materials

Article
Influence of Chlorination and Choice of Materials on
Fouling in Cooling Water System under Brackish
Seawater Conditions
Pauliina Rajala 1, *, Malin Bomberg 1 , Elina Huttunen-Saarivirta 1 , Outi Priha 1 , Mikko Tausa 2
and Leena Carpén 1
1 VTT Technical Research Centre of Finland, Espoo 02044-VTT, Finland; malin.bomberg@vtt.fi (M.B.);
elina.huttunen-saarivirta@vtt.fi (E.H-S.); outi.priha@vtt.fi (O.P.); leena.carpen@vtt.fi (L.C.)
2 Teollisuuden Voima Oyj, Eurajoki 27160, Finland; mikko.tausa@tvo.fi
* Correspondence: pauliina.rajala@vtt.fi; Tel.: +358-20-722-4451

Academic Editor: Hideyuki Kanematsu

Received: 31 March 2016; Accepted: 31 May 2016; Published: 15 June 2016

Abstract: Cooling systems remove heat from components and industrial equipment. Water cooling,
employing natural waters, is typically used for cooling large industrial facilities, such as power
plants, factories or refineries. Due to moderate temperatures, cooling water cycles are susceptible to
biofouling, inorganic fouling and scaling, which may reduce heat transfer and enhance corrosion.
Hypochlorite treatment or antifouling coatings are used to prevent biological fouling in these
systems. In this research, we examine biofouling and materials’ degradation in a brackish seawater
environment using a range of test materials, both uncoated and coated. The fouling and corrosion
resistance of titanium alloy (Ti-6Al-4V), super austenitic stainless steel (254SMO) and epoxy-coated
carbon steel (Intershield Inerta160) were studied in the absence and presence of hypochlorite.
Our results demonstrate that biological fouling is intensive in cooling systems using brackish seawater
in sub-arctic areas. The microfouling comprised a vast diversity of bacteria, archaea, fungi, algae
and protozoa. Chlorination was effective against biological fouling: up to a 10–1000-fold decrease
in bacterial and archaeal numbers was detected. Chlorination also changed the diversity of the
biofilm-forming community. Nevertheless, our results also suggest that chlorination enhances
cracking of the epoxy coating.

Keywords: biofouling; microbial influenced corrosion; Baltic Sea; biofilm; materials science

1. Introduction
Cooling water systems remove heat from components and industrial equipment. Water cooling
is typically used for cooling large industrial facilities, such as power plants, chemical factories and
petroleum refineries. Cooling systems operate by transferring heat from a heat source, such as a power
plant or industrial equipment, to a heat sink, typically water. The most common cooling water systems
are once-through systems and open recirculating cooling towers. The former is a common choice,
where available water resources are abundant, and thus, they usually use natural water from a lake,
river or sea. However, materials are susceptible to fouling in natural waters. Fouling consists of
biofouling, corrosion products and precipitation. When seawater is the cooling fluid, the phenomenon
is accentuated fundamentally due to the strong corrosive nature of salt water and to its elevated
biological activity. Biofouling, inorganic fouling and scaling can reduce heat transfer and enhance
corrosion. Corrosion reduces the lifetime of the systems and also further increases scaling and fouling
by providing uneven surfaces more favorable for the micro-organisms to attach. Fouling on the other
hand reduces the heat exchanging capacity of the system.

Materials 2016, 9, 475; doi:10.3390/ma9060475 www.mdpi.com/journal/materials

Materials 2016, 9, 475 2 of 21

Natural water sources contain diverse species of micro-organisms: bacteria, algae and fungi.
The interaction between the micro-organisms and the cooling system material surfaces challenges the
operational performance of the system by such phenomena as microbial-influenced corrosion (MIC)
and microfouling. Microbes can generate conditions that increase corrosion through the alteration of
pH and redox potential, the excretion of corrosion-inducing metabolites, direct or indirect enzymatic
reduction or oxidation of corrosion products and the formation of biofilms that create corrosive
microenvironments [1]. Scaling, where insoluble chemical compounds precipitate on the surfaces,
is also possible, although not directly connected with the presence of micro-organisms. Nevertheless,
all of these surface processes impair the transfer of heat in the system, thus decreasing the cooling
efficiency. Microfouling and inorganic scaling precedes the attachment and growth of macrofouling
organisms, such as mussels and macroalgae. Macro-organisms are known to favor surfaces where
biofilm is already formed [2,3]. Macrofouling reduces the flow rate of cooling water systems and may
reduce also the cooling capacity.
Micro-organisms are typically combated by the use of biocides; in the seawater environment, the
common approach is chlorination [4] by, e.g., the addition of hypochlorite compounds [5,6]. However,
the proper dosing and timing is a balance between the efficiency of the treatment and the avoidance
of the corrosive effect and environmental stress caused by the toxic effect of chlorination treatment.
Corrosion may be induced by hypochlorite compounds because they are strong oxidizing agents that
easily accelerate the anodic oxidizing reaction and induce the localized attack of passivating alloys,
such as stainless steels.
Besides using chemicals, microfouling may be combated by the application of coatings.
Special antifouling coatings have been developed [7], with the focus on either surface characteristics,
such as hydrophobicity or superhydrophobicity [8], that aim to reduce the contact between the material
and its environment, or antimicrobial tendency, i.e., the inherent property to influence the microbial
counts through interaction between the surface and the micro-organisms.
Seawater is a challenging environment for many metallic materials because of saline content
and biological activity. Titanium alloys are commonly-used materials especially in heat exchangers
because of their high corrosion resistance, strength and light weight [9]. Steel or coated steel is often
used as a pipe material. In this research, we examine biofouling and materials’ degradation in a
brackish seawater environment using a range of test materials, both uncoated and coated. Here,
the fouling and corrosion resistance of titanium alloy (Ti-6Al-4V), super austenitic stainless steel
(254SMO) and epoxy-coated carbon steel (Intershield Inerta160) were studied in the absence and
presence of hypochlorite.
This is one of the very few contributions that involves brackish seawater as the studied
environment and, thus, improves the understanding of the interactions of micro-organisms typical for
such a low-salt water with cooling system material surfaces.

