- Email: [email protected]
Microbial corrosion of galvanized steel in a simulated recirculating cooling tower system
Corrosion Science 52 (2010) 161–171
Contents lists available at ScienceDirect
Corrosion Science journal homepage: www.elsevier.com/locate/corsci
Microbial corrosion of galvanized steel in a simulated recirculating cooling tower system Esra Ilhan-Sungur *, Aysßın Çotuk Istanbul University, Faculty of Science, Department of Biology, 34134 Vezneciler, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 26 September 2008 Accepted 26 August 2009 Available online 31 August 2009 Keywords: A. Zinc B. SEM C. Microbiological corrosion
a b s t r a c t In this study, mixed species biofilm formation including sulphate reducing bacteria (SRB) on the galvanized steel surfaces and also microbiologically influenced corrosion (MIC) of galvanized steel were observed in a model recirculating cooling water system during 10 months. A biofilm which had a heterogeneous structure formed on galvanized steel coupons. The results suggested that galvanized steel was corroded by microorganisms as well as SRB in the biofilm. Extracellular carbohydrate was degraded and quantities of carbohydrate were positively correlated with the weight loss. The concentrations of zinc in the biofilm showed significant correlations with weight loss, carbohydrate amount and SRB count. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Cooling towers are an integral part of any power plant. The conditions of cooling towers are very suitable for microbial growth and biofilm formation [1]. A biofilm is a population of cells embedded in a thick mucilaginous matrix of extracellular polymeric substances (EPS) which may consist of 90% or more of polysaccharides [2]. Microbial biofilm and corrosion in cooling systems are the most common problems that damage expensive equipment, cause loss of production and increase maintenance costs [1,3]. Sulphate reducing bacteria were considered as the major bacterial group involved in microbiologically influenced corrosion [4]. Although numerous experiments are now available demonstrating microbiologically influenced corrosion of several metals under laboratory and in situ conditions [5–9], there are only one study about MIC of galvanized steel by SRB carried out in laboratory conditions [10]. However, to date we have not been able to find any published reports on the MIC of galvanized steel in a cooling water system. Galvanized steel is frequently used in the construction of cooling towers and water containers, owing to its good resistance to corrosion and biofauling. In spite of the well-known anticorrosive and antifouling properties of galvanized steel, Ilhan-Sungur and Cotuk [11] reported high SRB counts (104 cells/ml) in cooling tower water and structural failures in some cooling towers because of pitting corrosion. Even though numerous experiments are now available demonstrating that zinc is toxic to SRB strains [12,13], in our previous study [10], we showed microbiologically influ-
* Corresponding author. Tel.: +90 212 455 57 00; fax: +90 212 528 05 27. E-mail address: [email protected] (E. Ilhan-Sungur). 0010-938X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.corsci.2009.08.049
enced corrosion of the galvanized steel by SRB in closed laboratory conditions. However we wondered whether in a cooling tower which has an open environment and potable water, by contrast, mixed species biofilm including SRB would form on the galvanized steel surfaces and microbiologically influenced corrosion of galvanized steel would occur. For these purposes, in this study, we have developed a model recirculating cooling tower system capable of developing reproducible steady-state bacterial biofilms in nonsupplemented potable water. We believe that it provides a realistic representation of the conditions and organisms that we wanted to study. Turetgen [14] compared the biofilm reproducibility of the surfaces taken from a similar model system with the full-scale cooling tower system and reported that there was no significant differences between HPC counts and reproducibility from tested coupons in the full-scale and model system. The aims of this study were to observe the mixed species biofilm formation including SRB and anaerobic heterotrophic bacteria on the galvanized steel surfaces and microbiologically influenced corrosion of galvanized steel in a model recirculating cooling water system during 10 months and, also establish the effects of biological parameters on MIC. 2. Experimental procedure 2.1. Test material Galvanized steel for use in cooling tower construction was used as test material containing 0.027% C, 0.148% Mn, 0.011% Si, 0.01% P, 0.059% Al, 0.008% S and 99.737% Zn. The thickness of the zinc coating covering the stainless steel was 5 lm. The coupons (50 25 0.5 mm) were prepared according to guidelines in ASTM
162
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
G1–72 [15]. They were weighed and the total surface area of each coupon was determined. The cut areas of all the coupons were coated with epoxy zinc phosphate primer (Moravia, Turkey) (grey) and then covered with epoxy finish coating (Moravia, Turkey) (black) to avoid the initiation of corrosion at these disturbed areas [10].
2.2. Model system The experimental study was performed using a 100-L polypropylene laboratory scale cooling tower model system running with 80 L bulk water under constant hydraulic conditions, which simulated the situation in a cooling tower. It was equipped with a recirculation pump (550 W, 40 L min1, Pedrollo, Italy) in the basin and a heater (AT-100, 100 W, Atman, Germany) to facilitate evaporation within the bulk water. Cover lid had openings to ensure fresh air and daylight entry (Fig. 1). A supply of potable water was used to replenish the water lost by evaporation and blowdown (partial draining). Throughout the experiment, the water temperature was kept constant at 28 °C. Coupons were inserted vertically into coupon–holders situated in the water basins (Fig. 1). No chemicals (disinfectant, pH regulators or anti-scaling agents) were added to the system in order to exclude their possible negative effects (such as their disinfecting effect) on the microorganisms and biofilm formation. The water samples were analyzed monthly for various physico-chemical parameters such as pH, conductivity (WTW LF 95 Conductivity meter, Germany), total dissolved solids (TDS), alkalinity, sulphates (SO42), orthophosphates (PO42), chlorides (Cl), hydrogen sulphides (H2S) and dissolved oxygen [16–18]. Control systems containing sterile potable water with a flow rate 7.3 L h1 and with 40 galvanized steel coupons were run simultaneously with the model system. Gentamicin (50 lg/L) (Fortigen, Turkey) was added to the control system to prevent contamination. Ten coupons were removed from the model system monthly during 10 months for enumeration of SRB and heterotrophic bacteria, quantification of biofilm formation, corrosion rate measurement, EPS extraction, and carbohydrate, iron and zinc analysis. In addition for field emission scanning electron microscopy (FESEM) and energy dispersive X-ray analysis (EDS) observation, two coupons were removed from model system after 11 days and 10 months.
2.3. Bacterial analysis To estimate the number of bacteria in biofilm (sessile bacteria), three coupons were removed monthly from the basin, dip-rinsed in sterile potable water to remove unattached cells. Biofilm was suspended in 10 ml sterile potable water [19]. The resulting suspension was then serially diluted from 101 to 1010. To show the count of bacteria in bulk water (planktonic bacteria), bulk water samples were concentrated by filtration through a 0.22 lm pore size polyamide filter (Sartolon, Sartorius AG, Goettingen, Germany), and then membrane filters were resuspended in 50 ml sterile tap water by stomacher (IUL Instruments) for 2 min [20]. The suspensions were used as inoculum for isolation and enumeration of SRB and heterotrophic bacteria. Sessile and planktonic SRB counts were determined by the most probable number (MPN) technique using Postgate’s medium B with the following composition: C3H5O3Na (3.5 g/L), KH2PO4 (0.5 g/L), NH4Cl (1.0 g/L), Ca2SO4 (1.0 g/L), MgSO47H2O (2.0 g/L), yeast extract (1.0 g/L), C6H7O6Na (0.1 g/L), C2H3O2SNa, (0.1 g/L), FeSO47H2O (0.5 g/L), C6H5O7Na3 (0.3 g/L) (pH adjusted to 7.2 by 10% NaOH) [21,22]. MPN tubes were incubated in the dark at 30 °C for 3 months. In each inoculated tube, growth of sulphate reducers was indicated by the formation of a black FeS precipitate and by turbidity. For aerobic heterotrophic plate count (HPC), samples of 100 lL from dilution series of biofilm homogenates and filtered water suspensions were spread-plated in triplicate onto R2A agar (Oxoid, UK) plates and incubated at 28 °C for 10 days [23]. After the incubation time, the number of colonies was enumerated under a colony counter (WTW, model BZG 30). For enumeration of anaerobic heterotrophic bacteria, Thioglycollate medium (Oxoid, UK) was used and the plates were incubated at 30 °C for 21 days. The number of colonies was enumerated under the colony counter. Anaerobic conditions were created using the ‘‘AnaeroGen’’ systems (Oxoid, UK).
2.4. Quantification of biofilm formation The quantification of biofilm formation was carried out according to the method of O’Toole and Kolter [24]. Two coupons were air dried and surface-attached cells were stained for 15 min with a
Fig. 1. Schematic diagram of the model recirculating water system. Arrows indicate the flow direction.
163
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
0.5% (wt/vol) solution of crystal violet (Merck, Germany). After rinsing with dH2O, bound dye was solubilized in 95% ethanol. Adherent cells were quantified by determining the absorbance of the solution at 562 nm with spectrophotometer (Shimadzu UV 150-02). 2.5. EPS extraction and carbohydrate analysis Three biofilm coated coupons were removed from the model system monthly and the recovery of EPS was performed according to the method of Zhang et al. [25]. The carbohydrate content of EPS was measured using the phenol/sulphuric-acid method. Standard aqueous solutions of glucose (10–100 mg/L) were used for instrument calibration [26]. 2.6. Weight loss measurement Eight coupons used for the other analyses were cleaned as ASTM G1–72 and weighed after drying [15]. The difference between the initial and final weight was reported as weight loss. The values of the corrosion rate were determined by applying the following equation proposed by the ASTM standard G1–72 [15]. 2
Corrosion rate ¼ ðK WÞ=ðA t DÞ mg=dm day: where: K = 2.40 106 D, t = time of exposure (h), A = coupon area (cm2), W = weight loss after cleaning (g), D = density (g/cm3). 2.7. Iron and zinc measurement To determine iron and zinc concentrations in the biofilm, two coupons were used. The biofilm was removed from the total surface of the one coupon exposed to the medium, suspended in distilled water and vortexed for 5 min. Then the biofilm suspension and water samples from model and control systems were treated with 3 N nitric acid (HNO3) [27]. The acidified samples (pH 3) were filtered through a 0.45 lm membrane filter (Millipore) to remove insoluble suspended particles. The filtered samples were analysed for Fe and Zn content using a Shimadzu AA-680 Flame Atomic Absorption Spectrophotometer. 2.8. FESEM and EDS analysis Corrosion products formed on coupons were analysed by field emission scanning electron microscopy at the end of the experimental duration of exposure of 11 days and 10 months. Coupons were fixed with 2.5% glutaraldehyde, followed by dehydration in a graded series of ethanol and air-drying [28]. The dried samples were coated with a palladium (15 nm) and imaged with a Jeol JSM-6335 field emission electron microscope. Chemical analysis was done by energy dispersive X-ray spectroscopy. 2.9. Statistical analysis of data Bacteria counts were log10 transformed and standard deviations (SD) of the means were calculated. Statistical evaluation of the results was carried out by Pearson product moment correlation coefficient test. One-way analysis of variance (ANOVA) was used to compare variations of Fe and Zn concentrations in biofilm, EPS and water samples followed by Tukey’s-b post hoc test using SPSS statistical package program. t-test was employed to detect statistically significant changes in the bacterial counts. Differences were considered statistically significant at P < 0.05.
