SİMÜLE EDİLMİŞ SOĞUTMA KULESİ SU SİSTEMİNDE GALVANİZLİ ÇELİĞİN
MİKROBİYOLOJİK KOROZYONU
Esra SUNGURa, Nurhan CANSEVERb ve Ayşın ÇOTUKa
a
İstanbul Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, 34118 Vezneciler, İstanbul, Türkiye,
Yıldız Teknik Üniversitesi, Kimya-Metalurji Fakültesi, Metalurji ve Malzeme Mühendisliği Bölümü,
Davutpaşa Kampüsü, 34210, Esenler, İstanbul, Türkiye
b
ÖZET: Galvanizli çelik sac, korozyona ve biyofaulinge karşı dirençli olması nedeni ile su depoları ve soğutma
kulelerinin yapımında sıklıkla tercih edilen bir malzemedir. Çinkonun birçok mikroorganizmaya karşı toksik
etkisi olduğunu gösteren çalışmalar olmasına rağmen, daha önceki çalışmamızda sülfat indirgeyen bakterilerin
(SRB) galvanizli çelik yüzeylere tutunabildikleri ve biyofilm tabakası oluşturabildikleri saptanmıştır. Bu
çalışmada model bir soğutma kulesi su sisteminde, galvanizli çeliğin mikrobiyolojik korozyonunu incelemek ve
ayrıca, biyolojik parametrelerin mikrobiyolojik korozyon üzerine etkilerini belirlemek hedeflenmiştir. Deneyler,
galvanizli çelik kuponları içeren soğutma kulesine benzer şekilde, suyun sürekli sirküle olduğu simüle edilmiş
soğutma kulesi su sisteminde ve kontrol düzeneğinde gerçekleştirilmiştir. Deney kuponları 10 ay süresince ayda
bir kez olmak üzere model su sirkülasyon sisteminden çıkartılmış ve her deney süresinin sonunda SRB ve
aerobik heterotrofik bakteri sayısı saptanmış, korozyon hızı hesaplanmış, hücre dışı polimerik maddelerin
karbonhidrat miktarı ölçülmüş ve alan emisyonlu tarama elektron mikroskobunda (FESEM) incelemeleri
yapılmıştır. FESEM analizleri, galvanizli çelik yüzeylerde biyofilm tabakasının oluştuğunu göstermiştir.
Çalışmamızdaki korozyon deneyleri sonucunda, mikroorganizmaların galvanizli çeliği korozyona uğratabildiği
saptanmıştır. Deney ve kontrol kuponlarının ağırlık kayıplarının ortalamaları arasındaki farkın anlamlı olduğu ve
deney kuponlarının ağırlık kaybının kontrol kuponlarınınkinden daha fazla olduğu saptanmıştır (t(17)= 3.856, P
0.001). Ayrıca deney kuponlarındaki ağırlık kaybı ile biyofilmdeki karbonhidrat miktarı arasında da aynı yönde
anlamlı bir ilişkinin varlığı tespit edilmiştir (P 0.05).
Anahtar Kelimeler: Galvanizli çelik, soğutma kulesi, mikrobiyolojik korozyon, sülfat indirgeyen bakteriler,
karbonhidrat
MICROBIAL CORROSION OF GALVANIZED STEEL IN A SIMULATED
RECIRCULATING COOLING TOWER WATER SYSTEM
ABSTRACT: Galvanized steel is frequently used in the construction of cooling towers and water containers,
owing to its good resistance to corrosion and biofauling. Even though numerous experiments are now available
demonstrating that zinc is toxic to a variety of microorganisms, our previous study showed that sulfate reducing
bacteria (SRB) could attach and form a biofilm layer on the galvanized steel surfaces. The aim of this study is to
observe microbiologically influenced corrosion (MIC) of galvanized steel in a model recirculating cooling water
system and, also establish the effects of biological parameters on MIC. The experiments were carried out in a
simulated recirculating cooling tower water system which is similar to cooling tower and control system with
galvanized steel coupons. Assay coupons were removed from the model recirculating water system monthly
during 10 months for enumeration of SRB and aerobic heterotrophic bacteria, corrosion rate measurement,
extracellular polysaccharide substances extraction, carbohydrate analysis and field emission scanning electron
microscope (FESEM) observation. FESEM analysis showed that biofilm was formed on the galvanized steel
surfaces. Our results indicated that microorganisms were responsible for the corrosion of galvanized steel. We
detected that there was statistically significant difference in the median of weight loss from test and control
coupons, and weight loss of test coupons were higher than the control coupons (t(17)= 3.856, P 0.001). Also,
there was a correlation between weight loss of test coupons and carbohydrate amount (P 0.05).
Keywords: Galvanized steel, cooling tower, microbial corrosion, sulfate reducing bacteria, carbohydrate
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, 2). 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 (3).
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, 4).
Sulfate reducing bacteria (SRB) were considered as the major bacterial group involved in
microbiologically influenced corrosion (5).