2. Results

2.1. Biofouling
Fouling was visually evident on all surfaces included in the non-chlorinated system, yet minor
fouling had also occurred on the specimens exposed to chlorinated seawater (Figure 1). After one
month of exposure, a yellowish deposit could be detected on the specimen surfaces, while after three
months of test duration, such a yellowish deposit was accompanied by the presence of individual
rod-shaped precipitates. In order to get an insight into the quantity and quality of fouling on the
surfaces, molecular biological and microscopy analyses were performed.
Materials 2016, 9, 475 3 of 21
Materials 2016, 9, 475 3 of 21
Materials 2016, 9, 475 3 of 21

Figure 1. Photographs, showing the specimen surfaces after the three-month test. (A,B) 254SMO;
Figure 1.1. Photographs,
Photographs,showing
showingthethespecimen surfaces
specimen afterafter
surfaces the three-month test. (A,B)
the three-month 254SMO;
test. (A,B) (C,D)
254SMO;
(C,D) Ti alloy; (E,F) Inerta160-coated steel. Non-chlorinated: (A,C,E); chlorinated: (B,D,F).
Ti alloy;
(C,D) Ti (E,F)
alloy;Inerta160-coated steel. Non-chlorinated:
(E,F) Inerta160-coated (A,C,E);
steel. Non-chlorinated: chlorinated:
(A,C,E); (B,D,F).
chlorinated: (B,D,F).
2.1.1. Quantification of Micro-Organisms
2.1.1. Quantification of Micro-Organisms
Micro-Organisms
The results from quantitative polymerase chain reaction (qPCR) characterizations revealed that
Thewere
there results from quantitative
approximately 107–10polymerase
8 bacteria, 10 chain
4–105 reaction
archaeons (qPCR)
and 10 characterizations
2–104 fungi per cm revealed
2 on the that
there were approximately 10 77–1088 bacteria, 1044 –1055 archaeons and 1022 –1044 fungi per cm22 on the
were which
coupons, approximately
had been in 10non-chlorinated
–10 seawaterarchaeons
for 1–3 months (Figure –10 fungi2). The material did
coupons,
not have which
which had been
had beenimpact
a significant ininnon-chlorinated
non-chlorinated
on the numbers seawater
seawater forfor
of attached1–3 1–3 months
months
microbes (Figure
(Figure 2).
TheThe
in the2).non-chlorinatedmaterial
material did
did not
havehave
notenvironment. a In
significant
a significant impact
impact on onthethe numbers
numbers ofof attached
attached microbes
microbes
the chlorinated environment, a decrease in microbial numbers was clearly seen in
in the non-chlorinated
non-chlorinated
environment.
compared toIn In
thethe
the chlorinated environment,
chlorinated
non-chlorinated environment,
environment.aaChlorination
decrease in decreased
decrease microbial the numbers
numbers wasofclearly
attachedseen
compared
bacteria andto the
the non-chlorinated
non-chlorinated
archaea by 2–3 and 1–2 environment.
environment. Chlorination
Chlorination
orders of magnitude, decreased
decreased
respectively. the numbers
Both decreases of attached
attached
were statistically
significant
bacteria
bacteria and(p
and ≤ 0.01).by
archaea
archaea Furthermore,
by2–3
2–3and
and1–2 1–2inorders
the chlorinated
orders of
ofmagnitude,environment
magnitude, after Both
respectively.
respectively. three months ofwere
Bothdecreases
decreases exposure,
were the
statistically
statistically
number of bacteria
significant (p ≤ď0.01). and archaea
0.01).Furthermore, on
Furthermore,ininthe the surface of
thechlorinated Inerta160-coated
chlorinatedenvironment
environmentafter carbon steel
after three was
three months lower compared
months of exposure, the
to other
number
number ofmaterials.
of bacteria
bacteriaand There
and were only
archaea
archaea on
onthe very
the few fungi
surface
surface attached to the surfaces,
ofofInerta160-coated
Inerta160-coated carbon
carbon and
steel
steel the
waswasdecrease
lower
lower of fungi to
compared
compared
caused
other by chlorination was not statistically significant. In all cases, the number
to other materials. There were only very few fungi attached to the surfaces, and the decrease of fungi
materials. There were only very few fungi attached to the surfaces, and of
the biofilm-forming
decrease of
micro-organisms
caused by chlorination increased
chlorination was not on coupons exposed
statistically
statistically for threeIn
significant. months as compared
all cases, the number to those
of being tested
of biofilm-forming
biofilm-forming
for only one month.
micro-organisms increased on coupons exposed for three months as compared to those being tested
for only one month.