3. Results 3.1. Physico-chemical characters of model system water The physico-chemical parameters of the recirculating water in the model system were presented in Table 1. Conductivity, TDS and sulphates were increased with time (respectively, P < 0.01, P < 0.01 and P < 0.05). The temperature of the system was stable at 28 °C for the most part of the study, although it increased slightly during spells of warmer weather. 3.2. Bacterial growth and biofilm formation A biofilm which had a heterogeneous structure formed on galvanized steel coupons and Fig. 2 illustrates the presence of bacteria cells on the galvanized steel surfaces after 11 days and 10 months. In addition, quantitative biofilm amount increased with time (P < 0.01) (Fig. 3). Growth curves for the planktonic and sessile aerobic heterotrophic bacterial populations were shown in Fig. 4. t-Test analysis revealed that aerobic heterotrophic bacteria counts in biofilm were higher than in bulk water during experiment (P < 0.05). It was determined that there was an inverse correlation between HPC in biofilm and bulk water (P < 0.05). The results for the planktonic and sessile anaerobic heterotrophic bacteria counts were presented in Fig. 5. t-Test analysis showed that anaerobic heterotrophic bacteria counts in biofilm were higher than in bulk water during the experiment (P < 0.05) and the cell concentrations of planktonic and sessile bacteria decreased after October. In addition, a positive correlation was detected between anaerobic HPC in biofilm and bulk water (P < 0.05). SRB counts in biofilm and bulk water were shown in Fig. 6. The SRB counts in biofilm increased to a maximum of 5.7 107 ± 2.9 107 cells/cm2 until July, then decreased slowly and reached plateau phase thereafter. t-Test analysis showed that SRB counts in biofilm were higher than in bulk water during the experiment (P < 0.001). It was established that there was a positive correlation between the counts of SRB in biofilm and bulk water (P < 0.01). Also a positive correlation was detected between the SRB counts and anaerobic HPC in biofilm (P < 0.01). 3.3. Carbohydrate analysis It was shown that extracellular carbohydrate was degraded in biofilm including mixed species. The concentration of total carbohydrate on the test coupons increased during first three months and reached 85.40 ± 3.10 lg/cm2 after June. After July and August, the concentration of total carbohydrate on the coupons decreased from 85.40 ± 3.10 lg/cm2 to 67.3 0 ± 3.39 lg/cm2 and from Table 1 Physico-chemical parameters of the recirculating water in the model system. Parameters
Unit
Mean values* ± SD (n = 20)
Temperature pH Conductivity Total dissolved solids Total alkalinity Dissolved oxygen Chloride Sulphate Ortophosphate Hydrogen sulphide
°C pH units lS/cm mg/L mg/L mg/L mg/L mg/L lg/L mg/L
28.5 ± 1.5 (27.0–30.0) 8.1 ± 0.4 (7.2–8.5) 587 ± 85 (382–724) 405 ± 59 (263–500) 101 ± 29 (18–125) 7.3 ± 0.7 (6.5–9.1) 5.3 ± 0.7 (3.8–6.4) 96.8 ± 18.7 (52.3–117.3) 3.9 ± 3.5 (<3–12.9) 6.4 ± 2.7 (3.0–12.1)
±, Standard deviation (SD). n, number of measurements. * Values given in the parenthesis are the minimum and maximum observed during the course of this study.
164
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Fig. 2. FESEM micrographs of the biofilm formed on the galvanized steel surfaces; (A and B) after 11 days; (C, D, E and F) after 10 months. (A) Bar = 1 lm. (B) Bar = 1 lm. (C) Bar = 10 lm. (D) Bar = 10 lm. (E) Bar = 1 lm. (F) Bar = 1 lm.
67.30 ± 3.39 lg/cm2 to 61.51 ± 3.78 lg/cm2, respectively. A maximum carbohydrate concentration of 85.7 ± 4.31 lg/cm2 was detected at September and, then it showed decreasing and increasing up to January, respectively (Fig. 7). Quantities of carbohydrate detected in biofilm appeared to be positively correlated with the weight loss of test coupons (P < 0.05) (Fig. 7). 3.4. Weight loss measurement The results of the weight loss study showed that microorganisms caused a mean increase in weight loss compared to the control. The variation in weight loss and the corrosion rate observed for the galvanized steel were shown in Table 2. The weight loss of the test cou-
pons increased with time (P < 0.01) and the maximum value determined was 7.23 ± 0.22 mg/cm2 after 10 months exposure. We detected that the weight loss of test coupons was statistically higher than the control coupons (P 6 0.001) and positively correlated with the number of sessile SRB cells and carbohydrate amount (respectively, P < 0.05 and P < 0.05) (Fig. 7). Corrosion rates of the test and control coupons were seen to decrease with time (respectively, P < 0.01 and P < 0.01). The difference in corrosion rates between test and control coupons was significant (P < 0.001) (Table 2). 3.5. FESEM and EDS analysis Fig. 8(A) provides the EDS of galvanized steel control coupon exposed to sterile tap water for 10 months wherein, Fe and S peaks
165
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
6
Absorbance (OD 562 )
5 4 3 2 1
Ja nu ar y
em be r
be r
De c
No ve m
O ct ob er
be r Se pt em
Au gu st
Ju ly
Ju ne
M ay
Ap ril
0
Months Fig. 3. Qualitative biofilm amounts during the experiment. Error bars represent the standard deviation.
Bulk water (CFU/ml)
Biofilm (cells/cm2)
8 7
HPC log 10
6 5 4 3 2 1
ry Ja n
ua
be em ec
D
N
O
Se
r
r ov em
ob ct
em
be
er
r be
t
y
us
pt
Au g
Ju l
e Ju n
ay M
il Ap r
M
ar ch
0
Months Fig. 4. Aerobic heterotrophic bacteria counts in bulk water and biofilm formed on the galvanized steel surfaces during 10 months. Error bars represent the standard deviation. CFU, colony forming unit; HPC, heterotrophic plate count.
Bulk water (CFU/ml)
Biofilm (CFU/cm2)
5.5 5
Anaerobic HPC log 10
4.5 4 3.5 3 2.5 2 1.5 1 0.5
ry
r be em
D ec
Ja nu a
r be ov em N
O ct ob er
be r pt em
t Se
Au gu s
Ju ly
e Ju n
ay M
il Ap r
M
ar
ch
0
Months Fig. 5. Anaerobic heterotrophic bacteria counts in bulk water and biofilm formed on the galvanized steel surfaces during 10 months. Error bars represent the standard deviation. CFU, colony forming unit; HPC, heterotrophic plate count.
166
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Bulk water (cells/ml)
Biofilm (cells/cm2)
9 8 7
SRB log 10
6 5 4 3 2 1
r ov em be r D ec em be r Ja nu ar y
ob e
r
N
Se
O
ct
t
em be pt
y
Au gu s
Ju l
e Ju n
ay M
il Ap r
M
ar ch
0
Months Fig. 6. SRB counts in bulk water and biofilm formed on the galvanized steel surfaces during 10 months. Error bars represent the standard deviation.
Weight loss 8
80
7
70
6
60
5
50
2
4
40 3 30 2
20
1
10
0 ar y Ja
nu
r
r
em be De c
be No ve m
O ct ob er
r Se
pt
em
be
Au gu st
Ju ly
Ju ne
M
ay
0 Ap ril
Weight loss (mg/cm )
2
Carbohydrate amount (μ g/cm )
Carbohydrate 90
Months Fig. 7. Quantities of carbohydrate detected in biofilm. Error bars represent the standard deviation.
Table 2 Weight loss and average corrosion rates of test and control galvanized steel coupons during 10 months. Months
Weight loss (mg/cm2) ± SD, test
Weight loss (mg/cm2) ± SD, control
Corrosion rate, test (mg/dm2 day)
Corrosion rate, control (mg/dm2 day)
Ratio (test/control)
April May June July August September October November December January
1.08 ± 0.06 2.23 ± 0.10 3.10 ± 0.20 3.39 ± 0.30 3.78 ± 0.17 4.31 ± 0.28 5.10 ± 0.18 5.85 ± 0.22 6.41 ± 0.10 7.23 ± 0.22
1.03 ± 0.05 1.15 ± 0.02 1.51 ± 0.01 1.61 ± 0.01 1.77 ± 0.15 1.82 ± 0.02 1.87 ± 0.03 1.89 ± 0.01 1.95 ± 0.09 2.06 ± 0.11
3.61 3.68 3.42 3.07 2.59 2.49 2.47 2.48 2.40 2.41
3.50 1.91 1.68 1.39 1.18 1.01 0.86 0.76 0.72 0.68
1.03 1.93 2.04 2.21 2.19 2.47 2.87 3.26 3.33 3.54
±, Standard deviations (SD).
were not observed. Fig. 8(B and C) shows the EDS profiles of the galvanized steel coupons exposed to bulk water in the model cooling system for 10 months and 11 days, respectively. Peaks for Ca, Zn, Fe, O and Si were observed. However S peaks were not determined.
After the biofilm and corrosion products were removed from the surface of the coupons, depletion of zinc in some regions of the test coupons and the appearance of steel were noted. Pitting corrosion of the steel was also observed (Fig. 9(B)). However we
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
167
Fig. 8. EDS analysis of the corrosion products formed on the galvanized steel surfaces; (A) the control coupon exposed to sterile tap water for 10 months; (B) the test coupon exposed to bulk water in the model recirculating water system for 10 months; (C) the test coupon exposed to bulk water in model recirculating water system for 11 days. (A– C) bar = 5 mm.
did not observe any pitting or depletion of zinc of the control coupons (Fig. 9(A)). 3.6. Iron and zinc measurement Table 3 shows the amounts of dissolved iron found in the bulk water, biofilm and EPS obtained from the model system. The maximum concentration (0.26 ± 0.02 lg/ml) of iron in the bulk water was measured at the beginning of experiment (March)
and then some variations in iron amount were observed. The amount of iron in the biofilm showed an increasing and decreasing one after the other, and a maximum iron concentration of 1.39 ± 0.05 lg/cm2 was detected at December. The maximum concentration of iron in the EPS was measured as 0.73 ± 0.03 lg/mL at April. The iron amount in biofilm was found statistically higher than in EPS and bulk water (P < 0.001). The iron concentrations in biofilm, EPS and bulk water were not correlated to each other.
