroding mild steel
terials appeared to be a common characteristic coupons occurred in two stages. The first phase
of the bacteria isolated from the corroding pipes was an indirect effect which arose by the bacte-
and crude oil of the Pembina oil field. The role rial modification of the physico-chemical envi-
of this commonly produced adhesive material ronment of the metal, and the second phase
would be the establishment of these environ- involved attachment to and colonization of the
mental organisms on the pipe surfaces. The me- metal surface.
diation of exopolysaccharidic materials in the
attachment of bacteria to surfaces has been
widely reported. Geesey et al. (8) reported that ACKNOWLEDGMENT
microorganisms in an alpine stream were at- This investigation was supported by the National Research
tached to surfaces by means of exopolysaccha- Council (Canada) Operating Grant (no. A 3687) of D.W.S.W.774 OBUEKWE ET AL. APPL. ENVIRON. MICROBIOL.
LITERATURE CITED 14. Mackie, E. B., K. N. Brown, J. Lam, and J. W. Cos-
terton. 1979. Morphological stabilization of capsules of
1. Baumgartner, A. W. 1973. Microbial corrosion-what group B streptococci, types Ia, Ib, II, and III, with
causes it and how it can be controlled. Water flooding. specific antibody. J. Bacteriol. 138:609-617.
Soc. Pet. Eng. of AIME Repr. Ser. no. 2a, p. 58-62. 15. Marshall, K. C., R. Stout, and R. Mitchell. 1971. Mech-
2. Berry, V. K., and L. E. Murr. 1978. Direct observations anism of initial events in the sorption of marine bacteria
of bacteria and quantitative studies of their catalytic to surfaces. J. Gen. Microbiol. 68:337-348.
role in the leaching of lowgrade, copper-bearing waste, 16. McCowan, R. P., K.-J. Cheng, and J. W. Costerton.
p. 104-136. In L. E. Murr, A. E. Torma, and J. E. 1980. Adherent bacterial populations on bovine rumen
Brierley (ed.), Metallurgical applications of bacterial wall: distribution patterns of adherent bacteria. Appl.
leaching and related microbiological phenomena. Aca- Environ. Microbiol. 39:233-241.
demic Press, Inc., New York. 17. Miller, J. D. A. (ed.). 1970. Microbial aspects of metal-
3. Booth, G. H., and A. K. Tiller. 1960. Polarization studies lurgy, p. 202. American Elsevier, New York.
of mild steel in cultures of sulphate-reducing bacteria. 18. Nalbandian, J., M. L. Freedman, J. M. Tanzier, and
Downloaded from http://aem.asm.org/ on March 25, 2021 by guest
Trans. Faraday Soc. 56:1689-1696. S. M. Loveland. 1974. Ultrastructure of mutants of
4. Booth, G. H., and A. K. Tiller. 1962. Polarization studies Streptococcus mutans with reference to agglutination,
of mild steel in cultures of sulphate-reducing bacteria. adhesion, and extraceilular polysaccharide. Infect. Im-
Part 3: halophilic organisms. Trans. Faraday Soc. 58: mun. 10:1170-1179.
2510-2516. 19. Ole1jord, L. 1975. ESCA studies of passive films formed
5. Costerton, J. W., and G. G. Geesey. 1979. Microbial in steel by oxidizing inhibitors, p. 434-439. In Seventh
contamination of surfaces, p. 211-221. In K. L. Mittal Scandinavian Corrosion Congress. Trondheim, Norway.
(ed.), Surface contamination, vol. 1. Plenum Publishing 20. Patterson, H., R. Irvin, J. W. Costerton, and K.-J.
Corp., New York. Cheng. 1975. Ultrastructure and adhesion properties of
6. Costerton, J. W., G. G. Geesey, and K.-J. Cheng. 1978. Ruminococcus albus. J. Bacteriol. 122:278-287.
How bacteria stick. Sci. Am. 238:86-95. 21. Postgate, J. R. 1979. The sulphate-reducing bacteria, p.
7. Fletcher, M., and G. D. Floodgate. 1973. An electron- 102-108. Cambridge University Press, Cambridge.
microscopic demonstration of an acidic polysaccharide 22. Purkiss, B. E. 1970. Corrosion in industrial situations by
involved in the adhesion of marine bacterium to solid mixed microbial floras, p. 107-128. In J. D. A. Miller
surfaces. J. Gen. Microbiol. 74:325-334. (ed.), Microbial aspects of metallurgy. American Else-
8. Geesey, G. G., W. T. Richardson, H. G. Yeomans, R. vier, New York.
T. Irvin, and J. W. Costerton. 1977. Microscopic 23. Sasaki, H., T. Nakahara, Y. Kanda, K. Osato, and H.
examination of natural sessile bacterial populations Togano. 1977. Possibility of hydrogen depolarization
from an alpine stream. Can. J. Microbiol. 23:1733-1736. phenomenon of mild steel by sulfate-reducing bacteria.
9. Gibbons, R., K. Berman, P. Knoettner, and B. Kap- Corr. Eng. (Tokyo) 26:125-132.
simalis. 1966. Dental caries and alveolar bone loss in 24. Spurr, A. R. 1969. A low-viscosity epoxy resin embedding
gnotobiotic rats infected with capsule forming strepto- medium for electron microscopy. J. Ultrastruct. Res.
cocci of human origin. Arch. Oral Biol. 11:549-560. 26:31-43.
10. Horvath, J., and M. Solti. 1959. Beitrg zum Mechnismus 25. von Wolzogen Kuhr, C. A. H. 1961. Unity of anaerobic
der anaeroben mikrobiologischen Korrosion der Metalle and aerobic iron corrosion process in the soil. Corrosion
im Boden. Werkst. Korros. 10:624-630. 17:119-125.
11. Hughes, D. E. 1963. The microbiology of corrosion, p. 73-
82. In L. L. Shreir (ed.), Corrosion I. Newness-Butter- 26. Wanklyn, J. N., and C. J. P. Spruit. 1952. Influence of
worth, London. sulphate-reducing bacteria on the corrosion potential of
12. Jones, H. C., I. L. Roth, and W. M. Sanders II. 1969. iron. Nature (London) 169:928-929.
Electron microscopic study of a slime layer. J. Bacteriol. 27. Zobell, C. E., and E. C. Allen. 1933. Attachment of
99:316-325. marine bacteria to submerged slides. Proc. Soc. Exper.
13. Luft, J. H. 1971. Ruthenium red and ruthenium violet. I. Biol. Med. 30:1409-1411.
Chemistry, purification, methods of use for electron 28. Zobell, C. E., and E. C. Allen. 1935. The significance of
microscopy and mechanism of action. Anat. Rec. 171: marine bacteria in fouling of submerged surfaces. J.
347-368. Bacteriol. 29:239-251.You can also read