Figure 2. Quantity of (a) bacteria; (b) archaea (c) and fungi, on the coupon surfaces, determined using
quantitative PCR. Bars show standard deviations.
Figure 2. Quantity of (a) bacteria; (b) archaea (c) and fungi, on the coupon surfaces, determined using
Figure 2. Quantity of (a) bacteria; (b) archaea (c) and fungi, on the coupon surfaces, determined using
2.1.2. Microscopy
quantitative PCR. Bars show standard deviations.
quantitative PCR. Bars show standard deviations.
Examination by scanning electron microscopy (SEM) revealed that, in the non-chlorinated
2.1.2. Microscopy
system,
2.1.2. the coupon surfaces were abundant with various organisms: algae, including diatoms,
Microscopy
protozoa and micro-organisms
Examination by scanning (Figure
electron3).microscopy
A discontinuous
(SEM)and heterogeneous
revealed that, inlayer
theofnon-chlorinated
extracellular
Examination by scanning electron microscopy (SEM) revealed that, in the non-chlorinated system,
polymeric
system, the substances could also
coupon surfaces be detected
were abundant on with
the surfaces.
variousInorganisms:
the presencealgae,
of surface irregularities,
including diatoms,
the coupon surfaces were abundant with various organisms: algae, including diatoms, protozoa and
such as the grain boundaries of the base metal, these were preferentially accommodated
protozoa and micro-organisms (Figure 3). A discontinuous and heterogeneous layer of extracellular by the
micro-organisms
biofilm. In the (Figure 3). system,
chlorinated A discontinuous
the extent and
of heterogeneous
fouling was clearlylayer of extracellular
reduced as compared polymeric
to non-
polymeric substances could also be detected on the surfaces. In the presence of surface irregularities,
substances could also be detected on the surfaces. In the presence of surface irregularities, such
such as the grain boundaries of the base metal, these were preferentially accommodated by the
as the grain boundaries of the base metal, these were preferentially accommodated by the biofilm.
biofilm. In the chlorinated system, the extent of fouling was clearly reduced as compared to non-
Materials 2016, 9, 475 4 of 21

Materials 2016, 9, 475 4 of 21

In the chlorinated system, the extent of fouling was clearly reduced as compared to non-chlorinated
cases, and very
chlorinated few and
cases, micro-organisms could be easily could
very few micro-organisms distinguished
be easilyondistinguished
the specimen on
surfaces by SEM
the specimen
examinations (Figure
surfaces by SEM 4).
examinations (Figure 4).

A B

4 µm 1 µm

C D

4 µm 1 µm

E F

4 µm 1 µm

Figure 3. SEM images, showing the surfaces of coupons from the non-chlorinated system after three
Figure 3. SEM images, showing the surfaces of coupons from the non-chlorinated system after three
months of exposure. (A,B) 254SMO; (C,D) Ti alloy; (E,F) Inerta160.
months of exposure. (A,B) 254SMO; (C,D) Ti alloy; (E,F) Inerta160.
Materials 2016, 9, 475 5 of 21
Materials 2016, 9, 475 5 of 21

A B

4 µm 1 µm

C D

4 µm 1 µm

E F

4 µm 1 µm

Figure 4. SEM images, showing the surface of coupons from the chlorinated system after three months
Figure 4. SEM images, showing the surface of coupons from the chlorinated system after three months
of exposure. (A,B) SMO254; (C,D) Ti alloy; (E,F) Inerta160.
of exposure. (A,B) SMO254; (C,D) Ti alloy; (E,F) Inerta160.

