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Evolution of oxygen reduction current and biofilm on stainless steels cathodically polarised in natural aerated seawater
Electrochimica Acta 54 (2008) 148–153
Contents lists available at ScienceDirect
Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Evolution of oxygen reduction current and biofilm on stainless steels cathodically polarised in natural aerated seawater ` Marco Faimali ∗ , Elisabetta Chelossi, Francesca Garaventa, Christian Corra, Giuliano Greco, Alfonso Mollica ISMAR–CNR, Via De Marini 6, 16149 Genoa, Italy
a r t i c l e
i n f o
Article history: Received 30 August 2007 Received in revised form 6 February 2008 Accepted 25 February 2008 Available online 14 March 2008 Keywords: Biofilm Cathodic polarisation Oxygen reduction Passive layer Stainless Steels
a b s t r a c t The aim of a series of works recently performed at ISMAR was to provide new useful information for a better understanding of the mechanisms by which bacteria settlement causes corrosion on Stainless Steels (SS) and similar active–passive alloys exposed to seawater. In this work, the evolutions of cathodic current, bacteria population, and electronic structure of the passive layer were investigated on SS samples polarised at fixed potentials during their exposure to natural seawater. It was found that, during the first phase of biofilm growth, cathodic current increase is proportional to the number of settled bacteria at each fixed potential. However, the proportionality factor between settled bacteria and cathodic current depends on imposed potential. In particular, the proportionality factor strongly decreases when the potential is increased above a critical value close to −150 mV Ag/AgCl. This effect seems to be correlated with the electronic structure of the passive layer. Indeed, the outer part of the passive layer on tested SS was found to behave like a conductor at potentials more active than −150 mV Ag/AgCl, and like an n-type semiconductor at more noble potentials. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction It is widely recognised that, in seawater, cathodic processes run faster on Stainless Steels (SS) when biofilm forms on the metal surface. It is also generally accepted that this phenomenon – called “cathodic depolarisation” – accounts for higher natural seawater corrosiveness, than sterile water, toward SS and similar active–passive alloys. However, although several hypotheses have been proposed [1–12], the mechanism by which the biofilm causes “cathodic depolarisation” is still unclear. Actually, the presence of hydrogen peroxide and/or low pH inside the biofilm [1,2,6,9,11], the effect of enzymes or metal–organic compounds [3,10,11,12], the role of specific bacteria like manganese oxidizing bacteria [5,7] were some of the factors suggested by different authors as responsible of “cathodic depolarisation”. The aim of a series of works performed at ISMAR was to provide field test evidence, in order to select, from among the proposed hypotheses, the most suitable ones accounting for SS cathodic behaviour in natural seawater.
∗ Corresponding author. Tel.: +39 0106475425; fax: +39 0106475400. E-mail address: [email protected] (M. Faimali). 0013-4686/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2008.02.115
In this work, in particular, the evolutions of cathodic current, bacteria population, and electronic structure of the passive layer on SS samples exposed to natural seawater and polarised at fixed cathodic potentials were concurrently investigated and then correlated. 2. Materials and methods 2.1. Polarisation Several experiments on SS exposed to natural seawater and cathodically polarised were carried out at the ISMAR Marine Station, located in the port of Genoa, Italy. SS samples were immersed in seawater contained in a tank of about 100 L; water in the tank was constantly changed, at a rate of about 1.5–2 L min−1 , with natural seawater directly pumped from the sea. Tests were performed during the January–July 2005 period when seawater had the following main characteristics: 12 < T < 27 ◦ C, 6 < dissolved oxygen < 8 mg/L, 8 < pH < 8.2, 33.5 < salinity < 35.5‰. The system employed for concurrent polarisation of several SS samples at a fixed cathodic potential is shown in Fig. 1. An Ag/AgCl electrode filled with KCl 3 M (INGOLD 103633040) was used as reference electrode. Stainless Steel, 1 mm thick samples (25 mm × 10 mm), cut from a plate of a commercial super-austenitic alloy (UNSS 31254:
M. Faimali et al. / Electrochimica Acta 54 (2008) 148–153
149
Fig. 1. Set-up used for concurrent polarisation of several SS samples.
Cr = 19.5–20.5%, Ni = 17.5–18.5%, Mo = 6–6.5%, N = 0.18–0.22%, Cu = 0.5–1%, S < 0.01%, Si < 0.8%, P < 0.03%, Mn < 1%, C < 0.02%, Fe = balance), drilled (Ø = 1.6 mm), screwed, and then treated with emery papers up to P1200, were used for all tests. Electrical connections between SS samples in the solution and wires outside, in turn connected to a potentiostat, were performed through Titanium rods (Ø = 2 mm) suitably screwed at one hand. Titanium rod surface exposed to seawater was a negligible fraction of connected SS specimen surface (STi /SSS < 5%). The current flowing from the nth polarised sample (see Fig. 1) is calculated from the ohmic drop Vn measured across the resistor Rn , whose value was chosen in order to measure a Vn less than 10 mV. During the tests, cathodic current was regularly measured and recorded on all polarised samples. 2.2. Biofilm observation and image analysis SS specimens, joined with the relevant titanium rods, were removed from the test tank, gently rinsed in seawater sterilised by filtration (Millipore, 0.22 m pore size) to remove any unattached cells, fixed with 2% paraformaldehyde solution for 30 min, and washed in filtered phosphate buffer saline (PBS). Then, titanium rods were detached and finally SS samples were stored at 4 ◦ C in PBS before staining and microscopic analysis. After staining of bacterial cells with DAPI (4 -6-diamidino2-phenylindole, Sigma) [13], samples were observed at 400× magnification using an Olympus BX41 epifluorescence microscope coupled with an UV filter block for DAPI. A digital camera CAMEDIA 5060 (Olympus) was used to acquire images of 30 areas of 11 500 m2 randomly chosen on the SS surface of each sample. Images were transformed to tiff format (RGB colour) and analyzed by means of “Image J” software [14]. The surface fraction covered by bacteria was measured and the number of settled bacteria was then obtained by dividing the covered area by the mean area covered by a single bacterial cell, in turn defined as the mean value of 60 measures.
