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Microbially influenced corrosion of zinc and aluminium – Two-year subjection to influence of Aspergillus niger
Corrosion Science 49 (2007) 4098–4112 www.elsevier.com/locate/corsci
Microbially influenced corrosion of zinc and aluminium – Two-year subjection to influence of Aspergillus niger Eimutis Juzeli unas *, Rimantas Ramanauskas, Albinas Lugauskas, Konstantinas Leinartas, Meilut_e Samulevicˇien_e, Aloyzas Sudavicˇius, Remigijus Jusˇk_enas Institute of Chemistry, A.Gosˇtauto 9, 01108 Vilnius, Lithuania Received 21 April 2006; accepted 3 May 2007 Available online 3 June 2007
Abstract Aspergillus niger. Tiegh., a filamentous ascomycete fungus, was isolated from the metal samples exposed to marine, rural and urban sites in Lithuania. Al and Zn samples were subjected to two-year influence of A. niger under laboratory conditions in humid atmosphere. Electrochemical impedance spectroscopy (EIS) ascertained microbially influenced corrosion acceleration (MICA) of Zn and inhibition (MICI) of Al. EIS data indicated a two-layer structure of corrosion products on Zn. The microorganisms reduced the thickness of the inner layer, whose passivating capacity was much higher when compared to that of the outer layer. An increase in aluminium oxide layer resistance but decrease in the layer thickness implied that MICI affected primarily the sites of localized corrosion of Al (pores, micro-cracks, etc.). X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) studies indicated that bioproducts (i.e. organic acids) did not form crystalline phases with corrosion products of zinc. The study suggested a hypothesis that microorganisms could be used as corrosion protectors instead of toxic chemicals, application of which tends to be increasingly restricted. 2007 Elsevier Ltd. All rights reserved. Keywords: A. Aluminium; A. Zinc; B. EIS; C. Microbiological corrosion
*
Corresponding author. Fax: +370 5 2649774. E-mail address: [email protected] (E. Juzeli unas).
0010-938X/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.corsci.2007.05.004
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1. Introduction Action of microorganisms on metals includes numerous phenomena, e.g. production of corrosive metabolites (inorganic and organic acids, sulphide, ammonia, carbon dioxide, nitrogen oxides, etc.), chelatization of metal cations, production of organic solvents (ethanol, propanol, butanol), etc. Due to non-uniform surface colonization, microorganisms may cause formation of concentration cells, which results in localized corrosion. The corrosion process is affected also by favoured water uptake by the biofilm [1]. In general, microorganisms may cause either microbially influenced corrosion acceleration (MICA) or inhibition (MICI). While MICA was recently widely studied [1–13], relatively little is known about MICI process. The remarkable protective effect of biofilms was observed in solutions for aluminium alloys and brass (70Cu/30Zn) [13–17]. It has been shown that a pronounced pitting attack took place in sterile medium, whereas in the solutions containing bacteria the pitting process was stopped after two days of exposure. The authors also performed experiments with genetically engineered bacteria capable of producing inhibitors – polyglutamate or polyaspartate. However, they came to the conclusion that even the bacteria, which are not engineered to produce inhibitors, passivated the surface. MICI effects were also determined under atmospheric conditions [18,19]. The conclusions were drawn from two-year aluminium studies under influence of single wild strains (e.g. Penicillium frequentans, Bacillus mycoides), which were isolated from the metals exposed under outdoor conditions [20,21]. Atmospheric corrosion studies of Al, Cu, Zn and steel samples were carried out in the different regions (marine, rural, urban) of Lithuania with the aim to evaluate the role of aerochemical parameters and mirobiological ambience on the metal corrosion [21]. The monitoring of wet and dry deposition rates of pollutants, time of wetness, identification of bacteria and fungal species on metals and in precipitations collected from the samples were carried out. Numerous microbiological species were detected, e.g. Alternaria alternate, Aspergilus niger, Aureobasidium pullulans, B. mycoides, Cladosporium cladosporioides, Cladosporium herbarum, Paecilomyces parvus, P. frequentans [21]. From this microbial diversity, one of the most stable populations appeared to be A. niger. A. niger requires ions of iron, copper, zinc, manganese, molybdenum, boron, vanadium, gallium and scandium [22–24]. Influence of cadmium, lead, chromium and molybdenum on production of citric acid by A. niger has been studied [25]. Cadmium was found to accelerate the metabolism of A. niger. The citric acid production by A. niger represents a highly efficient bioprocess, which can be considered as a model for basic studies of other fungal fermentation processes that will become a key part of future biorefinery, where organic acids and ethanol, are produced from renewable biomass [26]. A. niger produces also numerous other acids: succinic, malic, oxalic, acetic, gluconic, glutaric, trans-glutaconic, cis-aconitic, glycolic, glyoxylic, pyruvic, galactonic, D-mannonic, sacchoric and Lascorbic. For the meantime, relatively little is known about A. niger in its role as an industrial organism for the production of organic acids and enzymes. A. niger is considered as an important model fungus for the study of eukaryotic protein secretion, the effects of environmental factors on the export of biomass degrading enzymes and the mechanisms of the control of fungal morphology [26]. Owing to a wide array of hydrolytic and oxidative enzymes, A. niger is involved in the breakdown of plant lingo-cellulose. Ability of A. niger to degrade minerals (apatite, galena
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and obsidian) has been studied [27]. The effects included mineral surface colonization, corrosion, modification through transformation into secondary minerals and penetration of the mineral substrate. The corrosion process was stimulated by glucose. Little is known up to now about influence of A. niger on electrochemical behaviour and biodegradation of metals, though some results in this field have been reported [18,28]. Localized corrosion of carbon steel wire rope induced by the fungi on wooden spools stored in a humid environment has been observed [28]. MIC is a complex interaction between the microbial population, the environment and the metal substrate; the latter could be of ultimate importance on character of MIC. Most of the studies of microbially influenced corrosion (MIC) were performed in aqueous solutions (artificial seawater, Luria Bertani medium, etc.) in the course of relatively fast formation of biofilm. The aim of the present study was to elucidate influence of A. niger on metal corrosion under atmosphere conditions. The subjects of investigation were aluminium and zinc, i.e. the metals of great technical importance with a different corrosive behaviour. Pure zinc corrodes more or less uniformly (deep pitting is not observed in many cases) with development of a thick (in the order of microns) and porous corrosion product layer with a low inhibiting capacity. By contrast, the localised corrosion is characteristic of aluminium; the passive layer on it is thin (in the order of nanometers) with a high insulating capability. Both metals are also of interest because they play an important role in the metabolism processes of A. niger [22–24]. Zinc ions activate glucose-6-phosphate dehydrogenase as well as cause incomplete oxidation of hydrocarbons and production of organic acid. Enhanced formation of A. niger conidia was determined in the medium with aluminium ions at a concentration of 0.001 mg/l, whereas at higher concentrations inhibition of fungal growth took place [29]. 2. Experimental The microorganisms were isolated from Al, Cu, Zn and steel samples exposed to atmosphere in outdoor stations arranged according to the standard ISO 9223, 8565 in the places covering different environmental conditions in Lithuania: marine – the Kuronian Spit, Preila (100 m from the shore of the Baltic Sea); rural – Kulioniuz and R ugsˇtelisˇkiuz villages in Mol_etuz and Utenos districts; urban – the central part of Vilnius (Institute of Chemistry) and suburb of Vilnius (Experimental Base of the Institute of Chemistry). Climatic parameters, air composition and water adsorption on metal samples were measured continuously at these sites (more details in [21]). The purity of the samples was Zn > 98.5%, Al > 99.5%, Cu > 99.5%, low carbon steel (C – 0.5–0.11%, Mn – 0.25–0.5%, Si < 0.05%, Cr – 0.1%). Fifteen samples of each metal (10 cm · 15 cm in size and 1–3 mm in thickness) were exposed unsheltered in the stands at 45 position to the horizon and the south orientation. The samples were degreased and disinfected before exposure using acetone and alcohol. Special vessels were placed under each sample to collect the rainwater, which rinsed the sample surface. Microorganisms were isolated from the metal samples in 2002 after 6, 9, and 12 months of exposure. The isolation was performed in two ways: (1) directly from corroding samples using a sterile metal loop and (2) preparing suspensions of different dilution from the rainwater, which rinsed the samples. The microorganisms were inoculated on two media: (1) solid malt, supplemented with antibiotics to suppress bacteria growth, for isolation of microscopic fungi, (2) beef-extract agar for bacteria isolation.
