Anaerobic Electrochemical Corrosion of Mild Steel in the Presence of

The corrosion of mild steel in a seawater medium containing extracellular polymeric substances (EPS) produced by sulfate-reducing bacteria (SRB) was s...
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Environ. Sci. Technol. 2002, 36, 1720-1727

Anaerobic Electrochemical Corrosion of Mild Steel in the Presence of Extracellular Polymeric Substances Produced by a Culture Enriched in Sulfate-Reducing Bacteria K W O N G - Y U C H A N , * ,† LI-CHONG XU,‡ AND HERBERT H. P. FANG‡ Department of Chemistry, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong

The corrosion of mild steel in a seawater medium containing extracellular polymeric substances (EPS) produced by sulfate-reducing bacteria (SRB) was studied by electrochemical experiments and atomic force microscopy (AFM). Under anaerobic conditions, the corrosion of mild steel increased up to 5-fold in the presence of a 1% (w/w) EPS solution but in the absence of SRB. The enhanced corrosion is mainly due to the oxidizing power of EPS with a reduction potential of E1/2 at -0.54 V (saturated calomel electrode), which is 0.4 V above that of hydrogen reduction. The electrochemical reduction of EPS provides a couple to iron oxidation, as demonstrated by H-shaped cell experiments in which the steel sample and EPS are not in physical contact but are ionically connected via the solution and electronically connected through an external wire. Fourier transformation infrared spectroscopy and X-ray photoelectron spectroscopy showed that EPS derived from SRB are comprised of 60% proteins, 37% polysaccharides, and 3% hydrocarbons. The XPS results showed that, upon corrosion, polysaccharide components were mostly converted to hydrocarbons.

Introduction In the aquatic environment, microorganisms tend to colonize on a solid surfaces to form a biofilm. Typically, a biofilm is composed of the microbial cells, extracellular polymeric substances (EPS) secreted by the cells, metabolic products, plus a variety of colloidal and dissolved substances. Collectively, the microbial cells form a biofilm layer to protect themselves from the external environment, but at the same time, they cause fouling of the underlying surfaces, such as those of pipelines, heat exchangers, marine structures, ship hulls, and teeth (1). Among all microbes, sulfate-reducing bacteria (SRB) have been studied most widely for their biofouling effect on metals (2), polymers (3), concrete (4), and other materials. SRB are able to use sulfate as an electron acceptor, which gives them the competitive edge over other anaerobes for substrates in the presence of sulfate ions. Thus, SRB are highly efficient in degrading organic pollutants (5, * Corresponding author phone: (852) 2859-7917; fax: (852) 28571586; e-mail: [email protected]. † Department of Chemistry. ‡ Department of Civil Engineering. 1720

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6) and, in the process, produce sulfide which can also complex metals in the environment by forming metal sulfide precipitates (7-9). SRB are also known to cause corrosion in the oil, gas, and shipping industries, as well as in sewer lines and polluted coastal waters (10). Such corrosion depends strongly on the microbiological activities as well as the chemical characteristics of the biofilm. The mechanism of biocorrosion is, however, not well-understood. The controlling factors could be the gradients of pH, oxygen, chloride, and metals in the biofilm. For SRB-related corrosion, an important factor could be cathodic depolarization, resulting from hydrogenase activity or the formation of a sulfide film. Recently, it was reported that EPS produced by SRB could accelerate the deterioration of mild steel, because of the ability of EPS to bind metal ions (11). Another study showed that the increased production of EPS in the presence of Cr3+ also accelerated the corrosion of mild steel in seawater (12). To determine whether the enhanced corrosion is caused by EPS alone, in the absence of other constituents of the SRB biofilm, corrosion experiments were conducted using isolated EPS.

