Formation of the Fe(II–III) hydroxysulphate green rust during
marine corrosion of steel associated to molecular detection
of dissimilatory sulphite-reductase
q
S. Pineau
a, R. Sabot
b, L. Quillet
c, M. Jeannin
b, Ch. Caplat
d, I. Dupont-Morral
a,
Ph. Refait
b,*aCorrodys, Centre de Corrosion Marine et Biologique, 55 Rue de Beuzeville, BP9, F-50120 Equeurdreville, France
bLaboratoire d’Etude des Mate´riaux en Milieux Agressifs, EA3167, Universite´ de La Rochelle, Baˆtiment Marie Curie, Avenue Michel Cre´peau,
F-17042 La Rochelle Cedex 01, France
cLaboratoire de Microbiologie du Froid, Equipe Biodiversite´ et Environnement, Universite´ de Rouen, F-76821 Mont St Aignan, France dEquipe de Recherche en Physico-Chimie et Biotechnologies, EA 3914, Universite´ de Caen Basse-Normandie, F-14032 Caen Cedex, France
Received 11 May 2007; accepted 7 November 2007 Available online 25 January 2008
Abstract
Accelerated corrosion phenomena of carbon steel constantly immersed in seawater could be simulated in situ via a galvanic coupling of the samples with steel port structures. Three harbours located on different seas and various conditions of immersion were considered so as to study the eventual correlation between dissimilatory sulphite-reductase genes and sulphate-containing corrosion products. In each case, after 6 or 12 months, the rust layers proved to be made of an inner black layer, close to steel surface, and an orange outer layer. Scanning electron microscopy, chemical analyses by inductively coupled plasma/atomic emission spectroscopy, X-ray diffraction and micro-Raman spectroscopy were used to obtain a detailed characterisation of these layers. The inner one proved to be mainly com-posed of iron sulphides FeS and Fe(II–III) hydroxysulphate green rust GRðSO2
4 Þ, the outer one of Fe(III) oxyhydroxides, with
lepido-crocite c-FeOOH as a major component. The molecular detection of dissimilatory sulphite-reductase, the key enzyme in dissimilatory sulphate reduction by micro-organisms, was applied for the first time to rust layers. This detection was positive in most cases, especially for the inner part of the rust layers. This demonstrates that sulphate reducing bacteria are associated to GRðSO2
4 Þ inside the rust layers,
GRðSO24 Þ more likely playing the role of a source of sulphate. The systematic presence of iron sulphides also testifies the activity of
sulphate reducing bacteria and/or thiosulphate reducing bacteria. Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: A. Steel; B. Raman spectroscopy; C. Marine corrosion; C. Rust; C. Sulphate-reducing bacteria
1. Introduction
Microbiologically influenced corrosion (MIC) of steel results from the combination of chemical, electrochemical, and biological factors. It is often observed in biologically active media, and in particular in seawater. The average
cor-rosion rates of steel in seawater, their dependence with the immersion zone, and their evolution with time is well known, and the steel thickness required for a structure that should last for decades can be reliably estimated. But the influence of micro-organisms can induce local drastic acceleration of the corrosion process that may lead in the most severe cases to the perforation of steel sheet piles in a few years[1–3].
During the last decade, several programs of accelerated corrosion study in harbour sites have been conducted around the world, especially in Europe[1,2]and Australia [3]. Most of them related to the accelerated low water
0010-938X/$ - see front matterÓ 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.corsci.2007.11.029
q
Part of the thesis presented at the Universite´ de Technologie de Compie`gne by S. Pineau.
*
Corresponding author. Tel.: +33 5 46 45 82 27; fax: +33 5 46 45 72 72. E-mail address:[email protected](Ph. Refait).
www.elsevier.com/locate/corsci Corrosion Science 50 (2008) 1099–1111
corrosion (ALWC) phenomenon. The main results demon-strated that the corrosion processes at ALWC sites were clearly different from those observed in NLWC (no low water corrosion) sites. The main factors influencing the accelerated corrosion seem to be surface roughness, water velocity, dissolved oxygen and micro-organisms communities.
Such accelerated corrosion phenomena are generally associated with sulphate reducing bacteria (SRB)[4,5]that have the ability to use sulphate as a terminal electron acceptor. The growth of SRB in marine environments causes significant modifications of many physicochemical parameters at the steel/seawater interface, including local changes in pH and redox potential values, variations in anion and cation concentrations and alteration of the com-position and structure of corrosion products. These effects often lead to significant changes in the corrosion behaviour of steel. The complexity of the local environment at the steel/seawater interface is increased by the presence of micro-organisms and their extracellular polymeric sub-stances (EPS). As a consequence of the biofilm heterogene-ity, areas with different ion concentrations are formed and the development of corrosion product layers of different protective characteristics occurs.
In the case of carbon steel, the rust layer is made up of two main parts: an outer orange layer mainly composed of iron oxyhydroxides and an inner black layer generally described as composed of iron sulphides. In fact, most of the sulphides produced by SRB are scavenged by reactive metal ions and promotes the formation of iron sulphides [6], such as pyrite FeS2[7], greigite Fe3S4[8]or
mackinaw-ite FeS [9]. The presence of these compounds on steel in seawater is then an indication that the corrosion process is influenced by SRB, since in seawater sulphur is only pres-ent as sulphate in the absence of bacterial activity. A few studies however reported an association between the Fe(II–III) hydroxysulphate, that is the sulphated green rust GRðSO24 Þ, and microbially influenced corrosion (MIC)
both in the laboratory[10], and on steel sheet piles in a har-bour[11,12].