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The microorganisms were grown at 26 ± 2 C. The grown colonies of fungi were counted after 3, 5 and 7 days. Pure culture was obtained from the grown microorganisms and was identified according to its physiological, cultural and morphological peculiarities by light and electronic scanning microscopy. Fungal species were identified according to various manuals [30–35]. The suspension of microorganisms was prepared using the following solution: NaNO3 – 2 g, KH2PO4 – 0.7 g, KCl – 0.5 g, MgSO4 Æ 7H2O – 0.5 g, FeSO4 Æ 7H2O – 0.01 g, distilled water – 1000 g (pH 6.0–6.5). Spore concentration was ca. 106 spores/ml, which was determined using a special counting chamber. The exposure vessels (with a volume of 5 l) contained 1 l of a saturated potassium sulphate solution, which maintained relative humidity at ca. 97%. The Al and Zn plates (4 cm2 in size and 1 mm in thickness) were treated with emery SiC paper (grade 1200), washed with acetone and alcohol and kept under ambient conditions for ca. 30 min. Then, they were put on a ceramic grid in a vertical position in the exposure vessels (four samples in each vessel) and infected by spraying them with a suspension containing a microbial population. The control sample was sprayed with the solution free of microorganisms, which was used for suspension preparation (the composition is given above). Then, the vessels were closed and kept in a glass container at a temperature of 26 ± 2 C. The samples were taken from the exposure vessels for scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) investigations at time intervals of 3, 7, 15 and 24 months. Different evolution stages of A. niger on Al are shown in Fig. 1. In addition, one of the withdrawn samples was checked for vitality of the microorganisms by taking replicas from its surface on different agar media: malt extract, maize and Czapek’s agar. The replica-samples were exposed at 26 ± 2 C temperature for 5 days and then inspected by microscopy. After all exposures (3, 7, 15 and 24 months), colonies of A. niger were grown from the taken replicas, which indicated vitality of the inoculated microorganisms (Fig. 2). Thus, the EIS studies relate to vital A. niger colonies on metals. After each withdrawal, to force the colony growth, the remaining samples were additionally infected by microorganisms as described above. For comparison of corrosion behaviour, measurements were also performed with pristine samples, preparation of which included sandpapering, degreasing and disinfecting with acetone and alcohol and 24-h exposure under ambient laboratory conditions. The EIS measurements were carried out using an IM6 apparatus (‘Zahner’) and an electrochemical glass cell equipped with holes for metal specimen as well as Ag/AgCl reference and Pt counter electrodes. The measurements were performed at open circuit potential applying a signal of amplitude of DE = ±5–10 mV. The sample was mounted in a special holder, placed in the electrochemical cell and EIS measurements were started after 5– 10 min. This time was needed to reach roughly constant electrode potential (quasi-steady state). No distinctive differences in the time of reaching of quasi-steady state were observed for inoculated and blank samples. The main part of EIS measurements was carried out in chloride solution although some was done also in sulphate solution. The chloride solution contained 0.6 M NaCl, which is commonly used for corrosion investigations (it is sufficiently conductive, the salinity is close to that of ocean and concentration of dissolved oxygen did not differ significantly from that in pure water). As will be shown in the results section, chloride ions penetrate surface layer and accelerate corrosion. (Note that the exposure vessels contained a
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Fig. 1. SEM micrographs of Al after 1.5 year exposure under influence of Aspergillus niger: (a) A site of initial colonization with preferential localization at microcracks; (b) and (c) fungi and mycelium growth; (d) formation of dense mycelium and conidia colony; (e) formation of new generation of fungi conidia on the head of conidiophores; (f) numerous conidia of new generation.
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Fig. 2. SEM micrographs of Aspergillus niger matured conidia and conidiaphores developed on replicas taken from a Zn sample on beef agar. Magnification ·1500.
chloride-free solution in order to achieve better conditions to reveal entirely biogenic factors of corrosion.) The X-ray photoelectron spectroscopy (XPS) data were recorded by an ‘Escalab MK’ spectrometer using X-radiation of Mg Ka (1253.6 eV, pass energy of 20 eV). The samples were etched in the preparation chamber by ionised argon at a vacuum of 5 · 104 Pa. A beam current of 20 lA/cm2 was used, which corresponds to an average etching rate of ca. 2.5 nm/min for Al (Al2O3) [36]. The analogous rate calculated for Zn (ZnO) is approximately twice as high as the rate for aluminium [37]. The etching rate of carbon is about ten times less than that of zinc. X-ray diffraction (XRD) investigations were performed using a D8 Advance diffractometer from Bruker AXS (Germany) with Cu Ka radiation selected by a secondary graphite monochromator. The step-scan mode with a step of 0.05 2h and a sampling time of 5 s/ step in the range 30 6 2h 6 70 was used. 3. Results and discussion The corrosion rate, by definition, is determined by electron transfer kinetics through a system of charged interfaces and different types of conductors: electronic (metal), semiconductor (corrosion product layers) and ionic (solution containing dissolved oxidiser(s) and corrosion products). Although MIC represents a complex interaction between the microbial population, the environment and the substrate, it does not mean any new type of corrosion and its rate is determined as the corrosion current density (jcorr, A cm2), which is in inverse proportion to the polarization resistance (Rp, X cm2) [38]. The parameter Rp can be determined from the voltammetric dependence as a ratio between the potential (E) and the current density (j) close to open circuit potential [Rp = (DE/Dj)DE!0], i.e. in the region where the voltammetric dependence could be approximated as a rectilinear one. Impedance (Z) measurements at low frequency (x) domain (Rp = Zx!0) or the equivalent circuit parameters used for the experimental data fitting provide another possibilities to obtain Rp. The polarization resistance method is frequently used for MIC studies as shown in the review [16].
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a 25
1
1 - Zn 2 - Aspergillus niger
Rp / kΩ cm2
20 15 10
2
5 0 0
b
5
10
15
20
25
1 - Al 2 - Aspergillus niger
Rp / kΩ cm2
150
100
2
50 1 0 0
c
5
10
25
15
20
25
Zinc
-25 -50 -75 Aluminium
O
Rp - Rpmicr / kΩ cm 2
0
-100 -125
Aspergillus niger 0
5
10
15
20
25
t / month Fig. 3. Polarization resistance change determined from EIS data (Rp = Zx!0) of abiotic samples (1) and those with Aspergillus niger, (2) during two-year exposure to humid atmosphere at 26 C.
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Fig. 3 shows the Rp change during two-year exposure of abiotic and biotic samples. (The data in Fig. 3 were determined as Rp = Zx!0, however, nearly identical values were obtained also from the equivalent circuits used for the data fitting.) It is obvious that the influence of microorganisms depends on the kind of the metal they colonize: corrosion acceleration is evident for zinc and inhibition for aluminium. When plotting the Rp data as a difference between the biotic and abiotic samples ðR0p Rmicr p Þ, it can be seen that the inhibition effect of Al corrosion is much stronger than that of acceleration of Zn corrosion (Fig. 3a). An increase in Rp with exposure time is observed for Zn samples, which means decrease in corrosion activity of both abiotic and biotic samples (Fig. 3a). This is due to development of corrosion product layer, whose inhibiting effect for the abiotic sample is more pronounced when compared to the biotic counterpart. The corrosion activity of abiotic aluminium does not change significantly after the twoyear exposure period (curve 1, Fig. 3b), while the samples with microorganisms decreased the activity markedly (curve 2). The inhibiting influence of A. niger reaches its maximum after 6–15 months of exposure, when the corrosion activity is reduced by more than one order of magnitude when compared to the abiotic counterpart. The EIS diagrams for Zn samples are given in Fig. 4. A pristine sample in NaCl solution exhibits two clearly pronounced time constants (curve 1, Fig. 4). Such behaviour is typical of zinc electrode [39]. To observe the two time constants, two conditions are of importance: (1) the sample has to be pre-exposed for some time to atmosphere and (2) immersed into chloride solution. Indeed, only one time constant is characteristic of the same pre-exposed sample if the EIS measurements were performed in sulphate solution (curve 3, Fig. 4). According to arguments provided in literature, the multi-time constant behaviour relates to layered structure of corrosion products on exposed zinc [40]. Thus, the data obtained in chloride solution (Fig. 4) imply a two-layer structure, the inner part of which is composed of the native corrosion products and the outer part is the layer penetrated by chloride. The second layer is not present in sulphate solution because, as commonly known, the penetration capability of chloride is much higher than that of sulphate. In agreement with the said above, the EIS data in sulphate solution fit well to an equivalent circuit consisting of one Rt-CPE element (charge transfer resistance-constant phase element) and the data in chloride solution fit to an equivalent circuit of two Rt-CPE elements (circuits and curves 1 and 3 in Fig. 4). (The constant phase element consists of capacitance (C, lF cm2) and frequency dispersion (n) a dimensionless parameter (n 6 1), which usually is correlated with porosity of corrosion products. If n is close to 1, this means CPE is most capacitive.) The fitting parameters are summarized in Table 1. The inner layer should be build of zinc(hydro)oxide and the outer layer of hydroxochloride. This assumption is supported by the literature data: ZnO, Zn(OH)2 and 4Zn(OH)2, ZnCl2 were detected on zinc in chloride solutions by FTIR and in situ Raman spectroscopy [41,42]. The EIS data for the Zn samples exposed for two years are shown in Fig. 4 (curves 2 and 4). Although there are distinctive differences between the abiotic and biotic samples, both of them correspond well to an equivalent circuit consisting of three Rt-CPE elements and RX in series (circuit 2 in Fig. 4 and the parameters in Table 1). Such results indicate a three-layer structure on zinc electrode.