Materials and Methods EPS Extraction and Characterization. The SRB seed was isolated from a marine sediment in the Victoria Harbor of Hong Kong and enriched at room temperature in a continuous stirred tank reactor for 6 months using a modified Postgate’s marine medium C (13). The seawater collected in the Victoria Harbor of Hong Kong was sterilized using a 0.45 µm membrane filter before use. The medium was prepared by adding, to each liter of seawater, 0.5 g of KH2PO4, 1 g of NH4Cl, 0.06 g of CaCl2‚6H2O, 0.06 g of MgSO4‚7H2O, 6 mL of sodium lactate (70%), 1 g of yeast extract, 0.004 g of FeSO4‚ 7H2O, and 0.3 g of sodium citrate. The seawater medium pH was adjusted to 7.2 ( 0.1 by titration with a 1 M NaOH solution. The medium was further autoclaved at 121 °C for 15 min. After autoclaving, the medium was purged with pure N2 to remove dissolved oxygen. The enriched SRB culture was used to inoculate a 20-L glass reactor with the same medium at pH 7.2-7.6, room temperature, and with an initial cell concentration of 1 × 106 cell mL-1. In the second day, the medium turned black because of the production of sulfides, and the sulfate concentration was analyzed daily by ion chromatography to monitor the activity of SRB. About 82% sulfate was reduced on the 10th day. To provide a supply of sulfate, half of the bulk solution was replaced every 10 days with a fresh oxygenfree sterile medium. The phylogenetic diversity of a biofilm produced by the same SRB culture was characterized on the basis of 16S rDNA (14). Results showed that most of the bacteria were affiliated with two families of SRB: Desulfovibrionaceae and Desulfobacteriaceae. EPS produced by the SRB culture were separated from the bulk solution after 30 days by centrifugation at 20 000g for 20 min at 4 °C. EPS in the supernatant were dialyzed for 48 h against distilled water using a 10 kDa molecular weight cutoff dialysis membrane (Pierce, Rockford, IL) at 4 °C and then lyophilized at -50 °C. A colorimetric method was applied to analyze the compositions of EPS. The polysaccharide and protein contents of EPS were measured using the phenol/sulfuric acid method (15) and the Lowry method (16), respectively. Glucose and bovine serum albumin were used as the corresponding standards. There was little cell lysis during EPS separation, as confirmed by comparing the DNA content in the SRB culture 10.1021/es011187c CCC: $22.00

 2002 American Chemical Society Published on Web 03/07/2002

and in the isolated EPS determined by established procedures (17). Results show that while the dry mass of the SRB culture samples contained 0.69 ( 0.05% DNA, the isolated EPS contained only 0.022 ( 0.002% DNA. A mixture of EPS and KBr powders was pressed into a pellet and analyzed by an FTS-7 Bio-Rad Fourier transformation infrared (FTIR) spectrometer. For X-ray photoelectron spectroscopic (XPS) analysis, dried EPS were pressed into a pellet and attached onto a carbon tape. The XPS spectra were recorded with a Physical Electronics Inc. Phi Quantum 2000 model using Al KR radiation at a base pressure below 5 × 10-9 Torr. Scanning was carried out over a wide binding energy range (0-1350 eV) at an emission angle of 45°. The elemental concentrations of C, N, O, Mg, P, S, Ca, and Fe were normalized. The binding energies of the peaks were determined using the carbon 1s position at 284.5 eV as a reference. Nonlinear least-squares curve fitting was performed with Phi Multipack software (Physical Electronics Inc., Eden Prairie, MN). Corrosion of Mild Steel Immersed in EPS-Containing Seawater. Mild steel coupons with dimensions 10 × 10 × 1.5 mm were used for the corrosion experiments. The elemental composition (by wt %) of the steel samples as analyzed by inductively coupled plasma optical emission spectrometry and spark optical emission spectrometry are as follows: Fe, 98.48%; C, 0.06%; Si, 0.13%; P, 0.03%; S, 0.045%; Mn, 0.51%; Cr, 0.10%; Mo, 0.02%; Ni, 0.14%; and Cu, 0.49%. The coupons were wet polished with a series of grit SiC papers (grades 220, 400, 600, and 800), followed by 0.3 µm alumina powder. All corrosion experiments were conducted anaerobically by N2 purge in simulated seawater media. The simulated seawater was prepared by adding NaCl (3 wt %) to deionized water. Two solutions were prepared: one dosed with 1% isolated EPS and one without EPS (EPS-free control). After stirring for a few minutes, EPS was partially dissolved and formed a colloidal suspension. Freshly polished mild steel coupons were immersed in the two seawater solutions for 60 days before examination with a Digital Instruments Nanoscope IIIA atomic force microscope (AFM). To expose the corrosion pits, the EPS film formed on the coupon was removed by a Clarke solution, in a similar way as described previously for the removal of an SRB biofilm (18). The coupons with the EPS film removed were dried in nitrogen before AFM microscopy work. Electrochemical Studies. The open circuit potential (OCP) measurement, potentiodynamic polarization, and cyclic voltammetry (CV) were conducted with a Princeton Applied Research model 263 potentiostat. The working electrode is a 1 cm2 circular disk mild steel electrode polished with SiC papers. The reference electrode is a standard calomel electrode (SCE) immersed with a Luggin capillary tube placed closely to the working electrode. The counter electrode is a 2 cm × 2 cm Pt electrode. The working, reference, and counter electrodes were immersed in an oxygen-free sealed solution chamber with external electric wires connected to the potentiostat. Potentiodynamic polarizations of mild steel in media were performed at a scan rate of 1 mV/s. CV was performed with a 1 cm × 1 cm Pt working electrode at the scan rate of 1000 mV/s. During the electrochemical measurements, the medium was purged with N2 to keep it anaerobic. H-Shaped Cell Experiments. To determine whether the effect of EPS on mild steel corrosion is purely electrochemical, corrosion experiments were repeated with mild steel coupons in H-shaped cells, as shown schematically in Figure 1. The H-shaped cell has two chambers separated by a fritted, sintered glass partition. The mild steel coupon was immersed in one chamber with EPS-free seawater solution. To measure the open circuit potential, a reference calomel electrode was immersed into the same chamber with a Luggin capillary

