First evaluation of the applicability of microbial extracellular polymeric substances for corrosion protection of metal substrates

Electrochimica Acta 54 (2008) 91–99

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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta

First evaluation of the applicability of microbial extracellular polymeric substances for corrosion protection of metal substrates R. Stadler a,∗ , W. Fuerbeth a , K. Harneit b , M. Grooters b , M. Woellbrink b , W. Sand b a b

DECHEMA e.V., Karl-Winnacker Institut, Theodor-Heuss-Allee 25, D-60486 Frankfurt am Main, Germany Biofilm Centre, University of Duisburg-Essen, Geibelstraße 41, D-47057 Duisburg, Germany

a r t i c l e

i n f o

Article history: Received 21 September 2007 Received in revised form 23 April 2008 Accepted 23 April 2008 Available online 4 May 2008 Keywords: Microbially influenced corrosion Biosurfactants Biofilm formation Extracellular polymeric substances Sulphate-reducing bacteria Microbially influenced corrosion inhibition Microbial footprints

a b s t r a c t In order to investigate the suitability of extracellular polymeric substances (EPS) produced by bacteria for corrosion inhibition, a system for simulation of microbially influenced corrosion (MIC) was established. Afterwards, various strains of bacteria including sulphate-reducing bacteria (SRB) and Pseudomonas were cultivated and their EPS harvested. These substances were tested in respect to their intrinsic corrosiveness towards different metal substrates. Based on the different behaviour of the EPS concerning their corrosiveness and on electrochemical results a choice of EPS suitable for prospective examinations was made. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction The fundamentals of metal corrosion are of electrochemical nature and characteristic examples for reactions at interfaces [1,2]. One prerequisite for the electrochemical process is the presence of local cells that is to say surface regions of anodic and cathodic properties. Biofilms as communities of various microorganisms embedded in extracellular polymeric substances (EPS) meet this requirement and also provide water which is necessary for electrolyte formation [3,4]. One often finds that the corrosion of metals is highly stimulated or even induced by these consortia of microorganisms. This is referred to as microbially influenced corrosion (MIC) [5,6]. Certainly, the deleterious behaviour of microorganisms can occur on any other material than metals [7]. Even glasses were found to be attacked, e.g. by fungi [8]. But in these cases, corrosion proceeds by different mechanisms. Another frequently used term is biofouling that also includes non-destructive effects of biofilms, e.g. clogging of pipelines or decreasing the performance of heatexchanging systems [9]. The opposite of MIC that is to say corrosion inhibition or a decrease of corrosion rate in the presence of biofilms is also

∗ Corresponding author. Tel.: +49 69 7564 248; fax: +49 69 7564 388. E-mail addresses: [email protected] (R. Stadler), [email protected] (W. Sand). 0013-4686/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2008.04.082

reported [10]. But the number of publications concerning microbially influenced corrosion inhibition (MICI) is few. As a high amount of information concerning MIC of metal substrates has already been published [11] a brief overview on the most important mechanisms shall be given [5–11]: the corrosion rate of metal substrates can be increased by the formation of an inhomogeneous surface, e.g. creation of corrosion cells by metabolic deposits. Indeed, some microorganisms are known to deposit metabolic substances of high anodic properties, e.g. manganese oxides [12]. Also sulphides, produced by sulphate-reducing bacteria (SRB) under anaerobic conditions, exhibit such anodic properties and hydrogen sulphide itself plays in important role in metal dissolution [13]. SRB are often found at sites exhibiting high amount of corrosion, especially on carbon steel which is frequently used for pipelines. In addition, other harmful metabolic substances like mineral acids or organic compounds exhibiting complex-binding properties can increase the corrosion rate. In contrast to low carbon steel stainless steel exhibits a higher resistance against corrosive impact. Nevertheless, the presence of a biofilm can speed up corrosion if the concentration of harmful anions like chloride beneath the biofilm is increased. The cathodic branch of the corrosion process is discussed to be stimulated by catalytic oxidation of hydrogen by bacteria, e.g. by SRB, that is to say an enzyme-driven cathodic depolarization. Two special groups of organic substances as components of a biofilm shall be mentioned here: enzymes and EPS—both excreted