168
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Fig. 9. SEM showing the surfaces of test coupons after biofilm and corrosion products of galvanized steel coupon in the model recirculating water system were removed after 5 months (A and B) and 10 months (C and D). Labelled arrows indicate exposed steel surface and corrosion pits. Bar = 10 lm, 100 nm, 10 lm and 10 lm, respectively. (E) The metal surface of control galvanized steel coupon in the sterile control system after 10 months, bar = 10 lm.
Table 3 The amounts of dissolved iron and zinc found in the bulk water, biofilm and EPS obtained from the model system during experiments. Months
March April May June July August September October November December January
Fe concentrations
Zn concentrations
Bulk water (lg/ml)
Biofilm (lg/cm2)
EPS (lg/cm2)
Bulk water (lg/ml)
Biofilm (lg/cm2)
EPS (lg/cm2)
0.26 ± 0.02 0.15 ± 0.02 0.07 ± 0.01 0.06 ± 0.01 0.04 ± 0.01 0.21 ± 0.02 0.07 ± 0.01 0.07 ± 0.01 0.03 ± 0.01 0.11 ± 0.01 0.14 ± 0.01
– 0.90 ± 0.05 0.67 ± 0.05 1.06 ± 0.03 0.39 ± 0.02 0.98 ± 0.02 0.59 ± 0.03 1.24 ± 0.07 0.46 ± 0.08 1.39 ± 0.05 0.48 ± 0.01
– 0.73 ± 0.03 0.28 ± 0.02 0.51 ± 0.05 0.35 ± 0.04 0.10 ± 0.01 0.32 ± 0.06 0.54 ± 0.04 0.33 ± 0.02 0.24 ± 0.03 0.69 ± 0.02
0.79 ± 0.01 1.03 ± 0.02 3.61 ± 0.01 1.95 ± 0.03 1.32 ± 0.01 2.15 ± 0.01 1.45 ± 0.01 1.42 ± 0.01 7.41 ± 0.05 0.47 ± 0.01 2.02 ± 0.01
– 162.96 ± 1.88 770.46 ± 2.18 1089.70 ± 6.10 888.60 ± 10.03 476.52 ± 10.52 902.56 ± 10.67 1121.33 ± 2.07 1124.03 ± 6.40 1166.77 ± 2.48 921.07 ± 9.65
–
±, Standard deviations (SD).
1.03 ± 0.69 7.81 ± 0.04 13.83 ± 0.18 12.46 ± 0.53 8.78 ± 0.42 3.42 ± 0.25 12.39 ± 0.33 3.70 ± 0.38 8.48 ± 0.86 5.29 ± 0.31
169
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Film layer
Control water
0.8
2.5
0.7 2
0.5
1.5 2
Fe (µg/cm )
Fe (µg/ml)
0.6
0.4 1
0.3 0.2
0.5 0.1
O
pt
ct
ob er N ov em be r D ec em be r Ja nu ar y
r be
st
em
Au gu
Se
Ju ly
Ju ne
M ay
il
0
Ap r
M ar ch
0
Months Fig. 10. Temporal changes in the amounts of iron in the water and film layers obtained from the control system. Error bars represent the standard deviation.
Fig. 10 represents the concentrations of iron found in the water and film layer formed on galvanized steel obtained from the control system. The concentration of iron in the control water and film layer decreased with time (respectively, P < 0.05 and P < 0.01). We detected a positive significant correlation between iron concentrations in the control water and film layer (P < 0.01). The maximum zinc concentrations in biofilm, EPS and bulk water were measured as 1166.77 ± 2.48 lg/cm2 at December, 13.83 ± 0.18 lg/cm2 at June and 7.41 ± 0.05 lg/mL at November, respectively (Table 3). The zinc amount in biofilm was found to be statistically higher than in the EPS and bulk water (P < 0.001). We could not detect any significant correlation between the zinc concentrations in biofilm, EPS and bulk water. However the concentrations of zinc in the biofilm showed significant correlations with weight loss, carbohydrate amount and SRB count in biofilm (respectively, P < 0.05, P < 0.01 and P < 0.01).
Fig. 11 shows the concentrations of zinc found in the water and film layer formed on galvanized steel obtained from the control system. The concentrations of zinc in the water and film layer reached peak values 93.2 ± 0.33 lg/mL and 549.14 ± 8.15 lg/mL at June, respectively, and then decreased and reached 3.43 ± 0.03 lg/mL at October and 117.58 ± 11.22 lg/mL at July, respectively, and then remained constant. The concentration of zinc in the control water showed a significant correlation within the film (P < 0.05). 4. Discussion In this work, we have developed a laboratory model system to study the effects of mixed species biofilm including SRB on the corrosion of galvanized steel. Although the model system was fed with tap water, the recirculating water had different water charac-
Film layer
Control water
100
600
90 500
80
400
60 50
300
40
Zn ( Ξ g/cm 2 )
Zn ( Ξ g/ml)
70
200
30 20
100
10
ua
ry
r be em ec
Ja n
r be D
ov em N
ob ct O
em be pt Se
er
r
t us Au g
y Ju l
e Ju n
M ay
Ap r
M
il
0 ar ch
0
Months Fig. 11. Temporal changes in the amounts of zinc in the water and film layers obtained from the control system. Error bars represent the standard deviation.
170
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
teristics than distributed tap water. The physico-chemical parameters of model system water were appropriate for the cooling water criteria. Even under a regular blowdown regime (3.5 L fresh water, daily), conductivity, TDS and sulphates which make the recirculating water more aggressive were increased with time. It could be resulted from circulation, heat and evaporation. Following previous field observations the water temperature was kept constant at 28 °C [29], during the study to eliminate the influence of temperature on the corrosion of galvanized steel. Our findings suggest that the planktonic bacteria cells including aerobic heterotrophic bacteria, anaerobic heterotrophic bacteria and SRB attached and formed mixed species biofilm on the galvanized steel surfaces, even though zinc is toxic to a variety of microorganisms [12,13,30,31]. The density of biofilm increased with ageing as shown by quantitative biofilm measurement. FESEM analysis validated the adhesion of bacteria cells on the galvanized steel surfaces. We found out that the growth rates both of the heterotrophic bacteria and SRB cells in the biofilm were greater than that of the planktonic cells. Beech et al. [32] reported the same result for SRB. Also Ellwood et al. [33] reported a similar result with respect to population increase of adhered cells. We determined an inverse correlation between HPC in the biofilm and bulk water in contrast to Kerr et al. [34] suggesting that the difference in environmental parameters can affect the rate of attachment and multiplication of heterotrophs. During the phase of biofilm development on galvanized steel, the increase in numbers of SRB and anaerobic heterotrophic bacteria in biofilm showed very high positive correlations with the numbers of them in the recirculating water. These indicated that anaerobic biofilm bacteria including SRB and heterotrophs were shedding continuously into the water phase and had not been able to establish themselves in the already developed biofilms. Similar results for SRB were reported by Power et al. [35,36]. Our results from the present study show that galvanized steel could be corroded by microorganisms as well as SRB in the mixed species biofilm. Corrosion rate of the test coupons was decreased with time, just as found by Ilhan-Sungur et al. [10]. However, although the results of corrosion rates reported by Ilhan-Sungur et al. [10] were generally higher than our study, the values of corrosion rate ratios (test/control) in our investigation were higher than reported by Ilhan-Sungur et al. [10] and increased with time. In the natural conditions, various biotic (EPS, quorum sensing, etc.) and abiotic (pH, temperature, etc.) factors can influence the corrosion course [37–39]. The weight loss of galvanized steel was correlated with the number of sessile SRB cells. However, since our study was not carried out with pure culture of SRB, and also various biotic and abiotic factors could influence the weight loss, we cannot say that weight loss of galvanized steel increases with SRB counts. IlhanSungur et al. [10], Beech et al. [32] and Angell and Urbanic [8] suggested that there was no correlation between numbers of SRB and corrosion rates or initiation of pitting. We detected that extracellular carbohydrate was degraded by mixed species of bacteria in biofilm. Zhang et al. [25] reported that mixed cultures degraded their own EPS material when they were in a starved state. Quantities of carbohydrate detected in biofilm appeared to be positively correlated with the weight loss and zinc concentration in the biofilm. It was reported that EPS can complex metal ions and, thus, affect the corrosion [40]. EDS analysis of the test galvanized steel coupons determined Fe and O in corrosion products. It was proposed that the corrosion products caused by SRB for iron will be either iron sulphides or phosphides which initiated a general type of corrosion or the combination of both [41]. However S peaks were not observed. Santegoeds et al. [42] showed that sulphide did not diffuse out of the
biofilm. Therefore, since EDS analyzer can enter maximum 1 lm within the biofilm, S peaks could not have been determined. The amount of iron in the biofilm showed an increasing and decreasing one after the other. Since all bacteria require metal ions for their growth, iron could be biosorbed by microorganisms such as SRB. Fe2+ is known as a relevant factor for SRBs which probably account for the biosynthesis of the iron-cytochromes in the respiratory chain [21]. Marchal et al. [39] showed that Fe2+ concentrations influenced growth of SRB and growth performances were significantly lower using a culture medium free of Fe2+. Other possibility is that the outer part of biofilm including iron could be detached and re-entered the bulk water. Although regular blowdown was carried out, sometimes an increase in the amount of iron in the bulk water was observed. This indicates that zinc was depleted and iron appeared was corroded. The concentrations of zinc in the biofilm showed significant correlations with weight loss and SRB count in the biofilm. It has been known that Zn is toxic and inhibitory to SRB [12,13]. Poulson et al. [13] and Ilhan-Sungur et al. [10] reported that a concentration of 13 lg/mL Zn and of 22.36 ± 0.007 lg/mL Zn are toxic to pure strains of Desulfovibrio desulfuricans and Desulfovibrio sp., respectively. We found, however, that SRB can survive in the mixed species biofilm with very high Zn concentrations (min 162.96 ± 1.88 max 1166.77 ± 2.48 lg/cm2). It appears that the effect of Zn on SRB vary depending on the type of SRB and their growth conditions. The natural environment may also influence bacterial metabolic activities and resistance to toxic agents. In addition we can say that toxicity studies carried out under laboratory conditions cannot mirror the facts. On the other hand the measurement of the dissolved metal concentration can serve as an indicator of the bioactivity of the SRB [12,13]. This is a first report on corrosion of galvanized steel by microorganisms as well as SRB in mixed species biofilm formed on galvanized steel in a simulated recirculating cooling tower system. No chemicals (disinfectant, pH regulators or anti-scaling agents) were added to the system in order to exclude their possible negative effects (such as their disinfecting effect) on the microorganisms and biofilm formation. However, microbiologically influenced corrosion of galvanized steel in a real cooling tower with biocide and corrosion inhibitory are currently under investigation.