FIGURE 1. Diagram of the H-shaped cell used for electrochemical measurements. placed closely to the steel coupon. The other chamber was filled with an EPS-containing solution, and a Pt electrode was immersed and electronically connected with the mild steel coupon through an external insulated copper wire which was isolated from the aqueous solution. The sintered glass prevented diffusion of EPS into the coupon containing chamber but allowed ions to be freely exchanged between the two cells. Two control experiments were conducted. The first control had no EPS chamber in either. The second control had EPS in the Pt electrode chamber but the steel and platinum were not electrically connected. The experiments were conducted in an anaerobic chamber, and all seawater solutions were autoclaved and purged with nitrogen prior to use. An anaerobic indictor (Oxoid, Basingstoke, Hants, U.K.) was placed in the chamber during the experiments. After 30 days, surfaces of the steel coupons were removed and examined by AFM.

Results and Discussion Corrosion of Mild Steel in EPS-Containing Seawater Medium. Pitting corrosion was observed in the mild steel coupons by AFM after 60 days. Two coupons were observed with AFM microscopy. For each coupon, 10 scan areas, each of 100 µm × 100 µm, were randomly selected for AFM imaging to provide statistical sampling of corrosion effects. The typical AFM images are shown in Figure 2a for steel coupons immersed in EPS-free medium and in Figure 2b for coupons in medium containing EPS, where larger and deeper pits are clearly observed. AFM has a higher resolution and provides a more accurate measurement in the vertical dimension than most other microscopic techniques. The pit depth and volume can be quantified from the digitized AFM images using section and bearing analysis, provided by the Nanoscope IIIA software. The detail procedure has been described elsewhere (18). The characteristic parameters of the pits were shown in Table 1, on the basis of the measurements of more than 50 pits for each coupon. Results showed that the average pit depth for the coupons immersed in EPS-containing seawater medium were 1.56 ( 0.55 µm, about 5 times greater than the coupons immersed in the EPS-free medium, with corresponding values of 0.31 ( 0.12 µm. Other parameters, such as pit area, volume, and total pit volume per scanned area also indicated that the steel was corroded largely in EPS. The corrosion due to EPS is confirmed without the presence of SRB and other biofilm constituents. The action of EPS can therefore be purely “chemical” or “electrochemical” and be decoupled from the other biological and metabolic activities of SRB. Electrochemical Studies of EPS. The anodic and cathodic behaviors of the mild steel coupon in 1% EPS solution and EPS-free solution are shown in Figure 3. A higher anodic current density was found in the EPS-free medium for all VOL. 36, NO. 8, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Measured Results of Pits in the Coupons Immersed in the EPS-Free and 1% EPS-Containing Media for 60 Days

a

sample

depth (µm/pit)

volume (µm3/pit)

area (µm2/pit)

total pit volume per scanned area (µm3/104 µm2)

ratio of V/ADa

EPS-free 1% EPS

0.31 ( 0.12 1.56 ( 0.55

0.74 ( 0.26 52.3 ( 39.2

7.3 ( 2.5 67.3 ( 30.4

17.4 ( 9.9 114.0 ( 38.7

0.33 0.50

V ) pit volume; A ) pit area; D ) pit depth.

