Effect of biofilm on cast iron pipe corrosion in drinking water distribution system: Corrosion scales characterization and microbial community structure investigation

Corrosion Science 50 (2008) 2816–2823

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Effect of biofilm on cast iron pipe corrosion in drinking water distribution system: Corrosion scales characterization and microbial community structure investigation F. Teng a, Y.T. Guan a,b,c,*, W.P. Zhu a a

Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, PR China Cooperative Research and Education Center for Environmental Technology, Tsinghua University and Kyoto University, Shenzhen 518055, PR China c Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China b

a r t i c l e

i n f o

Article history: Received 9 March 2008 Accepted 16 July 2008 Available online 25 July 2008 Keywords: A. Iron B. XRD B. XPS C. Microbiological corrosion

a b s t r a c t Effect of biofilm on corrosion scales of cast iron pipe was studied with the biofilm community structure investigated by PCR-DGGE to give an explanation to MIC from the viewpoint of microbial phase. Corrosion scales were identified with XRD and XPS. It was demonstrated that biofilm can greatly affect element composition and crystalline phase of corrosion scales. Biofilm can accelerate corrosion in 7 d, but inhibit corrosion after 7 d, which was due to iron bacteria and iron reducing bacteria (IRB), respectively. DGGE fingerprinting gave a well explanation to this transition, which might be contributed to the change of biofilm microbial diversity. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction The corrosion of cast iron pipe in drinking water distribution system (DWDS) will cause many problems such as clogging [1], offering habit for pathogen or opportunity pathogen [2], bringing unpleasant color, lowering the pressure resistance, engendering water supply accident and so on, thereby accelerating water quality deterioration and aggrandizing the threat to human health [1,3–6]. Consequently cast iron, especially grey cast iron, is prohibited in many developed countries. However, grey cast iron pipes have been widely used for decades and for economic reason it is difficult to substitute them with plastic or stainless steel pipes immediately. Thus, their usage in drinking water industry will still last for some years, and the research about the corrosion of cast iron pipes is still of great importance. There have been many reports about the metal corrosion including both electrochemical corrosion and microbiologically influenced corrosion (MIC) [7–9]. The biofilm was reported to contribute to the corrosion cell formation on the surface of metal surface due to the growth of microorganism [10–13] and the biofilm developed by many bacteria genus was found to accelerate metal corrosion. Schiffrin and de Sánchez [14] found that the genus Pseu* Corresponding author. Address: Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, PR China. Tel.: +86 755 26036702; fax: +86 755 26032454. E-mail address: [email protected] (Y.T. Guan). 0010-938X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.corsci.2008.07.008

domonas could modify the oxygen reduction kinetics. Sulfatereducing bacteria (SRB) were reported to increase corrosion rate by some reports [15,16]. Furthermore, a few researchers attempted to give a reasonable explanation about MIC. The enzymatic activity within biofilm was proved to play an important role in the ennoblement of metals [17] and H2O2 produced by the microorganism was demonstrated to increase the open circuit potential [18]. On the basis of electrochemical observation and theoretical deduction Busalmen et al. [19] described the catalysis role of hydroperoxidase. All these reports above had sorted to illustrate the rule about MIC and reinforced that the kinetics of electrochemical reactions was accelerated by microbiological activities. The promotive effects of microbiological activities on electrochemical corrosion were proved in these studies. However, some findings suggested that biofilm can protect the metal from corrosion under certain conditions [20,21]. The findings contradicted with the common sense that microbes could promote metal corrosion and also aroused a question that what kind of effect the biofilms common in DWDS would have on the corrosion of cast iron pipe. It was still a question whether the corrosion behavior in the high corrosive seawater with high salinity was as same as in DWDS. And although a few possible mechanisms about the promotion effect of biofilm were proposed, as far as the authors know, few researches had been reported about the effect of biofilm in a complicate community on the element composition and crystalline phase of corrosion scales. Also researchers above mainly focused on the corrosion behavior with electrochemical methods.

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And little importance was attached to the effect of the microbial phase of a complicate environment on the corrosion process. The application of molecular microbiological ecology techniques would help to answer the questions above. Study about the microbial diversity and community structure in the biofilm can disclose the dominant species that work in the corrosion inhibition or promotion. Once the microbial species are related with the environmental parameters, a better understanding about the corrosion reaction from the viewpoint of microbial phase could be obtained. Use of certain molecular markers, such as 16S rRNA or its encoding gene, to explore the microbial diversity and to analyze the structure of microbial communities [22] has already been applied to in various environment including soil [23], seawater [24], sediments [25], anaerobic granular sludge [26] and so on. But the community structure of biofilm on cast iron pipe has been studied rarely due to the difficulties accompanied with DNA extraction and PCR. Among the molecular methods including molecular cloning, T-RFLP, AFLP, RAPD and so on, PCR-DGGE (Denaturing gradient gel electrophoresis) is excellently suited to investigate the temporal and spatial distribution of bacterial populations due to its easiness, reproducibility, reliability, and speed [22]. In this paper, it is chosen as a tool to analyze the change of microbial community in biofilm microorganism in the corrosion scales under different development time. Herein, we sort to investigate (1) whether the biofilm in the water distribution system will promote or inhibit the corrosion of cast iron pipe; (2) whether or not the biofilm can affect the element composition and crystalline phase of the corrosion scales; (3) the change of microbial community structure of biofilm with time; (4) whether there would be some relationship between the community structure and the corrosion of cast iron. 2. Materials and methods