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by the cells. Enzymes can cause an increase of corrosion even after isolation from a biofilm and application as pure substance [14,15]. Extraction from the biofilm and adsorption on a substrate obviously do not necessarily cause the deactivation of the enzyme. In this context enzymes for catalytic reduction of oxygen to hydrogen peroxide or water are discussed to play an important role in biocorrosion [14]. EPS are of high significance for cell adhesion and biofilm formation [16,17]: they wrap single cells in the planktonic state, can reduce repulsive properties of surfaces and thus facilitate the process of cell adhesion. EPS are also constituents for the construction of the biofilm. The composition and the structure of these polymeric substances – mainly composed of sugars and lipids – do not only depend on the biological nature of the cells but also on the circumstances of their growth. Some EPS are widely applied in food industry, e.g. dextran produced by Leuconostoc spp. Depending on the chemical composition and structure of the EPS, these substances can exhibit high ability for complex-binding metal ions and thus are discussed to promote corrosion, e.g. in the case of EPS extracted from sulphate-reducing bacteria examined by Beech et al. [18,19]. Concerning the importance of EPS one has to recall the observations of Marshall and co-workers of the 1970s [20]; they found that mixtures of polymeric substances still remain on the surface even after deliberate removal of the cells or the entire biofilm. For this, these substances were termed as “footprints”. Some of these substances were also found to exhibit surface active properties (e.g. [21,22]) as little amounts of the isolated substances were able to induce desorption of microorganisms. Later, a more general concept of “microbial footprints” was proposed [23,24] including that EPS are intentionally applied by the microbes for labelling surfaces. Indeed, it was found that in some cases on such labelled surface spots no re-adhesion by cells of the labelling microorganism itself takes place [16]. EPS of different strains of the Pseudomonas genus – applied as conditioning layers – were shown to strongly influence the adhesion behaviour of single Pseudomonas cells [25]. Depending on the choice of substrate and EPS-concentration an increase or decrease of the number of adsorbed cells was found. But how to apply microorganisms for means of corrosion inhibition? As mentioned above, reports on MICI of metal substrates are few [10]. In most cases, living cells were found to be required [26–28]. Metal surfaces have been reported to be protected by a bacteriogenic layer of iron phosphates [29]. Even damaged surfaces could be repaired by this bacterial activity. Corrosion protection due to consumption of deleterious oxygen by the biofilm, either in a direct [30] or an indirect way [31], was also observed. In some of these cases, contributions of other substances of the biofilm, e.g. exo-enzymes, were discussed [30]. Microorganisms were genetically manipulated in order to produce substances with antibiotic properties, e.g. gramicidin-S [32]. Biofilms of SRB were found to protect stainless steel by blocking the surface against the attack of harmful chloride [33]. Staphylococcus-biofilms were found to decrease the number of adherent cells of Listeria, a pathogenic microorganism often found in dairy industry [34]. Keeping in mind that was mentioned above concerning EPS, the stabilization of corrosion products inside the biofilm also must be considered. In the presence of biofilms, the free corrosion potential Ecorr of the substrate often is found to be increased, a process that is termed as “ennoblement” [35,36]. The direct application of isolated EPS in a sense of microbial footprint also seems to be a promising approach which at the present is only little exploited. For example, isolated and purified EPS were found to prevent the adhesion of bacterial cells thus inhibiting the evolution of harmful biofilms as was shown by Beech et al.

Table 1 Strains of bacteria and growth media applied for experiments Strain

DSMZ item

DSM-media for cultivation

Desulfovibrio indonesiensis Desulfovibrio vulgaris Desulfovibrio alaskensis Pseudomonas flava Pseudomonas fragi Pseudomonas cichorii Pseudomonas putida Pseudomonas fluorescens Rhodococcus opacus Lactobacillus fermentum Lactobacillus acidophilus Citrobacter freundii Enterobacter aerogenes Arthrobacter spec.

15121 644 16109 619 3456 50259

63b 63a 383 234b 234b 234b

8531 20052 20079

234b 11c 11c

a b c

` [43]. Medium modified according to von Rege Media modified: no agar, no yeast. Media modified: no meat extract, no casein peptone.

who applied purified EPS of different Pseudomonas strains as mentioned above [25]. Recently, EPS of SRB were reported by Magalhaes de Paiva to exhibit similar effects [37]. Meylheuc and co-workers applied EPS of Lactobacillus and Pseudomonas in the sense of biosurfactants with repellent properties [38,39]. In some of these cases the EPS were found to induce changes of the chemical composition of the surface, e.g. an increase of the chromium content of stainless steel [38,39]. EPS of Pseudomonas were also found to decrease the number of adsorbed cells of pathogenic Enterococcus [40]. Nevertheless, a short time ago the successful application of some EPS as additives for paint to induce MICI was reported by Ferrari and Breur [41]. These findings support the view that the direct application of EPS can offer the key for the development of a new method of corrosion inhibition for metal substrates. Suitable EPS can be regarded as renewable substances that will meet increasingly stricter environmental directives. In addition, the new method provides an intelligent utilisation of resources as microorganisms can be applied in terms of renewable resources. For the successful application as biosurfactants, at least three demands must be met by the EPS: low intrinsic corrosiveness towards the substrate, efficient adhesiveness on the surface and low biodegradability by other microorganisms. This paper reports on the system for evaluating the applicability of EPS for MICI comprising a system for the reproducible simulation of MIC by SRB, harvesting and isolation EPS and their rating. Electrochemical results of EPS analysis are also given. 2. Experimental 2.1. Cultivation of bacteria and harvesting of EPS The bacteria strains applied for cultivation and EPS production at the Biofilm Centre are listed in Table 1. Strains marked with a DSMZ item were obtained from the German Collection of Microorganism and Cell Cultures (DSMZ1 ), other strains have been available at the Biofilm Centre. All strains were cultivated in appropriate media, e.g. recommended by DSMZ. Desulfovibrio vulgaris as a representative organism strain of sulphate reducing bacteria was also cultivated at DECHEMA and applied for experiments simulating MIC. As this strain is of high importance for the project, the composition of the