FIGURE 3. (a) Anodic and (b) cathodic potentiodynamic polarization curves of mild steel electrodes in EPS-free and EPS-containing seawater media at scan rate of 1 mV/s. FIGURE 2. AFM images of two mild steel surfaces after immersion for 60 days in seawater media: (a) EPS-free and (b) containing 1% EPS. anodic polarizations. The OCP of the steel coupons were measured to be -0.72 V (SCE) in EPS-free medium and -0.59 V (SCE) in 1% EPS medium. These results are surprising and appear to contradict the AFM results, because a material with a lower OCP would have a higher tendency to oxidize and corrode. One possible cause may be the acidity of EPS, which may also enhance corrosion. Lewandowski et al. (19) reported the pH profile on a metal surface covered with a simulated biopolymer as measured by an iridium oxide microelectrode, and they found that the low pH regions were identified with the corroded areas. In our experiments with 1% EPS, the medium had a pH of 3.9 as compared to 6.4 in the EPS-free medium. The acidic nature is probably due to the carboxylic acid group associated with uronic acid sugars of the polysaccharides and the amino acids of the protein components of the EPS. The OCP of mild steel coupons in EPS-free medium, however, did not change when pH was adjusted by inorganic acid in the range of 3.5-6.5. The other possible cause of increase in OCP by EPS could be its own 1722

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redox activity, resulting in a mixed OCP representing the combined redox effect of steel and EPS. To confirm this, the OCP of steel was measured in an H-shaped cell with 1% EPS and a platinum counter electrode in the other chamber. When the platinum electrode and the steel coupon were electronically connected, the OCP measured -0.59 V (SCE) but dropped back to -0.72 V (SCE) when the electrical connection was open. It is reasonable to expect that the platinum electrode be inert in the absence of dissolved oxygen and that the redox activity to raise the OCP should be due to EPS. The hydrogen evolution reaction at -0.8 V (SCE) or lower would not raise the OCP of mild steel. From the cathodic polarizations of the steel coupon in Figure 3b, a much higher cathodic current is observed in the EPS medium for the initial region near OCP. This further indicates the possibility of electrochemical reduction of EPS. To further investigate the electrochemical character of the EPS medium, CV experiments were separately performed with a platinum electrode in EPS-free and 1% EPS solution. Figure 4 clearly shows the reduction peak at -0.62 V (SCE) and the corresponding oxidation peak at -0.3 V (SCE) in the medium containing EPS. The OCP of Pt in 1% EPS is approximately -0.45 V (SCE). Therefore, it is reasonable to conclude that the OCP of steel in 1% EPS at -0.59 V (SCE)

FIGURE 4. Cyclic voltammetry curves of the Pt electrode in EPSfree and EPS-containing seawater media at a scan rate of 1000 mV/s. is the result of a mixed potential. The enhanced corrosion of steel in the presence of EPS can therefore be expected to be corrosion in which the EPS reduction with E1/2 of -0.54 V (SCE) provides the cathodic reaction. From Figure 4, the redox potential of oxygen is at 1.1 V (SCE) and will be the preferred cathodic couple for steel corrosion if dissolved oxygen is present. Under anaerobic conditions, however, EPS reduction is preferred (E1/2 ) -0.54 V (SCE)) to hydrogen reduction (-1.1 V (SCE)). The corrosion was clearly demonstrated by the H-shaped cell experiments where the metal coupons were immersed in a chamber with EPS-free solution. The coupons, though not in physical contact with the EPS solution, are ionically connected to the EPS medium through the sintered glass and electrically connected to the platinum counter electrode immersed in the other chamber. The EPS in the other chamber will receive electrons through the Pt electrode and the external circuit, and sodium and chloride ions will provide the ion transport in the solution. The AFM images of the steel samples after immersion in the H-shaped cells for 30 days were examined and compared with the control samples where either the external circuit was disconnected or the 1% EPS medium in the other chamber was replaced by the EPSfree medium. The steel coupon immersed in an H-shaped cell with the other chamber filled with 1% EPS medium was largely corroded, as shown in Figure 5b, giving a larger surface roughness (601 ( 62 nm) as compared to the control sample’s roughness (35 ( 11 nm). It should be mentioned that the corrosion of mild steel in the H-shaped cell was uniform across the surface and that the top layer of steel surface was easily peeled off. The AFM images clearly confirmed the enhanced corrosion of steel in the H-shaped cell. Besides AFM measurements, further measurements of corrosion current versus time using zero-resistance ammeter with the potentiostat would be desirable. Using the weight-loss method can also confirm the corrosion effects on a longer timescale. These could be interesting in further studies. In the case where the coupons were in actual physical contact with the EPS, it was observed that the EPS in solution forms a film on the steel surface. The electrochemical reduction may cause the precipitation of EPS on steel surface. EPS, however, also have the binding tendency to metal surfaces. It is difficult to identify which process is predominant. Insitu electrochemical AFM may be applied to reveal this detail but is beyond the scope of this study. In the presence of EPS, pitting corrosion was observed, probably because of the presence of cathodic and anodic regions on the same surface. Chemical Analysis of EPS. The electrochemical results suggest that the reducing property of EPS is contributing to the corrosion of steel. It will be desirable to investigate what constituents in EPS are giving the electrochemical behavior.