Green rusts (GRs) are Fe(II–III) hydroxy salts charac-terised by a crystal structure that consists of the stacking of Fe(OH)2-like layers carrying a positive charge due to
the presence of Fe(III), and of interlayers constituted of water molecules and anions that restore the electrical neutrality of the crystal [13,14]. Several GRs are known, and in particular those based on the main anions found in seawater, that is GR(Cl), GRðCO23 Þ and GRðSO
2 4 Þ.
The chemical composition of GRðSO24 Þ is for instance
FeII 4Fe
III
2 ðOHÞ12SO4 8H2O, [14] sometimes developed as
½FeII4Fe III
2 ðOHÞ12 2þ
½SO4 8H2O2 to remind that the
crys-tal structure is built on positive hydroxide layers alternat-ing with negative interlayers. In seawater, the [Cl]/ [SO24 ] and [SO24 =½HCO3 molar ratios are about 19 and
12, respectively (computed from [15]). Laboratory experi-ments demonstrated that GRðSO2
4 Þ formed instead of
GR(Cl) [16,17], as the layered structure of GRs presents
a strong affinity for divalent anions. GRs transform into Fe(III) (oxyhydr)oxides in the presence of O2and would
only persist for long periods if environmental conditions are anaerobic. This could explain the association of GRðSO24 Þ with SRB, since most of these micro-organisms
are obligate anaerobes. In a previous study, where GRðSO24 Þ was found in 25 years old rust layers formed
on steel in seawater, it was suggested that GRðSO24 Þ could
facilitate the colonisation of the surface by SRB since it constitutes a source of sulphate ions[16].
Early studies of SRB were based on culture techniques allowing the growth of micro-organisms by use of selective media. More recent studies have used molecular biology techniques to take into account the micro-organisms that cannot be cultivated when they are isolated from the envi-ronment[18]. As these micro-organisms constitute a heter-ogeneous group, including members of several phyla and domains, it is difficult to use the 16S rRNA gene as a molecular marker. However, the dsrAB genes that encode the dissimilatory sulphite-reductase, the key enzyme in dis-similatory sulphate reduction in these micro-organisms, may be an appropriate marker [19–21]. This approach of dsrAB detection has been used for the quantification of sul-phate reducing bacteria in sediments with success[22,23].
The aim of this work was to simulate accelerated corro-sion phenomena of carbon steel constantly immersed in seawater. Three harbours located on different seas and var-ious conditions of immersion were considered so as to study the eventual correlation between dissimilatory sulph-ite-reductase genes and GRðSO24 Þ. Corrosion layers grown on steel coupons were then carefully analysed by scanning electron microscopy (SEM) coupled with electron disper-sive spectrometry (EDS), X-ray diffraction (XRD), micro-Raman spectroscopy and chemical analyses by inductively coupled plasma/atomic emission spectroscopy (ICP/AES). The main steps of the general corrosion process of carbon steel in seawater could be detailed and the synergistic effects between redox cycles of iron and sulphur via the metabolism of micro-organisms could then be discussed. 2. Experimental methods
2.1. In situ accelerated corrosion methodology
In situ experimentations were conducted in three French ports: Nantes-Saint-Nazaire (Estuarine area, Atlantic Ocean), Le Havre (English Channel) and Marseille (Medi-terranean Sea). In order to establish the general trends of the corrosion process, several sites with high variability of water quality, environmental conditions and maritime activities, and several immersion depths (immersed zone, sub-surface zone, low water zone) were considered in each harbour (Table 1). In this work, the steel samples were con-stantly immersed in water. The case of cyclic conditions of immersion will be treated in another article. The choice of the sites was done according to the type of installation (age, type of structure, corrosion damage, etc.), the water quality
(three maritime coasts including estuarine area), the tide effect and the industrial activities. Fig. 1 illustrates the localization of each harbour.
The carbon steel samples were coupons (50 60 4 mm) produced by the blast-furnace route, having a weight percent composition of 0.380 Mn, 0.120 C, 0.037 Al, 0.023 Ni, 0.017 Cr, 0.010 P, 0.007 Si, 0.007 Cu, 0.004 S, 0.001 Mo, and Fe for the rest. The surface was shot blasted (Sa 2.5, angular shot) to obtain a roughness value of 50–70 lm, degreased with acetone and dried. The corro-sion process was accelerated via an electrical connection between the coupons and the port structure. This accelera-tion is due to the electrochemical disequilibrium between the highly reactive blast cleaned surface of the coupons and the somehow protected (by macrofouling and/or stabi-lised corrosion deposits) large metallic surface of the quay structure. The coupons were disposed in a Teflon holder
(Fig. 1), the electrical connection being maintained by weld point between each coupon. To prevent any crevice corro-sion phenomenon in the sliding channel of the Teflon holder, the right and left sides of the coupons were pro-tected with polyurethane painting. Twenty test units were fixed on a carbon steel unit holder that was welded to the quay structure by professional divers. The holders were immersed in November 2004, and the sampling has been conducted after 6 months (May 2005) and 12 months (November 2005) of exposure by professional divers. 2.2. Characterisation of the rust layers
Scanning electron microscopy (SEM), X-ray diffraction (XRD) and micro-Raman spectroscopy were used to charac-terise the rust layers. Energy dispersive X-ray spectrometry (EDS), coupled with SEM, and inductively coupled
Fig. 1. (a) Map of France showing the localisation of the three harbours considered in this study. (b) Principle of the modular experimental sampling mount.