5. Conclusion The following conclusions can be drawn from this study: (1) FESEM analysis showed that planktonic bacteria cells attached and formed the mixed species biofilm on the galvanized steel surfaces, even though zinc is toxic to a variety of microorganisms. (2) It is suggested from the present study that anaerobic biofilm bacteria including SRB and heterotrophs were shedding continuously into the water phase and had not been able to establish themselves in the already developed biofilms. (3) Our study showed that galvanized steel was corroded by microorganisms as well as SRB in the mixed species biofilm. (4) The weight loss of galvanized steel was correlated with the number of sessile SRB cells (P < 0.05). However, since our study was not carried out with pure culture of SRB, and also various biotic and abiotic factors could influence the weight loss, we cannot say that weight loss of galvanized steel increases with SRB counts. (5) Extracellular carbohydrate could be degraded by bacteria in biofilm including mixed species. The amount of total carbohydrate detected in the biofilm appeared to be positively correlated with the weight loss (P < 0.05). This suggested
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
that there was a direct correlation between corrosion of galvanized steel and carbohydrate amount. (6) We found, however, that SRB can survive in the mixed species biofilm with very high Zn concentrations (min 162.96 ± 1.88 max 1166.77 ± 2.48 lg/cm2). It appears that the effect of Zn on SRB vary depending on the type of SRB and their growth conditions. Also we can say that toxicity studies carried out under laboratory conditions cannot mirror the external reality. Acknowledgements The authors are grateful to Prof. Nurhan Cansever for fruitful discussions during the preparation of this paper. Msc. Bihter Minnosß and Dr. Nihal Dog˘ruöz are gratefully acknowledged. The model system was donated by Dizayn Teknik Plastic Pipes & Fittings Co. This study was supported by the Research Fund of Istanbul University (Project No: T-528/21102004). References [1] S.G. Choudhary, Emerging microbial control issues in cooling water systems, Systems Hydrocarb. Process. 77 (5) (1998) 91–102. [2] K. Lewis, Riddle of biofilm resistance, Antimicrob. Agents Chemother. 45 (2001) 999–1007. [3] W.A. Hamilton, Microbially influenced corrosion as a model system for the study of metal microbe interactions: a unifying electron transfer hypothesis, Biofouling 19 (1) (2003) 65–76. [4] W.A. Hamilton, Sulphate reducing bacteria and anaerobic corrosion, Ann. Rev. Microbiol. 39 (1985) 195–217. [5] A.K. Lee, M.G. Buehler, D.K. Newman, Influence of a dual-species biofilm on the corrosion of mild steel, Corros. Sci. 48 (1) (2006) 165–178. [6] D. Çetin, S. Bilgiç, S. Dönmez, G. Dönmez, Determination of biocorrosion of low alloy steel by sulfate-reducing Desulfotomaculum sp. isolated from crude oil field, Mater. Corros. 58 (11) (2007) 841–847. [7] T.S. Rao, T.N. Sairam, B. Viswanathan, K.V.K. Nair, Carbon steel corrosion by iron oxidising and sulphate reducing bacteria in a freshwater cooling system, Corros. Sci. 42 (8) (2000) 1417–1431. [8] P. Angell, K. Urbanic, Sulphate-reducing bacterial activity as a parameter to predict localized corrosion of stainless alloys, Corros. Sci. 42 (2000) 897–912. [9] D. Çetin, M.L. Aksu, Corrosion behavior of low-alloy steel in the presence of Desulfotomaculum sp., Corros. Sci. 51 (2009) 1584–1588. [10] E. Ilhan-Sungur, N. Cansever, A. Çotuk, Microbial corrosion of galvanized steel by a freshwater strain of sulphate reducing bacteria (Desulfovibrio sp.), Corros. Sci. 49 (2007) 1097–1109. [11] E. Ilhan-Sungur, A. Cotuk, Characterization of sulfate reducing bacteria isolated from cooling towers, Environ. Monit. Assess. 104 (2005) 211–219. [12] V.P. Utgikar, H.H. Tabak, J.R. Haines, R. Govind, Quantification of toxic and inhibitory impact of copper and zinc on mixed cultures of sulfate-reducing bacteria, Biotechnol. Bioeng. 82 (3) (2003) 306–312. [13] S.R. Poulson, P.J.S. Colberg, J.I. Drever, Toxicity of heavy metals (Ni, Zn) to Desulfovibrio desulfuricans, Geomicrobiol. J. 14 (1997) 41–49. [14] I. Turetgen, Comparison of free residual chlorine and monochloramine for efficacy against biofilms in model and full scale cooling towers, Biofouling 20 (2004) 81–85. [15] American Society for Testing and Material, Standard recommended practice for preparing, cleaning and evaluating corrosion test specimens, in: Annual Book of ASTM standards, Designation: G1-72, American Society for Testing Materials, Philadelphia, 1975, pp. 626–629. [16] APHA, Standards methods for the examination of water and wastewater, American Public Health Association, Washington, DC, 0-87553-091-5, 1981. [17] G. Bianucci, E.R. Bianucci, L’analisi chimica delle acque naturali ed inquinate, Hoepli, Milano 88203 (1987) 1987.
171
[18] C.E. Boyd, C.S. Tucker, Water quality and pond soil analyses for aquaculture, Alabama Agricultural Experiment Station, Auburn University, Alabama, 9780817307219, 1992. [19] G.A. Gagnon, R.M. Slawson, An efficient biofilm removal method for bacterial cells exposed to drinking water, J. Microbiol. Met. 34 (1999) 203–214. [20] E. Sungur, B. Minnos, N. Dogruoz, Isolation of aerobic heterotrophic and anaerobic sulphate reducing bacteria from model water system by filtration method, IUFS J. Biol. 67 (1) (2008) 33–38. [21] J.R. Postgate, The Sulphate Reducing Bacteria, 2nd ed., Cambridge University Press, Cambridge, Great Britain, 1984. [22] The Institute of Petroleum, Determination of the viable microbial content of fuels and fuel components boiling below 390 °C – Filtration and Culture Method, 1995, IP Method Number 385/95. [23] D.J. Reasoner, E.E. Geldrich, A new medium for the enumeration and subculture of bacteria from potable water, Appl. Environ. Microbiol. 49 (1985) 1–7. [24] G.A. O’toole, R. Kolter, Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis, Mol. Microbiol. 28 (3) (1998) 449–461. [25] X. Zhang, P.L. Bishop, B.K. Kinkle, Comparison of extraction methods for quantifying extracellular polymers in biofilms, Water Sci. Tech. 39 (7) (1999) 211–218. [26] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [27] Y.M. Nelson, W. Lo, L.W. Lion, M.L. Shuler, W.C. Ghirorse, Lead distribution in a simulated aquatic environment: effects of bacterial biofilms and iron oxide, Water Res. 29 (1995) 1934–1944. [28] C. Campanac, L. Pineau, A. Payard, G. Baziard-Mouysset, C. Roques, Interactions between biocide cationic agents and bacterial biofilms, Antimicrob. Agents Chemother. 46 (5) (2002) 1469–1474. [29] I. Turetgen, E. Ilhan-Sungur, A. Cotuk, Enumeration of Legionella pneumophila in cooling tower water systems, Environ. Monit. Assess. 100 (1–3) (2005) 53– 58. [30] G.M. Gadd, A.J. Griffiths, Microorganisms and heavy metal toxicity, Microbiol. Ecol. 4 (1978) 303–317. [31] I. Cohen, R. Bitan, Y. Nitzan, The effect of zinc and cadmium ions on Escherichia coli, Microbios 68 (1991) 157–168. [32] I.B. Beech, C.W. Sunny Cheung, C.S. Patrick Chan, M.A. Hill, R. Franco, A.R. Lino, Study of parameters implicated in the biodeterioration of mild steel in the presence of different species of sulphate-reducing bacteria, Int. Biodeter. Biodegr. 34 (1994) 289–303. [33] D.C. Ellwood, C.W. Keevil, P.D. Marsh, C.M. Brown, J.N. Wardell, Surfaceassociated growth, Philos. T. Roy. Soc. B 297 (1982) 517–532. [34] C.J. Kerr, K.S. Osborn, G.D. Robson, P.S. Handley, The relationship between pipe material and biofilm formation in a laboratory model system, J. Appl. Microbiol. 85 (1999) 29–38. [35] M.E. Power, J.R. Van Der Meer, H. Harms, O. Wanner, Colonization of aerobic biofilms by sulfate-reducing bacteria, Biofouling 17 (2001) 275–288. [36] M.E. Power, J.C. Araujo, J.R. Van Der Meer, H. Harms, O. Wanner, Monitoring sulfate-reducing bacteria in heterotrophic biofilms, Water Sci. Technol. 39 (1999) 49–56. [37] I.B. Beech, C.C. Gaylarde, Microbial polysaccharides and corrosion, Int. Biodeter. 27 (1991) 95–107. [38] C. Peng, J.K. Park, R.W. Patenaude, Statistics-based classification of microbially influenced corrosion in freshwater systems, Water Res. 28 (4) (1994) 951–959. [39] R. Marchal, B. Chaussepied, M. Warzywoda, Effect of ferrous ion availability on growth of a corroding sulfate-reducing bacterium, Int. Biodeter. Biodegr. 47 (2001) 125–131. [40] I.B. Beech, C.W. Sunny Cheung, Interactions of exopolymers produced by sulphate-reducing bacteria with metal ions, Int. Biodeter. Biodegr. 35 (1–3) (1995) 59–72. [41] W.P. Iverson, Mechanism of anaerobic corrosion of steel by sulphate reducing bacteria, Mater. Perform. 23 (3) (1984) 28–30. [42] C.M. Santegoeds, T.G. Ferdelman, G. Muyzer, De Beer, Structural and functional dynamics of sulfate-reducing populations in bacterial biofilms, Appl. Environ. Microbiol. 64 (1998) 3731–3739.