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E. Juzeliunas et al. / Corrosion Science 49 (2007) 4098–4112 CPE1
CPE2
1 R1 CPE1
RΩ
R2 CPE2
CPE3
2, 4 R1
R2
CPE1
RΩ
3
R3
RΩ
R1
2 10000
1 - pristine, NaCl 2 - exposed, NaCl 3 - pristine, Na2SO4 4 - A. niger , NaCl
Z / Ω cm2
4
1000
1 3 100
10
0
3
-10
1
phase / deg
-20 -30
2
-40 -50
4 -60 -70 10-2
10-1
100
101
102
103
104
105
f / Hz Fig. 4. EIS diagrams of pristine Zn (1,3) and abiotic (2) and biotic (3) samples exposed for two years to humid atmosphere at 26 C. Solutions: 1,2,4 – 0.6 M NaCl; 3 – 0.1 M Na2SO4. The experimental results (symbols) were fitted (lines) assuming the shown equivalent circuits and the fitting parameters are summarized in Table 1.
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Table 1 Fitting parameters for EIS data (Figs. 4 and 7) obtained for Zn and Al pristine samples and those exposed for two years with and without microorganisms to humid atmosphere Sample
C1, lF cm2
C2, lF cm2
C3, lF cm2
R1, kX cm2
R2, kX cm2
R3, kX cm2
Zn, pristine Zn, exposed Zn + Aspergillus niger Al, pristine Al, exposed Al + Aspergillus niger
1730 (n = 0.96) 11.9 (0.74) 59.5 (0.84) 3.6 (0.85) 2.2 (0.83) 14.7 (0.86)
9.4 (n = 0.77) 0.5 (0.54) 0.52 (0.83) – – 2.0 (0.76)
– 0.8 (0.66) 0.74 (0.59) – – –
0.33 17.5 4.70 5.9 8.2 59.9
0.24 0.12 0.84 – – 22.5
– 8.8 3.9 – – –
10000
5
4
8
3
2
Zn(002)
* - Zn (OH) Cl H O + - Zn CO (OH) H O 2
6
2
# 1000
+
#
*
+ 2
+
+
+
ZnO (002)
ZnO (100)
Intensity / cps
# - NaZn(OH)33H2O
Zn(100)
The X-ray diffraction (XRD) measurements indicate crystalline hydroxide, carbonate and chloride species to be developed both on abiotic and biotic Zn exposed to NaCl solution (Fig. 5). The difference in widths of the XRD peaks indicates that the grain size of the chloride species is much greater than that of the carbonate ones: the full width at half maximum for chloride is FWHM = 0.139 at 2H = 11.2 while the analogous value for carbonate is FWHM = 0.925 (2H = 12.9). No symptomatic differences were found between XRD patterns for blank zinc and that with A. niger. It means that biogenic products do not form crystalline phases with the zinc corrosion products. The surface analysis by X-ray photoelectron spectroscopy (XPS) and surface sputtering by ionised argon shows significant content of organic carbon within the studied layer (Fig. 6). The highest content of organic carbon was found on the top of the corrosion product layer (ca. 40 at.%), which may originate from the dehydrated microorganisms (under vacuum conditions) and their metabolites. The noticeable concentrations of
1 100 10
20
30
40
2Θ Fig. 5. XRD patterns for abiotic Zn sample (2) and that with A. niger (1). The samples were exposed for two years to humid atmosphere at 26 C and for 1 h to 0.6 M NaCl solution.
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Carbonate 6
content / at.%
80
4 2
60
S2- + SO42-
Oxygen
Cl-
0 0
40
Zinc
20
Carbon (org)
10 20 30 40 50
Zn+Aspergillus niger 0 0
10
20
30
40
50
sputtering time / min
Carbonate
2.0
120
1.5
content / at.%
100
1.0
80
Carbon (org)
S2- + SO42-
Cl-
0.5 0.0
0
60
5
10 15 20
Oxygen
40 Aluminium
20 0
Al+Aspergillus niger 0
5
10
15
20
sputtering time / min Fig. 6. Concentration profiles of organic carbon, oxygen (O2) and metals [Zn + Zn(II) and Al + Al(III)] on zinc (a) and aluminium (b) subjected for two years to influence of Aspergilus niger in humid atmosphere. The concentrations (in at.%) were determined by X-ray photoelectron spectroscopy (XPS) using surface sputtering by ionised argon. The insertions show distribution of lower concentrations: carbonate, chloride, sulphide and sulphate.
organic carbon in deeper levels (8–10 at.%) have to be considered as the result of the organic acid penetration into the corrosion product layer. Since the carbon was not detected by XRD (Fig. 5), it follows that carbon compounds do not form crystalline phases with the corrosion products. The same could be said about sulphides and sulphates minor amounts of which were detected by XPS (insertion in Fig. 6), but not by XRD.
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It should be noted that the entire corrosion product layer is much thicker than the depth profiles in Fig. 6. The sputtering time of 50 min and the used beam current of 20 lA/cm2 correspond to an average depth of 250 nm assuming the etching rate to be ca. 5 nm/min for Zn (ZnO) [37]. Thus, the analysis data relate to the depth of several hundreds nanometers, whereas the entire corrosion product layer is much thicker (according to literature, the thickness of corrosion products on zinc developed under atmospheric conditions ranges from 1 to 25 lm per year [43,44]). The XPS method in respect to the studies of layered non-homogeneous structures seems to be problematic due to non-uniformity of surface etching by ionised argon (for instance, the etching rate for Zn and C may differ about ten times). EIS measurements provide better opportunity to detect layered structures as was demonstrated above. It follows from EIS, XRD and XPS data that the top layer of the three-layer structure on Zn (R3C3) is composed of the hydroxide-carbonate-chloride species, which originate from the interaction of corrosion products (ZnO–Zn(OH)2) with carbon dioxide during exposure to the atmosphere and then with NaCl solution during exposure to an electrochemical cell. The intermediate layer (R2C2) is the rest of the hydroxide-carbonate, which was not penetrated by the chloride. The inner layer (i.e. next to the metal, R1C1) is typically composed of a zinc oxide. Since the outer layer (R3C3), which develops in chloride solution, may be considered as an experimental artefact, development of a two-layer structure in atmosphere may be concluded. The analogous structure was determined by other authors, who studied the zinc samples exposed under outdoor conditions and came to a conclusion that corrosion products consisted of a compact ZnO inner layer and a porous 2CO3Zn Æ 3Zn(OH)2 outer layer [40]. The layer thickness (d) may be characterised by its capacitance, which is in inverse proportion to the thickness (Table 1). The fitting parameters in Table 1 indicate that the inner layer on pristine Zn (R1C1) is much thinner as compared with the outer layer (C1 = 1730 lF cm2 vs. C2 = 9.4 lF cm2). At the same time, the resistance of this much thinner layer is not less (even greater) when compared to the resistance of the thick outer layer (R1 = 0.33 kX cm2 vs. R2 = 0.24 kX cm2). This clearly shows a high inhibiting capability of the native oxide layer, which is produced by corrosion process in atmosphere. (Strictly speaking, the above comparison of d will be correct assuming that there is no great difference in the relative permittivities of the films. However, this aspect may be neglected due to a great difference between C1 and C2). The EIS data in Fig. 3 and Table 1 provide information that corrosion acceleration by A. niger is caused primarily by diminution in the thickness of the first layer (C1R1), which, as shown above, has the greatest inhibiting capability. The capacitance C1 in the presence of A. niger is 5 times higher (the layer is thinner) when compared to that of the abiotic counterpart. The R1 value for the sample with microorganisms is accordingly less. It is interesting to refer here to analogous conclusion drawn from the data obtained with the strains B. mycoides and P. frequentans [18,19]. When returning back to Al samples, let us consider the EIS diagrams in Fig. 7 and the derived data in Table 1. The data indicate one corrosion product layer on the abiotic Al and appearance of a second layer in case of microorganisms, as seen from a certain contribution in the low frequency region (curve 2, Fig. 7). The resistance of the first layer developed under influence of A. niger is much greater when compared to that developed on the abiotic sample (59.9 kX cm2 and 8.2 kX cm2, respectively). At the same time, according to the capacitance values, the layer on the biotic sample is much thinner
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10 5
CPE1
1
2
RΩ
R1
10 4
1
CPE1
CPE2
Z / Ω cm2
2 10 3
R1
RΩ
R2
10 2
1 - Al 2 - A.niger 10 1
10 0
1
phase / deg
0
-20
2 -40
-60
-80 10-2
10-1
100
101
102
103
104
105
f / Hz Fig. 7. EIS diagrams for abiotic Al (1) and that with A. niger (2) in 0.6 M NaCl solution. The samples were exposed for two years to humid atmosphere at 26 C. The equivalent circuits, which were used for experimental data (symbols) fitting (lines), are shown in the figure. The fitting parameters are summarised in Table 1.