2.1.3. Effect of Chlorination on the Richness and Diversity of the Biofilm

2.1.3. Effect of Chlorination on the Richness and Diversity of the Biofilm
The total number of bacterial sequences obtained was 313,947, varying between zero and 15,459
The total number of bacterial sequences obtained was 313,947, varying between zero and
per sample and a mean of 6825 (±4136 standard deviation, STD) sequences per sample. The number
15,459 per sample and a mean of 6825 (˘4136 standard deviation, STD) sequences per sample.
of observed Operational Taxonomic Units (OTUs) at the 97% similarity level calculated from the non-
The number of observed Operational Taxonomic Units (OTUs) at the 97% similarity level calculated
rarefied number of sequence reads was between 561 and 2377 OTUs/sample in the non-chlorinated
from the non-rarefied number of sequence reads was between 561 and 2377 OTUs/sample in the
biofilms and 635 and 3416 in the chlorinated biofilms. The number of OTUs estimated by the Chao1
non-chlorinated biofilms and 635 and 3416 in the chlorinated biofilms. The number of OTUs estimated
richness index was between 952 and 5752 in the non-chlorinated biofilms and 455 and 3333 in the
by the Chao1 richness index was between 952 and 5752 in the non-chlorinated biofilms and 455 and
chlorinated biofilms. The number of bacterial OTUs detected, the Chao1 richness index and the
3333 in the chlorinated biofilms. The number of bacterial OTUs detected, the Chao1 richness index
Shannon diversity index were not statistically significantly different in the non-chlorinated
and the Shannon diversity index were not statistically significantly different in the non-chlorinated
treatments compared to the chlorinated ones, except the OTU number between the chlorinated and
treatments compared to the chlorinated ones, except the OTU number between the chlorinated and
non-chlorinated three-month exposure on the Inerta160, where the OTU number in the chlorinated
non-chlorinated three-month exposure on the Inerta160, where the OTU number in the chlorinated
treatment was higher than in the non-chlorinated treatment (p < 0.03) (Figure 5). In addition, the
treatment was higher than in the non-chlorinated treatment (p < 0.03) (Figure 5). In addition, the
Shannon diversity indices were also significantly higher in the non-chlorinated one-month exposure
Shannon diversity indices were also significantly higher in the non-chlorinated one-month exposure
with Inerta160 (p < 0.0003) and Ti alloy (p < 0.05) compared to the chlorinated counterparts.
with Inerta160 (p < 0.0003) and Ti alloy (p < 0.05) compared to the chlorinated counterparts.
The total number of archaeal sequences obtained was 972,618, varying between four and 41,687
per sample and a mean of 20,262 (±1955 STD) sequences per sample. The number of observed OTUs
at the 97% similarity level calculated from the non-rarefied number of sequence reads was between
1325 and 2739 OTUs/sample in the non-chlorinated biofilms and 57 and 2230 in the chlorinated
 biofilms. The number of OTUs estimated by the Chao1 richness index was between 2929 and 5752 in
the non-chlorinated biofilms and 57 and 5134 in the chlorinated biofilms. The material of the coupons
or the exposure time did not affect the archaeal alpha diversity values, but the chlorination strongly
affected the alpha diversity of the archaeal communities (Figure 5). The number of archaeal OTUs
detected,
Materials 2016, 9,the
475 Chao1 richness index and the Shannon diversity index were statistically significantly
6 of 21
higher in the non-chlorinated treatments compared to the chlorinated ones (p < 0.05–0.0009).

Figure 5. The number of observed Operational Taxonomic Units (OTUs) (A–C); the number of
Figure 5. The number of observed Operational Taxonomic Units (OTUs) (A–C); the number of
estimated OTUs according to the Chao1 richness estimators (D–F); and the Shannon diversity index
estimated
(G–I) of OTUs according
the coupon to the Chao1
samples. richness
The columns showestimators (D–F);ofand
mean values thesamples
three Shannonbased diversity index
on the
(G–I) of the coupon
normalized numbersamples. The columns
of sequences read pershow mean
sample, andvalues of three
the error samplesthe
bars indicate based on the
standard normalized
deviation.
number
Values of calculated
sequencesforread per sample,
the bacterial, and and
archaeal the fungal
error bars indicate
data are the in
presented standard deviation.
the left, center Values
and right
calculated
columns,for the bacterial,
respectively. Grey archaeal and fungal
columns present data are and
non-chlorinated presented in thechlorinated
black columns left, center and right
samples.
columns, respectively.
Statistically Greydifferent
significantly columnssample
present non-chlorinated
pairs andvs.
(non-chlorinated black columnsare
chlorinated) chlorinated
indicated samples.
with
stars: * p < 0.05, ** p < 0.005, *** p < 0.0005.
Statistically significantly different sample pairs (non-chlorinated vs. chlorinated) are indicated with
stars: * p < 0.05, ** p < 0.005, *** p < 0.0005.
The total number of internal transcribed spacer (ITS) sequences obtained was 222,360, varying
between zero and 11,711 per sample and a mean of 4941 (±2182 STD) sequences per sample. The
The total number of archaeal sequences obtained was 972,618, varying between four and 41,687 per
number of observed OTUs at the 97% similarity level calculated from the non-rarefied number of
sample and a mean of 20,262 (˘1955 STD) sequences per sample. The number of observed OTUs
sequence reads was between 177 and 374 OTUs/sample in the non-chlorinated biofilms and 69 and
at the
30597% similarity
in the chlorinatedlevel calculated
biofilms. from theofnon-rarefied
The number fungal ITS OTUs number of sequence
detected, the Chao1 reads wasindex
richness between
1325andandthe2739 OTUs/sample in the non-chlorinated biofilms and 57 and
Shannon diversity index were not statistically significantly different in the non-chlorinated2230 in the chlorinated
biofilms. The number
treatments compared ofto
OTUs estimatedones.
the chlorinated by the Chao1 richness index was between 2929 and 5752 in
the non-chlorinated biofilms and 57 and 5134 in the chlorinated biofilms. The material of the coupons
or the exposure time did not affect the archaeal alpha diversity values, but the chlorination strongly
affected the alpha diversity of the archaeal communities (Figure 5). The number of archaeal OTUs
detected, the Chao1 richness index and the Shannon diversity index were statistically significantly
higher in the non-chlorinated treatments compared to the chlorinated ones (p < 0.05–0.0009).
The total number of internal transcribed spacer (ITS) sequences obtained was 222,360, varying
between zero and 11,711 per sample and a mean of 4941 (˘2182 STD) sequences per sample.
The number of observed OTUs at the 97% similarity level calculated from the non-rarefied number of
sequence reads was between 177 and 374 OTUs/sample in the non-chlorinated biofilms and 69 and
305 in the chlorinated biofilms. The number of fungal ITS OTUs detected, the Chao1 richness index
and the Shannon diversity index were not statistically significantly different in the non-chlorinated
treatments compared to the chlorinated ones.
Materials 2016, 9, 475 7 of 21

