The role of Pseudomonas aeruginosa on the localized corrosion of 304 stainless steel

Corrosion Science, Voi. 34, No. 9, pp. 1531-1540, 1993

0010-938X/93 $6.00 + 0.00 © 1993 Pergamon Press Ltd

Printed in Great Britain.

THE ROLE OF PSEUDOMONAS AERUGINOSA LOCALIZED CORROSION OF 304 STAINLESS

ON THE STEEL

J. MORALES, P. ESPARZA, S. GONZ,~LEZ, R . SALVAREZZA* a n d M . P. ARI~VALO'~ Dpto de Ouimica Ffsica, Universidad de La Laguna, Avda. de la Trinidad s/n, 38205 La Laguna, Tenerife, Spain * Instituto de Investigaciones Fisicoqulmicas Te6ricas y Aplicadas (INIFTA), Suc. 4, C.C. 16, 1900 La Plata, Argentina t Cfitedra de Medicina Preventiva y Social, Facultad de Medicina. Universidad de La Laguna, Tenerife, Spain

Abstract--The influence of Pseudomonas aeruginosa on the corrosion resistance of 304 stainless steel in neutral buffered solutions containing 0.5 M NaCI was studied by using potentiodynamic and potentiostatic techniques complemented with scanning electron microscopy. Results showed that the growth of Pseudomonas aeruginosa in the medium decreased the resistance of the localized corrosion of 304 stainless steel specimens. This effect can be explained by the formation of microcrevices under the biological deposits and by passive film thinning due to the production of aggressive metabolites.

INTRODUCTION

MICRO-ORgANiSMS are involved in different corrosion processes of metals and alloys in aqueous media. 1 Microbial-induced corrosion seems to be related to the production of aggressive metabolites such as sulphides, 2 acid 3 or oxidant substances. 4 These metabolites decrease the resistance of the protective oxide layers 2 or increase the cathodic currents leading in both cases to the breakdown of passivity. 5 The action of metabolites is particularly intense in a relatively narrow region near the metal surface where preferential microbial growth occurs. Micro-organisms attached by extracellular products to the metal surface form complex biofilms. In contrast to the role of metabolites on the corrosion process, the role of the biofilm itself is not yet well understood. 6 This layer may promote crevice corrosion, increase the oxygen reduction, 7 and, in some cases, decrease the corrosion rate of metals. In this paper the role of a biofilm forming bacterium on the corrosion of 304 stainless steel was studied using electrochemical techniques. Stainless steel specimens exposed for 7 days to a Pseudomonas aeruginosa inoculated medium, free of chloride anions, exhibited a decrease in the resistance to localized corrosion when they were subjected to potentiodynamic runs in sterilized medium containing 0.5 M NaCI. The results suggested that the formation of biological deposit and/or protective film thinning by microbial metabolites were responsible for the decrease in the localized corrosion resistance of the 304 stainless steel. EXPERIMENTAL METHOD Working electrodes consisted of 304 stainless steel (SS) rods (0.6 cm diameter) with a circular exposed area of 0.28 cm2. The metal surface was polished with fine grained emery paper up to 600 grit. The

Manuscript received 9 October 1992. 1531

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J. MORALESet al.

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E(V(SCB)) FIG. 1. Current density (j) vs potential (E) profile recorded at 0 . 0 2 V s -1 after 1 h in (a) 0.1 M NaH2PO 4 + 0.05 M Na2B407, pH 7.5 and (b) 0.1 M NaH2PO 4 + 0.05 M N a 2 B 4 0 7 4- 10 -2 M sucrose, pH 7.5.