(14.7 lF cm2 and 2.2 lF cm2). This suggests an idea that A. niger may affect primarily the sites of localised corrosion, i.e. micropores and microcracks. A support for this assumption provides SEM data, which show preferential colonization of microcracks on the Al surface, most probably, owing to preferential accumulation of water in these sites (Fig. 1a).
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Aluminium is known as a metal, which corrodes not uniformly because of preferential attack on the grain boundaries. The localized corrosion of Al alloys was indicated by EIS as an appearance of a second time constant in the low frequency region [45]. The authors pointed out that the effect is usually partially masked by the scatter of the data at low frequencies. We also observed the same typical discrepancy of the phase angle from one time constant model for a pure pristine Al sample (the data not shown in this paper). However, the interpretation was problematic for the exposed samples due to wide scatter of the experimental data below 1 Hz. The data on chemical composition of the outer layer on Al developed during MIC are shown in Fig. 6. (The average sputtered profile is ca. 50 nm assuming that the used beam current of 20 lA/cm2 corresponds to an average etching rate of ca. 2.5 nm/min for Al (Al2O3) [36]). Organic carbon, carbonates, sulphates, sulphides and chlorides were determined within the outer corrosion product layer alongside to aluminium oxide. About 80 at.% of the top of the entire structure is composed of organic carbon, which is from microorganisms and their metabolites. At deeper levels, the content of carbon drops down to lower values, which may be attributed to weak penetration of organic acids into the aluminium oxide structure. The results obtained demonstrate that A. niger may act either as the corrosion accelerators or the inhibitors depending upon the metal they colonize. The microorganisms were capable of disintegrating of the thick corrosion product layers on zinc, while the aluminium oxide layer, in general, was stable against the microbiological action. Probably, microbial colonisation of the sites of localized attack led to a ‘‘healing’’ effect. The obtained results show that microorganisms could be considered as an alternative to chemicals, application of which for corrosion protection tends to be increasingly restricted. 4. Conclusions The wild strain A. niger acted either as corrosion accelerator or inhibitor depending upon the metal it colonized. Microbially induced corrosion acceleration was ascertained for zinc while corrosion inhibition was typical of aluminium. The EIS data indicated two-layer structures developed on Zn and Al under influence of A. niger. The corrosion acceleration of Zn lies primarily in diminishing of the thickness of the inner layer (next to the metal), which has the greatest passivating capacity. For Al, an increase in charge transfer resistance, but not in layer thickness, implied that the fungi might promote passivity at sites of localized corrosion (micropores, microcracks, etc.). The XRD studies ascertained crystalline hydroxide and carbonate species developed during MIC of Zn. No distinctive differences were found between XRD patterns for abiotic zinc and that with A. niger. XPS data showed that biogenic compounds penetrated into the corrosion product layer, but they did not form crystalline phases with the corrosion products of zinc. Acknowledgements This study was carried out as a part of the complex research program supported by the Lithuanian Science and Study Foundation (Contract KP-059). The authors remember with great respect Dr. E. Matulionis who passed away in 2003. He carried out SEM investigations at the Institute of Chemistry, Vilnius.
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References [1] D. Thierry, W. Sand, Microbially influenced corrosion, in: P. Marcus, J. Oudar (Eds.), Corrosion Mechanism in Theory and Practice, Marcel Dekker, New York, 1995, p. 457. [2] T.E. Cloete, Mater. Corros. 54 (2003) 520. [3] P. Linhardt, Mater. Corros. 55 (2004) 158. [4] X. Shi, R. Avci, M. Geiser, Z. Lewandowski, Corros. Sci. 45 (2003) 2577. [5] D. Wagner, A.H.L. Chamberlain, Biodegradation 8 (1997) 177. [6] R.S. Dubey, S.N. Upadhyay, Bull. Electrochem. 15 (1999) 266. [7] B.J. Pitonzo, P. Castro, P.S. Amy, G. Southam, D.A. Jones, D. Ringelberg, Corrosion 60 (2004) 64. [8] D.A. Jones, P.S. Amy, Corrosion 58 (2002) 638. [9] M.J. H Gayosso, G.Z. Olivares, N.R. Ordaz, C.J. Ramirez, R.G. Esquivel, A.P. Viveros, Electrochim. Acta 49 (2004) 4295. [10] G. Huang, K.-Y. Chan, H.H.P. Fang, J. Electrochem. Soc. 151 (2004) B434. [11] J.P. Busalmen, M. Vazquez, S.R. Sanchez, Electrochim. Acta 47 (2002) 1857. [12] R.P. George, D. Marshall, R.C. Newman, Corros. Sci. 45 (2003) 1999. [13] D. Ornek, A. Jayaraman, T.K. Wood, Z. Sun, C.H. Hsu, F. Mansfeld, Corros. Sci. 43 (2001) 2121. [14] F. Mansfeld, C.H. Hsu, D. Ornek, T.K. Wood, B.C. Syrett, J. Electrochem. Soc. 149 (2002) B130. [15] D. Ornek, T.K. Wood, C.H. Hsu, F. Mansfeld, Corros. Sci. 44 (2002) 2291. [16] F. Mansfeld, Mater. Corros. 54 (2003) 489. [17] R. Zuo, E. Kus, F. Mansfeld, T.K. Wood, Corros. Sci. 47 (2005) 279. [18] E. Juzeli unas, R. Ramanauskas, A. Lugauskas, M. Samulevicˇien_e, K. Leinartas, Electrochem. Commun. 7 (2005) 305. [19] E. Juzeli unas, R. Ramanauskas, A. Lugauskas, K. Leinartas, M. Samulevicˇien_e, A. Sudavicˇius, Electrochim. Acta 51 (2006) 6085. [20] A. Lugauskas, D. Bridzˇiuvien_e, A. Narkevicˇius, E. Ivasˇkevicˇ, Mikol. Fitopatol. 38 (5) (2004) 54. [21] R. Ramanauskas, E. Juzeli unas, A. Narkevicˇius, D. Bucˇinskien_e, A. Lugauskas, D. Pecˇiulyt_e, L. Levinskait_e, V. Ulevicˇius, D. Jasinevicˇien_e, Chemija 16 (2005) 25. [22] S.K. Gupta, K.K. Maggon, T.A. Venkitasubramanian, J. Gen. Microbiol. 99 (1977) 43. [23] A.K. Banik, Folia Microbiol. 21 (2) (1976) 139. [24] K.H. Domsch, W. Gams, T.-H. Anderson, Compendium of Soil Fungi, Academic Press, London, 1980. [25] A.R. Angumeenal, P. Kamalakannan, H.J. Prabhu, D. Venkappayya, Indian J. Chem. Technol. 9 (2002) 508. [26] S. Baker, J. Magnuson, Z. Dai, E. Panisko, C. Wend, L. Lasure.