2.1.4. Microbial Community Composition

The majority of bacterial sequences belonged to Proteobacteria, 42%–95% in the non-chlorinated
and 50%–80% in the chlorinated system (Figure 6A,B), of which Alphaproteobacteria formed the largest
group. From non-chlorinated biofilm samples, 254SMO 28%–55%, Inerta160 42%–76% and Ti alloy
38%–54% of the bacterial communities belonged to Alphaproteobacteria. Similarly, from chlorinated
biofilm, 254SMO 24%–54%, Inerta160 26%–55% and Ti alloy 43%–60% were Alphaproteobacteria.
In chlorinated samples, the most abundant alphaproteobacterial species belonged to families
Kordiimonadaceae, Rhodobacteraceae and Erythrobacteraceae (Table S1). In addition, on the surfaces
of Inerta160, species belonging to the Phyllobacteriaceae family were abundant. The Rhodobacteraceae
family was abundant on the surfaces of non-chlorinated samples.
In addition to Alphaproteobacteria, Gammaproteobacteria and Betaproteobacteria were detected
on the surfaces. In chlorinated samples, the relative abundance of Gammaproteobacteria was greater
than in non-chlorinated samples. The diversity of gammaproteobacterial species was also higher
in chlorinated samples. In the chlorinated samples, undetermined Alteromonadaceae, Oleibacter
and Pseudoalteromonas dominated, especially after one month (Table S1). Pseudoalteromonas was
the most common gammaproteobacterial family in chlorinated 254SMO and Ti alloy samples
after one month, forming 20%–48% of the bacterial community, while Alteromonadaceae was
the dominating gammaproteobacterial group on Inerta160 samples. In the three-month exposure,
Gammaproteobacteria had decreased, and Alphaproteobacteria dominated by Rhodobacteraceae
groups had increased. In addition, the chlorinated samples contained Cytophaga in all coupon
samples and groups of Betaproteobacteria and Epsilonproteobacteria especially in the one-month
samples, which were present at only very low relative abundances in the non-chlorinated samples.
Hetero-organotrophic Roseivirgra species belonging to the Bacteroidetes were common in the
chlorinated 254SMO and Ti alloy samples contributing 4%–17% of the bacterial sequence reads, but
were uncommon in the chlorinated Inerta160 and the non-chlorinated samples. Chloroplasts belonging
to Chlorophyta (eukaryotic green algae) were detected in the bacterial 16S rRNA gene sequencing and
contributed with high relative abundances especially in the three-month exposure.
In the PCA analysis, there was a clear division between the chlorinated and non-chlorinated
samples (Figure 6C).
The archaeal community composition did not differ significantly between the different materials
or exposure times, but was affected by the chlorination. The majority (85%–100%) of the identified
archaeal sequences belonged to the phylum Crenarchaeota (Figure 7A,B). In the non-chlorinated
treatments, the majority of the archaea belonged to the thaumarchaeotal Nitrosopumilus and the
Miscellaneous Crenarchaeotal Group (MCG) linages B10 and pGrfC26 with small inclusions of
Methanomicrobia (mostly Methanosarcina), Parvarchaeota and Thermoplasmatales (Figure 7A,
Table S2). In the chlorinated treatments, the distribution of different archaeal groups was more
heterogeneous. The relative abundance of Parvarchaeota increased especially in the chlorinated water;
the MCG archaea were only detected from eight of the 18 coupon samples; and the Methanomicrobia
were not detected at all. In addition, from almost all non-chlorinated samples sequence reads belonging
to Marine Benthic Group B (MBGB), Methanomicrobia and Thermoplasmatales archaea were identified.
In the PCA plot, the chlorinated and non-chlorinated samples mostly separated from each other
(Figure 7C).
The fungal communities were diverse and differed in composition between the non-chlorinated
and chlorinated environments (Figure 8A,B). On the non-chlorinated coupons, Ascomycota,
Basidiomycota, Rozellomycota and Zygomycota dominated, but after three months, the fungal
communities had changed to consist of mostly Chytridiomycota and Ascomycota. Small amounts
of Cercozoa protists were also detected. In the chlorinated environments, the difference between the
exposure times was not as clear. The fungal communities in the chlorinated treatments consisted mostly
of Ascomycetes and Monoblepharidomycetes with a growing relative abundance of unidentified fungal
groups, as well as Cercozoa protists. In the PCA analysis, the one-month Ti alloy and Inerta160 samples
Materials 2016, 9, 475 8 of 21

Materials 2016, 9, 475 8 of 21

grouped closely to the samples from the chlorinated treatments, but all of the three-month samples fell
three-month samples fell clearly to the other half of the plot into a separate group from the chlorinated
clearly to the
samples other8C).
(Figure half of the plot into a separate group from the chlorinated samples (Figure 8C).