The sampling process was kept under a strict aseptic environment so that the bacteria would not be introduced into the control. And light was avoided to simulate a dark environment in DWDS. Except for the inner wall that contacted with water, the other sections of the coupons were all coated with epoxy resins. After sampling, the coupons were put in glass bottles and dried under N2 atmosphere protection. After the coupons were dried, the corrosion products were scraped from the inner wall of the coupons with clean shaver blade. Then the scraped scales were used for crystalline phase identification and element composition investigation. The labels for the samples were listed in Table 2. 2.2. Chemical analysis X-ray powder diffractometer (XRD) as a versatile, non-destructive analytical technique was exploited for identification of the crystalline phases [27]. XRD analysis was carried out in D/max-IIIA powder diffractometer using Cu Ka radiation (k = 1.5418 Å) at a scanning range of 2h = 10–70° under a speed of 6°/min. The XRD patterns were analyzed with Materials Data Inc. Jade 5.0 program and PDF 2002 database was used for crystalline phase identification. X-ray photoelectron spectroscope (XPS) scan was performed to measure the chemical state of the surface species of corrosion product using PHI ESCA 5700 instrument, with a Al Ka X-ray source (1486.6 eV) and pass energy of 29.5 eV operated at a pressure of 7  1010 Torr. The binding energies were calibrated with respect to the signal of C 1s (binding energy = 284.82 eV). The background of the spectrum was removed with the Shirley method. Non-linear least squares curve fitting was performed using a Gaussian/Lorentzian peak shape after background removal with the XPSPeak software (http://www.phy.cuhk.edu.hk/~surface/ XPSPEAK, 2000). The concentration of ferrous ion and total iron in bulk water was determined by hydroxylamine hydrochloride method according to the standard method [28].

2.1. Sampling 2.3. DNA extraction Twelve cast iron coupons (length  width  thickness = 20 cm  2 cm  0.5 cm) were disinfected preliminarily and immersed in a covered 2 L glass bottle filled with drinking water from Tsinghua University with groundwater as source water. To investigate the effect of biofilm on corrosion scales, unsterilized raw drinking water was used in one group to have biofilm developed on the coupons; the water passed through 0.22 lm filter was used in another group as the control. The typical water quality was shown in Table 1. After 7, 15, 30 d, four coupons of each group were taken out carefully and the water was displaced with new tap water or sterile tap water to simulate the intermittent water flow environment in the actual pipes. Two of the sampled coupons were used for biofilm analysis and the other two for corrosion product analysis. The bulk water was sampled at a regular interval to measure the concentration of ferrous ion and total iron. All the samples were analyzed in parallel. Table 1 Typical water quality used for biofilm development and the control Water quality parameter

Value

pH Total dissolved solid (mg/L) Turbidity (NTU) Cl (mg/L) SO2 4 (mg/L) NO 3 (mg/L) Total Fe (mg/L) CODMn (mg/L) Total hardness (mg/L as CaCO3) Colorness Total alkalinity (mg/L as CaCO3)

7.46 632 <1 37.9 61.8 1.45 <0.05 0.91 320 <5 300

To character the microbial phase, corrosion scales with biofilm were removed with a small sterile brush and dissolved in 2 ml autoclaved raw water, which was then filtered onto 0.22 lm filter under vacuum condition to concentrate the cells. The filter was cut into pieces and put into a 10 ml centrifuge tube. Two millilitre lysis solution containing 2 mg/ml of lysozyme was added and the mixture was incubated at 37 °C for 2 h. Then 1 ml of buffer (0.1 M NaCl, 0.5 M Tris-HCl (pH 8.0), 10% SDS (w/v)) were added followed by three freeze (5 min in liquid nitrogen) and thaw (10 min under 65 °C) cycles. Proteinase K at a final concentration of 1 mg/ml was added and the resultant solution was incubated under 55 °C for 1 h. DNA in the supernatant was extracted twice with identical volume of Tris-equilibrated phenol (pH 8.0):chloroform:isoamylalcohol (25:24:1) by slightly mixing and centrifuging at 10000g for 20 min at 4 °C, followed by 3 ml chloroform:isoamylalcohol (24:1) extraction and centrifugation at 10000g for 20 min at 4 °C. DNA in the supernatant was precipitated in 2 ml isopropanol overnight and then centrifuged at 12000g for 40 min at 4 °C. The pellet Table 2 Labels for corrosion scales and bulk water with/without biofilm grown on the coupons Samples

Scales

Bulk water

With biofilm growth on 7 d Without biofilm growth on 7 d With biofilm growth on 15 d Without biofilm growth on 15 d With biofilm growth on 30 d Without biofilm growth on 30 d