Microbially influenced corrosion of zinc and aluminium – Two-year subjection to influence of Aspergillus niger Eimutis Juzeli unas *, Rimantas Ramanauskas, Albinas Lugauskas, Konstantinas Leinartas, Meilut_e Samulevicˇien_e, Aloyzas Sudavicˇius, Remigijus Jusˇk_enas Institute of Chemistry, A.Gosˇtauto 9, 01108 Vilnius, Lithuania Received 21 April 2006; accepted 3 May 2007 Available online 3 June 2007
Abstract Aspergillus niger. Tiegh., a filamentous ascomycete fungus, was isolated from the metal samples exposed to marine, rural and urban sites in Lithuania. Al and Zn samples were subjected to two-year influence of A. niger under laboratory conditions in humid atmosphere. Electrochemical impedance spectroscopy (EIS) ascertained microbially influenced corrosion acceleration (MICA) of Zn and inhibition (MICI) of Al. EIS data indicated a two-layer structure of corrosion products on Zn. The microorganisms reduced the thickness of the inner layer, whose passivating capacity was much higher when compared to that of the outer layer. An increase in aluminium oxide layer resistance but decrease in the layer thickness implied that MICI affected primarily the sites of localized corrosion of Al (pores, micro-cracks, etc.). X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) studies indicated that bioproducts (i.e. organic acids) did not form crystalline phases with corrosion products of zinc. The study suggested a hypothesis that microorganisms could be used as corrosion protectors instead of toxic chemicals, application of which tends to be increasingly restricted. 2007 Elsevier Ltd. All rights reserved. Keywords: A. Aluminium; A. Zinc; B. EIS; C. Microbiological corrosion
*
Corresponding author. Fax: +370 5 2649774. E-mail address: [email protected] (E. Juzeli unas).
0010-938X/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.corsci.2007.05.004
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1. Introduction Action of microorganisms on metals includes numerous phenomena, e.g. production of corrosive metabolites (inorganic and organic acids, sulphide, ammonia, carbon dioxide, nitrogen oxides, etc.), chelatization of metal cations, production of organic solvents (ethanol, propanol, butanol), etc. Due to non-uniform surface colonization, microorganisms may cause formation of concentration cells, which results in localized corrosion. The corrosion process is affected also by favoured water uptake by the biofilm [1]. In general, microorganisms may cause either microbially influenced corrosion acceleration (MICA) or inhibition (MICI). While MICA was recently widely studied [1–13], relatively little is known about MICI process. The remarkable protective effect of biofilms was observed in solutions for aluminium alloys and brass (70Cu/30Zn) [13–17]. It has been shown that a pronounced pitting attack took place in sterile medium, whereas in the solutions containing bacteria the pitting process was stopped after two days of exposure. The authors also performed experiments with genetically engineered bacteria capable of producing inhibitors – polyglutamate or polyaspartate. However, they came to the conclusion that even the bacteria, which are not engineered to produce inhibitors, passivated the surface. MICI effects were also determined under atmospheric conditions [18,19]. The conclusions were drawn from two-year aluminium studies under influence of single wild strains (e.g. Penicillium frequentans, Bacillus mycoides), which were isolated from the metals exposed under outdoor conditions [20,21]. Atmospheric corrosion studies of Al, Cu, Zn and steel samples were carried out in the different regions (marine, rural, urban) of Lithuania with the aim to evaluate the role of aerochemical parameters and mirobiological ambience on the metal corrosion [21]. The monitoring of wet and dry deposition rates of pollutants, time of wetness, identification of bacteria and fungal species on metals and in precipitations collected from the samples were carried out. Numerous microbiological species were detected, e.g. Alternaria alternate, Aspergilus niger, Aureobasidium pullulans, B. mycoides, Cladosporium cladosporioides, Cladosporium herbarum, Paecilomyces parvus, P. frequentans [21]. From this microbial diversity, one of the most stable populations appeared to be A. niger. A. niger requires ions of iron, copper, zinc, manganese, molybdenum, boron, vanadium, gallium and scandium [22–24]. Influence of cadmium, lead, chromium and molybdenum on production of citric acid by A. niger has been studied [25]. Cadmium was found to accelerate the metabolism of A. niger. The citric acid production by A. niger represents a highly efficient bioprocess, which can be considered as a model for basic studies of other fungal fermentation processes that will become a key part of future biorefinery, where organic acids and ethanol, are produced from renewable biomass [26]. A. niger produces also numerous other acids: succinic, malic, oxalic, acetic, gluconic, glutaric, trans-glutaconic, cis-aconitic, glycolic, glyoxylic, pyruvic, galactonic, D-mannonic, sacchoric and Lascorbic. For the meantime, relatively little is known about A. niger in its role as an industrial organism for the production of organic acids and enzymes. A. niger is considered as an important model fungus for the study of eukaryotic protein secretion, the effects of environmental factors on the export of biomass degrading enzymes and the mechanisms of the control of fungal morphology [26]. Owing to a wide array of hydrolytic and oxidative enzymes, A. niger is involved in the breakdown of plant lingo-cellulose. Ability of A. niger to degrade minerals (apatite, galena
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and obsidian) has been studied [27]. The effects included mineral surface colonization, corrosion, modification through transformation into secondary minerals and penetration of the mineral substrate. The corrosion process was stimulated by glucose. Little is known up to now about influence of A. niger on electrochemical behaviour and biodegradation of metals, though some results in this field have been reported [18,28]. Localized corrosion of carbon steel wire rope induced by the fungi on wooden spools stored in a humid environment has been observed [28]. MIC is a complex interaction between the microbial population, the environment and the metal substrate; the latter could be of ultimate importance on character of MIC. Most of the studies of microbially influenced corrosion (MIC) were performed in aqueous solutions (artificial seawater, Luria Bertani medium, etc.) in the course of relatively fast formation of biofilm. The aim of the present study was to elucidate influence of A. niger on metal corrosion under atmosphere conditions. The subjects of investigation were aluminium and zinc, i.e. the metals of great technical importance with a different corrosive behaviour. Pure zinc corrodes more or less uniformly (deep pitting is not observed in many cases) with development of a thick (in the order of microns) and porous corrosion product layer with a low inhibiting capacity. By contrast, the localised corrosion is characteristic of aluminium; the passive layer on it is thin (in the order of nanometers) with a high insulating capability. Both metals are also of interest because they play an important role in the metabolism processes of A. niger [22–24]. Zinc ions activate glucose-6-phosphate dehydrogenase as well as cause incomplete oxidation of hydrocarbons and production of organic acid. Enhanced formation of A. niger conidia was determined in the medium with aluminium ions at a concentration of 0.001 mg/l, whereas at higher concentrations inhibition of fungal growth took place [29]. 2. Experimental The microorganisms were isolated from Al, Cu, Zn and steel samples exposed to atmosphere in outdoor stations arranged according to the standard ISO 9223, 8565 in the places covering different environmental conditions in Lithuania: marine – the Kuronian Spit, Preila (100 m from the shore of the Baltic Sea); rural – Kulioniuz and R ugsˇtelisˇkiuz villages in Mol_etuz and Utenos districts; urban – the central part of Vilnius (Institute of Chemistry) and suburb of Vilnius (Experimental Base of the Institute of Chemistry). Climatic parameters, air composition and water adsorption on metal samples were measured continuously at these sites (more details in [21]). The purity of the samples was Zn > 98.5%, Al > 99.5%, Cu > 99.5%, low carbon steel (C – 0.5–0.11%, Mn – 0.25–0.5%, Si < 0.05%, Cr – 0.1%). Fifteen samples of each metal (10 cm · 15 cm in size and 1–3 mm in thickness) were exposed unsheltered in the stands at 45 position to the horizon and the south orientation. The samples were degreased and disinfected before exposure using acetone and alcohol. Special vessels were placed under each sample to collect the rainwater, which rinsed the sample surface. Microorganisms were isolated from the metal samples in 2002 after 6, 9, and 12 months of exposure. The isolation was performed in two ways: (1) directly from corroding samples using a sterile metal loop and (2) preparing suspensions of different dilution from the rainwater, which rinsed the samples. The microorganisms were inoculated on two media: (1) solid malt, supplemented with antibiotics to suppress bacteria growth, for isolation of microscopic fungi, (2) beef-extract agar for bacteria isolation.