1 DSMZ = Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, www.dsmz.de.

R. Stadler et al. / Electrochimica Acta 54 (2008) 91–99 Table 2 Composition of medium for Desulfovibrio vulgaris (Postgate C, modified according to ` [43]) von Rege Substance

Amount

Unit

Supplier/grade

Part A K2 HPO4 NH4 Cl Na2 SO4 CaCl2 × 2H2 O MgSO4 × 7H2 O Lactic acid (90%) Yeast extract (Difco) Resazurine, sodium salt H2 O deion

0.50 1.00 4.50 40 60 4.2 1.00 1 Ad 950

g g g mg mg ml g mg ml

Riedel de Haen/puriss. p.a Merck/p.a. Merck/p.a. Merck/p.a. Merck/p.a. Fluka/purum Roth Riedel-de Haen

Part B FeSO4 × 7H2 O Tri-sodium citrate × 2H2 O Ascorbic acid Deionized water

0.004 0.30 0.10 Ad 50.00

g g g ml

Merck/p.a. Merck/p.a. Merck/p.a.

appropriate medium is given in Table 2. The composition of this medium is based on the medium DSMZ 63 which is also known as Postgate C-medium. The EPS were harvested by centrifugation and purified by osmosis, details given in [42]. 2.1.1. Preparation of the medium The pH of part A is adjusted to 9.5 by addition of 4 M NaOH solution. Then the mixture is sterilized by autoclaving for 30 min at 121 ◦ C and 1.5 bar. After cooling down to room temperature, part B is added under sterile conditions. Finally the pH is adjusted to 7.5 by addition of pre-sterilized 4 M NaOH solution. Before application as nutrient the medium is flushed with N2 under sterile conditions in order to establish anaerobe conditions. The original growth medium DSMZ 63 does not contain citrate (cf. www.dsmz.de). But it normally contains thioglycolic acid (or sodium sulphide, respectively) in order to establish appropriate redox-conditions of the solution for a rapid growth of the SRB in case of low cell numbers (cells ml−1 ). For the experiments presented here, these compounds were moved in order to avoid interferences with MIC. As at the beginning of each experiment efficiently high numbers of bacteria cells were added to the solution, none of these sulphur-containing compounds were necessary. Characteristic for this medium is the sulphate content of 30 mmol l−1 . Sulphate is used by the bacteria as electron acceptor and finally reduced to hydrogen sulphide. 2.2. Metal substrates Three different kinds of metal substrate were applied, the compositions listed below (Table 3). All metal samples were treated with emery paper (SiC, P180 type, Struers) on both sides, rinsed with deionized water (H2 Odeion ) and subsequently degreased in ethanol and acetone using an ultrasonic bath.

Table 3 Chemical composition of the metal substrates Substrate

Pure iron (Armco) Carbon steel (ST 37) Stainless steel (SS 304)

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Table 4 Composition of PBS (phosphate buffer solution) Substance

Amount

Unit

Supplier/grade

NaCl KCl Na2 HPO4 KH2 PO4 H2 O deion

4 0.1 0.72 0.1 Ad 1000

g g g g ml

Merck/p.a. Riedel de Haen/puriss. p.a. Sigma–Aldrich Merck/p.a.

2.3. Simulation of MIC For simulation of MIC by Desulfovibrio vulgaris a system according to the setup of Eul was tested [44]. So-called coupons (30 mm × 20 mm × 1 mm) of the metal substrates were mounted to special TeflonTM holders manufactured at the mechanical workshop of DECHEMA. The setup was placed in a glass beaker, covered with aluminium foil and sterilized by heat-treatment for 9 h at 130 ◦ C. Further work requiring sterile conditions was performed in a clean-bench: two holders were then mounted to a presterilized rubber stopper normally used for freeze-drying. The chemical formulation of these stoppers did match the requirement of prevention of oxygen permeation from atmosphere. Afterwards, the stoppers were mounted to bottles containing sterilized and nitrogen-purged growth medium. Each bottle contained only one kind of substrate. The bottles were then inoculated by a suspension (1%, v/v) containing about 5 × 108 cells ml−1 . The number of cells was regularly checked by microscopy using a Neubauer-type counting chamber (Marienfeld). The inoculated setup was stored at 31 ◦ C in the dark without being agitated. Each term of 14 days at least three coupons were taken off the media for analysis. The rods with the remaining coupons were mounted to another bottle containing fresh growth medium. 1% (v/v) of the bacteria containing old solution was transferred to the new setup in order to increase bacteria growth. 2.4. Fluorescence dye staining for evaluation of sessile cells For the determination of the number of adsorbed cells some of the coupons were taken off the solution after a shorter period of 7 days and were treated under sterile and anaerobic conditions with a solution of the fluorescence dye DAPI (4 -6 diamidino-2phenylindole). DAPI selectively binds to DNA and RNA and can be applied for labelling all nucleic acid containing cell structures for the (quantitative) visualization by fluorescence microscopy (e.g. [45]). For the staining procedure, the coupons were rinsed with some ml of PBS (phosphate buffer solution, composition given in Table 4) and covered with 1 ml of a 0.01% DAPI solution (composition see below in Table 5) in a petri dish. Afterwards, the sample was incubated in the covered petri dish for 10 min in the dark at room temperature. After this period the liquid was pipetted off and the sample was rinsed with PBS solution and finally dried an air stream. The coupons were then analyzed under a fluorescence microscope (ZEISS Axioimager.Z1m, filter set no. 01). At least 24 photos of different spots are taken for each sample in order to get some qualitative information of the whole surface.