7W 7N 15 W 15 N 30 W 30 N

7 WW 7 NW 15 WW 15 NW 30 WW 30 NW

F. Teng et al. / Corrosion Science 50 (2008) 2816–2823

2.4. PCR and DGGE A nested PCR was used to amplify the extracted DNA. In the first round the whole length region of bacteria 16S rDNA was amplified with the universal 8-f (50 -TCCGGTTGATCCTGCC-30 ) [29] and 1492-r (50 -GGTTACCTTGTTACGACTT-30 ) [30] primers with the following program: 95 °C for 5 min followed by 35 cycles of 95 °C for 1 min, 52 °C for 45 s, 72 °C for 90 s and an additional extension step for 7 min. The product was diluted and used as the template for the next PCR step. In the second round a specific region of eubacterial 16S rDNA (V6–V8 region) was amplified using 968-f (50 -AAC GCG AAG AAC CTT AC-30 ) [31] with a 40-bp GC clamp (CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G) added to its 50 -end and 1401-r (50 -CGG TGT GTA CAA GAC CC-30 ) primers [32]. A touch-down PCR program was used including an initial denaturation at 95 °C for 5 min and 14 cycles of denaturation at 95 °C for 1 min, annealing at 65 °C (with the temperature decreasing 0.5 °C each cycle) for 30 s, and extension at 72 °C for 1 min, followed by 20 cycles of 95 °C for 1 min, 58 °C for 30 s, and 72 °C for 1 min. During the last cycle, the length of the extension step was increased to 7 min. An aliquot of the PCR product was electrophoresed in 1.2% agarose gel, stained with ethidium bromide for 10 min and photographed. In each amplification protocol the sterile water was used as negative control. DGGE was performed with a De-code system (Bio-Rad). Electrophoresis was performed with 8% polyacrylamide gels (ratio of acrylamide to bisacrylamide, 37.5:1) submerged in 1  TAE buffer (40 mM Tris, 40 mM acetic acid, 1 mM EDTA; pH 7.4) at 60 °C. 20 ll PCR product (about 400 ng) was loaded to each DGGE lane. The following electrophoresis conditions were chosen based on the optimization of denaturant concentration gradient: 12 h at 100 V in a linear 40–65% denaturant agent gradient (100% denaturant agent was defined as 7 M urea and 40% deionized formamide). The gels were stained for 30 min in 1  TAE buffer with SybrGold nucleic acid stain (Molecular Probes) and visualized with UV radiation by UVP equipment.

and 30NW respectively (Fig. 1b). As a higher total iron concentration in the bulk water were found in the group with biofilm in 7 d and in the control without biofilm in 15 d and 30 d, a conclusion can drawn that biofilm could speed the corrosion in 7 d but inhibit the corrosion after 7 d. It also meant that there must be a change after 7 d which lead to the transfer from corrosion acceleration to inhibition. The transition above resulted in the changed total iron concentration in bulk water (Fig. 1b). The Fe2+ concentration in the bulk water in the group with biofilm was lower before 7 d but higher after 7 d than that in the control without biofilm growth (Fig. 1a). Clearly, Fe2+ and total Fe exhibited a reverse change tendency in the supernatant with and without biofilm. 3.2. XRD patterns The XRD patterns under different time were very similar. Representative XRD patterns of corrosion products on cast iron coupons of different incubation time were shown in Fig. 2. The PDF Card No. and chemical formula of the identified peaks were listed in Table 3. c-Fe2O3 was found in all samples except 30 N in which a-Fe2O3 was observed. a-FeOOH existed in all samples with biofilm and in 15 N also. Iron oxide hydroxide (FeOOH) was found in 7 N and 30 N. Since the intensity for minor peaks were so weak, only the major peaks were identified. Calcite and aragonite were found in all samples except that aragonite was not found in 30 N. The vari-

a

8

Without biofilm With biofilm

7 6 5 4

2+

was washed with 70% alcohol and centrifuged at 12000g for 20 min at 4 °C. Then the pellet was desiccated for 30 min under room temperature and dissolved in 50 ll DNase-free 1  TE. The extracted DNA was used as template for PCR.

Fe (mg/l)

2818

3 2 1

2.5. Statistical analysis of DGGE profiles

0 0

5

10

15

20

25

30

time(d)

b 160 With biofilm Without biofilm

140 120

Total Fe (mg/l)

For statistical analysis of the DGGE patterns, the background was firstly removed and then the band intensity was recognized by the Labworks software both automatically and manually (UVP, USA). The band intensity which was not used for statistical analysis, was exported to Excel (Microsoft, USA), and was transformed to 1 if the band existed and 0 when no band existed by IF function. The transformation could eliminate the error of the band intensity by the automatic recognition and the minor error caused by the concentration difference of the loaded PCR products during DGGE. The protocol was repeated for three times and the average values were used as raw data for Principal components analysis (PCA) by Canoco 4.0 and cluster analysis by SPSS 15.0 (Erkrath, Germany). Cluster analysis was performed using hierarchical clustering according to the Ward method [33].

100 80 60 40 20

3. Results and discussion 3.1. Total iron and Fe2+ Before 7 d the total Fe concentration was higher in 7WW than in 7NW, but in 15WW and 30WW it was lower than that of 15NW

0 0

5

10

15

20

25

30

time (d) Fig. 1. Fe2+ and total Fe concentration in bulk water under different time: (a) Fe2+, (b) total Fe.