Figure 6. The bacterial community composition of (A) the non-chlorinated and (B) chlorinated
Figure 6. The
samples; (C) abacterial community
PCA plot based composition
on the bacterial of (A)
community the non-chlorinated
(family level). In (C), openand (B) indicate
symbols chlorinated
samples;
chlorinated and solid symbols non-chlorinated samples. Component 1 explains 39.2% symbols
(C) a PCA plot based on the bacterial community (family level). In (C), open and
indicate chlorinated
Component and26.2%
2 explains solid of
symbols non-chlorinated
the variance, samples.
with Actinobacteria, Component
Chloroplasts, 1 explains 39.2% and
Alphaproteobacteria
Component 2 explains 26.2%dominating
and Epsilonproteobacteria of the variance, with
the axis Actinobacteria, Chloroplasts, Alphaproteobacteria
loadings.
and Epsilonproteobacteria dominating the axis loadings.
Materials 2016, 9, 475 9 of 21
Materials 2016, 9, 475 9 of 21

Figure 7. The archaeal community composition of (A) the non-chlorinated and (B) chlorinated
Figure 7. The archaeal community composition of (A) the non-chlorinated and (B) chlorinated samples;
samples; (C) a PCA plot based on the archaeal community (family level). In (C), open symbols indicate
(C) a PCA plot based on the archaeal community (family level). In (C), open symbols indicate
chlorinated andsolid
chlorinated and solid symbols
symbols non-chlorinated
non-chlorinated samples.
samples. Component
Component 1 67.8%
1 explains explains 67.8% and
and Component
Component 2 explains
2 explains 35.1% 35.1% with
of the variance, of the variance, withtheThaumarchaeota,
Thaumarchaeota, the Miscellaneous
Miscellaneous Crenarchaeotal Group
Crenarchaeotal Group (MCG) and Parvarchaea dominating
(MCG) and Parvarchaea dominating the axis loadings. the axis loadings.
Materials 2016, 9, 475 10 of 21
Materials 2016, 9, 475 10 of 21

Figure 8. The fungal community composition of (A) the non-chlorinated and (B) chlorinated samples;
Figure 8. The fungal community composition of (A) the non-chlorinated and (B) chlorinated samples;
(C) a PCA plot based on the fungal community (family level). In (C), open symbols indicate
(C) a PCA plot based on the fungal community (family level). In (C), open symbols indicate chlorinated
chlorinated and solid symbols non-chlorinated samples. Component 1 explains 36.7% and
and solid symbols non-chlorinated samples. Component 1 explains 36.7% and Component 2 explains
Component 2 explains 22.6% of the variance of the variance with Monoblepharidomycetes,
22.6% of the variance of the variance with Monoblepharidomycetes, unidentified Chytridiomycota and
unidentified Chytridiomycota and unidentified Fungi determining the loadings of PC1 and
unidentified Fungi determining the loadings of PC1 and unidentified Ascomycota determining the
unidentified Ascomycota determining the loadings of PC2.
loadings of PC2.
Materials 2016, 9, 475 11 of 21

2.2. Inorganic Fouling

To obtain an insight into the nature of inorganic fouling, EDS analyses were conducted on the
surfaces. In addition to organic fouling and carbon on the surfaces, also high amounts of silicon,
sodium, aluminum, magnesium and chlorine were detected along with oxygen and elements from the
alloy. Inorganic scaling particularly on the surfaces of 254SMO was silicon-rich, but the contribution
of calcium, aluminum and magnesium was higher and that of carbon much lower than in the
non-chlorinated systems (Figure S1). On the surfaces of Ti alloy, only an organic carbon-containing
deposit was found at small amounts in addition to elements from the alloy itself (Figure S1).

2.3. Materials and Their Performance

The morphology of the epoxy coating (Figure S2) was typical of paints applied by brush, with
groove-like features being evident. The coating was continuous, and no pores or cracks could be
observed. Cross-sectional examinations revealed a very heterogeneous composite structure of the
unexposed coated coupon. The coating matrix contained plenty of fillers, the length scale of which
ranged roughly from 100 nm to 10 µm. According to EDS analyses, the most common fillers were oxides
of magnesium and silicon, but also niobium, tantalum, molybdenum and barium were occasionally
detected (Figure S2). The thickness of the coating was approximately 400–420 µm. Contact angle
measurements gave an average of 84 ˘ 4˝ ; thus, the coating was very slightly hydrophilic (contact
angle < 90˝ ).
In the case of alloys, no indications of material degradation could be detected in SEM
investigations. However, the grain boundaries in 254SMO became visible during exposure to seawater.
In turn, the examination of Inerta160-coated specimens revealed that the coating occasionally contained
some cracks or pores after the exposure. In the non-chlorinated system, the size of the cracks after
three months was approximately 100–200 nm. The specimens that were exposed for one month only
were also studied with respect to such cracks and pores, and there were only a few of them, confirming
their appearance as the seawater exposure proceeds. In the chlorinated system, the Inerta160 coating
was cracked to a significantly higher degree than in the non-chlorinated system (e.g., Figure 4F).
Furthermore, the size of cracks was greater: the crack lengths of up to 2–3 µm were frequently
discovered in the specimens exposed for three months.