Table 1
Characteristic features of the harbour sites considered in this study: ‘‘Port Autonome du Havre” (PAH); ‘‘Port Autonome de Nantes-Saint-Nazaire” (PANSN); ‘‘Port Autonome de Marseille” (PAM)
Port Site activity Hydrology Zonesa Structure (year)
Comment PAH Coaler terminal High tide
(8 m)
LWZ Sheet pilling (1978)
Cases of ALWC, High shipping and hydrodynamic activity, coal discharge
Chemical industry
Basin SSZ Sheet pilling (1930)
Industrial discharge of S, low hydrodynamic activity, periodic supply of freshwater
PANSN Food-processing
Average tide (4 m)
LWZ Piles (1982) Estuarine area, high shipping and hydrodynamic activity, organic matter supply
Shipbuilding dock
Basin SSZ Dock door (1932)
Moving structure (door), pollutant from shipbuilding
PAM Food-processing
Low tide (50 cm)
SSZ + IMZ Sheet pilling (1976)
Unexplained thickness losses (immersion zone), periodic supply of freshwater, organic matter supply
Containers terminal Low tide (50 cm) SSZ Sheet pilling (1975)
Normal corrosion state, high shipping activity
plasma-atomic emission spectrometry (ICP-AES) were used to determine the chemical composition of the rust layers.
An observation of the macroscopic morphology was realised before the coupons were dried for SEM and EDS analyses. The rust layers were carefully removed by scrap-ing so that analyses of the steel surface, the outer surface and the inner surface of the rust layer could be achieved. The thickness of these layers was typically between 1 and 5 mm before they dry. SEM pictures were obtained on a Cambridge Stereoscan 240 (Leo Microscopy) and coupled microanalyses were performed using an energy dispersive X-ray spectrometer Synergy 4 without specific calibration (energy of the electron beam = 20 keV). The efficiency of the in situ experimentation process for accelerated corro-sion simulation has been evaluated by weight loss and thickness loss, and by observations of the morphology of the rust layers and of the state of the steel surface. The thickness loss was determined via micrometric screw measurements.
For X-ray diffraction and micro-Raman spectroscopy analyses, corrosion layers were extracted from steel surface after that the coupons were frozen at80 °C. This step of freeze was necessary to preserve unstable compounds such as green rusts and amorphous iron sulphides from oxida-tion and transformaoxida-tion. During the first time of defrost-ing, slices of the corrosion layers were obtained by sterile scalpel cut. These slices were also used for SEM-EDS anal-yses. In most cases, it proved possible to isolate an inner dark layer from an outer orange-brown layer that could be analysed separately. Simultaneously, a sample of each layer (inner black layer and outer orange layer) was extracted by scalpel and ground to a powder as thin as pos-sible for X-ray diffraction analysis. This last method was also used for DNA extraction process. XRD experiments were carried out with a classical powder diffractometer (Brucker AXSÒ D8-Advance), using Cu Ka wavelength (k = 0.15406 nm) in Bragg–Brentano geometry. The sam-ples were coated with glycerol before analysis. This proce-dure limits the oxidation of compounds sensitive to O2,
such as GRs, during the acquisition of the pattern [24]. Micro-Raman analyses were performed on a Jobin Yvon High Resolution Raman spectrometer (LabRAM HR) equipped with a microscope (Olympus BX 41) and a Pel-tier-based cooled charge coupled device (CCD) detector. The analysed zone had a diameter of6 lm. Spectra were recorded with the acquisition LabSpec software at room temperature with a resolution of2 cm1. Excitation was provided by a He–Ne laser (632.8 nm). Its power was var-ied between 1.94 and 0.07 mW to prevent an excessive heating that could have induced the transformation of the analysed sample into hematite a-Fe2O3[25]. First, the
surface of the coupons and lamella of corrosion products have been analysed. Then, slices of corrosion products were put on a glass plate. A motorized XY stage was used to focus on the different oxide layers and to estimate their thickness.
Two coupons of each sample holder were devoted to the chemical analysis, by ICP-AES, of the corrosion products. The orange rust layers, not adherent, were extracted with porcelain spatula, whereas an ultrasonication scaler was required for the more adherent inner black layers. The average values of the two measurements were used for a principal components analysis.
ICP-AES analyses were performed on a Varian Vista MPX equipped with axially-viewed plasma. True simulta-neous measurement from the single CCD detector delivers maximum sensitivity for many applications. Major, minor and trace elements were determined in seawater after acid-ification (HNO31%). Dried (35°C) corrosion products had
to be dissolved which required an acid digestion procedure before analysis. A mixture of nitric (Normapur 69%) and hydrochloric (Normapur 37%) acid was used to dissolve solid samples in controlled conditions of temperature and pressure within a microwave oven. 5 mL of the acid mix-ture were added to 0.1–0.4 g of dried samples and placed in teflon bombs during one night at 40°C. A three-step program of temperature and pressure was applied to the bombs. The steps were: (1) increase of the temperature up to 180°C during 10 min at 80% of the higher pressure available (Pmax); (2) temperature and pressure kept
con-stant during 15 min; (3) decrease of the temperature down to 100°C during 5 min at 40% Pmax. The solutions to be
analysed were finally obtained after a dilution in 50 mL of ultrapure water (18.2 MX cm). An analytical blank was prepared for each series. All the glassware was cleaned by immersion in HNO3(10%) one night before use.
Detec-tion limits were under 1 lg L1 for Al, Ca, Cd, Fe, Mg, Mn, Sr, Ti, and Zn, under 5 lg L1 for Cu, Co, Cr, Ni, and Mo, and under 10 lg L1for P, Pb, S, Se, Si, and Sn. A statistical approach was envisioned to interpret the numerous experimental data given by the ICP-AES analy-ses. The Spad 3.01 software was used for the data process-ing required to perform the Principal Components Analysis (PCA). PCA involves a mathematical procedure that trans-forms a number of correlated variables into a smaller num-ber of uncorrelated variables called principal components. The first principal component accounts for as much of the variability in the data as possible, and each succeeding component accounts for as much of the remaining variabil-ity as possible. The aim of the PCA was to identify new meaningful underlying variables among metallic elements quantified in corrosion layers.