At each potential step (E), a sinusoidal wave (10 mV amplitude, 1590 Hz) was added for electrode impedance measurement (Schlumberger Mod. 1286 and 1255) and then capacitance C(E) calculation. For reasons that will be explained later on in this work, data will be plotted in [C(−350 mV)/C(E)]2 vs. E form instead of the usual Mott–Schottky representation ([1/C(E)]2 vs. E). At the end of each test, the following models were applied to fit relevant experimental data. 2.4. Model applied to describe cathodic current evolution Model [16] is based on the following assumptions (Fig. 2): (a) current density iE (t), measured at time t on a SS sample polarised at a fixed potential E, is the result of two oxygen reduction currents, concurrently running on complementary surface fractions: a slow cathodic current density, i1,E , which flows initially on the whole SS surface, and a fast one, i2,E , which flows on the surface fraction covered by “active sites for oxygen reduction” whose extent, (t), increases in time until complete coverage of the SS surface. It follows: iE (t) = i1,E [1 − (t)] + i2,E (t) = i1,E + [i2,E − i1,E ](t)
0 ≤ (t) ≤ 1
(1a)
(b) For (t), the following form of logistic expression was used: (t) =
2((t−t0 )/c ) [2((t−t0 )/c ) + 999]
(1b)
2.3. Passive layer electronic structure Information on the evolution of the passive layer electronic structure during SS polarisation at a fixed potential was obtained through the Mott–Schottky approach [15]. An SS sample normally polarised at E0 was temporarily submitted to fast potentiodynamic polarisation ranging from −350 to 350 mVAg/AgCl in steps of 50 mV every 0.2 s.
Fig. 2. Trend of cathodic current on polarised SS in natural seawater foreseen by the model. (Eqs. (1a) and (1b) continuous line; Eq. (1c) dotted line).
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Fig. 3. Trends of bacterial population density foreseen by the model (Eq. (2a) continuous line; Eq. (2c) dotted line).
in which “incubation time” t0 , defined here as the time necessary to reach (t0 ) = 10−3 coverage, and “current duplication time” c appear. The values of four parameters (i1,E , i2,E , t0 , and c ) must hence be fixed, when the model is applied to fit data measured during cathodic current evolution on polarised samples. A continuous black line in Fig. 2 shows the general trend of cathodic current foreseen by the model and the meaning of the mentioned four parameters. It can be shown that, when 10−3 (t) 1, the following simplified expression can be applied (dotted line in Fig. 2): iE (t) = a2(t/c )
(1c)
2.5. Model applied to describe the evolution of the first phases of bacteria settlement The evolution of settled bacteria density on SS samples polarised at a fixed potential E was described with a model based on the assumption that a biofilm initially develops according to the following steps (Fig. 3): (a) planktonic bacteria in seawater can settle on the SS surface with a given probability p, (b) a settled bacteria can, later on, generate another bacteria which settles in turn, being b the apparent duplication time.
Fig. 4. Evolution of cathodic current delivered by 20 SS samples polarised at −200 mVAg/AgCl in natural seawater at T = 12–13 ◦ C. The continuous line in the figure was calculated from Eqs. (1a) and (1b) assuming: i1 = 0.06 A cm−2 ; i2 = 40 A cm−2 ; t0 = 3.2 days; c = 0.5 days. The dotted line is calculated from the Eq. (1c) assuming the same value of the duplication time for the current, c = 0.5 days.