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3.3. XPS

20

30

40

50

60

70

2-Theta (deg.) Fig. 2. Representative XRD patterns of corrosion products scraped from the surface of cast iron coupons and the crystalline phase identified in PDF 2002 database (Incubation time = 15 d). For sample labels 15 N and 15 W, see Table 2. A major peak of Fe2O3 was found in XRD patterns of the corrosion scales with biofilm at all incubation time, while a major peak of CaCO3 was discovered in the control without biofilm grown.

Table 3 Crystalline phase of XRD patterns Crystalline phase

Chemical formula

PDF card no.

7W

Aragonite Calcite Goethite Maghemite

CaCO3 CaCO3 a-FeOOH c-Fe2O3

75-2330 83-0578 29-0713 24-0081

7N

Aragonite Calcite Iron oxide hydroxide Maghemite-C

CaCO3 CaCO3 FeOOH c-Fe2O3

75-2230 86-2339 75-1594 39-1346

15 W

Aragonite Calcite Goethite Maghemite-C

CaCO3 CaCO3 a-FeOOH c-Fe2O3

71-2396 81-2027 29-0713 24-0081

15 N

Aragonite Calcite Goethite Maghemite-Q

CaCO3 CaCO3 a-FeOOH c-Fe2O3

76-0606 86-2334 29-0713 25-1402

30 W

Aragonite Calcite Goethite Maghemite-C

CaCO3 CaCO3 a-FeOOH c-Fe2O3

76-0606 86-2334 81-0464 25-1402

30 N

Calcite Iron oxide hydroxide Hematite

CaCO3 FeOOH a-Fe2O3

72-1652 75-1594 72-0469

ation of crystalline phase proved that biofilm had effect on crystalline growth. When there was biofilm grown, the major peaks of the XRD patterns were always characterized by Fe2O3 (Fig. 2), while for the control the major peaks were always identified as CaCO3. The relative content of these crystalline were affect by the existence of biofilm as the species characterized by the major peaks changed from Ca in samples without biofilm to Fe in samples with biofilm. Consequently, relative element percentage of Ca and Fe which was contained in the crystalline was affected by biofilm growth. The crystalline phase production of Fe oxide was promoted by the biofilm since the peak intensity of Fe always exceeded that of Ca in the pattern of samples with biofilm. As shown in Fig. 2, the existence of biofilm could affect the crystalline phase and element composition in the scales.

1000

800

600

Fe3s -Mg2s

10

400

Fe3p -Mg2P O2s

0

Si2s

3

1

1

2 2 4

4

20

3

1

40

3

1

60

1

1

1

80

C1s

70

Ca2p3 -Ca2p1 -Ca2p

60

Mg KLL

Without Biofilm(15N)

100

50

Ca2s

40

O 1s

30

Fe LMM1

2

4 4

4 20

Fe2p3 -Fe2p1 -Fe2p

Goethite(FeO(OH))

O Kll

4

The XPS technique was very surface sensitive and can provide information on chemical changes that occurred at the interface between water and corrosion scales on pipe surface [27]. XPS spectra of corrosion products without biofilm grown (7 N) were presented in Fig. 3 as a typical spectrum. The atomic ratio of main elements detected in the corrosion products were listed in Table 4. It was presumed that all of these atomic ratios came from a homogeneously mixed sample. Fe atomic ratio of 7 W was 17.87% about three times that of 7 N, which meant Fe content in 7 W was much higher than that in 7 N. But after 7 d, the iron atomic ratio in scales without biofilm exceeded that in scales with biofilm (Table 4). The iron atomic percentage coincided with the tendency of total iron in bulk water and testified that the corrosion of cast iron pipe was promoted by the existence of biofilm in 7 d, but inhibited after 7 d. The atomic ratios of Ca in scales with biofilm were however, lower than those in scales without biofilm all the time. The higher Ca content was found in scales without biofilm growth (Table 4). The difference of Ca and Fe atomic ratio between scales with and without biofilm agreed with the fact that biofilm had effect on element composition as shown by XRD patterns. As the corrosion behavior was mainly concerned in this paper, only the spectrum of Fe(2p) was fitted with XPSPeak software. The binding energies were calibrated with respect to the signal of C 1s (binding energy = 284.82 eV). The standard Fe(2p3/2) peak position of Fe2O3 and FeOOH was fixed at 710.8 eV and 711.8 eV, respectively and FWHM was both fixed at 3.8 eV [27]. As the differences of binding energy of between various iron phases were significant (>0.3 eV), they could be differentiated [27]. The area percentages of fitted peaks were listed in Table 5. A shake up peak

Fe LMM2 Fe LMM1 Fe2s

Maghemite(Fe2O3)

3

10

2

0 120

Calcite(CaCO3)

344

3 4

20

2 3

4

4

2

4

40

1

Intensity (Arab. Units)

60

Aragonite(CaCO3)

4

80

1

Counts (arab. Units)

With Biofilm(15W)

2

3

100

200

0

Bingding Energy (eV) Fig. 3. Representative XPS spectrum of corrosion scales scraped from the surface of cast iron coupons (incubation time = 7 d, without biofilm grown). XPS spectrum of corrosion scales under different incubation time with and without biofilm were very similar. Fe(2p) spectrum was fitted with XPSPeak software.