2.3. Molecular detection of sulphate-reducing micro-organisms
The molecular detection of the dissimilatory sulphite-reductase (dsrAB) genes of sulphate-reducing micro-organ-isms was achieved by Direct and Nested PCR (polymerase chain reaction) from bacterial DNA.
Total DNA was extracted from 0.5 g of the rust layers (wet weight) by use of a Bio-101 FastDNA Spin kit in com-bination with the FastPrep FP120 bead beating system
(Bio-101, USA) according to the manufacturer’s instruc-tions. The concentration of the resulting DNA was esti-mated by UV spectrometer. The dsrAB genes were Direct PCR-amplified with the dsr-1F (50
-AC[CG]CACTGGAA-GCACG-30, positions 421–436) and dsr-4R (50
-GTGTAG-CAGTTACCGCA-30, positions 2347–2363) primers, as
previously described by Wagner et al. [20]. Each reaction tube (50 lL) contained 0.125 lL of each primer (100 pmol lL1), 2 lL of dNTP (20 mM each), 36.3 lL of ultra-pure H2O, 5 lL of 10 PCR buffer (Eurogentec), 1.25 lL of
10% (w/v) BSA, 3 lL of MgCl2(25 mM), 0.2 lL (1 U) of
Taq polymerase (Red Goldstar, Eurogentec) and 2 lL of DNA template (approx. 50 ng lL1). PCR was carried out in a Perkin–Elmer thermocycler (Gene Amp PCR sys-tem 6700) as follows: 3 min at 94°C for initial denaturation; 30 cycles of 15 s at 94°C, 20 s at 54 °C, 2 min at 72 °C; and a final extension for 1 min at 72°C. For the Nested PCR pro-cess, the dsr-1F primer was coupled with the dsr-1R1 primer (5’-CGGTSAGYTCRTCCTG-3’, position 836–852). The Nested PCR mixture was similar to Direct PCR. The DNA template used was 1 lL from the direct PCR DNA amplificated and the reaction cycles as follows: 3 min at 94°C for initial elongation; 30 cycles of 15 s at 94 °C, 20 s at 54°C, 30 s at 72 °C; and a final extension for 7 min at 72°C.
PCR products were electrophorezed through a 0.5–2% (w/v) agarose gel in 0.5 Tris/acetate/EDTA containing ethidium bromide (0.05 lg mL1). DNA bands were visu-alized by UV illumination.
3. Results
3.1. Morphology of the degradation and estimation of the kinetics of corrosion
Fig. 2presents a schematic representation of the various morphologies of the corrosion profiles obtained from all the experimental conditions (ports and immersion zones). All the coupons were subject to uniform corrosion. In the case of coupons exposed in sub-surface zones (basins of
Nantes-St-Nazaire and Le Havre) a typical profile of ‘‘ter-race” corrosion or ‘‘beach mark” corrosion [3] has been observed. This form of corrosion could have been induced by the experimental system. Micro-craters and macro-cra-ters were observed, especially under corrosion tubercles.
The average results of weight and thickness loss after 12 months of exposure are reported in Table 2. Firstly, they show that the corrosion rates are much higher (0.35– 1.93 mm/year depending on the site and on the immersion zone) than the natural corrosion rate measured on the respective port structures. The natural corrosion rate was about 0.1–0.15 mm/year, as usually observed for steel dur-ing the first year of exposure in LWZ. These high values confirm the efficiency of the in situ accelerated corrosion methodology. Secondly, it can be seen that the corrosion rate is rather homogeneous, comprised between 1.06 and 1.93 mm/year, excepted for one site, where it was measured at 0.35 mm/year. This peculiar site corresponds to an estu-arine situation, which implies that the salinity is often much lower than the average salinity of seawater.
3.2. SEM observations and chemical analyses of the rust layers
In most cases, 4–6 analyses were performed on each sam-ple. Typical SEM photographs of the inner and outer sides of a typical rust layer are presented inFig. 3, together with the corresponding EDS spectra. The image of the outer part shows acicular or lath-like crystals. This morphology is rather typical of orthorhombic Fe(III) oxyhydroxides such as goethite a-FeOOH and lepidocrocite c-FeOOH [26]. Other grains with cauliflower morphology are also seen. The EDS analysis shows that the main elements are Fe and O. Other minor elements are present, namely Na, Cl, Mg, S, Si, and Ca. They probably come from seawater. The image of the inner part shows overlapping hexagonal platelets. Such a morphology was previously attributed to iron sulphides [27]and to GRðSO24 Þ[16], that is to com-pounds where Fe and S are combined. The corresponding EDS analysis confirms that sulphur is, in the inner part of
Fig. 2. Schematic representation of the various morphologies of the corrosion process and corresponding photographs of the steel surface. (a) General corrosion, (b) micro-craters form of localised corrosion, (c) macro-craters form of localised corrosion, (d) ‘‘terrace” form of general corrosion.
the rust layers, a major element together with iron and oxygen.
X-ray mappings of various elements were performed on cross sections of rust layers. A typical example is shown in Fig. 4. It can be seen that chloride is mainly present in the outer part of the rust layer. The same behaviour was observed for Na (not represented). The outer part is loose and very porous, and the pores were filled with seawater. So NaCl crystals formed after drying of the rust layer. In contrast, the inner part contains sulphur and much less chloride. The electrolyte present in the pores was probably significantly different from seawater.