FIGURE 5. Three-dimensional AFM images of Fe electrode surface when the Pt electrode was immersed in (a) EPS-free and (b) EPScontaining seawater solutions for 30 days. EPS comprised of complex mixtures of chemical compounds and separation of EPS into its different chemical constituents for further electrochemical tests would be a formidable task. Here, XPS analyses were obtained for EPS before and after corrosion experiments. The results can suggest broadly what reactions and chemical changes have taken place during the corrosion of steel in EPS-containing medium. Figure 6 shows the FTIR spectrum of EPS. The peak assignments are listed in Table 2. The results suggest the possible presence of polysaccharides, carboxylic acids, proteins, and hydrocarbon groups, as suggested in the literature (21, 22). Figure 7 illustrates a wide XPS spectrum of EPS, showing the primary elements C, O, N, Mg, P, S, and Ca. The relative elemental compositions calculated were 60.7%, 30.9%, 7.3%, 0.1%, 0.1%, 0.4%, and 0.4% for C, O, N, Mg, P, S, and Ca, respectively; no Fe was detected. Calcium and magnesium (both less than 0.5%) are probably present in biochemical compounds. Therefore, the corrosion behavior of steel in EPS solution cannot be due to metals in EPS. Some functional groups can be identified from the carbon, oxygen, nitrogen, and sulfur peaks (24). The carbon peaks can be generally categorized into three types of carbon: (i) carbon bonded to carbon and hydrogen, C-(C, H), at a binding energy of 284.2 eV; (ii) carbon singly bonded to VOL. 36, NO. 8, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Characteristic IR Bands in EPS region hydrocarbons proteins polysaccharides

wavenumber (cm-1)

band assignment

literature cited

3400 2928 2855 1654 1542 1380, 1319 1239 1049

O-H stretch CH2 asymmetric stretch CH2 symmetric stretch CdO of amide I C-N of amide II C-O from carboxylic acid PdO C-O, C-O-C stretch from polysaccharides

20, 21 22 22 22 22 22, 23 20, 21 20, 21

TABLE 3. Characterization of XPS Spectra of Various Components in EPS C 1s parameter position (eV) fwhma (eV) atomic conc (%) a

FIGURE 6. FTIR spectrum of EPS/KBr powder.

FIGURE 7. XPS spectrum of EPS pellet. oxygen or nitrogen, C-(O, N), at a binding energy of 285.9 eV; and (iii) carbon doubly bonded with one oxygen, CdO, or singly bonded to two oxygen atoms, O-C-O, at a binding energy of 287.6 eV. The oxygen peaks can be decomposed into two overlapping peaks. The first oxygen peak, denoted O-C, was attributed to the hydroxyl group (C-OH) in acetal, or to hemiacetal (C-O-C), at a binding energy 532.4 eV; the second one was attributed to the oxygen to carbon double bond (OdC) in carboxylic acid, carboxylate, ester, carbonyl, or amide at a binding energy of 531.5 eV. Nitrogen appeared at a binding energy of 399.5 eV, attributed to unprotonated amine or amide functions (25). The S peaks at the binding energies of 161.9 and 162.6 eV were indicative of cysteine and methionine, respectively (26), which are constituents of protein. XPS spectra also provide quantitative information on various components. Elemental concentration ratios can be calculated from the peak areas. The parameters of carbon 1s and oxygen 1s, as well as the concentration ratios of various components in EPS, are summarized in Table 3. 1724

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O 1s

CdO, C-(C, H) C-(O, N) O-C-O

OdC

O-C

284.2 1.70 37.9

531.5 2.24 71.4

532.4 1.78 28.6

285.9 1.76 42.5

287.6 1.85 19.6

fwhm: full width at half-maximum.