3. Results and discussion In the example shown in Fig. 4, field data describing cathodic current increase on 20 SS samples polarised at −200 mV Ag/AgCl and exposed to seawater at T = 12–13 ◦ C are reported. The continuous line in the figure shows the best data fit obtained from Eqs. (1a) and (1b) calculated assuming i1 = 0.06 A cm−2 ; i2 = 40 A cm−2 ; t0 = 3.2 days; c = 0.5 days. It can be seen that the model based on the idea that the area covered by “active sites for fast oxygen reduction” develops with time until a complete metal surface coverage can be used to successfully fit the trend of cathodic currents observed on polarised samples. Further, it can be seen that the dotted line, calculated from the simplified Eq. (1c) with the same c = 0.5 days, is sufficient to correctly describe cathodic current trend in between 4 < t < 9 days. Fig. 5 shows settled bacteria density progression as observed on SS samples taken in the mentioned exposure time interval (4 < t < 9
From these assumptions, it follows that the number, NE (t), of settled bacteria at a given time t is: NE(t) =
pb [2(t/b ) − 1] ln(2)
(2a)
The values of two parameters (p, b ) must, hence, be fixed when the model is used to fit the experimental data describing bacteria settlement evolution. Eq. (2a) is valid if no other factors – such as mutual disturbance between settled bacteria due to excessive biofilm development – hinder biofilm growth. Eq. (2a) can, hence, be used for a relatively low surface biofilm coverage. Fig. 3 shows the trend of bacterial population density foreseen by the model. Depending on considered exposure time, Eq. (2a) can be approximated by NE (t) = bt
if t b
(2b)
and by NE (t) = c2(t/b )
if t b
(2c)
Fig. 5. Evolution of settled bacteria density on SS samples polarised at −200 mVAg/AgCl in natural seawater at T = 12–13 ◦ C; data are represented as M ± S.E., N = 30. The line in the figure was calculated from the simplified Eq. (2c) assuming b = 0.5 days as apparent duplication time of the settled bacteria.
M. Faimali et al. / Electrochimica Acta 54 (2008) 148–153
Fig. 6. Cathodic current vs. settled bacteria on SS samples polarised at −200 mVAg/AgCl in natural seawater at T = 12–13 ◦ C; the line in the figure was calculated assuming ˛ = 2 × 10−7 A bacteria−1 as proportionality factor. Bacterial density is represented as M ± S.E., N = 30.
days); the continuous line in the figure was calculated from the simplified Eq. (2c) assuming b = 0.5 days as apparent duplication time of settled bacteria. Being duplication time for the current equal to duplication time for the settled bacteria ( c = b = 0.5 days) it follows, from Eqs. (1c) and (2c), that 2t/0.5 =
iE (t) NE (t) = a c
(3)
and, hence, that, at each instant during the first phases of biofilm growth, the current is simply proportional to the number of settled bacteria: iE = ˛NE
(4)
This is confirmed by the graph in Fig. 6, in which the relevant data taken from Figs. 4 and 5 are concurrently plotted: the line in the figure was calculated from Eq. (4) assuming ˛ = 2 × 10−7 A bacteria−1 as proportionality factor. Additional tests were organised to verify whether the proportionality between settled bacteria and cathodic current observed on SS samples polarised at −200 mV Ag/AgCl is confirmed at other imposed potentials. For this purpose, groups of SS samples were polarised, at −300, −200, −100, 0 and 100 mV Ag/AgCl, respectively; seawater temperature and quality were the same for all tests. For each group of polarised samples, data plotted in Fig. 7 show the cathodic current measured on a specimen just before being taken out of the exposure tank and the bacterial population later observed on the same sample. Data measured at a fixed potential are fitted by a line whose slope is always unitary in the log (i) − log(bacteria density) plot as foreseen by Eq. (4); however, the more shifted is the line towards the graph bottom, the more noble is the potential. This means that, while cathodic current is always proportional to biofilm amount, the proportionality factor ˛ (A bacteria−1 ) decreases if the imposed potential is ennobled. In Fig. 8, the proportionality factors, calculated from the data shown in Fig. 7 as well as from the results of additional tests not reported here, are plotted vs. imposed potential. The line in Fig. 8, which looks like a cathodic polarisation curve related to a single settled bacteria, shows that the mean “cathodic
151
Fig. 7. Cathodic current vs. settled bacteria on SS samples polarised at −300, −200, −100, 0 and 100 mV Ag/AgCl in natural seawater at T = 12–13 ◦ C. Bacterial density is represented as M ± S.E., N = 30.
efficiency” of a bacteria sharply decreases when the potential is ennobled by a value ranging between −200 and −100 mV Ag/AgCl. An attempt was made to verify if this effect could be correlated, as suggested by previous tests [17], to a change in the passive layer electronic structure induced by a change in imposed potential. To get information about the passive layer electronic structure, the Mott–Schottky approach – a fast and not destructive technique applicable in situ – was used. Fig. 9 shows two examples of Mott–Schottky plots obtained on the SS type used in this work: the line connecting triangles shows the plot obtained on a sample polarised for some days at E0 = 100 mV Ag/AgCl (i.e. the most noble potential tested in this work), whereas the line connecting square symbols shows part of the plot obtained on the same sample some hours after the imposed potential was brought to −300 mV Ag/AgCl (i.e. the most active potential tested in this work).
Fig. 8. Proportionality factor, ˛ (A bacteria−1 ), between cathodic current and settled bacteria vs. imposed potential.
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The interpretation of the graphs in Fig. 9 is the following [15]:
Fig. 9. Mott–Schottky plots on SS UNSS31254 after 3 days of polarisation at E0 = 100 mV Ag/AgCl (triangles) and some hours since the imposed potential was changed to −300 mV Ag/AgCl (squares).