Graphic representations given by PCA analysis of the composition of the outer and inner parts of the rust layers are given inFig. 5. For the outer part (Fig. 5a), the metallic elements are scattered on the graph. No particular groups of elements are seen, which could mean that the layers are unstable or in constant evolution. In contrast, for the
inner part (Fig. 5b), elements present in seawater, such as Ca and Sr, are gathered in the same part of the graph. They are opposed (compared to the centre) to those present in steel like Cr, Mo, Cu, Mn, Ni, Cd, and Fe. Such groups characterise the various behaviours of metallic elements during the corrosion process. These behaviours are linked to the origin, repartition and physico-chemical properties of the elements. The existence of a third group may be pro-posed. It gathers Al and Si among others. These are ele-ments present in steel and in seawater (clay). Finally, the PCA graph presents S as a separated element, presenting no relationship with other elements associated to seawater or steel since its position is at 90° of the others. It can then be deduced that S is characterised by a peculiar chemical behaviour and/or a peculiar origin.
3.3. XRD and l-Raman analyses of the rust layers
Examples of XRD and l-Raman analyses performed on the outer part of the rust layers are presented inFig. 6. The most intense diffraction lines visible on the XRD pattern presented here are those of lepidocrocite c-FeOOH (ICDD file no. 44-1415). Besides, the main diffraction lines of goe-thite a-FeOOH (ICDD file no. 81-0464) are also seen. These compounds are the Fe(III) oxyhydroxides commonly found in rust, and mainly responsible for its characteristic colour. Since l-Raman spectroscopy analyses a local zone, with a diameter of a few micrometers, it is possible to distinguish the various phases present and measure the spectrum of each one. This is really true for the dominant compound. However, if small crystals of a minor phase are dispersed in the major one, it should be more difficult to isolate the spectrum of the minor phase from that of the major phase. In our case, lepidocrocite was predominant in most of the analysed samples. For instance, the Raman spectrum given inFig. 6b displayed only the vibration bands of c-FeOOH
Table 2
Corrosion rate (mm year1) obtained by 2 methods from samples exposed on various test-sites, after 12 months of immersion: ‘‘Port Autonome du Havre” (PAH); ‘‘Port Autonome de Nantes-Saint-Nazaire” (PANSN); ‘‘Port Autonome de Marseille” (PAM)
Port Zonesa Corrosion rateb
Screw Weight Average PAH LWZ 1.68 1.74 1.71 SSZ 1.47 1.44 1.45 PANSN LWZ 0.35 0.34 0.345 SSZ 1.97 1.89 1.93 PAM SSZ + IMZ 1.19 1.26 1.22 SSZ 0.99 1.14 1.06
a LWZ, low water zone; SSZ, sub-surface zone; IMZ, immersion zone. bScrew = thickness loss deduced by micrometric screw; Weight =
weight loss measurements; Average = average of corrosion rate from the two methods.
Fig. 3. SEM photographs and EDS spectra of a rust layer. (a) Outer side, close to seawater and (b) inner side, close to steel surface. The corresponding steel coupon remained 12 months in the low water zone in the harbour of Nantes-Saint Nazaire.
[28–30], two intense ones at 252 and 380 cm1and smaller others at 218, 311, 350, 529, and 652 cm1. Goethite could also be detected in the outer part of the corrosion layer. Its spectrum is made of two main bands, at 299 and 385 cm1, accompanied by four minor ones, at 243, 479, 550 and 685 cm1[28]. The spectrum obtained here (Fig. 6c), is con-sistent with this description, but the first peak located at 253 cm1 is abnormally intense. This peak is more likely partially due to lepidocrocite, the major component that have its main band at 252 cm1. Similarly, the intense band observed at 388 cm1may have a contribution due to the second intense Raman band of lepidocrocite usually found at 380 cm1. Finally, magnetite Fe3O4was often detected,
and in some cases akaganeite (b-FeOOH) was also found. Fig. 7 presents two typical Raman spectra and one XRD pattern of the main compounds identified in the inner part of the rust layers. The XRD pattern given here is that of the Fe(II–III) hydroxysulphate GRðSO24 Þ, that
have been identified sometimes in rust layers formed on steel in seawater [11,12,16]. It is characterised by three intense lines at 2h = 8.03°, 16.04°, and 24.17° [14,16,30]. They correspond to interplanar distances of 1.10 nm, 0.552 nm, and 0.368 nm that are d001, d002 and d003 [14].
In most of the samples, only GRðSO2
4 Þ could be detected.
In some cases, two very broad lines, maybe corresponding to an amorphous or nanocrystalline compound, were observed. In the peculiar case presented in Fig. 7 three additional sharp diffraction lines are seen. Their positions correspond to those of the three main lines of the Fe(II– III) hydroxycarbonate GRðCO23 Þ. It is generally admitted
that the layered structure of GRs is characterised by a strong affinity for divalent anions [31–33]. This explains why it is GRðSO24 Þ that forms and not GR(Cl
). It is also
generally admitted that, among the divalent anions, CO23 is preferred, since it gives the highest stability to this kind of structure [31–33]. The ½SO24 =½HCO
3 molar ratio is
high, close to 12, which explains why GRðSO2
4 Þ is the
main GR obtained.
While XRD detected only GRðSO24 Þ (except this small
amount of GRðCO23 Þ detected in one case), l-Raman
spectroscopy allowed us to identify two distinct phases in the inner part of the rust layers. In fact, analysis after anal-ysis, it appeared that the inner part of the rust layer, visu-ally distinguished from the outer part, was itself subdivided in two layers. In the external part of the inner layer, what we could then call the central layer, only GRðSO2
4 Þ was
found. Its Raman spectrum is displayed inFig. 7b. It shows the two intense bands at 430 and 500 cm1that character-ise all GR compounds[30,34–38].
The last Raman spectrum (Fig. 7c) was obtained in a zone closer to the metal. It is totally different, only com-posed of two sharp Raman peaks, at 208 and 283 cm1. These two bands were also observed by Hansson et al. [39] during an anodic polarisation of an iron electrode in carbonated media. Using in situ micro-Raman spectros-copy, they detected these bands 5 min after the injection of a Na2S solution. They assumed that these two
vibra-tion bands corresponded to amorphous FeS, as already observed by Boughriet et al. [40] in anoxic sediments. As XRD did not show any diffraction line of crystalline varie-ties such as mackinawite or pyrrhotite, this compound may rather be amorphous or nanocrystalline. So, we conclude that vibration bands at 208 and 283 cm1are characteristic of amorphous FeS.