electrodes were rinsed with twice distilled water and dried in air at room temperature. Hanging meniscus electrodes were used in order to avoid crevice corrosion of the 304 SS electrodes. The electrochemical measurements were made in a conventional glass cell using a large platinum plate as counter electrode and a saturated calomel electrode (SCE) as a reference electrode. The electrolyte solution used for either culture medium or for electrochemical measurements was 0.1 M NaH2PO 4 4-0.05 M NaEB407 + 10-2M sucrose pH 7.5 prepared from p.a. reagents and twice distilled water. This electrolyte was used since the electrochemical behaviour of stainless steel in 0.1 M NaH2PO 4 + 0.05 M Na2B407 is relatively well known.8 Sucrose addition causes no effect on the shape and potentials of the current peaks Ia-IIa/IIc (Fig. 1) related to the electroformation and electroreduction of the FeEOa.xH20 outer layer on a pre-existing Cr203 layer, s'9 as revealed by voltammetry at 0.02 V s-1 . Besides, peak IIIa, assigned to the electro-oxidation of Cr(III) species to Cr(VI), and peak III~, assigned to the electroreduction of the Cr(IV) species to Cr(III),8'9 remain unchanged in the presence of sucrose. Microbial cultures were prepared by inoculating sterilized Erlenmeyer flasks containing 200 ml of the phosphate-borate buffer (pH 7.5) + 10-2 M sucrose as carbon source. Inocula were made from Pseudomonas aeruginosa cultures in nutrient agar. Bacterial growth was followed through oxygen consumption measured with conventional oxygen electrode (Fig. 2). After 24 h, the bacterial culture reached the stationary phase of growth with a bacterial concentration close to I x 105 ml- 1. After 7 days the microbial culture remained active (demonstrated by inoculating nutrient agar with a drop of the culture medium). The 304 stainless steel (SS) specimens were placed in Erlenmeyer flasks with or without bacteria for 7 days. They were removed and immediately placed in the electrochemical cell containing de-aerated (by argon bubbling) and sterilized 0.1 M NaH2PO 4 + 0.05 M Na2B407 4- 10-2 M sucrose + 0.5 M NaCl in order to determine breakdown potentials and induction times for pitting corrosion. Breakdown potentials

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w e r e determined from apparent current density (j) vs potential (E) plots recorded at 1.6 x 10.4 V s 1 from -0.70 V in the positive direction. The value of E b was determined in the j/E profiles as the most positive potential value exhibiting a passive current value. Induction times, ti , for localized corrosion were measured by holding the 304 SS electrode for 30 min at E = -0.70 V(SCE) and then stepping the potential to a preset value more positive than E b and recording the corresponding current transients. Then, t i values were measured from the minimum of the current in the current transients. (Eb)

EXPERIMENTAL RESULTS

Open circuit experiments S p e c i m e n s of 304 SS w e r e p l a c e d for 7 d a y s in E r l e n m e y e r flasks c o n t a i n i n g sterile p h o s p h a t e - b o r a t e b u f f e r + 10 -2 M sucrose o1" t h e s a m e m e d i u m i n o c u l a t e d with Pseudomonas aeruginosa. T h e i n s p e c t i o n , with an optical m i c r o s c o p e , o f the s p e c i m e n s k e p t in c o n t a c t with the i n o c u l a t e d m e d i u m r e v e a l e d m i c r o b i a l g r o w t h at c e r t a i n sites of t h e m e t a l surface (Fig. 3). T h e m i c r o b i a l d e p o s i t s a r e f o r m e d as jellylike c o l o n i e s which are typical of Pseudomonas aeruginosa. T h e m i c r o s c o p i c o b s e r v a t i o n s s h o w no e v i d e n c e of c o r r o s i o n e i t h e r with o r w i t h o u t b a c t e r i a . T h e c o r r e s p o n d i n g o p e n circuit p o t e n t i a l s r e m a i n e d stable in the - 0 . 0 4 to - 0 . 0 8 V ( S C E ) p o t e n t i a l r a n g e i n d i c a t i n g t h a t t h e m e t a l surface was c o m p l e t e l y p a s s i v a t e d . 7 S i m i l a r e x p e r i m e n t s w e r e m a d e with the a d d i t i o n o f 0.5 M N a C l to the m e d i a . In this case, localized c o r r o s i o n was d e t e c t e d on s e v e r a l 304 SS s p e c i m e n s in c o n t a c t with t h e m e d i u m c o n t a i n i n g Pseudomonas aeruginosa, w h e r e a s the s p e c i m e n s in c o n t a c t with the sterile m e d i u m r e m a i n e d free of c o r r o s i o n . In b o t h cases the o p e n circuit p o t e n t i a l s w e r e also in the - 0 . 0 4 to - 0 . 0 8 V ( S C E ) r a n g e . T h e s e e x p e r i m e n t s d e m o n s t r a t e t h a t the p r e s e n c e of g r o w i n g Pseudomonas aeruginosa e n h a n c e s passivity b r e a k d o w n c a u s e d b y the aggressive C1- anions. ~0