• starting from the left of the graph, an initial 1/C(E)2 linear decrease vs. potential is observed. This behaviour is due to the presence on the steel surface of a passive layer whose inner part, rich in chromium, behaves like a p-type semiconductor; • it follows a potential range, from roughly −600 to −400 mV Ag/AgCl, where the graph is almost flat; • a further potential increase causes an increase in 1/C(E)2 . This trend describes the behaviour of an n-type semiconductor due to the outer part of the passive layer which is rich in iron compounds. The higher the initial slope of the graph, the less defective is the semiconductor, being the slope inversely proportional to charge carrier density; • the slope of Mott–Schottky plot at a potential higher than −400/−350 mV Ag/AgCl provides, therefore, information on charge carrier density in the external layer of the passive film. Carrier density depends, in turn, on iron oxidation degree (␥-Fe2 O3 behaves like an n-type semiconductor, whereas Fe3 O4 behaves as a metallic conductor). Therefore, it depends on the potential E0 normally imposed during the test (see slope decrease due to more active potential imposition). In order to get information on the role of imposed potential and, eventually, of biofilm growth on the electronic structure of the outer
Fig. 10. Top-left side: cathodic current evolution plot on five SS samples polarised at −300, −200, −100, 0 and 100 mV Ag/AgCl, respectively. Mott–Schottky plots were obtained on all samples after 0.1, 0.2, 1, 2, 5, 15, and 39 days since the beginning of the test (see symbols on the central line).
M. Faimali et al. / Electrochimica Acta 54 (2008) 148–153
part of the passive layer, the Mott–Schottky approach was repeatedly applied (Fig. 10) on five SS samples polarised at −300, −200, −100, 0 and 100 mV Ag/AgCl, respectively. On the top-left side of Fig. 10, cathodic current trends observed on the polarised samples are shown; under all tested potentials, cathodic current starts to increases within the first 10 days of exposure as a result of biofilm growth. To obtain data during each phase of biofilm growth, measures for the construction of Mott–Schottky plots were performed on all tested samples after 0.1, 0.2, 1, 2, 5, 15, and 39 days since the beginning of the test. Fig. 10 shows that, at each of the tested potentials, the shape of the plots was essentially the same throughout the test: therefore, charge carrier density in the external layer of the passive film does not depend on the presence or absence of biofilm on the SS surface. Conversely, the electronic structure of the outer part of the passive layer depends on imposed potential. An initial clearly positive slope of the plots – describing the behaviour of n-type semiconductor and hence the prevailing presence of Fe3+ in the outer part of passive layer can be observed only if the imposed potential is greater than −200/−100 mV Ag/AgCl. This fact confirms that a change in the electronic structure of the outer part of the passive layer (Fig. 10) takes place at the same potential level which separates high from low “cathodic efficiency” associated with a single settled bacteria (Fig. 8). 4. Conclusions Field tests on cathodically polarised SS in natural aerated seawater showed that: • at each imposed potential, cathodic current increase is proportional to the amount of settled bacteria during the first phases of biofilm development; • the proportionality factor between settled bacteria and cathodic current depends on imposed potential and, in particular, it
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strongly decreases when the potential is ennobled above a critical value close to −150 mV Ag/AgCl. This effect appears correlated to the electronic structure of the outer part of the passive layer on polarised SS samples; • impedance measurements (Mott–Schottky plots) showed, in fact, that the outer part of the passive layer on SS behaves like a conductor, if it has developed at potentials more active than −150 mV Ag/AgCl, and like an n-type semiconductor at more noble potentials. Acknowledgement This work was partly supported by the EA-Biofilms NEST508866 Project of the 6th European FP. References [1] S. Dexter, S.H. Lin, Proc. 7th Int. Congress on Marine Corrosion and Fouling, Valencia, Spain, 1988. [2] P. Chandrasekaran, S. Dexter, Corrosion/93, paper N◦ 493, NACE, Houston, TX, 1993. [3] V. Scotto, R. DiCintio, G. Marcenaro, Corros. Sci. 25 (1985) 185. [4] M. Eashwar, S. Maruthamuthu, Biofouling 8 (1995) 203. [5] W.H. Dickinson, F. Caccavo, Z. Lewandowski, Corros. Sci. 38 (1996) 1407. [6] H. Amaya, H. Miyuki, Corros. Eng. 46 (1997) 567. [7] W.H. Dickinson, F. Caccavo Jr., B. Olesen, Z. Lewandowski, Appl. Environ. Microbiol. 63 (1997) 2502. [8] V. Scotto, M. Rapallino, Second seminar on Enzymes and Corrosion, Paris, France, 2000. [9] N. Washizu, Y. Katada, T. Kodama, Corros. Sci. 46 (2004) 1291. [10] R. Johnsen, E. Bardal, Corrosion 41 (1985) 296. ´ [11] I. Dupont, D. Feron, G. Novel, Int. Biodeter. Biodegr. 41 (1998) 13. [12] V. Scotto, M.E. Lai, Corros. Sci. 40 (1) (1998) 1007. [13] K. Takata, H. Hirano, Acta Histochem. Cytochem. 23 (1990) 679. [14] Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, 1997–2005, http://rsb.info.nih.gov/ij/. [15] E. Hakiki, S. Boudin, B. Rondot, M. Da Cunha Belo, Corros. Sci. 37 (1995) 1809. [16] Mollica, E. Traverso, D.Thierry, Publications N◦ 22 in European Federation of Corrosion Series, Institute of Materials, London, 1997, p. 51. [17] A. Mollica, E. Bardal, Second seminar on Enzymes and Corrosion, Paris, France, 2000.