In conclusion, the rust layers, whatever the harbour, whatever the depth of immersion, whatever the zone in the harbour, proved to be composed, after 6 months or after 12 months, of three main compounds. Amorphous FeS forms a first layer close to the steel surface. It is cov-ered by a second layer of GRðSO24 Þ and finally a thicker
layer of Fe(III) oxyhydroxides (mainly lepidocrocite) is found. The average oxidation number of Fe then increases from steel to seawater (0, +2, +2.33 and +3). It is possible that magnetite, often observed as a minor component, forms an additional very thin layer between GR and FeO-OH, since the average oxidation number for Fe in Fe3O4is
Fig. 4. SEM photograph (side view) and elemental X-ray mapping of Cl and S of a rust layer. The corresponding steel coupon remained 12 months in the low water zone in the harbour of Nantes-Saint Nazaire. Top: sea side, bottom: steel side.
+2.67. Note finally that SEM observations and EDS anal-yses are in agreement with the Raman and XRD study. Fe and O are the main elements found in the outer part of the rust layers that is mainly composed of FeOOH phases. S is found together with Fe and O as a major element in the inner part of the rust layers, and this part proved to be composed of FeS and FeII4FeIII2 ðOHÞ12SO4 8H2O. The
large hexagonal platelets observed in the inner layer should correspond to GRðSO24 Þ, the only crystalline phase
pres-ent. This is actually the typical morphology of GRðSO2 4 Þ
crystals[16,41].
3.4. Molecular detection of dissimilatory sulphite-reductase in rust layers
The average of total DNA rate obtained from corrosion deposit is 50 ng lL1. This quantity of DNA is a low value compared to total bacteria (about 108bacteria per gram of humid deposit) estimated by direct epifluorescence filter technique (DEFT) with acridine orange.
The dsrAB amplification products were obtained from six of the 12 samples by the Nested PCR approach (Nos. 1, 2, 5, 8, 9 and 11) including three positive detections with Direct PCR approach (Table 3).
With the inner parts of the rust layers, the dsrAB ampli-fication products were obtained for the low water zones of the ports of Le Havre (Nos. 1 and 2) and Nantes-St-Naz-aire (No. 5), mainly after 6 months of immersion. The sam-ples of the port of Marseille showed the most important response to molecular detection by Direct PCR, in immer-sion zone (No. 9) and sub-surface zone (No. 11), only after 6 months of test.
With the outer parts of the rust layers, only two dsrAB amplification products were obtained, in low water zone of the port of Le Havre (No. 1) and in immersion zone of the port of Marseille (No. 9, including a Direct PCR detec-tion). Thus, if SRB are mainly present inside the inner part of the rust layers, they are not absent of the outer part. 4. Discussion
4.1. Accelerated corrosion processes of steel immersed in seawater
The main information given by this study is that, what-ever the ocean or sea, whatwhat-ever the harbour and localisa-tion in this harbour, whatever the depth of immersion, the main trends of the degradation of steel constantly immersed in seawater are the same. Moreover, in our experimental conditions, the process was always partially influenced by bacterial activity, and mainly by that of SRB. The process is in fact the consequence of (i) the chem-istry of iron species in seawater and (ii) the interaction between these chemical processes and biological activity. The different steps leading to the stratification observed after 6 or 12 months of accelerated marine corrosion are summarised in Fig. 8. The process begins of course with the dissolution of Fe and the reduction of dissolved O2:
Fe! Fe2þþ 2e ð1Þ
1=2O2þ H2Oþ 2e! 2OH ð2Þ
The corrosion process is uniform. Cathodic (2) and anodic (1) reactions take place on the whole surface. The accumu-lation of Fe2þaq and OHions at the steel/seawater interface rapidly leads to the precipitation of Fe(OH)2 hydroxide
layers. These monolayers tend to assemble, which would lead to the Fe(II) hydroxide Fe(OH)2. Very sensitive to
the oxidizing action of O2, Fe(OH)2 would rapidly be
Fig. 5. PCA analyses of ICP-AES results performed on the various rust layers sampled on steel coupons immersed 6 or 12 months in the three harbours considered in this study. (a) Outer layer and (b) inner layer; the arrow shows the independence of element S compared to main subgroups (surrounded by ellipses).
transformed into GRðSO24 Þ[16]. However, in the presence
of dissolved O2, the Fe 2+
cations of the Fe(OH)2
monolay-ers are rapidly oxidized into Fe3+ cations, which leads to ½FeII
1xFe III x ðOHÞ2
xþ
positively charged hydroxide monolay-ers. In this case, negatively charged interlayers made of SO24 anions and water molecules would form on the hydroxide monolayers, and GRðSO24 Þ would then be
ob-tained directly without the formation of Fe(OH)2as a
pre-cursor, as observed in laboratory experiments [13]. Fe(OH)2 could not be observed in this study because it
did not form or because it was oxidised too rapidly for being detected. We will assume for simplification that GRðSO24 Þ is the first solid obtained according to:
6Fe2þþ 10OHþ SO24 þ 1=2O2þ 9H2O
! FeII 4Fe
III
2 ðOHÞ12SO4 8H2O ð3Þ
During the very first stages of the corrosion process, the GRðSO24 Þ layer is in contact with seawater. It is oxidized
rapidly into lepidocrocite c-FeOOH. Note that the oxida-tion of GRs can also lead to goethite a-FeOOH and mag-netite Fe3O4. Lepidocrocite is however the main product of
the oxidation of GRðSO24 Þ in seawater like media[16]and
is favoured by large oxygen flows[42] and moderate tem-peratures (T 6 20°C) [43,44]. Our analyses demonstrate that lepidocrocite is the main final product of the oxidation
of Fe, even if goethite and magnetite were detected some-times. Thus, in the first days the rust layer consists essen-tially of a thin layer of GRðSO24 Þ under a thick layer of
lepidocrocite. As the rust layer is loose and porous, it does not constitute a physical barrier and the access of dissolved O2to the steel surface is not hindered significantly and the
rust layer continues to grow.