2.4. Seawater
Seawater temperature varied between 17–21 ˝ C for the first 67 days, after which the temperature
gradually decreased to 8 ˝ C by the end of the experiment period (Figure S3). Phosphorus, chlorophyll,
nitrogen and total organic carbon (TOC) concentrations were determined in the beginning of the
experiment, (tot-P 17 µg¨ L´1 , a-chlorophyll 2.2 µg¨ L´1 , tot-N 310 µg¨ L´1 , TOC 4.2–4.5 mg¨ L´1 ).
The microbial numbers in seawater were, on average, 106 bacteria, 103 archaeons and <104 fungi
per mL (Table 1). The chlorination decreased the number of microorganisms in the beginning of the
chlorination, but later, after the chlorine dose had stabilized, microbial numbers did not differ between
chlorinated and non-chlorinated water (Table 1).
The bacterial community in the non-chlorinated seawater at the beginning of the experiment
consisted of proteobacteria (33%–38%), cyanobacteria (38%–39%), Actinobacteria (11%–12%) and
Bacteroidetes (11%–12%) (Figure 7, Table S1). The chlorination affected the bacterial community at
the beginning of the experiment, and the majority of the 16S rRNA gene reads detected belonged
to Chlorophyta chloroplasts (94% of the sequence reads). Over time, the bacterial community
changed to resemble that of the non-chlorinated input water at one and three months, where the
dominating bacterial families belonged to Acidimicrobiia, Actinobacteria, Synechococcophycideae and
Alphaproteobacteria after one month and Acidimicrobiia and Actinobacteria after three months.
The PCA analysis also set the bacterial communities of the water separately from the biofilm
communities (Figure 7C).
Materials 2016, 9, 475 12 of 21

Table 1. Microbial numbers in sea water, as determined by quantitative PCR, using Escherichia coli,
Halobacterium salinarum and Aspergillus versicolor as external standards.

Bacteria, cfu/mL Archaeons, cells/mL Fungi, spores/mL

Non-Chlorinated Chlorinated Non-Chlorinated Chlorinated Non-Chlorinated Chlorinated
beginning 7.7 ˆ 106 1.8 ˆ 103 3.2 ˆ 103 <1 2300 3
1 month 8.3 ˆ 106 5.6 ˆ 106 8.8 ˆ 103 6.8 ˆ 103 500 380
3 months 5.8 ˆ 106 8.8 ˆ 106 1.3 ˆ 104 6.8 ˆ 103 360 710

The archaea in the non-chlorinated seawater belonged mostly to Thaumarchaeota and MCG
archaea, but in the chlorinated water, the archaeal community consisted almost solely of Parvarchaeota.
The archaeal communities in both non-chlorinated and chlorinated water changed over time, and
while the MCG archaea formed a great part of the non-chlorinated water, the archaeal community
in both the chlorinated and non-chlorinated water were dominated by Thaumarchaeota. In the PCA
analysis plot, the archaeal non-chlorinated water samples fell close to the non-chlorinated biofilm
samples, while the chlorinated outgoing water samples from the beginning of the experiment fall
separately from the rest of the samples (Figure 8C).
The fungal community of the non-chlorinated seawater at the beginning of the experiment
consisted mostly of unidentified Ascomycota and of Sordariomycetes fungi, and the fungal community
became more diverse over time. The relative abundance of Sordariomycetes decreased and was
replaced by Basidiomycota, Chytridiomycota, Zygomycota and Rozellomycota. During the final
sampling, Chytridiomycota was the dominating fungal group, and the relative abundance of Cercozoa
protists had increased. In the chlorinated water, the Sordariomycetes almost disappeared due to
the chlorination already at the beginning of the experiment, and unidentified Ascomycota was the
dominating fungal group. At the one-month sampling point, the proportion of unidentified fungi
had increased to over 50% of the community, and the Ascomycota had dramatically decreased.
At the end of the experiment, the Ascomycota had recovered and formed again approximately 40%
of the fungal community, and the relative abundance of Chytridiomycota and Dothideomycetes had
increased. The Cercozoa protists were also well represented in the chlorinated outgoing water at
the end of the experiment period (Figure 8A,B). The PCA analysis placed the non-chlorinated water
samples from the beginning and one-month time point separate from the rest of the samples and the
corresponding chlorinated water samples close to the biofilm samples from the chlorinated and the
non-chlorinated one-month samples. After three months, the non-chlorinated water samples fell with
the non-chlorinated three-month biofilm samples (Figure 8C).

3. Discussion
Cooling water systems utilizing natural waters are very demanding applications with respect to
the reliable long-term performance of materials and coatings. The results obtained here demonstrated
that biological fouling is intensive in cooling systems using brackish seawater in sub-arctic areas
during the summer period. The microfouling comprised a vast diversity of bacteria, archaea, fungi,
algae and protozoa. Our results demonstrate that chlorination was effective against biological fouling;
up to a 10–1000-fold decrease in bacterial and archaeal number was detected. When interpreting the
results from the quantitative PCR assay, it has to be borne in mind that the gene copy numbers vary in
different microbial species. Thus, the results do not give absolute numbers, but still give an overview
estimate of the trend and can be well used for comparing different samples. The coating, Inerta160,
combined with chlorination further improved the antifouling effect. However, in non-chlorinated
environments, the material selection did not have any significant effect on biofouling.
In addition to reducing the number of micro-organisms, the chlorination also had an
effect on species diversity attached to surfaces compared to the non-chlorinated environment.
Principal component analysis (PCA) was utilized to visualize the difference in sample clustering
according to the species detected. In the PCA analysis, there was a clear division of biofouling species
Materials 2016, 9, 475 13 of 21