Contents lists available at ScienceDirect
Corrosion Science journal homepage: www.elsevier.com/locate/corsci
Microbial corrosion of galvanized steel in a simulated recirculating cooling tower system Esra Ilhan-Sungur *, Aysßın Çotuk Istanbul University, Faculty of Science, Department of Biology, 34134 Vezneciler, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 26 September 2008 Accepted 26 August 2009 Available online 31 August 2009 Keywords: A. Zinc B. SEM C. Microbiological corrosion
a b s t r a c t In this study, mixed species biofilm formation including sulphate reducing bacteria (SRB) on the galvanized steel surfaces and also microbiologically influenced corrosion (MIC) of galvanized steel were observed in a model recirculating cooling water system during 10 months. A biofilm which had a heterogeneous structure formed on galvanized steel coupons. The results suggested that galvanized steel was corroded by microorganisms as well as SRB in the biofilm. Extracellular carbohydrate was degraded and quantities of carbohydrate were positively correlated with the weight loss. The concentrations of zinc in the biofilm showed significant correlations with weight loss, carbohydrate amount and SRB count. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Cooling towers are an integral part of any power plant. The conditions of cooling towers are very suitable for microbial growth and biofilm formation [1]. A biofilm is a population of cells embedded in a thick mucilaginous matrix of extracellular polymeric substances (EPS) which may consist of 90% or more of polysaccharides [2]. Microbial biofilm and corrosion in cooling systems are the most common problems that damage expensive equipment, cause loss of production and increase maintenance costs [1,3]. Sulphate reducing bacteria were considered as the major bacterial group involved in microbiologically influenced corrosion [4]. Although numerous experiments are now available demonstrating microbiologically influenced corrosion of several metals under laboratory and in situ conditions [5–9], there are only one study about MIC of galvanized steel by SRB carried out in laboratory conditions [10]. However, to date we have not been able to find any published reports on the MIC of galvanized steel in a cooling water system. Galvanized steel is frequently used in the construction of cooling towers and water containers, owing to its good resistance to corrosion and biofauling. In spite of the well-known anticorrosive and antifouling properties of galvanized steel, Ilhan-Sungur and Cotuk [11] reported high SRB counts (104 cells/ml) in cooling tower water and structural failures in some cooling towers because of pitting corrosion. Even though numerous experiments are now available demonstrating that zinc is toxic to SRB strains [12,13], in our previous study [10], we showed microbiologically influ-
* Corresponding author. Tel.: +90 212 455 57 00; fax: +90 212 528 05 27. E-mail address: [email protected] (E. Ilhan-Sungur). 0010-938X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.corsci.2009.08.049
enced corrosion of the galvanized steel by SRB in closed laboratory conditions. However we wondered whether in a cooling tower which has an open environment and potable water, by contrast, mixed species biofilm including SRB would form on the galvanized steel surfaces and microbiologically influenced corrosion of galvanized steel would occur. For these purposes, in this study, we have developed a model recirculating cooling tower system capable of developing reproducible steady-state bacterial biofilms in nonsupplemented potable water. We believe that it provides a realistic representation of the conditions and organisms that we wanted to study. Turetgen [14] compared the biofilm reproducibility of the surfaces taken from a similar model system with the full-scale cooling tower system and reported that there was no significant differences between HPC counts and reproducibility from tested coupons in the full-scale and model system. The aims of this study were to observe the mixed species biofilm formation including SRB and anaerobic heterotrophic bacteria on the galvanized steel surfaces and microbiologically influenced corrosion of galvanized steel in a model recirculating cooling water system during 10 months and, also establish the effects of biological parameters on MIC. 2. Experimental procedure 2.1. Test material Galvanized steel for use in cooling tower construction was used as test material containing 0.027% C, 0.148% Mn, 0.011% Si, 0.01% P, 0.059% Al, 0.008% S and 99.737% Zn. The thickness of the zinc coating covering the stainless steel was 5 lm. The coupons (50 25 0.5 mm) were prepared according to guidelines in ASTM
162
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
G1–72 [15]. They were weighed and the total surface area of each coupon was determined. The cut areas of all the coupons were coated with epoxy zinc phosphate primer (Moravia, Turkey) (grey) and then covered with epoxy finish coating (Moravia, Turkey) (black) to avoid the initiation of corrosion at these disturbed areas [10].
2.2. Model system The experimental study was performed using a 100-L polypropylene laboratory scale cooling tower model system running with 80 L bulk water under constant hydraulic conditions, which simulated the situation in a cooling tower. It was equipped with a recirculation pump (550 W, 40 L min1, Pedrollo, Italy) in the basin and a heater (AT-100, 100 W, Atman, Germany) to facilitate evaporation within the bulk water. Cover lid had openings to ensure fresh air and daylight entry (Fig. 1). A supply of potable water was used to replenish the water lost by evaporation and blowdown (partial draining). Throughout the experiment, the water temperature was kept constant at 28 °C. Coupons were inserted vertically into coupon–holders situated in the water basins (Fig. 1). No chemicals (disinfectant, pH regulators or anti-scaling agents) were added to the system in order to exclude their possible negative effects (such as their disinfecting effect) on the microorganisms and biofilm formation. The water samples were analyzed monthly for various physico-chemical parameters such as pH, conductivity (WTW LF 95 Conductivity meter, Germany), total dissolved solids (TDS), alkalinity, sulphates (SO42), orthophosphates (PO42), chlorides (Cl), hydrogen sulphides (H2S) and dissolved oxygen [16–18]. Control systems containing sterile potable water with a flow rate 7.3 L h1 and with 40 galvanized steel coupons were run simultaneously with the model system. Gentamicin (50 lg/L) (Fortigen, Turkey) was added to the control system to prevent contamination. Ten coupons were removed from the model system monthly during 10 months for enumeration of SRB and heterotrophic bacteria, quantification of biofilm formation, corrosion rate measurement, EPS extraction, and carbohydrate, iron and zinc analysis. In addition for field emission scanning electron microscopy (FESEM) and energy dispersive X-ray analysis (EDS) observation, two coupons were removed from model system after 11 days and 10 months.
2.3. Bacterial analysis To estimate the number of bacteria in biofilm (sessile bacteria), three coupons were removed monthly from the basin, dip-rinsed in sterile potable water to remove unattached cells. Biofilm was suspended in 10 ml sterile potable water [19]. The resulting suspension was then serially diluted from 101 to 1010. To show the count of bacteria in bulk water (planktonic bacteria), bulk water samples were concentrated by filtration through a 0.22 lm pore size polyamide filter (Sartolon, Sartorius AG, Goettingen, Germany), and then membrane filters were resuspended in 50 ml sterile tap water by stomacher (IUL Instruments) for 2 min [20]. The suspensions were used as inoculum for isolation and enumeration of SRB and heterotrophic bacteria. Sessile and planktonic SRB counts were determined by the most probable number (MPN) technique using Postgate’s medium B with the following composition: C3H5O3Na (3.5 g/L), KH2PO4 (0.5 g/L), NH4Cl (1.0 g/L), Ca2SO4 (1.0 g/L), MgSO47H2O (2.0 g/L), yeast extract (1.0 g/L), C6H7O6Na (0.1 g/L), C2H3O2SNa, (0.1 g/L), FeSO47H2O (0.5 g/L), C6H5O7Na3 (0.3 g/L) (pH adjusted to 7.2 by 10% NaOH) [21,22]. MPN tubes were incubated in the dark at 30 °C for 3 months. In each inoculated tube, growth of sulphate reducers was indicated by the formation of a black FeS precipitate and by turbidity. For aerobic heterotrophic plate count (HPC), samples of 100 lL from dilution series of biofilm homogenates and filtered water suspensions were spread-plated in triplicate onto R2A agar (Oxoid, UK) plates and incubated at 28 °C for 10 days [23]. After the incubation time, the number of colonies was enumerated under a colony counter (WTW, model BZG 30). For enumeration of anaerobic heterotrophic bacteria, Thioglycollate medium (Oxoid, UK) was used and the plates were incubated at 30 °C for 21 days. The number of colonies was enumerated under the colony counter. Anaerobic conditions were created using the ‘‘AnaeroGen’’ systems (Oxoid, UK).
2.4. Quantification of biofilm formation The quantification of biofilm formation was carried out according to the method of O’Toole and Kolter [24]. Two coupons were air dried and surface-attached cells were stained for 15 min with a
Fig. 1. Schematic diagram of the model recirculating water system. Arrows indicate the flow direction.
163
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
0.5% (wt/vol) solution of crystal violet (Merck, Germany). After rinsing with dH2O, bound dye was solubilized in 95% ethanol. Adherent cells were quantified by determining the absorbance of the solution at 562 nm with spectrophotometer (Shimadzu UV 150-02). 2.5. EPS extraction and carbohydrate analysis Three biofilm coated coupons were removed from the model system monthly and the recovery of EPS was performed according to the method of Zhang et al. [25]. The carbohydrate content of EPS was measured using the phenol/sulphuric-acid method. Standard aqueous solutions of glucose (10–100 mg/L) were used for instrument calibration [26]. 2.6. Weight loss measurement Eight coupons used for the other analyses were cleaned as ASTM G1–72 and weighed after drying [15]. The difference between the initial and final weight was reported as weight loss. The values of the corrosion rate were determined by applying the following equation proposed by the ASTM standard G1–72 [15]. 2
Corrosion rate ¼ ðK WÞ=ðA t DÞ mg=dm day: where: K = 2.40 106 D, t = time of exposure (h), A = coupon area (cm2), W = weight loss after cleaning (g), D = density (g/cm3). 2.7. Iron and zinc measurement To determine iron and zinc concentrations in the biofilm, two coupons were used. The biofilm was removed from the total surface of the one coupon exposed to the medium, suspended in distilled water and vortexed for 5 min. Then the biofilm suspension and water samples from model and control systems were treated with 3 N nitric acid (HNO3) [27]. The acidified samples (pH 3) were filtered through a 0.45 lm membrane filter (Millipore) to remove insoluble suspended particles. The filtered samples were analysed for Fe and Zn content using a Shimadzu AA-680 Flame Atomic Absorption Spectrophotometer. 2.8. FESEM and EDS analysis Corrosion products formed on coupons were analysed by field emission scanning electron microscopy at the end of the experimental duration of exposure of 11 days and 10 months. Coupons were fixed with 2.5% glutaraldehyde, followed by dehydration in a graded series of ethanol and air-drying [28]. The dried samples were coated with a palladium (15 nm) and imaged with a Jeol JSM-6335 field emission electron microscope. Chemical analysis was done by energy dispersive X-ray spectroscopy. 2.9. Statistical analysis of data Bacteria counts were log10 transformed and standard deviations (SD) of the means were calculated. Statistical evaluation of the results was carried out by Pearson product moment correlation coefficient test. One-way analysis of variance (ANOVA) was used to compare variations of Fe and Zn concentrations in biofilm, EPS and water samples followed by Tukey’s-b post hoc test using SPSS statistical package program. t-test was employed to detect statistically significant changes in the bacterial counts. Differences were considered statistically significant at P < 0.05.