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The microorganisms were grown at 26 ± 2 C. The grown colonies of fungi were counted after 3, 5 and 7 days. Pure culture was obtained from the grown microorganisms and was identified according to its physiological, cultural and morphological peculiarities by light and electronic scanning microscopy. Fungal species were identified according to various manuals [30–35]. The suspension of microorganisms was prepared using the following solution: NaNO3 – 2 g, KH2PO4 – 0.7 g, KCl – 0.5 g, MgSO4 Æ 7H2O – 0.5 g, FeSO4 Æ 7H2O – 0.01 g, distilled water – 1000 g (pH 6.0–6.5). Spore concentration was ca. 106 spores/ml, which was determined using a special counting chamber. The exposure vessels (with a volume of 5 l) contained 1 l of a saturated potassium sulphate solution, which maintained relative humidity at ca. 97%. The Al and Zn plates (4 cm2 in size and 1 mm in thickness) were treated with emery SiC paper (grade 1200), washed with acetone and alcohol and kept under ambient conditions for ca. 30 min. Then, they were put on a ceramic grid in a vertical position in the exposure vessels (four samples in each vessel) and infected by spraying them with a suspension containing a microbial population. The control sample was sprayed with the solution free of microorganisms, which was used for suspension preparation (the composition is given above). Then, the vessels were closed and kept in a glass container at a temperature of 26 ± 2 C. The samples were taken from the exposure vessels for scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) investigations at time intervals of 3, 7, 15 and 24 months. Different evolution stages of A. niger on Al are shown in Fig. 1. In addition, one of the withdrawn samples was checked for vitality of the microorganisms by taking replicas from its surface on different agar media: malt extract, maize and Czapek’s agar. The replica-samples were exposed at 26 ± 2 C temperature for 5 days and then inspected by microscopy. After all exposures (3, 7, 15 and 24 months), colonies of A. niger were grown from the taken replicas, which indicated vitality of the inoculated microorganisms (Fig. 2). Thus, the EIS studies relate to vital A. niger colonies on metals. After each withdrawal, to force the colony growth, the remaining samples were additionally infected by microorganisms as described above. For comparison of corrosion behaviour, measurements were also performed with pristine samples, preparation of which included sandpapering, degreasing and disinfecting with acetone and alcohol and 24-h exposure under ambient laboratory conditions. The EIS measurements were carried out using an IM6 apparatus (‘Zahner’) and an electrochemical glass cell equipped with holes for metal specimen as well as Ag/AgCl reference and Pt counter electrodes. The measurements were performed at open circuit potential applying a signal of amplitude of DE = ±5–10 mV. The sample was mounted in a special holder, placed in the electrochemical cell and EIS measurements were started after 5– 10 min. This time was needed to reach roughly constant electrode potential (quasi-steady state). No distinctive differences in the time of reaching of quasi-steady state were observed for inoculated and blank samples. The main part of EIS measurements was carried out in chloride solution although some was done also in sulphate solution. The chloride solution contained 0.6 M NaCl, which is commonly used for corrosion investigations (it is sufficiently conductive, the salinity is close to that of ocean and concentration of dissolved oxygen did not differ significantly from that in pure water). As will be shown in the results section, chloride ions penetrate surface layer and accelerate corrosion. (Note that the exposure vessels contained a
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Fig. 1. SEM micrographs of Al after 1.5 year exposure under influence of Aspergillus niger: (a) A site of initial colonization with preferential localization at microcracks; (b) and (c) fungi and mycelium growth; (d) formation of dense mycelium and conidia colony; (e) formation of new generation of fungi conidia on the head of conidiophores; (f) numerous conidia of new generation.
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Fig. 2. SEM micrographs of Aspergillus niger matured conidia and conidiaphores developed on replicas taken from a Zn sample on beef agar. Magnification ·1500.
chloride-free solution in order to achieve better conditions to reveal entirely biogenic factors of corrosion.) The X-ray photoelectron spectroscopy (XPS) data were recorded by an ‘Escalab MK’ spectrometer using X-radiation of Mg Ka (1253.6 eV, pass energy of 20 eV). The samples were etched in the preparation chamber by ionised argon at a vacuum of 5 · 104 Pa. A beam current of 20 lA/cm2 was used, which corresponds to an average etching rate of ca. 2.5 nm/min for Al (Al2O3) [36]. The analogous rate calculated for Zn (ZnO) is approximately twice as high as the rate for aluminium [37]. The etching rate of carbon is about ten times less than that of zinc. X-ray diffraction (XRD) investigations were performed using a D8 Advance diffractometer from Bruker AXS (Germany) with Cu Ka radiation selected by a secondary graphite monochromator. The step-scan mode with a step of 0.05 2h and a sampling time of 5 s/ step in the range 30 6 2h 6 70 was used. 3. Results and discussion The corrosion rate, by definition, is determined by electron transfer kinetics through a system of charged interfaces and different types of conductors: electronic (metal), semiconductor (corrosion product layers) and ionic (solution containing dissolved oxidiser(s) and corrosion products). Although MIC represents a complex interaction between the microbial population, the environment and the substrate, it does not mean any new type of corrosion and its rate is determined as the corrosion current density (jcorr, A cm2), which is in inverse proportion to the polarization resistance (Rp, X cm2) [38]. The parameter Rp can be determined from the voltammetric dependence as a ratio between the potential (E) and the current density (j) close to open circuit potential [Rp = (DE/Dj)DE!0], i.e. in the region where the voltammetric dependence could be approximated as a rectilinear one. Impedance (Z) measurements at low frequency (x) domain (Rp = Zx!0) or the equivalent circuit parameters used for the experimental data fitting provide another possibilities to obtain Rp. The polarization resistance method is frequently used for MIC studies as shown in the review [16].
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a 25
1
1 - Zn 2 - Aspergillus niger
Rp / kΩ cm2
20 15 10
2
5 0 0
b
5
10
15
20
25
1 - Al 2 - Aspergillus niger
Rp / kΩ cm2
150
100
2
50 1 0 0
c
5
10
25
15
20
25
Zinc
-25 -50 -75 Aluminium
O
Rp - Rpmicr / kΩ cm 2
0
-100 -125
Aspergillus niger 0
5
10
15
20
25
t / month Fig. 3. Polarization resistance change determined from EIS data (Rp = Zx!0) of abiotic samples (1) and those with Aspergillus niger, (2) during two-year exposure to humid atmosphere at 26 C.
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Fig. 3 shows the Rp change during two-year exposure of abiotic and biotic samples. (The data in Fig. 3 were determined as Rp = Zx!0, however, nearly identical values were obtained also from the equivalent circuits used for the data fitting.) It is obvious that the influence of microorganisms depends on the kind of the metal they colonize: corrosion acceleration is evident for zinc and inhibition for aluminium. When plotting the Rp data as a difference between the biotic and abiotic samples ðR0p Rmicr p Þ, it can be seen that the inhibition effect of Al corrosion is much stronger than that of acceleration of Zn corrosion (Fig. 3a). An increase in Rp with exposure time is observed for Zn samples, which means decrease in corrosion activity of both abiotic and biotic samples (Fig. 3a). This is due to development of corrosion product layer, whose inhibiting effect for the abiotic sample is more pronounced when compared to the biotic counterpart. The corrosion activity of abiotic aluminium does not change significantly after the twoyear exposure period (curve 1, Fig. 3b), while the samples with microorganisms decreased the activity markedly (curve 2). The inhibiting influence of A. niger reaches its maximum after 6–15 months of exposure, when the corrosion activity is reduced by more than one order of magnitude when compared to the abiotic counterpart. The EIS diagrams for Zn samples are given in Fig. 4. A pristine sample in NaCl solution exhibits two clearly pronounced time constants (curve 1, Fig. 4). Such behaviour is typical of zinc electrode [39]. To observe the two time constants, two conditions are of importance: (1) the sample has to be pre-exposed for some time to atmosphere and (2) immersed into chloride solution. Indeed, only one time constant is characteristic of the same pre-exposed sample if the EIS measurements were performed in sulphate solution (curve 3, Fig. 4). According to arguments provided in literature, the multi-time constant behaviour relates to layered structure of corrosion products on exposed zinc [40]. Thus, the data obtained in chloride solution (Fig. 4) imply a two-layer structure, the inner part of which is composed of the native corrosion products and the outer part is the layer penetrated by chloride. The second layer is not present in sulphate solution because, as commonly known, the penetration capability of chloride is much higher than that of sulphate. In agreement with the said above, the EIS data in sulphate solution fit well to an equivalent circuit consisting of one Rt-CPE element (charge transfer resistance-constant phase element) and the data in chloride solution fit to an equivalent circuit of two Rt-CPE elements (circuits and curves 1 and 3 in Fig. 4). (The constant phase element consists of capacitance (C, lF cm2) and frequency dispersion (n) a dimensionless parameter (n 6 1), which usually is correlated with porosity of corrosion products. If n is close to 1, this means CPE is most capacitive.) The fitting parameters are summarized in Table 1. The inner layer should be build of zinc(hydro)oxide and the outer layer of hydroxochloride. This assumption is supported by the literature data: ZnO, Zn(OH)2 and 4Zn(OH)2, ZnCl2 were detected on zinc in chloride solutions by FTIR and in situ Raman spectroscopy [41,42]. The EIS data for the Zn samples exposed for two years are shown in Fig. 4 (curves 2 and 4). Although there are distinctive differences between the abiotic and biotic samples, both of them correspond well to an equivalent circuit consisting of three Rt-CPE elements and RX in series (circuit 2 in Fig. 4 and the parameters in Table 1). Such results indicate a three-layer structure on zinc electrode.