Contents lists available at ScienceDirect
Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Evolution of oxygen reduction current and biofilm on stainless steels cathodically polarised in natural aerated seawater ` Marco Faimali ∗ , Elisabetta Chelossi, Francesca Garaventa, Christian Corra, Giuliano Greco, Alfonso Mollica ISMAR–CNR, Via De Marini 6, 16149 Genoa, Italy
a r t i c l e
i n f o
Article history: Received 30 August 2007 Received in revised form 6 February 2008 Accepted 25 February 2008 Available online 14 March 2008 Keywords: Biofilm Cathodic polarisation Oxygen reduction Passive layer Stainless Steels
a b s t r a c t The aim of a series of works recently performed at ISMAR was to provide new useful information for a better understanding of the mechanisms by which bacteria settlement causes corrosion on Stainless Steels (SS) and similar active–passive alloys exposed to seawater. In this work, the evolutions of cathodic current, bacteria population, and electronic structure of the passive layer were investigated on SS samples polarised at fixed potentials during their exposure to natural seawater. It was found that, during the first phase of biofilm growth, cathodic current increase is proportional to the number of settled bacteria at each fixed potential. However, the proportionality factor between settled bacteria and cathodic current depends on imposed potential. In particular, the proportionality factor strongly decreases when the potential is increased above a critical value close to −150 mV Ag/AgCl. This effect seems to be correlated with the electronic structure of the passive layer. Indeed, the outer part of the passive layer on tested SS was found to behave like a conductor at potentials more active than −150 mV Ag/AgCl, and like an n-type semiconductor at more noble potentials. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction It is widely recognised that, in seawater, cathodic processes run faster on Stainless Steels (SS) when biofilm forms on the metal surface. It is also generally accepted that this phenomenon – called “cathodic depolarisation” – accounts for higher natural seawater corrosiveness, than sterile water, toward SS and similar active–passive alloys. However, although several hypotheses have been proposed [1–12], the mechanism by which the biofilm causes “cathodic depolarisation” is still unclear. Actually, the presence of hydrogen peroxide and/or low pH inside the biofilm [1,2,6,9,11], the effect of enzymes or metal–organic compounds [3,10,11,12], the role of specific bacteria like manganese oxidizing bacteria [5,7] were some of the factors suggested by different authors as responsible of “cathodic depolarisation”. The aim of a series of works performed at ISMAR was to provide field test evidence, in order to select, from among the proposed hypotheses, the most suitable ones accounting for SS cathodic behaviour in natural seawater.
∗ Corresponding author. Tel.: +39 0106475425; fax: +39 0106475400. E-mail address: [email protected] (M. Faimali). 0013-4686/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2008.02.115
In this work, in particular, the evolutions of cathodic current, bacteria population, and electronic structure of the passive layer on SS samples exposed to natural seawater and polarised at fixed cathodic potentials were concurrently investigated and then correlated. 2. Materials and methods 2.1. Polarisation Several experiments on SS exposed to natural seawater and cathodically polarised were carried out at the ISMAR Marine Station, located in the port of Genoa, Italy. SS samples were immersed in seawater contained in a tank of about 100 L; water in the tank was constantly changed, at a rate of about 1.5–2 L min−1 , with natural seawater directly pumped from the sea. Tests were performed during the January–July 2005 period when seawater had the following main characteristics: 12 < T < 27 ◦ C, 6 < dissolved oxygen < 8 mg/L, 8 < pH < 8.2, 33.5 < salinity < 35.5‰. The system employed for concurrent polarisation of several SS samples at a fixed cathodic potential is shown in Fig. 1. An Ag/AgCl electrode filled with KCl 3 M (INGOLD 103633040) was used as reference electrode. Stainless Steel, 1 mm thick samples (25 mm × 10 mm), cut from a plate of a commercial super-austenitic alloy (UNSS 31254:
M. Faimali et al. / Electrochimica Acta 54 (2008) 148–153
149
Fig. 1. Set-up used for concurrent polarisation of several SS samples.
Cr = 19.5–20.5%, Ni = 17.5–18.5%, Mo = 6–6.5%, N = 0.18–0.22%, Cu = 0.5–1%, S < 0.01%, Si < 0.8%, P < 0.03%, Mn < 1%, C < 0.02%, Fe = balance), drilled (Ø = 1.6 mm), screwed, and then treated with emery papers up to P1200, were used for all tests. Electrical connections between SS samples in the solution and wires outside, in turn connected to a potentiostat, were performed through Titanium rods (Ø = 2 mm) suitably screwed at one hand. Titanium rod surface exposed to seawater was a negligible fraction of connected SS specimen surface (STi /SSS < 5%). The current flowing from the nth polarised sample (see Fig. 1) is calculated from the ohmic drop Vn measured across the resistor Rn , whose value was chosen in order to measure a Vn less than 10 mV. During the tests, cathodic current was regularly measured and recorded on all polarised samples. 2.2. Biofilm observation and image analysis SS specimens, joined with the relevant titanium rods, were removed from the test tank, gently rinsed in seawater sterilised by filtration (Millipore, 0.22 m pore size) to remove any unattached cells, fixed with 2% paraformaldehyde solution for 30 min, and washed in filtered phosphate buffer saline (PBS). Then, titanium rods were detached and finally SS samples were stored at 4 ◦ C in PBS before staining and microscopic analysis. After staining of bacterial cells with DAPI (4 -6-diamidino2-phenylindole, Sigma) [13], samples were observed at 400× magnification using an Olympus BX41 epifluorescence microscope coupled with an UV filter block for DAPI. A digital camera CAMEDIA 5060 (Olympus) was used to acquire images of 30 areas of 11 500 m2 randomly chosen on the SS surface of each sample. Images were transformed to tiff format (RGB colour) and analyzed by means of “Image J” software [14]. The surface fraction covered by bacteria was measured and the number of settled bacteria was then obtained by dividing the covered area by the mean area covered by a single bacterial cell, in turn defined as the mean value of 60 measures.