3. Results 3.1. Physico-chemical characters of model system water The physico-chemical parameters of the recirculating water in the model system were presented in Table 1. Conductivity, TDS and sulphates were increased with time (respectively, P < 0.01, P < 0.01 and P < 0.05). The temperature of the system was stable at 28 °C for the most part of the study, although it increased slightly during spells of warmer weather. 3.2. Bacterial growth and biofilm formation A biofilm which had a heterogeneous structure formed on galvanized steel coupons and Fig. 2 illustrates the presence of bacteria cells on the galvanized steel surfaces after 11 days and 10 months. In addition, quantitative biofilm amount increased with time (P < 0.01) (Fig. 3). Growth curves for the planktonic and sessile aerobic heterotrophic bacterial populations were shown in Fig. 4. t-Test analysis revealed that aerobic heterotrophic bacteria counts in biofilm were higher than in bulk water during experiment (P < 0.05). It was determined that there was an inverse correlation between HPC in biofilm and bulk water (P < 0.05). The results for the planktonic and sessile anaerobic heterotrophic bacteria counts were presented in Fig. 5. t-Test analysis showed that anaerobic heterotrophic bacteria counts in biofilm were higher than in bulk water during the experiment (P < 0.05) and the cell concentrations of planktonic and sessile bacteria decreased after October. In addition, a positive correlation was detected between anaerobic HPC in biofilm and bulk water (P < 0.05). SRB counts in biofilm and bulk water were shown in Fig. 6. The SRB counts in biofilm increased to a maximum of 5.7 107 ± 2.9 107 cells/cm2 until July, then decreased slowly and reached plateau phase thereafter. t-Test analysis showed that SRB counts in biofilm were higher than in bulk water during the experiment (P < 0.001). It was established that there was a positive correlation between the counts of SRB in biofilm and bulk water (P < 0.01). Also a positive correlation was detected between the SRB counts and anaerobic HPC in biofilm (P < 0.01). 3.3. Carbohydrate analysis It was shown that extracellular carbohydrate was degraded in biofilm including mixed species. The concentration of total carbohydrate on the test coupons increased during first three months and reached 85.40 ± 3.10 lg/cm2 after June. After July and August, the concentration of total carbohydrate on the coupons decreased from 85.40 ± 3.10 lg/cm2 to 67.3 0 ± 3.39 lg/cm2 and from Table 1 Physico-chemical parameters of the recirculating water in the model system. Parameters
Unit
Mean values* ± SD (n = 20)
Temperature pH Conductivity Total dissolved solids Total alkalinity Dissolved oxygen Chloride Sulphate Ortophosphate Hydrogen sulphide
°C pH units lS/cm mg/L mg/L mg/L mg/L mg/L lg/L mg/L
28.5 ± 1.5 (27.0–30.0) 8.1 ± 0.4 (7.2–8.5) 587 ± 85 (382–724) 405 ± 59 (263–500) 101 ± 29 (18–125) 7.3 ± 0.7 (6.5–9.1) 5.3 ± 0.7 (3.8–6.4) 96.8 ± 18.7 (52.3–117.3) 3.9 ± 3.5 (<3–12.9) 6.4 ± 2.7 (3.0–12.1)
±, Standard deviation (SD). n, number of measurements. * Values given in the parenthesis are the minimum and maximum observed during the course of this study.
164
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Fig. 2. FESEM micrographs of the biofilm formed on the galvanized steel surfaces; (A and B) after 11 days; (C, D, E and F) after 10 months. (A) Bar = 1 lm. (B) Bar = 1 lm. (C) Bar = 10 lm. (D) Bar = 10 lm. (E) Bar = 1 lm. (F) Bar = 1 lm.
67.30 ± 3.39 lg/cm2 to 61.51 ± 3.78 lg/cm2, respectively. A maximum carbohydrate concentration of 85.7 ± 4.31 lg/cm2 was detected at September and, then it showed decreasing and increasing up to January, respectively (Fig. 7). Quantities of carbohydrate detected in biofilm appeared to be positively correlated with the weight loss of test coupons (P < 0.05) (Fig. 7). 3.4. Weight loss measurement The results of the weight loss study showed that microorganisms caused a mean increase in weight loss compared to the control. The variation in weight loss and the corrosion rate observed for the galvanized steel were shown in Table 2. The weight loss of the test cou-
pons increased with time (P < 0.01) and the maximum value determined was 7.23 ± 0.22 mg/cm2 after 10 months exposure. We detected that the weight loss of test coupons was statistically higher than the control coupons (P 6 0.001) and positively correlated with the number of sessile SRB cells and carbohydrate amount (respectively, P < 0.05 and P < 0.05) (Fig. 7). Corrosion rates of the test and control coupons were seen to decrease with time (respectively, P < 0.01 and P < 0.01). The difference in corrosion rates between test and control coupons was significant (P < 0.001) (Table 2). 3.5. FESEM and EDS analysis Fig. 8(A) provides the EDS of galvanized steel control coupon exposed to sterile tap water for 10 months wherein, Fe and S peaks
165
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
6
Absorbance (OD 562 )
5 4 3 2 1
Ja nu ar y
em be r
be r
De c
No ve m
O ct ob er
be r Se pt em
Au gu st
Ju ly
Ju ne
M ay
Ap ril
0
Months Fig. 3. Qualitative biofilm amounts during the experiment. Error bars represent the standard deviation.
Bulk water (CFU/ml)
Biofilm (cells/cm2)
8 7
HPC log 10
6 5 4 3 2 1
ry Ja n
ua
be em ec
D
N
O
Se
r
r ov em
ob ct
em
be
er
r be
t
y
us
pt
Au g
Ju l
e Ju n
ay M
il Ap r
M
ar ch
0
Months Fig. 4. Aerobic heterotrophic bacteria counts in bulk water and biofilm formed on the galvanized steel surfaces during 10 months. Error bars represent the standard deviation. CFU, colony forming unit; HPC, heterotrophic plate count.
Bulk water (CFU/ml)
Biofilm (CFU/cm2)
5.5 5
Anaerobic HPC log 10
4.5 4 3.5 3 2.5 2 1.5 1 0.5
ry
r be em
D ec
Ja nu a
r be ov em N
O ct ob er
be r pt em
t Se
Au gu s
Ju ly
e Ju n
ay M
il Ap r
M
ar
ch
0
Months Fig. 5. Anaerobic heterotrophic bacteria counts in bulk water and biofilm formed on the galvanized steel surfaces during 10 months. Error bars represent the standard deviation. CFU, colony forming unit; HPC, heterotrophic plate count.
166
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Bulk water (cells/ml)
Biofilm (cells/cm2)
9 8 7
SRB log 10
6 5 4 3 2 1
r ov em be r D ec em be r Ja nu ar y
ob e
r
N
Se
O
ct
t
em be pt
y
Au gu s
Ju l
e Ju n
ay M
il Ap r
M
ar ch
0
Months Fig. 6. SRB counts in bulk water and biofilm formed on the galvanized steel surfaces during 10 months. Error bars represent the standard deviation.
Weight loss 8
80
7
70
6
60
5
50
2
4
40 3 30 2
20
1
10
0 ar y Ja
nu
r
r
em be De c
be No ve m
O ct ob er
r Se
pt
em
be
Au gu st
Ju ly
Ju ne
M
ay
0 Ap ril
Weight loss (mg/cm )
2
Carbohydrate amount (μ g/cm )
Carbohydrate 90
Months Fig. 7. Quantities of carbohydrate detected in biofilm. Error bars represent the standard deviation.
Table 2 Weight loss and average corrosion rates of test and control galvanized steel coupons during 10 months. Months
Weight loss (mg/cm2) ± SD, test
Weight loss (mg/cm2) ± SD, control
Corrosion rate, test (mg/dm2 day)
Corrosion rate, control (mg/dm2 day)
Ratio (test/control)
April May June July August September October November December January
1.08 ± 0.06 2.23 ± 0.10 3.10 ± 0.20 3.39 ± 0.30 3.78 ± 0.17 4.31 ± 0.28 5.10 ± 0.18 5.85 ± 0.22 6.41 ± 0.10 7.23 ± 0.22
1.03 ± 0.05 1.15 ± 0.02 1.51 ± 0.01 1.61 ± 0.01 1.77 ± 0.15 1.82 ± 0.02 1.87 ± 0.03 1.89 ± 0.01 1.95 ± 0.09 2.06 ± 0.11
3.61 3.68 3.42 3.07 2.59 2.49 2.47 2.48 2.40 2.41
3.50 1.91 1.68 1.39 1.18 1.01 0.86 0.76 0.72 0.68
1.03 1.93 2.04 2.21 2.19 2.47 2.87 3.26 3.33 3.54
±, Standard deviations (SD).
were not observed. Fig. 8(B and C) shows the EDS profiles of the galvanized steel coupons exposed to bulk water in the model cooling system for 10 months and 11 days, respectively. Peaks for Ca, Zn, Fe, O and Si were observed. However S peaks were not determined.
After the biofilm and corrosion products were removed from the surface of the coupons, depletion of zinc in some regions of the test coupons and the appearance of steel were noted. Pitting corrosion of the steel was also observed (Fig. 9(B)). However we
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
167
Fig. 8. EDS analysis of the corrosion products formed on the galvanized steel surfaces; (A) the control coupon exposed to sterile tap water for 10 months; (B) the test coupon exposed to bulk water in the model recirculating water system for 10 months; (C) the test coupon exposed to bulk water in model recirculating water system for 11 days. (A– C) bar = 5 mm.
did not observe any pitting or depletion of zinc of the control coupons (Fig. 9(A)). 3.6. Iron and zinc measurement Table 3 shows the amounts of dissolved iron found in the bulk water, biofilm and EPS obtained from the model system. The maximum concentration (0.26 ± 0.02 lg/ml) of iron in the bulk water was measured at the beginning of experiment (March)
and then some variations in iron amount were observed. The amount of iron in the biofilm showed an increasing and decreasing one after the other, and a maximum iron concentration of 1.39 ± 0.05 lg/cm2 was detected at December. The maximum concentration of iron in the EPS was measured as 0.73 ± 0.03 lg/mL at April. The iron amount in biofilm was found statistically higher than in EPS and bulk water (P < 0.001). The iron concentrations in biofilm, EPS and bulk water were not correlated to each other.