Potentiodynamic measurements T o i n v e s t i g a t e t h e role o f m i c r o b i a l d e p o s i t s on the l o c a l i z e d c o r r o s i o n r e s i s t a n c e of 304 SS w i t h o u t i n t e r f e r e n c e o f m e t a b o l i t e s , Eb a n d t i m e a s u r e m e n t s for s p e c i m e n s k e p t in c o n t a c t with sterile a n d Pseudomonas aeruginosa-inoculated m e d i a w e r e m a d e in sterile p h o s p h a t e - b o r a t e b u f f e r + 0.5 M NaC1. F o r b o t h Eb a n d t i d e t e r m i -

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nations the starting potential value was - 0 . 7 V ( S C E ) , a potential at which the original Fe203 layer is completely electroreduced (see Fig. 6 below). This procedure allows the separation of the effect of microbial deposits on the corrosion resistance of 304 SS from differences in the passive film properties induced by metabolites. For this purpose, specimens of 304 SS kept in contact with phosphate-borate buffer with 10 -2 M sucrose for 7 days at open circuit were transferred to an electrochemical cell containing phosphate-borate buffer + 0.5 M NaC1 purged with purified argo n. It should be noted that in this case the elimination of oxygen is required in order to obtain reliable values of Eu from potentiodynamic runs. The current 0)-potential (E) profiles recorded at v = 1.6 x 10 -4 V s -1 from - 0 . 7 V(SCE) upwards showed a passive region extending up to a certain critical value, Eb (Fig. 4). At potentials more positive than Eb, the j/E profiles exhibit a marked current increase due to the nucleation and growth of pits on the metal surface, s The dynamics of this process involve current bursts due to the initiation and repassivation of pits even at potentials more negative than Eb .8 During pitting a white solid, probably ferrous phosphate, precipitates from the pitted regions of the specimens falling to the bottom of the cell. Localized corrosion is a complex process in both time s and space 11 so that it can be better described as a stochastic rather than a deterministic process due to the large scatter in the localized corrosion parameters such as Eu and t i. A statistical analysis is required to obtain a reliable interpretation of the localized corrosion process. As previously reported 8 a useful parameter is the probability of forming at least one stable localized corrosion center (Pn~>l) on the metal surface which is defined as:

Pn>~l = Ni(E)/Nt

(1)