Galvanized steel is frequently used in the construction of cooling towers and water
containers, owing to its good resistance to corrosion and biofauling. Even though numerous
experiments are now available demonstrating that zinc is toxic to a variety of microorganisms,
our previous study showed that SRB (Desulfovibrio sp.) could attach and form a biofilm layer
on the galvanized steel surfaces (6).
The aim of this study was to observe microbiologically influenced corrosion (MIC) of
galvanized steel in a model recirculating cooling water system and, also establish the effects
of biological parameters on MIC. For these purposes, ten coupons were removed from model
system monthly during 10 months for enumeration of SRB and heterotrophic bacteria,
corrosion rate measurement, EPS extraction and carbohydrate analysis. In addition for
FESEM observation, two coupons were removed from model system after 11 days and 10
months.
2. EXPERIMENTAL METHODS
2.1. Test material
Galvanized steel for use in cooling tower construction was used as test material. The
thickness of the zinc coating covering the steel was 5 m. The coupons (50 x 25 x 0.5 mm)
were prepared according to guidelines in ASTM G1-72 (7). 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 (6).
2.2. Model system
The experimental study was performed using a 100-liter 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 min-1, Pedrollo, Italy) in the basin and a heater (AT-100, 100W, Atman,
Germany) to facilitate evaporation within the bulk water (Figure 1). Throughout the
experiment, the water temperature was kept constant at 28°C. Coupons were inserted
vertically into coupon–holders situated in the water basins (Figure 1). No chemicals
(disinfectant, pH regulators or anti-scaling agents) were added to the system as to exclude
their possible negative effects (such as their disinfecting effect) on the microorganisms and
biofilm formation. Control systems without SRB containing sterile potable water with 40
galvanized steel coupons were run simultaneously with test systems. Gentamicin (50 g/l)
was added to the control system to prevent contamination.
Figure 1. Schematic diagram of model recirculating water system full stop, arrows indicate the flow direction.
2.3. Bacterial analysis
To estimate the number of sessile bacteria, three coupons were removed monthly from
the basin, dip-rinsed in sterile potable water to remove unattached cells. Biofilm was removed
from the total surface of the one coupon with a sterile cotton swab that was then immersed in
10 ml sterile potable water, vortexed for 60 s (8). The resulting suspension was then serially
diluted from 10-1 to 10-10.
To show the count of planktonic bacteria, bulk water samples were concentrated by
filtration through a 0.22 μm 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 1 minute (9). 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. pH adjusted to 7.2 by 10% NaOH (10, 11).
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.
For aerobic heterotrophic plate count (HPC), samples of 100 µl from dilution series of
biofilm homogenates and filtered water suspensions were spread-plated in triplicate onto R2A
agar (OxoidTM) plates and incubated at 28°C for 10 days (12).
For enumeration of anaerobic HPC, Thioglycollate medium (Oxoid, UK) was used and
the plates were incubated at 30°C for 21 days (13). Anaerobic conditions were created using
the ‘‘AnaeroGen’’ systems (Oxoid, UK).
2.4. EPS extraction and carbohydrate analysis
EPS extraction was carried out according to Zhang et al. (14). 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 (15).
2.5. Weight loss measurement
Eight coupons used for other analyses were cleaned as ASTM G1-72 and weighed after
drying (7). 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 G 1-72 (7).
Where:
Corrosion rate= (K x W)/ (A x t x D) mg/dm2.day
K= 2.40x106xD
t= time of exposure (h)
A= coupon area (cm2)
W= weight loss after cleaning (g)
D= density (g/cm3)
2.6. FESEM analysis
Coupons were fixed with 2.5% glutaraldehyde, followed by dehydration in a graded
series of ethanol and air-drying (16). The dried samples were coated with a palladium (15 nm)
and imaged with a Jeol JSM-6335 field emission scanning electron microscope.
2.7. Statistical analysis of data
Bacterial counts were log10 transformed and standard deviations of the means were
calculated. Statistical evaluation of the results was carried out by Pearson product moment
correlation coefficient test. t-test was employed to detect statistically significant changes in
the bacterial counts.
3. RESULTS
3.1. Bacterial growth
Figure 2 illustrates the presence of bacteria cells on the galvanized steel surfaces after 10
months.
a
b
Figure 2. FESEM micrograph of biofilm formed on galvanized steel surfaces after 10 months. A) Arrow
indicates EPS; B) Arrow indicates coccoidal-shaped bacterial cell.
Growth curves for planktonic and sessile aerobic heterotrophic bacterial populations were
shown in Figure 3. It was determined that there was an inverse correlation between HPC in
biofilm and bulk water (r = -0.636, P 0.05). t-test analysis revealed that there was a
statistically significant difference in the mean of R2A plate counts obtained from galvanized
steel and bulk water.