168
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Fig. 9. SEM showing the surfaces of test coupons after biofilm and corrosion products of galvanized steel coupon in the model recirculating water system were removed after 5 months (A and B) and 10 months (C and D). Labelled arrows indicate exposed steel surface and corrosion pits. Bar = 10 lm, 100 nm, 10 lm and 10 lm, respectively. (E) The metal surface of control galvanized steel coupon in the sterile control system after 10 months, bar = 10 lm.
Table 3 The amounts of dissolved iron and zinc found in the bulk water, biofilm and EPS obtained from the model system during experiments. Months
March April May June July August September October November December January
Fe concentrations
Zn concentrations
Bulk water (lg/ml)
Biofilm (lg/cm2)
EPS (lg/cm2)
Bulk water (lg/ml)
Biofilm (lg/cm2)
EPS (lg/cm2)
0.26 ± 0.02 0.15 ± 0.02 0.07 ± 0.01 0.06 ± 0.01 0.04 ± 0.01 0.21 ± 0.02 0.07 ± 0.01 0.07 ± 0.01 0.03 ± 0.01 0.11 ± 0.01 0.14 ± 0.01
– 0.90 ± 0.05 0.67 ± 0.05 1.06 ± 0.03 0.39 ± 0.02 0.98 ± 0.02 0.59 ± 0.03 1.24 ± 0.07 0.46 ± 0.08 1.39 ± 0.05 0.48 ± 0.01
– 0.73 ± 0.03 0.28 ± 0.02 0.51 ± 0.05 0.35 ± 0.04 0.10 ± 0.01 0.32 ± 0.06 0.54 ± 0.04 0.33 ± 0.02 0.24 ± 0.03 0.69 ± 0.02
0.79 ± 0.01 1.03 ± 0.02 3.61 ± 0.01 1.95 ± 0.03 1.32 ± 0.01 2.15 ± 0.01 1.45 ± 0.01 1.42 ± 0.01 7.41 ± 0.05 0.47 ± 0.01 2.02 ± 0.01
– 162.96 ± 1.88 770.46 ± 2.18 1089.70 ± 6.10 888.60 ± 10.03 476.52 ± 10.52 902.56 ± 10.67 1121.33 ± 2.07 1124.03 ± 6.40 1166.77 ± 2.48 921.07 ± 9.65
–
±, Standard deviations (SD).
1.03 ± 0.69 7.81 ± 0.04 13.83 ± 0.18 12.46 ± 0.53 8.78 ± 0.42 3.42 ± 0.25 12.39 ± 0.33 3.70 ± 0.38 8.48 ± 0.86 5.29 ± 0.31
169
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
Film layer
Control water
0.8
2.5
0.7 2
0.5
1.5 2
Fe (µg/cm )
Fe (µg/ml)
0.6
0.4 1
0.3 0.2
0.5 0.1
O
pt
ct
ob er N ov em be r D ec em be r Ja nu ar y
r be
st
em
Au gu
Se
Ju ly
Ju ne
M ay
il
0
Ap r
M ar ch
0
Months Fig. 10. Temporal changes in the amounts of iron in the water and film layers obtained from the control system. Error bars represent the standard deviation.
Fig. 10 represents the concentrations of iron found in the water and film layer formed on galvanized steel obtained from the control system. The concentration of iron in the control water and film layer decreased with time (respectively, P < 0.05 and P < 0.01). We detected a positive significant correlation between iron concentrations in the control water and film layer (P < 0.01). The maximum zinc concentrations in biofilm, EPS and bulk water were measured as 1166.77 ± 2.48 lg/cm2 at December, 13.83 ± 0.18 lg/cm2 at June and 7.41 ± 0.05 lg/mL at November, respectively (Table 3). The zinc amount in biofilm was found to be statistically higher than in the EPS and bulk water (P < 0.001). We could not detect any significant correlation between the zinc concentrations in biofilm, EPS and bulk water. However the concentrations of zinc in the biofilm showed significant correlations with weight loss, carbohydrate amount and SRB count in biofilm (respectively, P < 0.05, P < 0.01 and P < 0.01).
Fig. 11 shows the concentrations of zinc found in the water and film layer formed on galvanized steel obtained from the control system. The concentrations of zinc in the water and film layer reached peak values 93.2 ± 0.33 lg/mL and 549.14 ± 8.15 lg/mL at June, respectively, and then decreased and reached 3.43 ± 0.03 lg/mL at October and 117.58 ± 11.22 lg/mL at July, respectively, and then remained constant. The concentration of zinc in the control water showed a significant correlation within the film (P < 0.05). 4. Discussion In this work, we have developed a laboratory model system to study the effects of mixed species biofilm including SRB on the corrosion of galvanized steel. Although the model system was fed with tap water, the recirculating water had different water charac-
Film layer
Control water
100
600
90 500
80
400
60 50
300
40
Zn ( Ξ g/cm 2 )
Zn ( Ξ g/ml)
70
200
30 20
100
10
ua
ry
r be em ec
Ja n
r be D
ov em N
ob ct O
em be pt Se
er
r
t us Au g
y Ju l
e Ju n
M ay
Ap r
M
il
0 ar ch
0
Months Fig. 11. Temporal changes in the amounts of zinc in the water and film layers obtained from the control system. Error bars represent the standard deviation.
170
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
teristics than distributed tap water. The physico-chemical parameters of model system water were appropriate for the cooling water criteria. Even under a regular blowdown regime (3.5 L fresh water, daily), conductivity, TDS and sulphates which make the recirculating water more aggressive were increased with time. It could be resulted from circulation, heat and evaporation. Following previous field observations the water temperature was kept constant at 28 °C [29], during the study to eliminate the influence of temperature on the corrosion of galvanized steel. Our findings suggest that the planktonic bacteria cells including aerobic heterotrophic bacteria, anaerobic heterotrophic bacteria and SRB attached and formed mixed species biofilm on the galvanized steel surfaces, even though zinc is toxic to a variety of microorganisms [12,13,30,31]. The density of biofilm increased with ageing as shown by quantitative biofilm measurement. FESEM analysis validated the adhesion of bacteria cells on the galvanized steel surfaces. We found out that the growth rates both of the heterotrophic bacteria and SRB cells in the biofilm were greater than that of the planktonic cells. Beech et al. [32] reported the same result for SRB. Also Ellwood et al. [33] reported a similar result with respect to population increase of adhered cells. We determined an inverse correlation between HPC in the biofilm and bulk water in contrast to Kerr et al. [34] suggesting that the difference in environmental parameters can affect the rate of attachment and multiplication of heterotrophs. During the phase of biofilm development on galvanized steel, the increase in numbers of SRB and anaerobic heterotrophic bacteria in biofilm showed very high positive correlations with the numbers of them in the recirculating water. These indicated that anaerobic biofilm bacteria including SRB and heterotrophs were shedding continuously into the water phase and had not been able to establish themselves in the already developed biofilms. Similar results for SRB were reported by Power et al. [35,36]. Our results from the present study show that galvanized steel could be corroded by microorganisms as well as SRB in the mixed species biofilm. Corrosion rate of the test coupons was decreased with time, just as found by Ilhan-Sungur et al. [10]. However, although the results of corrosion rates reported by Ilhan-Sungur et al. [10] were generally higher than our study, the values of corrosion rate ratios (test/control) in our investigation were higher than reported by Ilhan-Sungur et al. [10] and increased with time. In the natural conditions, various biotic (EPS, quorum sensing, etc.) and abiotic (pH, temperature, etc.) factors can influence the corrosion course [37–39]. The weight loss of galvanized steel was correlated with the number of sessile SRB cells. However, since our study was not carried out with pure culture of SRB, and also various biotic and abiotic factors could influence the weight loss, we cannot say that weight loss of galvanized steel increases with SRB counts. IlhanSungur et al. [10], Beech et al. [32] and Angell and Urbanic [8] suggested that there was no correlation between numbers of SRB and corrosion rates or initiation of pitting. We detected that extracellular carbohydrate was degraded by mixed species of bacteria in biofilm. Zhang et al. [25] reported that mixed cultures degraded their own EPS material when they were in a starved state. Quantities of carbohydrate detected in biofilm appeared to be positively correlated with the weight loss and zinc concentration in the biofilm. It was reported that EPS can complex metal ions and, thus, affect the corrosion [40]. EDS analysis of the test galvanized steel coupons determined Fe and O in corrosion products. It was proposed that the corrosion products caused by SRB for iron will be either iron sulphides or phosphides which initiated a general type of corrosion or the combination of both [41]. However S peaks were not observed. Santegoeds et al. [42] showed that sulphide did not diffuse out of the
biofilm. Therefore, since EDS analyzer can enter maximum 1 lm within the biofilm, S peaks could not have been determined. The amount of iron in the biofilm showed an increasing and decreasing one after the other. Since all bacteria require metal ions for their growth, iron could be biosorbed by microorganisms such as SRB. Fe2+ is known as a relevant factor for SRBs which probably account for the biosynthesis of the iron-cytochromes in the respiratory chain [21]. Marchal et al. [39] showed that Fe2+ concentrations influenced growth of SRB and growth performances were significantly lower using a culture medium free of Fe2+. Other possibility is that the outer part of biofilm including iron could be detached and re-entered the bulk water. Although regular blowdown was carried out, sometimes an increase in the amount of iron in the bulk water was observed. This indicates that zinc was depleted and iron appeared was corroded. The concentrations of zinc in the biofilm showed significant correlations with weight loss and SRB count in the biofilm. It has been known that Zn is toxic and inhibitory to SRB [12,13]. Poulson et al. [13] and Ilhan-Sungur et al. [10] reported that a concentration of 13 lg/mL Zn and of 22.36 ± 0.007 lg/mL Zn are toxic to pure strains of Desulfovibrio desulfuricans and Desulfovibrio sp., respectively. We found, however, that SRB can survive in the mixed species biofilm with very high Zn concentrations (min 162.96 ± 1.88 max 1166.77 ± 2.48 lg/cm2). It appears that the effect of Zn on SRB vary depending on the type of SRB and their growth conditions. The natural environment may also influence bacterial metabolic activities and resistance to toxic agents. In addition we can say that toxicity studies carried out under laboratory conditions cannot mirror the facts. On the other hand the measurement of the dissolved metal concentration can serve as an indicator of the bioactivity of the SRB [12,13]. This is a first report on corrosion of galvanized steel by microorganisms as well as SRB in mixed species biofilm formed on galvanized steel in a simulated recirculating cooling tower system. No chemicals (disinfectant, pH regulators or anti-scaling agents) were added to the system in order to exclude their possible negative effects (such as their disinfecting effect) on the microorganisms and biofilm formation. However, microbiologically influenced corrosion of galvanized steel in a real cooling tower with biocide and corrosion inhibitory are currently under investigation.