Table 4 Atomic ratio (mol%) of the corrosion product under different incubation time Samples

C (%)

O (%)

Mg (%)

Ca (%)

Fe (%)

7N 7W 15 N 15 W 30 N 30 W

51.58 25.12 40.88 55.02 31.18 44.01

36.06 53.50 42.50 33.14 48.05 40.53

3.17 2.2 0.46 0.31 0.68 0.30

2.19 1.50 2.61 2.27 2.03 1.73

6.03 17.87 8.44 5.06 12.40 8.22

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Table 5 Curve fit summary of the deconvolution of Fe(2p3/2) peak of cast iron

FeOOH Fe2O3 Shake up peak

7 N (%)

15 W (%)

15 N (%)

30 W (%)

30 N (%)

43.9 34.0 22.1

35.8 64.2 –a

44.3 22.9 32.8

66.7 33.3 –

43.4 34.5 22.1

64.4 35.6 –

The shake up peak does not exist.

Fe2O3

Counts (a. b.)

FeO(OH)

700

700

710

Fe2O3

720

7N

Fe2p1/2

Fe2p3/2

a

7 W (%)

730

7W

FeO(OH) Shake up

710

720

730

Binding Energy (eV) Fig. 4. Representative curve fitting of the Fe(2p) spectra of corrosion scales (incubation time = 7 d). For labels 7 N and 7 W, see Table 2. A shake up peak in the range from 713 eV to 715.5 eV existed in all Fe(2p3/2) spectra of corrosion products with biofilm grown.

[34] in the range from 713 eV to 715.5 eV [35] existed in all Fe(2p3/2) spectra of corrosion products with biofilm (Fig. 4), which might be the peak of FeSO4 or Fe2(SO4)3 [27,35]. 3.4. Fingerprinting of DGGE patterns The DGGE fingerprinting of the PCR products of biofilm microorganisms amplified with 968-GC-f/1401-r primers was shown in Fig. 5. PCR products were loaded on the lanes in triplicate. Lanes 1–3, 4–6, 7–9 showed the DGGE pattern for biofilm on 7 d, 15 d, 30 d, respectively. Two kinds of microbial diversity index, the Shannon–Wiener index and the Simpson index were calculated and the results were shown in Table 6. From the DGGE fingerprinting, it was clearly shown that species represented by bands 14, 17 and 21 existed in biofilm for 7 d but disappeared in biofilm for 15 d (Fig. 5). Since the quantity of species was represented by the band intensity in DGGE patterns, it indicated that species represented by bands 7, 8, 10 and 19 increased while that represented by bands 14, 17 and 21 decreased from 7 d to 15 d in quantity. As indicated in Table 6, the Shannon–Wiener index and the Simpson index had the similar rule with the lowest values on 15 d and highest on 30 d, while indexes on 7 d were in the middle. A transition of the microbial diversity index existed from 7 d to 15 d. As presented above, the biofilm promoted corrosion in an incubation time of 7 d but inhibited corrosion after 7 d, which also had a change after 7 d. Since this tendency of the Shannon–Wiener index and the Simpson index were so similar to that of total iron in the bulk water and the Fe atomic ratio in the scales which also had a change happened from 7 d to 15 d, it was implied that there might be some relationship between the transition from accelerating to inhibiting corrosion and the change of biofilm microbial diversity.

Fig. 5. DGGE fingerprinting of the PCR products of biofilm microorganisms amplified with 968-GC-F/140-r primers. Lanes 1–3, 4–6, 7–9 represented patterns for biofilm of 7 d, 15 d, 30 d, respectively.

F. Teng et al. / Corrosion Science 50 (2008) 2816–2823 Table 6 Shannon–Wiener index and Simpson index calculated according to the DGGE fingerprinting Lanes no.

Simpson index

Shannon–Wiener index

Lane Lane Lane Lane Lane Lane Lane Lane Lane

0.908 0.911 0.909 0.874 0.874 0.856 0.917 0.919 0.885

2.428 2.494 2.468 2.225 2.219 2.07 2.636 2.668 2.282

1 2 3 4 5 6 7 8 9

(7 d) (7 d) (7 d) (15 d) (15 d) (15 d) (30 d) (30 d) (30 d)

Fig. 6. Cluster analysis of the transformed data (0–1) derived from the DGGE patterns with SPSS 15.0. For lanes and DGGE patterns, see Fig. 5. Cluster analyses revealed a closer relative distance of the microbial species between biofilm of 7 d and 15 d.