Biofilm (cells/cm2)
Bulk water (CFU/ml)
8
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HPC log10
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Figure 3. Aerobic heterotrophic bacteria counts in bulk water and biofilm formed on galvanized steel surfaces
during 10 months. Error bars represent the standard deviation. CFU: Colony Forming Unit, HPC: Heterotrophic
Plate Count.
SRB counts in biofilm and bulk water were shown in Figure 4. t-test analysis showed that
SRB counts in biofilm were higher than in bulk water during experiment (t(19)= -3.905, P
0.001). It was established that there was a positive correlation between counts of SRB in
biofilm and bulk water (r = 0.830, P 0.01).
3.2. Weight loss measurement
The variation in weight loss and the corrosion rate observed for the galvanized steel were
shown in Table 1 and Figure 5. Our results indicated that microorganisms were responsible
for the corrosion of galvanized steel. We detected that weight loss of test coupons was
statistically higher than the control coupons (t(17)= 3.856, P 0.001) and positively correlated
with the number of sessile SRB cells (r = 0.683, P 0.05). Corrosion rate was seen to decrease
with time (P 0.01) (Figure 5).
Biofilm (cells/cm2)
Bulk water (cells/ml)
9
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7
SRB log 10
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5
4
3
2
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Au
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Figure 4. SRB counts in bulk water and biofilm formed on galvanized steel surfaces during 10 months. Error
bars represent the standard deviation.
Table 1. 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
April
1.08 ± 0.06
1.03 ± 0.05
3.61
3.50
1.03
May
2.23 ± 0.10
1.15 ± 0.02
3.68
1.91
1.93
June
3.10 ± 0.20
1.51 ± 0.01
3.42
1.68
2.04
July
3.39 ± 0.30
1.61 ± 0.01
3.07
1.39
2.21
August
3.78 ± 0.17
1.77 ± 0.15
2.59
1.18
2.19
September
4.31 ± 0.28
1.82 ± 0.02
2.49
1.01
2.47
October
5.10 ± 0.18
1.87 ± 0.03
2.47
0.86
2.87
November
5.85 ± 0.22
1.89 ± 0.01
2.48
0.76
3.26
December
6.41 ± 0.10
1.95 ± 0.09
2.40
0.72
3.33
January
7.23 ± 0.22
2.06 ± 0.11
2.41
0.68
3.54
2
mdd: mg/dm .day
±: Standard deviation
Corrosion rate, Corrosion rate,
test (mdd)
control (mdd)
Ratio
(test/control)
Test
Control
4
3,5
Corrosion rate (mdd)
3
2,5
2
1,5
1
0,5
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Figure 5. Evaluation of the corrosion rate of test and control galvanized steel coupons over exposure time. Error
bars represent the standard deviation.
3.3. Carbohydrate analysis
It was shown that extracellular carbohydrate was degraded by mixed species of bacteria
in biofilm. Quantities of carbohydrate detected in biofilm appeared to be positively correlated
with the weight loss (r = 0.649, P 0.05) (Figure 6).
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2
90
Weight loss (mg/cm )
Weight loss
2
Carbohydrate amount ( g/cm )
Carbohydrate
Months
Figure 6. Quantities of carbohydrate detected in biofilm formed on galvanized steel surfaces during 10 months.
4. DISCUSSION
Our findings suggest that the planktonic bacteria cells including aerobic 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 (17–19). FESEM analysis validated
the adhesion of bacteria cells on the galvanized steel surfaces. Ilhan-Sungur et al. (6) reported
that SRB could attach and form a biofilm layer on 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. (20) was reported same
result for SRB. Also Ellwood et al. (21) reported a similar result with respect to population
increase of adhered cells.
Although numerous experiments are now available demonstrating microbiologically
influenced corrosion of several metals under laboratory and in situ conditions (22, 23), there
are only one study about MIC of galvanized steel and it was carried out in laboratory
conditions (6). However, to date we have not been able to find any published reports on the
MIC of galvanized steel in a cooling water system.
Our results from the present study show that galvanized steel could be corroded by
microorganisms. 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 can not say that
weight loss of galvanized steel increase with SRB counts. Ilhan-Sungur et al. (6), Beech et al.
(20) and, Angell and Urbanic (23) suggested that there was no correlation between number of
SRB and corrosion rates or initiation of pitting.
We detected that extracellular carbohydrate was degraded by mixed species of bacteria in
biofilm. Zhang and Bishop (24) 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. It was reported that EPS can complex metal ions
and, thus, affect the corrosion (20).
5. CONCLUSION
* Microorganisms were responsible for the corrosion of galvanized steel.
* The weight loss of galvanized steel was correlated with the number of sessile SRB cells (r =
0.683, P 0.05).
* Extracellular carbohydrate could be degraded by bacteria in biofilm including mixed
species. The amount of total carbohydrate detected in the biofilm was appeared to be
positively correlated with the weight loss (r = 0.649, P 0.05).
ACKNOWLEDGEMENTS: 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).
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