5. Conclusion The following conclusions can be drawn from this study: (1) FESEM analysis showed that planktonic bacteria cells attached and formed the mixed species biofilm on the galvanized steel surfaces, even though zinc is toxic to a variety of microorganisms. (2) It is suggested from the present study that anaerobic biofilm bacteria including SRB and heterotrophs were shedding continuously into the water phase and had not been able to establish themselves in the already developed biofilms. (3) Our study showed that galvanized steel was corroded by microorganisms as well as SRB in the mixed species biofilm. (4) The weight loss of galvanized steel was correlated with the number of sessile SRB cells (P < 0.05). However, since our study was not carried out with pure culture of SRB, and also various biotic and abiotic factors could influence the weight loss, we cannot say that weight loss of galvanized steel increases with SRB counts. (5) Extracellular carbohydrate could be degraded by bacteria in biofilm including mixed species. The amount of total carbohydrate detected in the biofilm appeared to be positively correlated with the weight loss (P < 0.05). This suggested
E. Ilhan-Sungur, A. Çotuk / Corrosion Science 52 (2010) 161–171
that there was a direct correlation between corrosion of galvanized steel and carbohydrate amount. (6) We found, however, that SRB can survive in the mixed species biofilm with very high Zn concentrations (min 162.96 ± 1.88 max 1166.77 ± 2.48 lg/cm2). It appears that the effect of Zn on SRB vary depending on the type of SRB and their growth conditions. Also we can say that toxicity studies carried out under laboratory conditions cannot mirror the external reality. Acknowledgements The authors are grateful to Prof. Nurhan Cansever for fruitful discussions during the preparation of this paper. Msc. Bihter Minnosß and Dr. Nihal Dog˘ruöz are gratefully acknowledged. The model system was donated by Dizayn Teknik Plastic Pipes & Fittings Co. This study was supported by the Research Fund of Istanbul University (Project No: T-528/21102004). References [1] S.G. Choudhary, Emerging microbial control issues in cooling water systems, Systems Hydrocarb. Process. 77 (5) (1998) 91–102. [2] K. Lewis, Riddle of biofilm resistance, Antimicrob. Agents Chemother. 45 (2001) 999–1007. [3] W.A. Hamilton, Microbially influenced corrosion as a model system for the study of metal microbe interactions: a unifying electron transfer hypothesis, Biofouling 19 (1) (2003) 65–76. [4] W.A. Hamilton, Sulphate reducing bacteria and anaerobic corrosion, Ann. Rev. Microbiol. 39 (1985) 195–217. [5] A.K. Lee, M.G. Buehler, D.K. Newman, Influence of a dual-species biofilm on the corrosion of mild steel, Corros. Sci. 48 (1) (2006) 165–178. [6] D. Çetin, S. Bilgiç, S. Dönmez, G. Dönmez, Determination of biocorrosion of low alloy steel by sulfate-reducing Desulfotomaculum sp. isolated from crude oil field, Mater. Corros. 58 (11) (2007) 841–847. [7] T.S. Rao, T.N. Sairam, B. Viswanathan, K.V.K. Nair, Carbon steel corrosion by iron oxidising and sulphate reducing bacteria in a freshwater cooling system, Corros. Sci. 42 (8) (2000) 1417–1431. [8] P. Angell, K. Urbanic, Sulphate-reducing bacterial activity as a parameter to predict localized corrosion of stainless alloys, Corros. Sci. 42 (2000) 897–912. [9] D. Çetin, M.L. Aksu, Corrosion behavior of low-alloy steel in the presence of Desulfotomaculum sp., Corros. Sci. 51 (2009) 1584–1588. [10] E. Ilhan-Sungur, N. Cansever, A. Çotuk, Microbial corrosion of galvanized steel by a freshwater strain of sulphate reducing bacteria (Desulfovibrio sp.), Corros. Sci. 49 (2007) 1097–1109. [11] E. Ilhan-Sungur, A. Cotuk, Characterization of sulfate reducing bacteria isolated from cooling towers, Environ. Monit. Assess. 104 (2005) 211–219. [12] V.P. Utgikar, H.H. Tabak, J.R. Haines, R. Govind, Quantification of toxic and inhibitory impact of copper and zinc on mixed cultures of sulfate-reducing bacteria, Biotechnol. Bioeng. 82 (3) (2003) 306–312. [13] S.R. Poulson, P.J.S. Colberg, J.I. Drever, Toxicity of heavy metals (Ni, Zn) to Desulfovibrio desulfuricans, Geomicrobiol. J. 14 (1997) 41–49. [14] I. Turetgen, Comparison of free residual chlorine and monochloramine for efficacy against biofilms in model and full scale cooling towers, Biofouling 20 (2004) 81–85. [15] American Society for Testing and Material, Standard recommended practice for preparing, cleaning and evaluating corrosion test specimens, in: Annual Book of ASTM standards, Designation: G1-72, American Society for Testing Materials, Philadelphia, 1975, pp. 626–629. [16] APHA, Standards methods for the examination of water and wastewater, American Public Health Association, Washington, DC, 0-87553-091-5, 1981. [17] G. Bianucci, E.R. Bianucci, L’analisi chimica delle acque naturali ed inquinate, Hoepli, Milano 88203 (1987) 1987.
171
[18] C.E. Boyd, C.S. Tucker, Water quality and pond soil analyses for aquaculture, Alabama Agricultural Experiment Station, Auburn University, Alabama, 9780817307219, 1992. [19] G.A. Gagnon, R.M. Slawson, An efficient biofilm removal method for bacterial cells exposed to drinking water, J. Microbiol. Met. 34 (1999) 203–214. [20] E. Sungur, B. Minnos, N. Dogruoz, Isolation of aerobic heterotrophic and anaerobic sulphate reducing bacteria from model water system by filtration method, IUFS J. Biol. 67 (1) (2008) 33–38. [21] J.R. Postgate, The Sulphate Reducing Bacteria, 2nd ed., Cambridge University Press, Cambridge, Great Britain, 1984. [22] The Institute of Petroleum, Determination of the viable microbial content of fuels and fuel components boiling below 390 °C – Filtration and Culture Method, 1995, IP Method Number 385/95. [23] D.J. Reasoner, E.E. Geldrich, A new medium for the enumeration and subculture of bacteria from potable water, Appl. Environ. Microbiol. 49 (1985) 1–7. [24] G.A. O’toole, R. Kolter, Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis, Mol. Microbiol. 28 (3) (1998) 449–461. [25] X. Zhang, P.L. Bishop, B.K. Kinkle, Comparison of extraction methods for quantifying extracellular polymers in biofilms, Water Sci. Tech. 39 (7) (1999) 211–218. [26] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [27] Y.M. Nelson, W. Lo, L.W. Lion, M.L. Shuler, W.C. Ghirorse, Lead distribution in a simulated aquatic environment: effects of bacterial biofilms and iron oxide, Water Res. 29 (1995) 1934–1944. [28] C. Campanac, L. Pineau, A. Payard, G. Baziard-Mouysset, C. Roques, Interactions between biocide cationic agents and bacterial biofilms, Antimicrob. Agents Chemother. 46 (5) (2002) 1469–1474. [29] I. Turetgen, E. Ilhan-Sungur, A. Cotuk, Enumeration of Legionella pneumophila in cooling tower water systems, Environ. Monit. Assess. 100 (1–3) (2005) 53– 58. [30] G.M. Gadd, A.J. Griffiths, Microorganisms and heavy metal toxicity, Microbiol. Ecol. 4 (1978) 303–317. [31] I. Cohen, R. Bitan, Y. Nitzan, The effect of zinc and cadmium ions on Escherichia coli, Microbios 68 (1991) 157–168. [32] I.B. Beech, C.W. Sunny Cheung, C.S. Patrick Chan, M.A. Hill, R. Franco, A.R. Lino, Study of parameters implicated in the biodeterioration of mild steel in the presence of different species of sulphate-reducing bacteria, Int. Biodeter. Biodegr. 34 (1994) 289–303. [33] D.C. Ellwood, C.W. Keevil, P.D. Marsh, C.M. Brown, J.N. Wardell, Surfaceassociated growth, Philos. T. Roy. Soc. B 297 (1982) 517–532. [34] C.J. Kerr, K.S. Osborn, G.D. Robson, P.S. Handley, The relationship between pipe material and biofilm formation in a laboratory model system, J. Appl. Microbiol. 85 (1999) 29–38. [35] M.E. Power, J.R. Van Der Meer, H. Harms, O. Wanner, Colonization of aerobic biofilms by sulfate-reducing bacteria, Biofouling 17 (2001) 275–288. [36] M.E. Power, J.C. Araujo, J.R. Van Der Meer, H. Harms, O. Wanner, Monitoring sulfate-reducing bacteria in heterotrophic biofilms, Water Sci. Technol. 39 (1999) 49–56. [37] I.B. Beech, C.C. Gaylarde, Microbial polysaccharides and corrosion, Int. Biodeter. 27 (1991) 95–107. [38] C. Peng, J.K. Park, R.W. Patenaude, Statistics-based classification of microbially influenced corrosion in freshwater systems, Water Res. 28 (4) (1994) 951–959. [39] R. Marchal, B. Chaussepied, M. Warzywoda, Effect of ferrous ion availability on growth of a corroding sulfate-reducing bacterium, Int. Biodeter. Biodegr. 47 (2001) 125–131. [40] I.B. Beech, C.W. Sunny Cheung, Interactions of exopolymers produced by sulphate-reducing bacteria with metal ions, Int. Biodeter. Biodegr. 35 (1–3) (1995) 59–72. [41] W.P. Iverson, Mechanism of anaerobic corrosion of steel by sulphate reducing bacteria, Mater. Perform. 23 (3) (1984) 28–30. [42] C.M. Santegoeds, T.G. Ferdelman, G. Muyzer, De Beer, Structural and functional dynamics of sulfate-reducing populations in bacterial biofilms, Appl. Environ. Microbiol. 64 (1998) 3731–3739.