The XPS spectra can be interpreted to give rough quantitative information about the molecular composition of the isolated EPS, as demonstrated by Rouxhet et al. (27) for the analysis of microbial cell surfaces. If we assume that proteins (Pr), polysaccharides (PS), and hydrocarbon-like compounds (HC) are the basic model constituents of EPS, the XPS spectra can be fitted to quantify the ratios of these three constituents, according to the intensities of the peaks of the characteristic functional groups, as shown in Table 4 together with the elemental concentration of carbon in each constituent. The data were computed from the model constituents, C6H10O5 for polysaccharides and CH2 for hydrocarbon-like compounds. Proteins have diverse complexities, but their elemental concentrations of C, N, and O vary in a very narrow range. The XPS analysis results for a series of proteins, such as albumin, γ-globulin, fibrinogen, hemoglobin, insulin, ferritin, and trypsin, showed that the elemental concentrations of C, N, and O were 65.2% ( 2.6%, 13.4% ( 0.9%, and 20.4% ( 3.0%, respectively (28). The parameters deduced by XPS spectra are functions of the concentrations of the model constituents on the basis of the three components of the carbon peak

(CdO + O-C-O)/C ) 0.225(CPr/Ct) + 0.167(CPS/Ct) (1) (C-(O, N))/C ) 0.263(CPr/Ct) + 0.833(CPS/Ct)

(2)

(C-(C, H))/C ) 0.512(CPr/Ct) + 1.000(CHC/Ct)

(3)

where CPr, CPS, and CHC are the carbon concentrations in the forms of proteins, polysaccharides, and hydrocarbons. Ct is the sum of these three components. Solving the equations provides the proportion of carbon associated with each molecular constituent: CPr/Ct, CPS/Ct, and CHC/Ct. These ratios can then be converted into weight fractions, using the carbon concentration of each constituent (Table 4). The weight fractions of proteins, polysaccharides, and hydrocarbons are 60%, 37%, and 3%, respectively, according to the elemental concentration of three components of the carbon peaks in Table 3. The fitted results show that the concentration of extracellular proteins in EPS was greater than that of polysaccharides. The presence of a large amount of proteins in the EPS produced by SRB suggests that they may play important roles in biofilm formation and possibly

TABLE 4. Relative Compositions of Model Constituents model constituent

(C-(C, H))/C

(C-(O, N))/C

(CdO)/C (O-C-O)/C

carbon conc (mmol of C/g)b

proteinsa polysaccharides hydrocarbons

0.512 0.000 1.000

0.263 0.833 0.000

0.225 0.167 0.000

47.7 37.0 71.4

a Data computed from the XPS analysis of some major proteins (28). mmol of carbon.

b

Carbon concentration: per gram weight of model constituents contains

FIGURE 9. XPS oxygen 1s spectra of EPS (a) before and (b) after interacting with steel.

FIGURE 8. XPS carbon 1s spectra of EPS (a) before and (b) after interacting with steel and (c) in the Pt electrode chamber of the H-shaped cell after steel corroded in the other chamber. in the resulting biocorrosion. Separate colorimetric analyses showed that proteins occupy about 69% (w/w) of total EPS

and polysaccharides only 31% (w/w). This is in good agreement with the computed results from XPS data. Further colorimetric analyses to assay uronic acid, such as using methydroxydiphenyl reagent (29) would also help to investigate the source of the reduction and the acidity. Although specific chemical changes of EPS cannot be inferred during corrosion, changes in the relative proportions of EPS constituents, given the XPS spectral changes, can suggest the functional groups that are electrochemically active. Figures 8 and 9 show the XPS spectra of carbon 1s and oxygen 1s of EPS before and after the corrosion of steel. The changes in the relative abundance of proteins, polysaccharides, and hydrocarbons, and the ratios of CPr/Ct, CPS/Ct, and CHC/Ct are summarized in Table 5. The changes in the carbon 1s spectra show that the relative abundance of CdO or O-C-O and C-(O, N) decreased, whereas the relative abundance of C-(C, H) increased in EPS after the steel was corroded (Figure 8b). The changes are similar in the H-shaped cell experiments where the EPS are not in contact with iron, as shown in Figure 8c. These calculated values strongly suggest that the CdO or O-C-O, in carboxylic acid, aldehyde, and acetal could be the sites of electrochemical reduction with possible products of C-O or C-(C, H). This is consistent VOL. 36, NO. 8, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 5. Calculated Values for Carbon and Oxygen Bonds and the Molecular Composition in EPS before and after Corrosion of Steel