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CPE2
1 R1 CPE1
RΩ
R2 CPE2
CPE3
2, 4 R1
R2
CPE1
RΩ
3
R3
RΩ
R1
2 10000
1 - pristine, NaCl 2 - exposed, NaCl 3 - pristine, Na2SO4 4 - A. niger , NaCl
Z / Ω cm2
4
1000
1 3 100
10
0
3
-10
1
phase / deg
-20 -30
2
-40 -50
4 -60 -70 10-2
10-1
100
101
102
103
104
105
f / Hz Fig. 4. EIS diagrams of pristine Zn (1,3) and abiotic (2) and biotic (3) samples exposed for two years to humid atmosphere at 26 C. Solutions: 1,2,4 – 0.6 M NaCl; 3 – 0.1 M Na2SO4. The experimental results (symbols) were fitted (lines) assuming the shown equivalent circuits and the fitting parameters are summarized in Table 1.
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Table 1 Fitting parameters for EIS data (Figs. 4 and 7) obtained for Zn and Al pristine samples and those exposed for two years with and without microorganisms to humid atmosphere Sample
C1, lF cm2
C2, lF cm2
C3, lF cm2
R1, kX cm2
R2, kX cm2
R3, kX cm2
Zn, pristine Zn, exposed Zn + Aspergillus niger Al, pristine Al, exposed Al + Aspergillus niger
1730 (n = 0.96) 11.9 (0.74) 59.5 (0.84) 3.6 (0.85) 2.2 (0.83) 14.7 (0.86)
9.4 (n = 0.77) 0.5 (0.54) 0.52 (0.83) – – 2.0 (0.76)
– 0.8 (0.66) 0.74 (0.59) – – –
0.33 17.5 4.70 5.9 8.2 59.9
0.24 0.12 0.84 – – 22.5
– 8.8 3.9 – – –
10000
5
4
8
3
2
Zn(002)
* - Zn (OH) Cl H O + - Zn CO (OH) H O 2
6
2
# 1000
+
#
*
+ 2
+
+
+
ZnO (002)
ZnO (100)
Intensity / cps
# - NaZn(OH)33H2O
Zn(100)
The X-ray diffraction (XRD) measurements indicate crystalline hydroxide, carbonate and chloride species to be developed both on abiotic and biotic Zn exposed to NaCl solution (Fig. 5). The difference in widths of the XRD peaks indicates that the grain size of the chloride species is much greater than that of the carbonate ones: the full width at half maximum for chloride is FWHM = 0.139 at 2H = 11.2 while the analogous value for carbonate is FWHM = 0.925 (2H = 12.9). No symptomatic differences were found between XRD patterns for blank zinc and that with A. niger. It means that biogenic products do not form crystalline phases with the zinc corrosion products. The surface analysis by X-ray photoelectron spectroscopy (XPS) and surface sputtering by ionised argon shows significant content of organic carbon within the studied layer (Fig. 6). The highest content of organic carbon was found on the top of the corrosion product layer (ca. 40 at.%), which may originate from the dehydrated microorganisms (under vacuum conditions) and their metabolites. The noticeable concentrations of
1 100 10
20
30
40
2Θ Fig. 5. XRD patterns for abiotic Zn sample (2) and that with A. niger (1). The samples were exposed for two years to humid atmosphere at 26 C and for 1 h to 0.6 M NaCl solution.
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Carbonate 6
content / at.%
80
4 2
60
S2- + SO42-
Oxygen
Cl-
0 0
40
Zinc
20
Carbon (org)
10 20 30 40 50
Zn+Aspergillus niger 0 0
10
20
30
40
50
sputtering time / min
Carbonate
2.0
120
1.5
content / at.%
100
1.0
80
Carbon (org)
S2- + SO42-
Cl-
0.5 0.0
0
60
5
10 15 20
Oxygen
40 Aluminium
20 0
Al+Aspergillus niger 0
5
10
15
20
sputtering time / min Fig. 6. Concentration profiles of organic carbon, oxygen (O2) and metals [Zn + Zn(II) and Al + Al(III)] on zinc (a) and aluminium (b) subjected for two years to influence of Aspergilus niger in humid atmosphere. The concentrations (in at.%) were determined by X-ray photoelectron spectroscopy (XPS) using surface sputtering by ionised argon. The insertions show distribution of lower concentrations: carbonate, chloride, sulphide and sulphate.
organic carbon in deeper levels (8–10 at.%) have to be considered as the result of the organic acid penetration into the corrosion product layer. Since the carbon was not detected by XRD (Fig. 5), it follows that carbon compounds do not form crystalline phases with the corrosion products. The same could be said about sulphides and sulphates minor amounts of which were detected by XPS (insertion in Fig. 6), but not by XRD.
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It should be noted that the entire corrosion product layer is much thicker than the depth profiles in Fig. 6. The sputtering time of 50 min and the used beam current of 20 lA/cm2 correspond to an average depth of 250 nm assuming the etching rate to be ca. 5 nm/min for Zn (ZnO) [37]. Thus, the analysis data relate to the depth of several hundreds nanometers, whereas the entire corrosion product layer is much thicker (according to literature, the thickness of corrosion products on zinc developed under atmospheric conditions ranges from 1 to 25 lm per year [43,44]). The XPS method in respect to the studies of layered non-homogeneous structures seems to be problematic due to non-uniformity of surface etching by ionised argon (for instance, the etching rate for Zn and C may differ about ten times). EIS measurements provide better opportunity to detect layered structures as was demonstrated above. It follows from EIS, XRD and XPS data that the top layer of the three-layer structure on Zn (R3C3) is composed of the hydroxide-carbonate-chloride species, which originate from the interaction of corrosion products (ZnO–Zn(OH)2) with carbon dioxide during exposure to the atmosphere and then with NaCl solution during exposure to an electrochemical cell. The intermediate layer (R2C2) is the rest of the hydroxide-carbonate, which was not penetrated by the chloride. The inner layer (i.e. next to the metal, R1C1) is typically composed of a zinc oxide. Since the outer layer (R3C3), which develops in chloride solution, may be considered as an experimental artefact, development of a two-layer structure in atmosphere may be concluded. The analogous structure was determined by other authors, who studied the zinc samples exposed under outdoor conditions and came to a conclusion that corrosion products consisted of a compact ZnO inner layer and a porous 2CO3Zn Æ 3Zn(OH)2 outer layer [40]. The layer thickness (d) may be characterised by its capacitance, which is in inverse proportion to the thickness (Table 1). The fitting parameters in Table 1 indicate that the inner layer on pristine Zn (R1C1) is much thinner as compared with the outer layer (C1 = 1730 lF cm2 vs. C2 = 9.4 lF cm2). At the same time, the resistance of this much thinner layer is not less (even greater) when compared to the resistance of the thick outer layer (R1 = 0.33 kX cm2 vs. R2 = 0.24 kX cm2). This clearly shows a high inhibiting capability of the native oxide layer, which is produced by corrosion process in atmosphere. (Strictly speaking, the above comparison of d will be correct assuming that there is no great difference in the relative permittivities of the films. However, this aspect may be neglected due to a great difference between C1 and C2). The EIS data in Fig. 3 and Table 1 provide information that corrosion acceleration by A. niger is caused primarily by diminution in the thickness of the first layer (C1R1), which, as shown above, has the greatest inhibiting capability. The capacitance C1 in the presence of A. niger is 5 times higher (the layer is thinner) when compared to that of the abiotic counterpart. The R1 value for the sample with microorganisms is accordingly less. It is interesting to refer here to analogous conclusion drawn from the data obtained with the strains B. mycoides and P. frequentans [18,19]. When returning back to Al samples, let us consider the EIS diagrams in Fig. 7 and the derived data in Table 1. The data indicate one corrosion product layer on the abiotic Al and appearance of a second layer in case of microorganisms, as seen from a certain contribution in the low frequency region (curve 2, Fig. 7). The resistance of the first layer developed under influence of A. niger is much greater when compared to that developed on the abiotic sample (59.9 kX cm2 and 8.2 kX cm2, respectively). At the same time, according to the capacitance values, the layer on the biotic sample is much thinner
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10 5
CPE1
1
2
RΩ
R1
10 4
1
CPE1
CPE2
Z / Ω cm2
2 10 3
R1
RΩ
R2
10 2
1 - Al 2 - A.niger 10 1
10 0
1
phase / deg
0
-20
2 -40
-60
-80 10-2
10-1
100
101
102
103
104
105
f / Hz Fig. 7. EIS diagrams for abiotic Al (1) and that with A. niger (2) in 0.6 M NaCl solution. The samples were exposed for two years to humid atmosphere at 26 C. The equivalent circuits, which were used for experimental data (symbols) fitting (lines), are shown in the figure. The fitting parameters are summarised in Table 1.