Cluster analyses revealed a closer association of the microbial species between biofilm of 7 d and 15 d. Principal components analysis (PCA) showed an incubation time pattern, with the main

2821

shifts in the bacterial species composition taking place after 15 d. The first two axes of the PCA analysis on the samples under different incubation time (Fig. 7) revealed a clear-cut time pattern with the samples almost perfectly circling anti-clockwise. The first principal component (PC1) on the samples with different incubation time (Fig. 7) corresponded to 47.7% of the variation in the samples with different incubation time. The second axis (PC2, 24.1% of the variation in the data) separated the difference between the triplicate samples. 3.5. Corrosion mechanism discussion The XRD patterns showed that biofilm had great effect on element composition and crystalline phase of corrosion scales. The same phenomena were also testified by XPS analysis since a notable Fe atomic ratio difference was observed between scales with and without biofilm. The concentration of total iron in bulk water indicated that the cast iron pipes were corroded to a higher extent in the control in 15 d and 30 d and in the group with biofilm in 7 d. The iron atomic ratio results testified these results as iron atomic ratio were also higher in scales of 7d with biofilm and in scales of 15 d and 30 d without biofilm. It also reinforced that biofilm growth would have effect on the element composition in scales. Fe2+ concentration in the bulk water, however, changed with a reverse direction from total Fe. However, Fe2+ can explain the protection mechanism by biofilm after 7 d which would be discussed later. The DGGE patterns reflected the change of dominant species with time. In our research before, by isolation and cultivation methods only two kinds of iron bacteria, namely Leptospirillum ferriphilum and Leptospirillum ferrooxidans were successfully separated from the drinking water distribution system which could respire Fe or Fe2+ in absence of organic matters [36]. What must be pointed out was that these iron bacteria, were strict aerobic organisms which needed O2 to grow [37,38]. Therefore before 7 d, because of a high O2 concentration, they might use the Fe as

Fig. 7. PCA analysis of DGGE banding patterns in the biofilms under different incubation time. The arrow indicated the general trend of development time. For lanes and bands, see the DGGE pattern (Fig. 5). Lanes 1–3, 4–6, 7–9 represented patterns for biofilm of 7 d, 15 d, 30 d, respectively. The first principal component (PC1) on the samples with different incubation time corresponded to 47.7% of the variation in the samples with different incubation time. The second axis (PC2, 24.1% of the variation in the data) separated the difference between the triplicate samples.

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F. Teng et al. / Corrosion Science 50 (2008) 2816–2823

electron supplier and CO2 or O2 and so on as electron acceptor to grow. But after 7 d, the oxygen was consumed by the bacterial growth and the oxidation of Fe(OH)2, these iron bacteria might not exist due to an absence of oxygen after 7 d as Sarin had shown that after 2 h under stagnation 90% O2 would be exhausted [4]. Thus, after 7 d the disappearance of these bacteria would be reflected by a decrease of species. The disappearance of some species after 7 d could be clearly testified by the change of Shannon–Wiener index and Simpson index (Table 6) and DGGE patterns (Fig. 5) from 7 d to 15 d. Species represented by bands 14, 17 and 21 did not exist in biofilm of 15 d and 30 d under which time the biofilm act as a protector against corrosion. Therefore, theoretically these three species might be the key microorganisms that caused corrosion which did not exist in biofilm after 7 d, though due to the technical reason we did not sequence the bands and identify them. And if they were in scarcity in biofilm after 7 d as deduced, the biofilm would not promote the corrosion as in 7 d.

Fe  Fe2þ þ 2e þ

ð1Þ



O2 þ 4H þ 4e  2H2 O

ð2Þ

Fe2þ þ 2OH  FeðOHÞ2ðsÞ 2þ

4Fe

3þ

þ

ð3Þ 3þ

þ 4H þ O2ðaqÞ  4Fe 

þ 2H2 O

ð4Þ

þ 3OH  FeðOHÞ3ðsÞ

ð5Þ

2FeðOHÞ3  Fe2 O3 þ H2 O

ð6Þ

FeðOHÞ3  FeOOH þ H2 O

ð7Þ

Fe

On the base of these electrochemical reactions, the corrosion promotion of biofilm on 7 d can be explained by Eqs. (1)–(7) [39]. Under an oxidation atmosphere, the corrosion cell was composed of an iron anode and an O2 cathode. Fe was oxidized to Fe2+ both by iron bacteria and O2, which incorporated with OH- into Fe(OH)2. The produced Fe(OH)2 was further oxidized to Fe(OH)3 which had a low solubility under a circumstance of pH 7. The Fe(OH)3 sediment was dehydrated to form Fe2O3 and FeOOH. Besides the electrochemical corrosion, there was also pitting corrosion caused by iron bacteria in biofilm which had been reported by many researches [10–13]. At this time the group with biofilm had a faster corrosion rate than the control. After 7 d, the corrosion rate in the control began to exceed that in the group with biofilm. The dominance shifted between different species with time and more species were found in biofilm of 30 d (Fig. 5). Though the iron bacteria would not multiply under an anaerobic environment which would resulted in a decreased corrosion speed, there might be some other species which might accelerate corrosion by anaerobic respiration of Fe. The most common species of that could exist under a strict anaerobic environment might be SRB which could corrode cast iron pipe as had been shown [15,16]. The SRB, however, were not detected by both isolation and culture method and FISH (fluorescence in situ hybridization) method in our works before [36]. In fact SRB which were heterotrophic would be hard to grow in ground water because of organic carbon scarcity. One kind of another species that was common under high concentration of iron might be the iron reducing bacteria (IRB), which was strictly anaerobic [40]. In the initial incubation time, the iron reducing bacteria could not be the main species because of an oxidative atmosphere, and the relatively high ORP in which the oxidation of Fe2+ and Fe(OH)2 dominated. After 7 d, the oxygen concentration decreased and there would be relatively beneficial environment for iron reducing bacteria to grow. After 15 d, the O2 would be completely exhausted and became an anaerobic condition which was more favorable for the iron reducing bacteria to multiply. The multiply of IRB would be reflected by the increase of new species or species quantity from 15 d to 30 d. The DGGE pat-