where Ni(E) is the number of specimens that develop pitting at potentials <~ E, a preset potential value, and N t is the total number of specimens. The Pn~>l vs E plot derived from thej/E data and calculated through equation (1) is shown in Fig. 5(a). It can be seen that, as E moves in the positive direction, Pn>~l increases, and for E > 0.3 V, Pn~l :=> 1. Similar runs were made for 304 SS specimens kept in contact with the phosphate-borate buffer + 10 -2 M sucrose containing Pseudomonas aeruginosa for 7 days (Fig. 5b). Despite the fact that the medium for these experiments is oxygen-free due to argon bubbling, no damage in the adhesion of microbial deposits formed on the 304 SS could be found. In this case, the Pn>>-ivs E plot showed a behaviour similar to that found for specimens in contact with the bacteria-free medium except that Pn~>l ~ 1 for E > 0.2 V (Fig. 5b). This result indicated that the breakdown of the passive film leading to localized corrosion was enhanced in the bacteria-containing medium. This conclusion is supported by measurements of corresponding induction times, 11 ti. For the 304 SS specimens kept for 7 days in a medium free of Pseudomonas aeruginosa, the mean value of li at E = 0.3 V(SCE) was 28.4 s (averaged over seven samples). For runs made with specimens kept for 7 days in a medium containing micro-organisms, at E = 0.2 V(SCE), fi = 17.6 s, despite the fact that this figure was obtained at a potential lower than that of specimens passivated in the absence of Pseudomonas aeruginosa. Possible changes in the passive film properties of specimens in contact with the microbial culture were studied from cathodic polarization curves run in sterile phosphate-borate to avoid the interference of metabolites in the medium. For 304 SS kept in contact with the phosphate-borate buffer + 1 0 - 2 M sucrose for 7 days, the cathodic polarization curves run at 0.1 V s -1 from the open circuit potential to

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(a) Microbial deposits formed on 304 SS after 7 days of exposure to a Pseudomonas aeruginosa inoculated media, (b) detail showing the structure of the deposit.

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FIG. 7. Micrographs showing pits formed on 304 SS in phosphate-borate buffer containing 0.5M NaCI: (a) specimens in contact for 7 days with sterile phosphate-borate buffer + 10-2M sucrose, (b) specimens in contact for 7 days with phosphate-borate buffer + 10 -2 M sucrose inoculated with Pseudomonas aeruginosa. FIG. 8.

SEM micrographs showing bacteria attached to the 304 SS surface.

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FIG. 4. j/E profiles for a 304 SS specimen recorded at 1.6 × 10 -.4 V s i in the p h o s p h a t e borate buffer + 0.5 M NaCI: (a) 304 SS kept in contact for 7 days with the sterile p h o s p h a t e borate buffer + 10 -2 M sucrose; (b) 304 SS kept in contact for 7 days with the phosphate-borate buffer + 10 -2 M sucrose inoculated with Pseudomonas aeruginosa.

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FIG. 5. P,,>~ VS E plots: (a) specimens in contact for 7 days with the sterile p h o s p h a t e borate buffer + 10 -2 M sucrose, (b) specimens in contact for 7 days with p h o s p h a t e - b o r a t e buffer + 10 -2 M sucrose inoculated with Pseudomonas aeruginosa.

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J. MORALESet al.

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-0.5 ~.(v(scB)) FIG. 6. Cathodic]/Eprofilesrecorded at 0.1 V s- 1: (a) specimensin contactfor 7 dayswith the sterile phosphate-borate buffer + 10-2 M sucrose, (b) specimensin contact for 7 days with phosphate-borate buffer + 10-2 M sucroseinoculatedwithPseudomonasaeruginosa. - 1 . 3 V(SCE) in the phosphate-borate buffer showed a broad peak extending from - 0 . 5 V up to the hydrogen evolution reaction potential region (Fig. 6a). This current peak corresponds to the electroreduction of Fe203.xH20 to Fe (II) species. 8'9 The mean value of the charge density (averaged over 18 specimens) is q ~ 5.7 mC cm -2, which corresponds to an apparent film thickness in the order of 8 nm. The cathodic polarization curves recorded for the 304 SS specimens immersed for 7 days in the Pseudomonas aeruginosa-containing medium (Fig. 6b) yield q = 4.2 mC cm -2. This figure suggests that the passive film thickness becomes slightly thinner for those specimens which have been kept in contact with the Pseudomonas aeruginosa culture. SEM observations The SEM micrographs of the 304 SS specimens in contact with the phosphateborate buffer + 0.5 M NaC1 for 7 days obtained after the potentiodynamic runs showed the presence of hemispherical pits with electropolished bottoms, randomly distributed on the metal surface (Fig. 7a). For the 304 SS specimens kept in contact with bacteria containing medium, the localized corrosion process results in irregular shaped pits (Fig. 7b). A closer inspection of the micrographs also revealed the presence of isolated bacteria (Fig. 8) attached to the metal surface as well as some crystals. The bacteria shape (1-3/~m length, 0.8/~m width) is close to that expected for Pseudomonas aeruginosa (1-2/~m length, 0.5 Izm width) considering that some distortion has occurred as no fixation procedure was used after the 304 SS specimen was removed from the medium. DISCUSSION The experimental results allow the conclusion that Pseudomonas aeruginosa enhances the breakdown of passivity of 304 SS in CI- ions-containing medium. This