Table 5 Composition of DAPI-solution

Chemical composition (%) (remaining: Fe) C

Si

Mn

Ni

Cr

Mo

Substance

Amount

Unit

Supplier/grade

Max. 0.015 0.2 0.07

Traces 0.31 1

Max. 0.08 1.4 2

– 0.5 8

– 0.12 18

– 0.03 –

DAPI Formaldehyde (37%) H2 O deion

10 5.4 94.6

mg ml ml

Sigma–Aldrich Fluka

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2.5. Spot test for evaluation of intrinsic corrosiveness A droplet of 50 ␮l of EPS-containing suspension (3 mg ml−1 ) was deposited on the surface of a well prepared coupon. The coupons were stored in a chamber in order to establish constant humidity and atmospherical composition. By this, desiccation of the droplet was avoided over the entire period of the experiments. In order to elucidate the influence of oxygen the experiments were performed with and without purging the atmosphere with nitrogen gas, respectively. The duration of the experiment was varied between 2 and 24 h. 2.6. Electrochemical examinations of EPS under aerobic conditions For the electrochemical experiments, rod-shaped samples were prepared (10 mm in diameter, 6 mm thick, surface area of 3.45 cm2 ) as described above. The specimens were electrically connected by a screw embedded in plastic material. A Viton® O-ring was used for sealing thus avoiding contact between screw and electrolyte and excluding contact corrosion of the substrate. A conventional threeelectrode setup was applied: a platinum sheet of 2 cm2 was used as counter electrode. Saturated calomel electrodes (sce) were used as reference electrodes and placed near to the working electrode via a Haber-Luggin capillary that was filled with 0.2 M K2 SO4 solution. The potential difference due to the capillary was also included when regularly checking the potential of the reference electrode applied against an external sce (245 mV vs. normal hydrogen electrode, nhe). All potentials are referred to sce. The electrode setup was mounted into a glass vessel containing a solution of 0.2 M potassium sulphate (K2 SO4 /Merck p.a.) and EPS (30 mg l−1 ). The low amount of bacterial EPS was due to the limited availability of the substances. The EPS were added to the electrolyte which then was vigorously stirred for 15 min. Even at this low EPSamount some of the EPS did not totally dissolve, in some cases unsolvable solids were observed. Additional experiments were performed by application of commercialized polymeric substances: alginic acid (sodium salt, from brown algae; Sigma–Aldrich), xanthan gum (Sigma–Aldrich) and dextran (from Leuconostoc ssp; Fluka). These substances were applied in different concentrations. During the experiments oxygen from air was allowed to enter the setup by diffusion as the glass vessel was not entirely covered. The solution was stirred with a magnetic stir bar only modestly at about 45 rpm. Under these conditions oxygen transport to the electrodes is assumed to be controlled by diffusion. The temperature – checked before and after experiment – was constant over period of one experiment but varied between 24 and 27 ◦ C for all. For the experimental procedure, the mounted working electrodes were kept for 60 min at their free corrosion potential Ecorr . Afterwards, measurements of polarization resistance Rp were performed and the current–potential curves were recorded at a potential sweep rate of 0.05 mV s−1 in anodic sweep direction. The experiments were performed in triplicate by application of Radiometer PGP 201 potentiostats and VoltaMaster4 software.