At each potential step (E), a sinusoidal wave (10 mV amplitude, 1590 Hz) was added for electrode impedance measurement (Schlumberger Mod. 1286 and 1255) and then capacitance C(E) calculation. For reasons that will be explained later on in this work, data will be plotted in [C(−350 mV)/C(E)]2 vs. E form instead of the usual Mott–Schottky representation ([1/C(E)]2 vs. E). At the end of each test, the following models were applied to fit relevant experimental data. 2.4. Model applied to describe cathodic current evolution Model [16] is based on the following assumptions (Fig. 2): (a) current density iE (t), measured at time t on a SS sample polarised at a fixed potential E, is the result of two oxygen reduction currents, concurrently running on complementary surface fractions: a slow cathodic current density, i1,E , which flows initially on the whole SS surface, and a fast one, i2,E , which flows on the surface fraction covered by “active sites for oxygen reduction” whose extent, (t), increases in time until complete coverage of the SS surface. It follows: iE (t) = i1,E [1 − (t)] + i2,E (t) = i1,E + [i2,E − i1,E ](t)
0 ≤ (t) ≤ 1
(1a)
(b) For (t), the following form of logistic expression was used: (t) =
2((t−t0 )/c ) [2((t−t0 )/c ) + 999]
(1b)
2.3. Passive layer electronic structure Information on the evolution of the passive layer electronic structure during SS polarisation at a fixed potential was obtained through the Mott–Schottky approach [15]. An SS sample normally polarised at E0 was temporarily submitted to fast potentiodynamic polarisation ranging from −350 to 350 mVAg/AgCl in steps of 50 mV every 0.2 s.
Fig. 2. Trend of cathodic current on polarised SS in natural seawater foreseen by the model. (Eqs. (1a) and (1b) continuous line; Eq. (1c) dotted line).
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M. Faimali et al. / Electrochimica Acta 54 (2008) 148–153
Fig. 3. Trends of bacterial population density foreseen by the model (Eq. (2a) continuous line; Eq. (2c) dotted line).
in which “incubation time” t0 , defined here as the time necessary to reach (t0 ) = 10−3 coverage, and “current duplication time” c appear. The values of four parameters (i1,E , i2,E , t0 , and c ) must hence be fixed, when the model is applied to fit data measured during cathodic current evolution on polarised samples. A continuous black line in Fig. 2 shows the general trend of cathodic current foreseen by the model and the meaning of the mentioned four parameters. It can be shown that, when 10−3 (t) 1, the following simplified expression can be applied (dotted line in Fig. 2): iE (t) = a2(t/c )
(1c)
2.5. Model applied to describe the evolution of the first phases of bacteria settlement The evolution of settled bacteria density on SS samples polarised at a fixed potential E was described with a model based on the assumption that a biofilm initially develops according to the following steps (Fig. 3): (a) planktonic bacteria in seawater can settle on the SS surface with a given probability p, (b) a settled bacteria can, later on, generate another bacteria which settles in turn, being b the apparent duplication time.
Fig. 4. Evolution of cathodic current delivered by 20 SS samples polarised at −200 mVAg/AgCl in natural seawater at T = 12–13 ◦ C. The continuous line in the figure was calculated from Eqs. (1a) and (1b) assuming: i1 = 0.06 A cm−2 ; i2 = 40 A cm−2 ; t0 = 3.2 days; c = 0.5 days. The dotted line is calculated from the Eq. (1c) assuming the same value of the duplication time for the current, c = 0.5 days.