(14.7 lF cm2 and 2.2 lF cm2). This suggests an idea that A. niger may affect primarily the sites of localised corrosion, i.e. micropores and microcracks. A support for this assumption provides SEM data, which show preferential colonization of microcracks on the Al surface, most probably, owing to preferential accumulation of water in these sites (Fig. 1a).
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Aluminium is known as a metal, which corrodes not uniformly because of preferential attack on the grain boundaries. The localized corrosion of Al alloys was indicated by EIS as an appearance of a second time constant in the low frequency region [45]. The authors pointed out that the effect is usually partially masked by the scatter of the data at low frequencies. We also observed the same typical discrepancy of the phase angle from one time constant model for a pure pristine Al sample (the data not shown in this paper). However, the interpretation was problematic for the exposed samples due to wide scatter of the experimental data below 1 Hz. The data on chemical composition of the outer layer on Al developed during MIC are shown in Fig. 6. (The average sputtered profile is ca. 50 nm assuming that the used beam current of 20 lA/cm2 corresponds to an average etching rate of ca. 2.5 nm/min for Al (Al2O3) [36]). Organic carbon, carbonates, sulphates, sulphides and chlorides were determined within the outer corrosion product layer alongside to aluminium oxide. About 80 at.% of the top of the entire structure is composed of organic carbon, which is from microorganisms and their metabolites. At deeper levels, the content of carbon drops down to lower values, which may be attributed to weak penetration of organic acids into the aluminium oxide structure. The results obtained demonstrate that A. niger may act either as the corrosion accelerators or the inhibitors depending upon the metal they colonize. The microorganisms were capable of disintegrating of the thick corrosion product layers on zinc, while the aluminium oxide layer, in general, was stable against the microbiological action. Probably, microbial colonisation of the sites of localized attack led to a ‘‘healing’’ effect. The obtained results show that microorganisms could be considered as an alternative to chemicals, application of which for corrosion protection tends to be increasingly restricted. 4. Conclusions The wild strain A. niger acted either as corrosion accelerator or inhibitor depending upon the metal it colonized. Microbially induced corrosion acceleration was ascertained for zinc while corrosion inhibition was typical of aluminium. The EIS data indicated two-layer structures developed on Zn and Al under influence of A. niger. The corrosion acceleration of Zn lies primarily in diminishing of the thickness of the inner layer (next to the metal), which has the greatest passivating capacity. For Al, an increase in charge transfer resistance, but not in layer thickness, implied that the fungi might promote passivity at sites of localized corrosion (micropores, microcracks, etc.). The XRD studies ascertained crystalline hydroxide and carbonate species developed during MIC of Zn. No distinctive differences were found between XRD patterns for abiotic zinc and that with A. niger. XPS data showed that biogenic compounds penetrated into the corrosion product layer, but they did not form crystalline phases with the corrosion products of zinc. Acknowledgements This study was carried out as a part of the complex research program supported by the Lithuanian Science and Study Foundation (Contract KP-059). The authors remember with great respect Dr. E. Matulionis who passed away in 2003. He carried out SEM investigations at the Institute of Chemistry, Vilnius.
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References [1] D. Thierry, W. Sand, Microbially influenced corrosion, in: P. Marcus, J. Oudar (Eds.), Corrosion Mechanism in Theory and Practice, Marcel Dekker, New York, 1995, p. 457. [2] T.E. Cloete, Mater. Corros. 54 (2003) 520. [3] P. Linhardt, Mater. Corros. 55 (2004) 158. [4] X. Shi, R. Avci, M. Geiser, Z. Lewandowski, Corros. Sci. 45 (2003) 2577. [5] D. Wagner, A.H.L. Chamberlain, Biodegradation 8 (1997) 177. [6] R.S. Dubey, S.N. Upadhyay, Bull. Electrochem. 15 (1999) 266. [7] B.J. Pitonzo, P. Castro, P.S. Amy, G. Southam, D.A. Jones, D. Ringelberg, Corrosion 60 (2004) 64. [8] D.A. Jones, P.S. Amy, Corrosion 58 (2002) 638. [9] M.J. H Gayosso, G.Z. Olivares, N.R. Ordaz, C.J. Ramirez, R.G. Esquivel, A.P. Viveros, Electrochim. Acta 49 (2004) 4295. [10] G. Huang, K.-Y. Chan, H.H.P. Fang, J. Electrochem. Soc. 151 (2004) B434. [11] J.P. Busalmen, M. Vazquez, S.R. Sanchez, Electrochim. Acta 47 (2002) 1857. [12] R.P. George, D. Marshall, R.C. Newman, Corros. Sci. 45 (2003) 1999. [13] D. Ornek, A. Jayaraman, T.K. Wood, Z. Sun, C.H. Hsu, F. Mansfeld, Corros. Sci. 43 (2001) 2121. [14] F. Mansfeld, C.H. Hsu, D. Ornek, T.K. Wood, B.C. Syrett, J. Electrochem. Soc. 149 (2002) B130. [15] D. Ornek, T.K. Wood, C.H. Hsu, F. Mansfeld, Corros. Sci. 44 (2002) 2291. [16] F. Mansfeld, Mater. Corros. 54 (2003) 489. [17] R. Zuo, E. Kus, F. Mansfeld, T.K. Wood, Corros. Sci. 47 (2005) 279. [18] E. Juzeli unas, R. Ramanauskas, A. Lugauskas, M. Samulevicˇien_e, K. Leinartas, Electrochem. Commun. 7 (2005) 305. [19] E. Juzeli unas, R. Ramanauskas, A. Lugauskas, K. Leinartas, M. Samulevicˇien_e, A. Sudavicˇius, Electrochim. Acta 51 (2006) 6085. [20] A. Lugauskas, D. Bridzˇiuvien_e, A. Narkevicˇius, E. Ivasˇkevicˇ, Mikol. Fitopatol. 38 (5) (2004) 54. [21] R. Ramanauskas, E. Juzeli unas, A. Narkevicˇius, D. Bucˇinskien_e, A. Lugauskas, D. Pecˇiulyt_e, L. Levinskait_e, V. Ulevicˇius, D. Jasinevicˇien_e, Chemija 16 (2005) 25. [22] S.K. Gupta, K.K. Maggon, T.A. Venkitasubramanian, J. Gen. Microbiol. 99 (1977) 43. [23] A.K. Banik, Folia Microbiol. 21 (2) (1976) 139. [24] K.H. Domsch, W. Gams, T.-H. Anderson, Compendium of Soil Fungi, Academic Press, London, 1980. [25] A.R. Angumeenal, P. Kamalakannan, H.J. Prabhu, D. Venkappayya, Indian J. Chem. Technol. 9 (2002) 508. [26] S. Baker, J. Magnuson, Z. Dai, E. Panisko, C. Wend, L. Lasure.