samples were added for MIC or not. Related to this, the concentrations of sulphate and lactic acid, respectively, decreased to levels of about 50% of the concentrations supplied at the beginning. Purging the solutions with nitrogen gas in order to remove cell-toxic hydrogen sulphide only led to a more rapid increase of the cell number but did not exceed 109 cells ml−1 . Under these conditions, the microorganisms were obviously not able to metabolize the nutrients more efficiently. When immersed in solutions containing Desulfovibrio vulgaris pure iron and carbon steel show comparable behaviour: on both metals, a black and slimy biofilm evolves within 7 or 8 days. Mass loss in the first period of 14 days was evaluated to 0.34 mg cm−2 . When analyzing samples taken off the solution after a longer immersion periods, no further mass loss was found. Microscopic analysis showed irregularly distributed pits in the surface. Samples of stainless steel, on the other hand, were not affected: they were covered only by a thin, light greenish film, easy to remove by rinsing. No significant mass loss could be determined for stainless steel. 3.2. Fluorescence dye staining for evaluation of sessile cells Of high interest is the number of sessile cells adsorbed on the surface as these cells represent nuclei for biofilm formation. A well established method for unambiguously labelling cells and thus distinguishing between surface areas covered by cells or biofilm and areas of bare surface is given by fluorescence dye staining and fluorescence microscopy (FM), as was described above. Fig. 1 gives representative FM–photographs of samples after staining with DAPI, showing that carbon steel and stainless steel exhibit different behaviour. As pure iron behaves like carbon steel, no figures are given for sake of brevity: after an immersion period of 7 days one found that the samples of pure iron and carbon steel as well exhibited areas of high fluorescence intensity due to adsorbed cells and black areas due to bare surface. Samples taken off the solution after an extended immersion period of 14 days still exhibited uncovered surface areas that are clearly distinguishable from biofilm-covered areas. It will be necessary to examine the relationship between the locations of corrosion impact and cell adhesion or biofilm formation, respectively. Samples of stainless steel, on the other hand, exhibit a complete biofilm even after 7 days of immersion, the number of cells attached

3. Results and discussion 3.1. Setup for simulation of MIC by SRB As MIC of iron and steel substrates by sulphate reducing bacteria is well studied the results of the experiments presented here shall only mentioned briefly: the number of the planktonic cells of Desulfovibrio vulgaris was found to increase up to a maximum value of about 109 cells ml within 3 or 4 days, regardless, whether metal

Fig. 1. Representative fluorescence-micrographs (corresponding to 67 ␮m × 105 ␮m) of samples of carbon steel (upper part) and stainless steel (lower part) taken after DAPI staining. Influence of immersion time in Desulfovibrio vulgaris containing medium. Left: sample taken off the solution after 7 days. Right: sample taken off the solution after 14 days.

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being too high for being determined quantitatively. For the evaluation of EPS as protecting materials, it will be necessary to study the cell adhesion (semi-) quantitatively on a shorter time-scale, e.g. of few hours. Nevertheless, these experimental results show that the system for studying MIC was established successfully. Desulfovibrio vulgaris will be applied as model organism for the evaluation of the applicability of EPS for MICI. 3.3. Spot test for evaluation of intrinsic corrosiveness Polymeric substances suitable for MICI must not be corrosive themselves towards metal substrates that is to say these substances must not exhibit intrinsic corrosiveness. In order to evaluate the suitability of the substances with regard to this feature, it was necessary to establish a setup for rating EPS providing quick and reliable results. The results of this ‘spot test’ system are given in Fig. 2 showing photographs of the different metal substrates after treatment with droplets of solutions containing EPS of the three strains of the Desulfovibrio genus. The photographs clearly show that in the presence of oxygen (left part of the figure) only stainless steel remains unaffected, whereas pure iron and carbon steel are attacked, regardless of the origin of the polymeric substances. The corrosive attack in the presence of oxygen is indicated by typical red-brownish spots on the surface. Any of these substances does not provide protection. Under anaerobic conditions, on the other hand, remarkable differences are observed: the EPS of Desulfovibrio alaskensis do only little affect (or not affect at all) pure iron as well as carbon steel. In the related photographs one only finds light grey spots on the surfaces, residues of dried the EPS-suspension. In contrast, the presence of EPS of Desulfovibrio indonesiensis and Desulfovibrio vulgaris causes black spots, indicating the formation of corrosion products. Similar experiments were performed with the commercialized polymeric substances mentioned above. Under aerobic conditions corrosion of all samples but stainless steel was observed. Under anaerobic conditions none of these substances exhibits corrosiveness regardless to their chemical composition. Under aerobic conditions the composition of the pure substances seems not to play a remarkable role. This was also discussed by Roe and co-workers, who studied alginate as well as agarose applied in experiments dealing with MIC of carbon steel under aerobic conditions [50]. In absence of bacteria cells, none of the substances exhibited protective properties as none of them were able to prevent diffusion of oxygen to the substrate. Alginate, though containing carboxylate functional groups did not cause higher degrees of corrosion than agarose. Concerning the inhibition of MIC, Xanthan and pure galacturonic acids have been applied for experiments dealing with MIC in regard to anion exclusion [51]: xanthan – composed of glucose, mannose and galacturonic acids – indeed was found to decrease the diffusion of anions that are required as nutrients. No distinct information was given concerning MIC. On the other hand, the importance of the chemical composition of the polymers was found for the adhesion behaviour of bacteria: Pseudomonas aeruginosa, for example, an alginate producing microorganism, normally forms proper biofilms. Defect mutants of this microorganism, that produce alginate polymer without Oacetyl groups only form thin biofilms as is reported by Flemming and co-workers [49]. The authors assume that the lack of the Oacetyl groups leads to weaker polymer||surface interactions thus preventing the formation of regular biofilms. The different behaviour of the EPS of the Desulfovibrio strains with respect to their intrinsic corrosiveness presented above can be related to the properties of the corresponding biofilms containing living cells [18,19,46–48] (and citations therein): Beech and co-workers reported that purified EPS as well as living biofilms of