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ion plays a relevant role as no corrosion was detected in the inoculated medium in its absence. No differences have been detected in the open circuit potential measurements between the inoculated and the sterile media. An increase in the oxygen electroreduction rate due to biofilm formation6 can be discarded. Thus, in principle, the bacteria-enhanced effect on the localized corrosion of 304 SS can be assigned to microbial deposits attached to the metal surface promoting localized corrosion and/ or the production of aggressive metabolites which can decrease the passive film resistance. The contribution of the first effect can be concluded considering the results involving 304 SS specimens kept for 7 days in sterile and Pseudomonas aeruginosa inoculated media. For both types of specimens, the j/E curves run at v -- 1.6 x 10 - 4 V s - 1 in the phosphate-borate buffer + 0.5 M'NaCI (Fig. 4) were initiated at -0.7 V(SCE), i.e. a potential at which the outer Fe203.xH20 layer is already completely electroreduced (Fig. 6). Then, as the potential moves in the positive direction the outer passive layer is equally reformed in both cases. For the specimens kept in Pseudomonas aeruginosa-inoculated media, the pitting probability is shifted negatively indicating that their resistance to localized corrosion was decreased. This conclusion was further supported by the ti measurements for 304 SS specimens held at -0.7 V(SCE) for 30 min. In this case, a marked decrease in the 6 value for those specimens kept in contact with the bacteria containing medium was observed. This decrease cannot be assigned to changes in the properties of the outer passive layer. In principle, changes of the inner C r 2 0 3 layer promoted by bacterial metabolites could be expected. There is no way to test for these effects. The irregular shape of pits formed on those specimens in contact with the bacteria-containing medium (Fig. 7b) suggests that localized corrosion starts at sites under microbial deposits (Fig. 3). A strong support for this possibility is obtained by comparing P,~ 1

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J. MORALESet al.

for specimens kept in contact with bacteria-containing media to that obtained for specimens with artificial crevices in the same experimental conditions 12 (Fig. 9). A good correlation is found between both curves indicating that biological deposits promote localized corrosion in a way similar to that observed in artificial crevices. It should be noted that the open circuit potentials in the bacteria-containing media lie in the potential region where crevice corrosion becomes possible according to the en>~l v s E plot (Fig. 9). The presence of bacteria caused as a second effect a certain apparent thinning of the external FeeO3.xH20 layer (Fig. 6). In this case one can observe a decrease in the electroreduction charge related to the outer passive layer, suggesting that bacterial growth promoted a decrease in the passive film resistance probably by producing complexing substances or a passive film with a more open structure. CONCLUSIONS

The present results suggest that Pseudomonas aeruginosa decreased the resistance of 304 SS in neutral buffered solutions by acting in two different ways: firstly by enhancing localized corrosion under microbial deposits, and secondly by thinning the external passive film layer due to metabolites production. Acknowledgements--The authors thank UNELCO S.A. (Union Electrica de Canarias S.A.) for the financial support of this work. R. C. Salvarezza thanks CONiCET (Argentina) for supporting the cooperation programme between La Laguna and INIFTA.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

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