terns (Fig. 5) showed that species represented by bands 4, 5, 6, 13 and 16 emerged in biofilm of 30 d, which did not exist in biofilm of 15 d. As the result of the growth of IRB, the formed Fe(III) would be reduced to Fe(II) resulting in an increased Fe2+ concentration (Fig. 1a) as indicated by Dubiel et al. [20]. Since Fe2+ was in a dissolved form covering the surface of the coupons, a reducing atmosphere was kept on the surface of the coupons which gave birth to an increased potential of Fe/Fe2+. Consequently the speed of Fe dissolution was reduced from 15 d to 30 d. As can be shown by cluster analysis (Fig. 6), species between 7 d and 15 d was closer and the microbial diversity in biofilm increased from 15 d to 30 d, thereby the microbial community of 7–15 d still was in a period of transition. In a transition period of 7–15 d, the iron reducing bacteria might not dominant in biofilm whose protection mechanism therefore should be contributed to the disappeared species represented by bands 14, 17 and 21 or to the barrier mechanism by biofilm as indicated by Thomas [41]. As shown above, the DGGE pattern could explain the reason for the change of the accelerating to the inhibiting corrosion by biofilm based on the mechanism above, though some points were still needed to be proved. Due to the difficulties of re-PCR to the excised bands in the DGGE patterns, we did not identify them. Some works were still in progress to characterize them. What should be emphasized was that the change of microbial diversity might have relationship with the change of the accelerating to the inhibiting corrosion by biofilm in essential

HCO3 þ OH  CO2 3 þ H2 O

ð8Þ

2þ CO2  CaCO3ðsÞ 3 þ Ca

ð9Þ

The XRD patterns (Fig. 2) of samples with biofilm all had a low intensity of CaCO3 peaks. While the area for CaCO3 peaks were larger for the control and CaCO3 were all the time identified as the major peak. The atomic ratio also coincided with the results by XRD. Atomic ratios of Ca in samples with biofilm were higher than that in the control. The mechanism for CaCO3 production can be illustrated by Eqs. (8) and (9) [39]. In the cathode of corrosion cell, H+ was exhausted by the reduction of O2 and OH- accumulated in the local area. Therefore, Eq. (8) tends to react towards the direction for CO2 3 production. Thus, CaCO3 will settle and became part of the corrosion scales. It was reported that extracellular polymeric substances can absorb Ca2+ [42]. Also Ca2+ was necessary for the bacteria growth which will consume much of Ca2+ (usually 103 mM). Hence the applicable Ca2+ for CaCO3 formation decreased greatly.

4. Conclusion (1) The existence of biofilm had great effect on the element composition and crystalline phase of the scales. (2) The existence of biofilm could speed the corrosion in 7 d but inhibit the corrosion after 7 d. Thus the role of biofilm in the corrosion process had relationship with the immersion time. (3) The corrosion promotion of biofilm in 7 d could be contributed to two kinds of iron bacteria, namely L. ferriphilum and L. ferrooxidan, which could respire Fe or Fe2+. The corrosion inhibition by biofilm after 7 d could be linked with some iron reducing bacteria, which could reduce Fe(III)–Fe(II) and result in corrosion inhibition. The transition from accelerating to inhibiting corrosion might be due to the change of biofilm microbial diversity. (4) PCR–DGGE, as a powerful molecular microbial ecology which documented the change of biofilm microbial community structure and explained the transfer of corrosion pro-

F. Teng et al. / Corrosion Science 50 (2008) 2816–2823

motion to corrosion inhibition, could be used widely in the research of MIC to explain the corrosion mechanism which had not been related with the activity of microorganisms.

Acknowledgement This work is supported by the National Natural Science Foundation of China (Project No. 50478011). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

[12] [13] [14] [15]