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Desulfovibrio indonesiensis were found to be more corrosive towards carbon steel when compared to Desulfovibrio alaskensis but in the case of pure EPS of Desulfovibrio indonesiensis the authors did apply oxic conditions [18]. Our results presented here clarify that for evaluating intrinsic corrosion, one has to apply an oxygen-free atmosphere. Otherwise general corrosion by oxygen and humidity will superimpose the corrosive attack exerted by EPS. The different behaviour of the Desulfovibrio strains is also reflected by the different abilities of the EPS to complex-bind metal ions as was reported by Beech and co-workers [19]. A strong relationship between the chemical composition of the EPS and their corrosiveness is likely to be expected. In addition, one has to keep in mind, that these EPS are mixtures of different substances probably including enzymes. Therefore, chemical analysis of these EPS should be performed. 3.4. Electrochemical examination of EPS under aerobic conditions Under aerobic conditions in electrolyte of neutral pH, one has to consider the reduction of O2 to hydrogen peroxide and finally to water giving rise to cathodic limiting currents. The current density can be calculated according to the Levich equation: iO2 ,

D

= −4FDO2

(CO2 )0 ı

(1)

where i is the diffusion limited current density of oxygen reduction, F the Faraday constant (96,485 A s mol−1 ), DO2 the diffusion coefficient of oxygen (assumed to 10−5 cm s−1 at 25 ◦ C), (CO2 )0 the concentration of oxygen near to the surface of the electrolyte exposed to ambient air (assumed to 2 × 107 mol cm−3 ) and ı is the thickness of the Nernst diffusion layer (assumed to 5 × 10−3 cm). For unstirred solutions the diffusion limited current can be calculated to 0.15 mA cm−2 (= 0.52 mA × 3.45 cm−2 ), assuming a transfer of four electrons resulting in H2 O and neglecting contributions of metal reduction for reasons of simplification. Deviations have to be taken into account with regard to the thickness of the Nernst diffusion layer that will decrease with increasing rate of oxygen transport. But under the experimental conditions applied above, contributions of convective oxygen transport will be neglected. Further studies could be performed by means of convective diffusion like rotating electrodes but are out of scope of this project. As described above, after being grinded and de-greased, the samples were mounted to the electrochemical cells containing EPS dissolved in 0.2 M K2 SO4 . After monitoring Ecorr for a period of 1 h, the polarization resistance Rp () was evaluated by varying the potential from Ecorr and calculated to corrosion rates (mm a−1 ) given in brackets. An example of potential and current as a function of polarization time is given in Fig. 3. To summarize, the values of Ecorr determined by polarization after 1 h of immersion were in the range between −700 and −650 mV sce, regardless of metal substrate or EPS. The values of Rp of pure iron were in a range from 95  (0.53 mm a−1 ) to 150  (0.33 mm a−1 ), with the exception of 443  (0.11 mm a−1 ) at high xanthan concentration. For carbon steel, one found Rp between 78  (0.66 mm a−1 ) for EPS of Desulfovibrio indonesiensis and 133  (0.38 mm a−1 ), also in this case with the exception of 409  (0.12 mm a−1 ) at high xanthan concentration. No significant values of Rp could be determined for stainless steel. After the polarization procedure, current–potential curves (CPC) were recorded at 0.05 mV s−1 in anodic sweep direction, starting at −1.2 V. 3.4.1. Stainless steel The current–potential curves of stainless steel in the presence of commercialized EPS are given in Figs. 4 and 5 (the curves related

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Fig. 2. Photographs of metal samples after exposure for 24 h to droplets of EPS-containing solution. Left: aerobic conditions. Right: anaerobic conditions. A = pure iron, B = carbon steel ST 37, C = stainless steel. H = H2 O deion; v = EPS of Desulfovibrio vulgaris; i = ∼Desulfovibrio indonesiensis; a = ∼Desulfovibrio alaskensis.

to dextran are similar to those of alginate and thus not given here). A significant influence can only be observed in the case of xanthan at higher concentration: the current densities due to oxygen reduction at potentials lower than −500 mV are decreased indicating that oxygen reduction is affected. This can mainly be contributed to changes of the viscosity which influences the diffusion coefficient as indicated by the Stokes–Einstein equation: D=

kB T 6R0

(2)

where kB is the Boltzmann constant (J K−1 ), T the temperature (K),  the dynamic viscosity (N s m−2 ) and R0 is the radius of diffusing particles (m). For water under standard conditions the dynamic viscosity  equals 1 but the dynamic viscosity, e.g. of honey can reach values up to 104 . No such influence is observed in the case of alginate at high concentration (and dextran as well). These solutions did not exhibit high viscosity. Also the onset of the anodic current at potentials of about 1150 mV is influenced by the presence of the polymeric substances indicating some inhibitive effect on the oxidation.