Meanwhile, a biofilm has developed, and the rust layer is itself covered by an outer layer mainly made of organic sub-stances, micro-organisms, and mineral substances origi-nated from seawater (clay particles, calcium carbonates, etc.), as revealed by the ICP-AES detection of elements such as Ca, Sr, Si, Mg, and Al. As the thickness of both rust lay-ers and biofilm increases, the diffusion of O2from seawater
to the steel surface becomes limited. Biological activity in the biofilm and in the outer part of the rust layer may also consume an important proportion of dissolved oxygen, leading to anaerobic conditions in the inner part of the rust layer. These conditions limit the oxidation of the green rust, then, if the kinetics of the oxidation of GRðSO24 Þ becomes
slower than that of the oxidation of steel into GRðSO24 Þ,
the green rust can accumulate on the steel surface. ICP-AES analyses also indicated that metallic elements from steel (Cu, Mn, Cr, Ni, Cd, and Mo) were accumulated inside the inner part of the rust layer. It does not seem that they influence the corrosion process as their concentrations
Fig. 6. XRD and l-Raman spectroscopy analysis of the outer part of a rust layer. (a) XRD pattern of the whole sample, (b) and (c) l-Raman spectra of the main components found in the layer. The corresponding steel coupon remained 6 months in the low water zone in the harbour of Le Havre.
in the carbon steel considered in this study are kept low. It could however be different with low- alloy steels.
At the GRðSO24 Þ/c-FeOOH interface, dissolved O2
pro-vokes the oxidation of green rust into Fe(III) oxyhydroxide according to the reaction[16]:
FeII4FeIII2 ðOHÞ12SO4 8H2Oþ 3=4O2
! 5FeOOH þ Fe2þþ SO2
4 þ 23=2H2O ð4Þ
Sulphate ions were accumulated inside the green rust when it formed. They are released at the GRðSO2
4 Þ/c-FeOOH
interface. O2 being consumed by the oxidation of the
GR, the electrolyte in the pores is close to an anaerobic medium. Then, locally, in the inner part of the rust layer near the GR/lepidocrocite interface, conditions favouring the development of SRB, that is presence of a reservoir of sulphate and low concentration of O2, are met.
SRB were detected inside the rust layers by dsrAB prim-ers with Direct PCR or Nested PCR molecular approaches. The main positive results of dsrAB detection were obtained with the inner part of the rust layers. This confirms that anaerobic or micro-aerobic conditions were met in the
Fig. 7. XRD and l-Raman spectroscopy analysis of the inner part of rust layers. (a) XRD pattern of a whole sample (the corresponding steel coupon remained 12 months in the low water zone in the harbour of Le Havre), (b) l-Raman spectrum of an outer zone (steel coupon immersed 6 months in the low water zone in the harbour of Le Havre) and (c) l-Raman spectrum of an inner zone, closer to the steel surface (steel coupon immersed 12 months in the low water zone in the harbour of Nantes-Saint-Nazaire). RS and RC are diffraction lines of sulphated and carbonated green rusts, respectively. RV are vibration bands of sulphated green rust.
Table 3
Summary of results for ‘‘direct” and ‘‘nested” PCR amplification of dissimilatory sulphite-reductase (dsrAB) gene from outer and inner part of corrosion deposits
Outer part of deposit Inner part of deposit
Porta PAH PANSN PAM PAH PANSN PAM
Sampleb 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 ‘‘Direct” PCR + + + + ‘‘Nested” PCR + + + + + + + + +, positive signal when amplification products were obtained with group-specific primers., negative signal when absence of a visible band of PCR products on an agarose gel.
a PAH (Le Havre), PANSN (Nantes-St-Nazaire), PAM (Marseille).
corresponding rust samples. These conditions could have developed homogeneously inside the overall rust layer or could have been favoured locally by the formation of bac-terial aggregates, leading to heterogeneous anaerobic con-ditions. However, the systematic presence of GRðSO2
4 Þ
in the inner part of the rust layers rather indicates that uni-form micro-aerobic or anaerobic conditions are met. This confirms that the inner part of the rust layer is a preferen-tial site for the growth of SRB, as it is constituted of a source of sulphate, i.e. GRðSO24 Þ, that also develops
pref-erentially under micro-aerobic conditions. Reaction (4) written above described how sulphates are released in solu-tion when GRðSO24 Þ is oxidised into FeOOH. However,
GRðSO24 Þ is necessarily in equilibrium with a solution that
contains sulphate ions. The consumption of the sulphate ions in solution by bacterial activity would shift the equilib-rium and induce progressively the dissolution of the GR. It is also possible that SRB are able to extract directly sul-phate ions from the GR structure, without any dissolution required. But it has not yet been proven. Note finally that the porosity of the outer part of the rust layers permits the transfer of mineral ions and organic matter required for bacterial growth.
Iron sulphide was systematically identified together with GRðSO24 Þ from this accelerated corrosion experiment. In
our experimental conditions, sulphide species can only have been generated by micro-organisms. Since the element S is almost integrally present as sulphate, it is the reduction of sulphates by SRB that produces sulphides, mostly under the form of HS, the stable species in slightly alkaline con-ditions. Their reaction with Fe2þaq ions, produced at the steel surface, then leads to the formation of FeS as observed experimentally.