3. Results and discussion In the example shown in Fig. 4, field data describing cathodic current increase on 20 SS samples polarised at −200 mV Ag/AgCl and exposed to seawater at T = 12–13 ◦ C are reported. The continuous line in the figure shows the best data fit obtained from Eqs. (1a) and (1b) calculated assuming i1 = 0.06 A cm−2 ; i2 = 40 A cm−2 ; t0 = 3.2 days; c = 0.5 days. It can be seen that the model based on the idea that the area covered by “active sites for fast oxygen reduction” develops with time until a complete metal surface coverage can be used to successfully fit the trend of cathodic currents observed on polarised samples. Further, it can be seen that the dotted line, calculated from the simplified Eq. (1c) with the same c = 0.5 days, is sufficient to correctly describe cathodic current trend in between 4 < t < 9 days. Fig. 5 shows settled bacteria density progression as observed on SS samples taken in the mentioned exposure time interval (4 < t < 9
From these assumptions, it follows that the number, NE (t), of settled bacteria at a given time t is: NE(t) =
pb [2(t/b ) − 1] ln(2)
(2a)
The values of two parameters (p, b ) must, hence, be fixed when the model is used to fit the experimental data describing bacteria settlement evolution. Eq. (2a) is valid if no other factors – such as mutual disturbance between settled bacteria due to excessive biofilm development – hinder biofilm growth. Eq. (2a) can, hence, be used for a relatively low surface biofilm coverage. Fig. 3 shows the trend of bacterial population density foreseen by the model. Depending on considered exposure time, Eq. (2a) can be approximated by NE (t) = bt
if t b
(2b)
and by NE (t) = c2(t/b )
if t b
(2c)
Fig. 5. Evolution of settled bacteria density on SS samples polarised at −200 mVAg/AgCl in natural seawater at T = 12–13 ◦ C; data are represented as M ± S.E., N = 30. The line in the figure was calculated from the simplified Eq. (2c) assuming b = 0.5 days as apparent duplication time of the settled bacteria.
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Fig. 6. Cathodic current vs. settled bacteria on SS samples polarised at −200 mVAg/AgCl in natural seawater at T = 12–13 ◦ C; the line in the figure was calculated assuming ˛ = 2 × 10−7 A bacteria−1 as proportionality factor. Bacterial density is represented as M ± S.E., N = 30.
days); the continuous line in the figure was calculated from the simplified Eq. (2c) assuming b = 0.5 days as apparent duplication time of settled bacteria. Being duplication time for the current equal to duplication time for the settled bacteria ( c = b = 0.5 days) it follows, from Eqs. (1c) and (2c), that 2t/0.5 =
iE (t) NE (t) = a c
(3)
and, hence, that, at each instant during the first phases of biofilm growth, the current is simply proportional to the number of settled bacteria: iE = ˛NE
(4)
This is confirmed by the graph in Fig. 6, in which the relevant data taken from Figs. 4 and 5 are concurrently plotted: the line in the figure was calculated from Eq. (4) assuming ˛ = 2 × 10−7 A bacteria−1 as proportionality factor. Additional tests were organised to verify whether the proportionality between settled bacteria and cathodic current observed on SS samples polarised at −200 mV Ag/AgCl is confirmed at other imposed potentials. For this purpose, groups of SS samples were polarised, at −300, −200, −100, 0 and 100 mV Ag/AgCl, respectively; seawater temperature and quality were the same for all tests. For each group of polarised samples, data plotted in Fig. 7 show the cathodic current measured on a specimen just before being taken out of the exposure tank and the bacterial population later observed on the same sample. Data measured at a fixed potential are fitted by a line whose slope is always unitary in the log (i) − log(bacteria density) plot as foreseen by Eq. (4); however, the more shifted is the line towards the graph bottom, the more noble is the potential. This means that, while cathodic current is always proportional to biofilm amount, the proportionality factor ˛ (A bacteria−1 ) decreases if the imposed potential is ennobled. In Fig. 8, the proportionality factors, calculated from the data shown in Fig. 7 as well as from the results of additional tests not reported here, are plotted vs. imposed potential. The line in Fig. 8, which looks like a cathodic polarisation curve related to a single settled bacteria, shows that the mean “cathodic
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Fig. 7. Cathodic current vs. settled bacteria on SS samples polarised at −300, −200, −100, 0 and 100 mV Ag/AgCl in natural seawater at T = 12–13 ◦ C. Bacterial density is represented as M ± S.E., N = 30.
efficiency” of a bacteria sharply decreases when the potential is ennobled by a value ranging between −200 and −100 mV Ag/AgCl. An attempt was made to verify if this effect could be correlated, as suggested by previous tests [17], to a change in the passive layer electronic structure induced by a change in imposed potential. To get information about the passive layer electronic structure, the Mott–Schottky approach – a fast and not destructive technique applicable in situ – was used. Fig. 9 shows two examples of Mott–Schottky plots obtained on the SS type used in this work: the line connecting triangles shows the plot obtained on a sample polarised for some days at E0 = 100 mV Ag/AgCl (i.e. the most noble potential tested in this work), whereas the line connecting square symbols shows part of the plot obtained on the same sample some hours after the imposed potential was brought to −300 mV Ag/AgCl (i.e. the most active potential tested in this work).
Fig. 8. Proportionality factor, ˛ (A bacteria−1 ), between cathodic current and settled bacteria vs. imposed potential.
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The interpretation of the graphs in Fig. 9 is the following [15]:
Fig. 9. Mott–Schottky plots on SS UNSS31254 after 3 days of polarisation at E0 = 100 mV Ag/AgCl (triangles) and some hours since the imposed potential was changed to −300 mV Ag/AgCl (squares).