Fig. 3. Polarization of carbon steel in 0.2 K2 SO4 solution containing 0.3% xanthan. Transients of potential and current density, E = Ecorr ± 10 mV, dE/dt = 0.1 mV s−1 .

The current–potential curves of stainless steel in presence of the EPS harvested from the various bacteria strains are given in Figs. 6–8. Whereas the EPS of the Desulfovibrio strains do not exhibit influence on the oxygen reduction, significant influence can be derived from the curves obtained in the solutions of the other bacteria strains. Among these EPS those two of the Pseudomonas strains (Pseudomonas cichorii and Pseudomonas flava) as well as Lactobacillus fermentum and Rhodococcus opacus exert an inhibitive influence on the oxygen reduction. As the concentration of 0.0067% is fairly low, this effect may be caused not only by changes of viscosity when compared to pure electrolyte. In addition, also the shapes of the related curves are different when compared to those obtained in pure electrolyte. A general inhibitive effect of all those EPS on the onset of the anodic oxidation is observed the onset potential being shifted for about 60 mV.

Fig. 4. Current–potential curves of stainless steel in 0.2 M K2 SO4 solution containing alginate at different concentrations (w/v). Anodic sweep direction only: 0.05 mV s−1 . Inset enlarged view. a (- - -) = 0.003%; b (. . .. . .) = 0.03%; c (-·-·-) = 0.3%; d (—) = no alginate.

R. Stadler et al. / Electrochimica Acta 54 (2008) 91–99

Fig. 5. Current–potential curves of stainless steel in 0.2 M K2 SO4 solution containing xanthan at different concentrations (w/v). Anodic sweep direction only, 0.05 mV s−1 . Inset enlarged view. a (- - -) = 0.003%; b (. . .. . .) = 0.03%; c (-·-·-) = 0.3%; d (—) = no xanthan.

The inhibitive influence of the Pseudomonas EPS under aerobic conditions is in line with other reports on biofilm or EPS of Pseudomonas showing an inhibitive effect on corrosion as mentioned above, as was for L. fermentum–EPS. As at the present no sufficient information on the chemical composition of the EPS is available, any further discussion would be speculative. 3.4.2. Carbon steel Figs. 9–11 give the CPC of carbon steel in EPS containing solutions. In contrast to pure iron, in the case of carbon steel the onset of the anodic current occurs at higher potential when compared to pure electrolyte. No remarkable differences are found when comparing the effect of the different commercialized EPS with exception of xanthan at high concentration. A more pronounced difference is found in the presence of the bacterial EPS. Interestingly, the curves recorded in presence of the EPS of Desulfovibrio vulgaris and L. fermentum, respectively, occur at the same potentials.

Fig. 6. Current–potential curves of stainless steel in 0.2 M K2 SO4 solution containing SRB–EPS (0.0067%, w/v). Anodic sweep direction only, 0.05 mV s−1 . Insets (a) and (b): enlarged views. a = Desulfovibrio indonesiensis; b = Desulfovibrio alaskensis; c = Desulfovibrio vulgaris; d = no EPS.

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Fig. 7. Current–potential curves of stainless steel in 0.2 M K2 SO4 solution containing Pseudomonas–EPS (0.0067%, w/v). Anodic sweep direction only: 0.05 mV s−1 . Insets (a) and (b): enlarged views. a = P. cichorii; b = P. flava; c = Pseudomonas fluorescens; d = Pseudomonas putida; e = P. fluorescens; f = no EPS (the two P. fluorescens strains were of different origin).

3.4.3. Pure iron The following Figs. 12–14 show the CPCs of pure iron EPS containing solutions. No characteristic current waves due to oxygen reduction can be observed in the potential range between One finds that regardless of the EPS applied the anodic current due to the oxidation of the electrode occurs at lower potential when compared to pure electrolyte. Whereas in the case of xanthan one can observe an influence of the concentration, no influence seems to be exerted in the case of alginate (the curves in the presence of dextran are similar to those of alginate). Remarkable differences are found in the presence of bacterial EPS: in presence of Desulfovibrio vulgaris–EPS the anodic current increases at lower potential when compared to EPS of L. fermentum. No or little influence is observed at potentials lower than −1 V, where oxygen reduction is expected to occur.

Fig. 8. Current–potential curves of stainless steel in 0.2 M K2 SO4 solution containing EPS of various bacteria (0.0067%, w/v). Anodic sweep direction only: 0.05 mV s−1 . Insets (a) and (b): enlarged views. a = Rhodococcus opacus; b = Lactobacillus fermentum; c = Citrobacter freundii; d = Arthrobacter spec.; e = Enterobacter aerogenes; f = no EPS.