4.2. Evolution with years of the rust layers
Steel structures immersed for decades in seawater are covered by thick rust layers mainly composed of an inner layer of magnetite Fe3O4and an outer layer of Fe(III)
oxy-hydroxides, a- and c-FeOOH. A careful study devoted to the role of GRðSO2
4 Þ allowed us to identify this compound
in the most outer part of the rust layer, that is between Fe(III) oxyhydroxides and the (mainly) organic layer that covers the corrosion products[16], indicating that the pro-cesses identified in this study somehow subsisted now and then inside the rust layers. Nevertheless, it is clear that as time passes, the composition of the inner part of the rust layer changes from a mixture of FeS and GRðSO24 Þ to
Fe3O4. These rust layers are rather homogeneous and can
play a positive role, somehow protecting the carbon steel structures [45]. But a localised failure of this stable layer induced by biologic and/or abiotic phenomenon could ini-tiate an accelerated localised corrosion process. SRB con-stitutes the main bacterial group suspected for the accelerated corrosion process of carbon steel in seawater. However, SRB is not the only bacterial group implicated in sulphide cycle in natural environment and in corrosion processes[46]. For example, thiosulphate-reducing bacteria (TRB) could be more aggressive than SRB [47]. Finally, it is generally recognized that synergistic relationships exist between bacterial communities such as SRB and SOB (sul-phide-oxidizing bacteria) and probably other metabolic groups[48,49]. Thus, this molecular approach with specific primers of SRB must be completed in the future with other ‘‘metabolic-targets” probes. Moreover, this study was con-ducted without quantitative analysis of SRB, which should also be made in the future.
4.3. Applications of molecular detection techniques for the microbiological study of rust layers
Only a low molecular detection of sulphate reducing micro-organisms could be obtained in the outer part of the rust. To explain this result, different hypotheses con-cerning the DNA extraction and amplification can be suggested.
DNA extraction is an essential step of molecular biology investigations. Actually, the extraction of DNA from the
studied samples was difficult. The concentrations obtained were relatively weak (40–60 ng/ll) despite the improve-ments of the purification techniques we developed. This problem could explain the absence of dsrAB signal for some samples.
We tried to solve this problem of weak concentration of total DNA by using a very significant technique of dsrAB gene amplification: the nested PCR. This technique made it possible to clearly improve the results for a certain number of samples.
Another explanation to explain the lack of results with some dsrAB amplifications could be related to the fact that rust layers probably corresponded to an environment con-taining a high rate of inhibitors for DNA PCR amplifica-tion, such as humic acids and sulphides.
A last hypothesis could be the choice of the primers used. Indeed, there are two sets of primers to amplify the dsrAB gene. The first one makes it possible to amplify the dsrAB gene specifically, but does not allow amplifying all the dsrAB genes[20]. The second one is more degener-ated and makes it possible to amplify all the genes dsrAB, but also ‘‘non dsrAB amplificates” [21].
The first set of primers was chosen in order to work under completely specific conditions with respect to the dsrAB gene. Therefore, it is possible that in some cases the sulfate-reducing micro-organisms present have a dsrAB gene that cannot be amplified by this primer set.
Another possibility is that in the inner part of the rust, the O2concentration is low, so that some SRB can grow
in these micro-aerobic conditions[50], but in the outer part of the rust, the O2concentration could be higher, and
unfa-vourable to the development of SRB. 5. Conclusion
GRðSO24 Þ and iron sulphides form the inner part of the
rust layers developing on steel permanently immersed in seawater whatever the corrosion profile observed and whatever the environmental conditions on port structures. SRB are also mainly located within the inner part of the rust layer. This association suggests a natural interrelation-ship between the corrosion process of carbon steel and the growth of SRB in the rust layers. GRðSO2
4 Þ, that forms
during the early stage of the corrosion process, can consti-tute a source of sulphate. The SRB can then easily produce the sulphide species that lead to the formation of FeS. Thus, the identification of SRB and FeS in corrosion deposits is not a sufficient clue to testify of a microbiolog-ically influenced localised accelerated corrosion process during industrial expertise. The direct molecular detection of dissimilatory sulphite-reductase gene from natural cor-rosion products, which was used for the first time in this study, showed the potential of molecular techniques for a quick investigation of target-metabolic groups.
The evolution with time of the system generally leads to uniform corrosion and produces ultimately a rust layer composed of magnetite (inner part) and Fe(III)
oxyhydrox-ides (outer part) that may be somewhat protective. The peculiar conditions that lead to catastrophic degradations of port structures are not completely understood. It seems clear however, that these conditions promote the develop-ment of SRB, thus favouring a high rate of sulphide produc-tion and hindering the formaproduc-tion of magnetite. They may be inherent to the heterogeneous nature of the initial biofilm that may lead to heterogeneities in the ‘‘rust-biofilm” sys-tem. These heterogeneities may not only be of physico-chemical nature but also of biological nature thus promot-ing unusual associations and synergistic effects between var-ious types of micro-organisms. In the future, molecular techniques should not be restricted to SRB but extended to other micro-organisms such as sulphide-oxidising or Fe(III)-reducing bacteria.
A similar study will be devoted to the corrosion of car-bon steel located in the tidal zone, therefore subject to a cyclic immersion in seawater.
Acknowledgements
This work was supported by the port of Nantes-Saint-Nazaire, the port of Marseille, the port of Le Havre, the Centre d’Etude Technique Maritime et Fluvial (CETMEF) and BAC Corrosion Control A/S. The experimental system has been developed and supported by the Research Centre (Sheet Piling Department) of Arcelor Profil Luxembourg S.A. Their contributions are gratefully acknowledged. References
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