C 1s in XPS O 1s in XPS calculated by eqs 1-3

a

constituents in EPS

mole fractions before corrosion

mole fractions after corrosion

C-(C, H)/C C-(O, N)/C CdO/C; (O-C-O)/C OdC/O O-C/O FeO/O proteins (CPr/C) polysaccharides (CPS/C) hydrocarbons (CHC/C)

0.38 0.42 0.20 0.71 0.29 n/aa 0.64 0.31 0.05

0.66 0.21 0.13 0.32 0.15 0.53 0.52 0.08 0.40

n/a: not applicable.

with the change of abundance ratio of OdC and O-C in the oxygen 1s spectra, which decreased from 2.50 to 2.07 after the corrosion of iron in EPS solution. The computed results show that polysaccharide content (CPS/Ct) decreased from 0.31 to 0.08 with a corresponding increase of hydrocarbon content, whereas proteins decreased slightly after corrosion. This suggests that the reducing components, possibly CdO, O-C-O, and C-O, mainly came from polysaccharides. The EPS-steel chemical interaction may help to enhance the anaerobic corrosion when the EPS are in physical contact with the steel coupons. However, as demonstrated by the H-shaped cell experiments, significant and equal corrosion behavior was observed when the EPS were not in contact with the steel coupons. Corrosion, due to reducing groups, possibly from polysaccharides, should be the major factor in enhanced corrosion under anaerobic conditions. This study only investigated the corrosion due to EPS “dissolved” in solution. With the presence of a biofilm, the role and action of EPS would be different. Because of the porosity and heterogeneity of the biofilm structure, it can be expected that the EPS will be concentrated there, in localized regions. The more even distribution of EPS in a biofilm may enhance the set of of polarities of electrochemical corrosion cells. On the other hand, EPS may be more attached to the biofilm and result in less contact with the metal surface. In-situ investigations in the presence of a biofilm are desirable but would call for more exotic techniques of in-situ analyses and the use of inserted microelectrodes.

Acknowledgments The authors thank the Hong Kong Research Grants Council for financial support of this study (Project No. HKU7004/ 00E). The authors also thank the Advanced Surface and Materials Analyses Center of Chinese University of Hong Kong for XPS characterization.

Appendix Example Calculation of Weight Fractions of EPS Constituents Based On C Peaks in XPS Analysis On the basis of the carbon 1s spectrum of XPS in Figure 8a, 19.6% of carbon bonds in EPS were CdO or O-C-O, 42.5% were C-O or C-N, and 37.9% were C-C or C-H. Substituting these values into eqs 1-3, we obtain

0.196 ) 0.225(CPr/Ct) + 0.167(CPS/Ct)

(4)

0.425 ) 0.263(CPr/Ct) + 0.833(CPS/Ct)

(5)

0.379 ) 0.512(CPr/Ct) + 1.000(CHC/Ct)

(6)

Solving these equations yields CPr/Ct ) 0.643, CPS/Ct ) 0.307, and CHC/Ct ) 0.05. Carbon concentrations for each constituent are 47.7 × 10-3 M carbon/g of protein, 37.0 × 10-3 M carbon/g of 1726

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polysaccharides, and 71.4 × 10-3 M carbon/g of hydrocarbon. The mass ratios of Pr, PS, and HC should therefore be Pr/ PS/HC ) (0.643/47.7)/(0.307/37.0)/(0.05/71.4) or 0.01348/ 0.00830/0.00070. After normalization, the mass fractions of Pr, PS, and HC were found to be 0.60, 0.37, and 0.03, respectively. Hence, the EPS comprised by weight 60% protein, 37% polysaccharide, and 3% hydrocarbon.

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Received for review August 2, 2001. Revised manuscript received January 25, 2002. Accepted January 29, 2002. ES011187C

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