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Fig. 9. Current–potential curves of carbon steel in 0.2 M K2 SO4 solution containing alginate at different concentrations (w/v). Anodic sweep direction only: 0.05 mV s−1 . Inset enlarged view. a (- - -) = 0.003%; b (. . .. . .) = 0.03%; c (-·-·-) = 0.3%; d (—) = no alginate.

Fig. 10. Current–potential curves of carbon steel in 0.2 M K2 SO4 solution containing xanthan at different concentrations (w/v). Anodic sweep direction only: 0.05 mV s−1 . Inset enlarged view. a (- - -) = 0.003%; b (. . .. . .) = 0.03%; c (-·-·-) = 0.3%; d (—) = no xanthan.

Fig. 11. Current–potential curves of carbon steel in 0.2 M K2 SO4 solution containing EPS of different origin (0.0067%, w/v). Anodic sweep direction only: 0.05 mV s−1 . a = no EPS; b = Pseudomonas fragi; c = P. cichorii; d = P. fluorescens; e = R. opacus; f = L. fermentum; g = Desulfovibrio vulgaris; h = Desulfovibrio indonesiensis.

Fig. 12. Current–potential curves of pure iron in 0.2 M K2 SO4 solution containing alginate at different concentrations (w/v). Anodic sweep direction only: 0.05 mV s−1 . Inset enlarged view. a (- - -) = 0.003%; b (. . .. . .) = 0.03%; c (-·-·-) = 0.3%; d (—) = no alginate.

Fig. 13. Current–potential curves of pure iron in 0.2 M K2 SO4 solution containing xanthan at different concentrations (w/v). Anodic sweep direction only: 0.05 mV s−1 . Inset enlarged view. a (- - -) 0.003%; b (. . .. . .) = 0.03%; c (-·-·-) = 0.3%; d (—) = no xanthan.

Fig. 14. Current–potential curves of pure iron in 0.2 M K2 SO4 solution containing EPS of different origin (0.0067%, w/v). Anodic sweep direction only: 0.05 mV s−1 . a = Desulfovibrio vulgaris; b = P. cichorii; c = R. opacus; d = L. fermentum; e = no EPS.

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4. Conclusions The results of simulating MIC by SRB show that a setup has been established successfully. This setup will be applied for evaluating the protective properties of EPS layers towards corrosive attack by SRB under anaerobic conditions. Further analysis will be performed, e.g. by AFM in order to evaluate the relationship between the adhesion sites of bacteria and occurrence of corrosion, e.g. pit formation. The key issue is to find EPS that can prevent the adhesion of bacterial cells on the metal surface. This will be focused on by application of staining methods on smaller time scales. In addition, special staining techniques for the biopolymers as well as for distinguishing microorganisms of different origin are already in progress. The latter will be necessary when applying mixed bacteria populations. Intrinsic corrosiveness of the EPS on pure iron and carbon steel under anaerobic conditions was observed in the case of the EPS of Desulfovibrio vulgaris and Desulfovibrio indonesiensis whereas no corrosive attack was observed by the EPS of Desulfovibrio alaskensis. The more corrosive behaviour of Desulfovibrio indonesiensis is in line with the observations of Beech et al. [19]. From this point of view the Desulfovibrio alaskensis–EPS are promising substances for corrosion inhibition by biopolymers and will be applied for further experiments. Nevertheless, it shall be studied whether ‘corrosive’ EPS can be applied in composite-layers, e.g. in combination with commercialized EPS, where no direct contact to the surface will be established. The electrochemical examinations of the EPS under aerobic conditions revealed that oxygen reduction is influenced on stainless steel only by few substances: EPS of P. cichorii, P. flava, R. opacus and L. fermentum. Among the commercialized substances under study only xanthan at high concentration shows an effect that can be explained by increase of the viscosity when compared to pure electrolyte. The onset of the anodic current at higher potentials indicating the oxidation of the sample is influenced by most of the substances. Some kind of inhibitive behaviour thus can be derived. The electrochemical data concerning mass loss and polarization resistance indicate no significant of the substances. More information will be collected when performing examinations under anaerobic conditions also in the presence of SRB. To summarize, among the EPS under examination only a few seem to be suitable according to the results presented above. A probable footprint-effect of the Desulfovibrio vulgaris EPS on the adhesion of the related cells is still under examination. Further examinations concerning the biodegradability of the EPS by SRB and the adhesiveness of suitable polymers are in progress and will be presented elsewhere. Acknowledgements This work was funded by the German Federal Ministry of Eco¨ Wirtschaft nomic Affairs and Employment (Bundesministerium fur und Arbeit, BMWA) via the German industrial research association “Arbeitsgemeinschaft industrieller Forschungsvereinigungen” (AiF, project number 178 ZN). The authors are grateful for the participa-

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