T.E. Larson Jr., F.R. Sollo, J. Am. Water Works Ass. 59 (1967) 1562. O.H. Tuovinen, J.C. Hsu, Appl. Environ. Microbiol. 44 (1982) 761. A. Kuch, Corros. Sci. 28 (1988) 221. P. Sarin, J. Bebee, K.K. Jim, M.A. Beckett, W.M. Kriven, J.A. Clemen, Water Res. 38 (2004) 1259. S.A. Imran, J.D. Dietz, G. Mutoti, J.S. Taylor, A.A. Randall, C.D. Cooper, J. Am. Water Works Ass. 97 (2005) 93. L.S. McNeill, M. Edwards, J. Am. Water Works Ass. 93 (2001) 88. W.P. Iverson, Int. Biodeter. Biodegr. 47 (2001) 63. M.M. Benjamin, Internal Corrosion of Water Distribution Systems, second ed., American Water Works Association Research Foundation, 1996. J.P. Lin, M. Ellaway, R. Adrien, Corros. Sci. 43 (2001) 2065. D. Starosvetsky, R. Armon, J. Yahalom, J. Starosvetsky, Int. Biodeter. Biodegr. 47 (2001) 79. B.J. Little, P.A. Wagner, Z. Lewandowski, Spatial relationships between bacteria and mineral surfaces, in: J.F. Banfield, K.H. Nealson (Eds.), Geomicrobiology: Interactions between Microbes and Minerals, Mineralogical Society of America, Washington, DC, 1997, pp. 123–159. J.D. Gu, M. Roman, T. Esselman, R. Mitchell, Int. Biodeter. Biodegr. 41 (1998) 25. J.E.G. González, F.J.H. Santana, J.C. Mirza-Rosca, Corros. Sci. 40 (1998) 2141. D.J. Schiffrin, S.R. de Sánchez, Corrosion 41 (1985) 31. A.D. Seth, R.G.J. Edyvean, The function of sulfate-reducing bacteria in corrosion of potable water mains, Int. Biodeter. Biodegr. 58 (2006) 108.

2823

[16] W.A. Hamilton, Sulfate-reducing bacteria and anaerobic corrosion, Ann. Rev. Microbiol. 39 (1985) 195–217. [17] I. Dupont, D. Fhon, G. Novel, Int. Biodeter. Biodegr. 41 (1998) 13. [18] N. Washizu, Y. Katada, T. Kodama, Corros. Sci. 46 (2004) 1291. [19] J.P. Busalmen, M. Vázquez, S.R. de Sánchez, Electrochim. Acta 47 (2002) 1857. [20] M. Dubiel, C.H. Hsu, C.C. Chien, F. Mansfeld, D.K. Newman, Appl. Environ. Microbiol. 68 (2002) 1440. [21] R. Zuo, E. Kus, F. Mansfeld, T.K. Wood, Corros. Sci. 47 (2005) 279. [22] G. Muyzer, K. Smalla, Anton. Leeuw. 73 (1998) 127. [23] A.K. Müller, K. Westergaard, S. Christensen, S.J. Sørensen, FEMS Microbiol. Ecol. 36 (2001) 11. [24] B. Díez, C. Pedrós-Alió, T.L. Marsh, R. Massana, Appl. Environ. Microbiol. 67 (2001) 2942. [25] J.P. GRAY, R.P. Herwig, Appl. Environ. Microbiol. 62 (1996) 4049. [26] M.A. Pereira, K. Roest, Alfons J.M. Stams, M. Mota, M. Alves, Antoon D.L. Akkermans, FEMS Microbiol. Ecol. 41 (2002) 95–103. [27] Z.J. Tang, S. Hong, W. Xiao, J. Taylor, Corros. Sci. 48 (2006) 322. [28] EPA of China, Analysis Method for Water and Waster Water, fourth ed., Press of Chinese Environmental Science, 2002. [29] N. Banning, F. Brock, J.C. Fry, R.J. Parkes, E.R.C. Hornibrook, A.J. Weightman, Environ. Microbiol. 7 (2005) 947. [30] P.J. Waldron, S.T. Petsch, Appl. Environ. Microbiol. 73 (2007) 4171. [31] E.G. Zoetendal, A. von Wright, T. Vilpponen-Salmela, K. Ben-Amor, Antoon D.L. Akkermans, Willem M. de Vos, Appl. Environ. Microbiol. 68 (2002) 3401. [32] U. Nübel, B. Engelen, A. Felske, J. Snaidr, A. Wieshuber, R.I. Amann, W. Ludwig, H. Backhaus, J. Bacteriol. 178 (1996) 5636. [33] F. Widmer, A. Fliebbach, E. Laczkó, J. Schulze-Aurich, J. Zeye, Soil Biol. Biochem. 33 (2001) 1029. [34] Z.D. Cui, S.L. Wu, S.L. Zhu, X.J. Yang, Appl. Surf. Sci. 252 (2006) 2368. [35] S.J. Yuan, S.O. Pehkonen, Colloid Surf. B: Biointerf. 59 (2007) 87. [36] J.H. Liu, Researching on Biofilms Attached to Water Supply Pipe-Microbial Community and its Influence, MS thesis, Tsinghua University, 2003. [37] N.J. Coram, D.E. Rawlings, Appl. Environ. Microbiol. 68 (2002) 838. [38] Víctor Parro, Mercedes Moreno-Paz, PNAS 100 (2003) 7885. [39] V.L. Snoeyink, D. Jenkins, Water Chemistry, John Wiley & Sons Inc., 1980. [40] Manuel Carmona, Eduardo Díaz, Mol. Microbiol. 58 (2005) 1210. [41] C.J. Thomas, R.G.J. Edyvean, R. Brook, Biofouling 1 (1988) 65–77. [42] T.D. Perry, V. Klepac-Ceraj, X.V. Zhang, C.J. Mcnamara, M.F. Polz, S.T. Martin, N. Berke, R. Mitchell, Environ. Sci. Technol. 39 (2005) 8770.