• starting from the left of the graph, an initial 1/C(E)2 linear decrease vs. potential is observed. This behaviour is due to the presence on the steel surface of a passive layer whose inner part, rich in chromium, behaves like a p-type semiconductor; • it follows a potential range, from roughly −600 to −400 mV Ag/AgCl, where the graph is almost flat; • a further potential increase causes an increase in 1/C(E)2 . This trend describes the behaviour of an n-type semiconductor due to the outer part of the passive layer which is rich in iron compounds. The higher the initial slope of the graph, the less defective is the semiconductor, being the slope inversely proportional to charge carrier density; • the slope of Mott–Schottky plot at a potential higher than −400/−350 mV Ag/AgCl provides, therefore, information on charge carrier density in the external layer of the passive film. Carrier density depends, in turn, on iron oxidation degree (␥-Fe2 O3 behaves like an n-type semiconductor, whereas Fe3 O4 behaves as a metallic conductor). Therefore, it depends on the potential E0 normally imposed during the test (see slope decrease due to more active potential imposition). In order to get information on the role of imposed potential and, eventually, of biofilm growth on the electronic structure of the outer
Fig. 10. Top-left side: cathodic current evolution plot on five SS samples polarised at −300, −200, −100, 0 and 100 mV Ag/AgCl, respectively. Mott–Schottky plots were obtained on all samples after 0.1, 0.2, 1, 2, 5, 15, and 39 days since the beginning of the test (see symbols on the central line).
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part of the passive layer, the Mott–Schottky approach was repeatedly applied (Fig. 10) on five SS samples polarised at −300, −200, −100, 0 and 100 mV Ag/AgCl, respectively. On the top-left side of Fig. 10, cathodic current trends observed on the polarised samples are shown; under all tested potentials, cathodic current starts to increases within the first 10 days of exposure as a result of biofilm growth. To obtain data during each phase of biofilm growth, measures for the construction of Mott–Schottky plots were performed on all tested samples after 0.1, 0.2, 1, 2, 5, 15, and 39 days since the beginning of the test. Fig. 10 shows that, at each of the tested potentials, the shape of the plots was essentially the same throughout the test: therefore, charge carrier density in the external layer of the passive film does not depend on the presence or absence of biofilm on the SS surface. Conversely, the electronic structure of the outer part of the passive layer depends on imposed potential. An initial clearly positive slope of the plots – describing the behaviour of n-type semiconductor and hence the prevailing presence of Fe3+ in the outer part of passive layer can be observed only if the imposed potential is greater than −200/−100 mV Ag/AgCl. This fact confirms that a change in the electronic structure of the outer part of the passive layer (Fig. 10) takes place at the same potential level which separates high from low “cathodic efficiency” associated with a single settled bacteria (Fig. 8). 4. Conclusions Field tests on cathodically polarised SS in natural aerated seawater showed that: • at each imposed potential, cathodic current increase is proportional to the amount of settled bacteria during the first phases of biofilm development; • the proportionality factor between settled bacteria and cathodic current depends on imposed potential and, in particular, it
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strongly decreases when the potential is ennobled above a critical value close to −150 mV Ag/AgCl. This effect appears correlated to the electronic structure of the outer part of the passive layer on polarised SS samples; • impedance measurements (Mott–Schottky plots) showed, in fact, that the outer part of the passive layer on SS behaves like a conductor, if it has developed at potentials more active than −150 mV Ag/AgCl, and like an n-type semiconductor at more noble potentials. Acknowledgement This work was partly supported by the EA-Biofilms NEST508866 Project of the 6th European FP. References [1] S. Dexter, S.H. Lin, Proc. 7th Int. Congress on Marine Corrosion and Fouling, Valencia, Spain, 1988. [2] P. Chandrasekaran, S. Dexter, Corrosion/93, paper N◦ 493, NACE, Houston, TX, 1993. [3] V. Scotto, R. DiCintio, G. Marcenaro, Corros. Sci. 25 (1985) 185. [4] M. Eashwar, S. Maruthamuthu, Biofouling 8 (1995) 203. [5] W.H. Dickinson, F. Caccavo, Z. Lewandowski, Corros. Sci. 38 (1996) 1407. [6] H. Amaya, H. Miyuki, Corros. Eng. 46 (1997) 567. [7] W.H. Dickinson, F. Caccavo Jr., B. Olesen, Z. Lewandowski, Appl. Environ. Microbiol. 63 (1997) 2502. [8] V. Scotto, M. Rapallino, Second seminar on Enzymes and Corrosion, Paris, France, 2000. [9] N. Washizu, Y. Katada, T. Kodama, Corros. Sci. 46 (2004) 1291. [10] R. Johnsen, E. Bardal, Corrosion 41 (1985) 296. ´ [11] I. Dupont, D. Feron, G. Novel, Int. Biodeter. Biodegr. 41 (1998) 13. [12] V. Scotto, M.E. Lai, Corros. Sci. 40 (1) (1998) 1007. [13] K. Takata, H. Hirano, Acta Histochem. Cytochem. 23 (1990) 679. [14] Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, 1997–2005, http://rsb.info.nih.gov/ij/. [15] E. Hakiki, S. Boudin, B. Rondot, M. Da Cunha Belo, Corros. Sci. 37 (1995) 1809. [16] Mollica, E. Traverso, D.Thierry, Publications N◦ 22 in European Federation of Corrosion Series, Institute of Materials, London, 1997, p. 51. [17] A. Mollica, E. Bardal, Second seminar on Enzymes and Corrosion, Paris, France, 2000.