between the chlorinated and non-chlorinated environment. Alphaproteobacteria dominated in the

biofilm in the non-chlorinated environment regardless of the material, and the Rhodobacteraceae
family was abundant on the surfaces of non-chlorinated samples. The relative abundance
of alphaproteobacteria increased with longer exposure time, and the biofilm became thicker
over time. In chlorinated samples, the majority of the alphaproteobacteria belonged to the
families Kordiimonadaceae, Rhodobacteraceae and Erythrobacteraceae. Many bacteria belonging
to Rhodobacteraceae are phototrophic iron oxidizing [10], whereas the Kordiimonadaceae family is
often detected from marine environments where they degrade hydrocarbons [11]. Members of the
Erythrobacteraceae family are also commonly detected from marine systems and may oxidize organic
carbon compounds [12]. Erythrobacteraceae may also oxidize manganese, a process that has been
linked to localized corrosion of stainless steels [13]. Bacteria belonging to both Rhodobacteraceae and
Erythrobacteraceae have previously been identified as forming biofilms on copper-based antifouling
coating in marine systems [14]. In fact, Erythrobacteraceae bacteria had 10–100-times higher relative
abundances in the biofilms of the chlorinated systems compared to the non-chlorinated ones (Table S2).
Gammaproteobacterial Pseudoalteromonas species were tolerant to chlorination and formed the
majority of the biofilm in the chlorinated environment. Some Pseudoalteromonas species produce
compounds that have antibacterial, algicidal, antifungal or antiviral activity and may thus have
a beneficial position when competing for space in biofilm [15]. On the other hand, certain
Pseudoalteromonas species attract other organisms to surfaces [16]. Pseudoalteromonas species originating
from marine water have been shown to cause corrosion of duplex stainless steel where the bacteria
induced local corrosion by decreasing the chromium content under the biofilm and accumulated
chloride ions on the metal surfaces [17]. The accumulation of chloride, as well as the apparent
enrichment of Pseudoalteromonas in the biofilms of the 254SMO and Ti alloy coupons of our chlorinated
systems indicate that this bacterium may not be susceptible to the chlorination treatment. The relative
abundance of Bacteroidetes in biofilm in a chlorinated environment was also higher compared to the
non-chlorinated system and the majority of these resembled hetero-organotrophic Roseivigra species.
After prolonged exposure time, the composition of the biofilms became more uniform on the surface
of different materials.
Most studies concentrate mainly on the bacterial fraction of biofouling, and thus, the role
of the archaeal community in biofouling is not well known. Archaeal diversity on surfaces was
significantly affected by chlorination, but although a small part of the archaeal community consisted
of hydrogenotrophic methanogenic archaea, in both the chlorinated and non-chlorinated treatments,
Crenarchaeotal lineages (Thaumarchaeota and MCG) dominated. Our results agree with previous
studies on the MIC of oil pipes [18,19] or carbon steel in marine environments [20], where the archaeal
counterparts causing corrosion have belonged to different hydrogenotrophic methanogenic species.
However, to the best of our knowledge, Crenarchaeotal (Thaumarchaeota and MCG) or Parvarchaeota
species have not been associated with the MIC of stainless steel or titanium, consistent with our
observations. It is very possible that these archaea do not primarily cause corrosion, but they may
facilitate MIC and biofilm formation by capturing inorganic carbon into the organic form and recycle
nitrogen compounds (ammonia) from decomposing senescent biofilms for the benefit of the other
microbial community [21,22] members that cause MIC. In addition, the MCG archaea have been shown
to have genes encoding extra-cellular protein-degrading enzymes [23], which would assist in the
degradation of organic matter, such as old biofilms, releasing ammonia for the Thaumarchaeota, which
may then produce nitrite and nitrate for the nitrite- and nitrate-reducing bacteria.
Fungi were generally detected at lower concentrations than the bacteria and archaea, but the
fungal communities on the specimen surfaces and in the water were surprisingly diverse. Despite their
low abundances, fungi may play a great role in the formation of biofilms, because through their
metabolism, the fungi consume oxygen efficiently, thus providing anoxic microhabitats for, e.g.,
anaerobic sulfate reducers or the methanogenic archaea to operate. Ascomycetes fungi, such as
Aspergillus niger, have been reported to cause corrosion on magnesium alloy in artificial seawater [24].
Materials 2016, 9, 475 14 of 21

Diverse fungi also caused severe corrosion damage to storage tanks and transporting pipelines for
oil [25]. Ascomycetes oxidize manganese [26], and fungi secrete different types of exudates, such
as organic acids and enzymes, which may locally lower the pH and induce localized corrosion and
cracking [27]. In agreement with our results, Pereira et al. [28] also found a variable effect of chlorination
on the inhibition of Ascomycetes fungi, and Ascomycetes were detected in both the chlorinated
and non-chlorinated systems. Clearly, species belonging to Chytridiomycota, Rozellomycota and
Zygomycota were more susceptible to chlorination than the Ascomycota, while species of the
Monoblepharidomycetes were detected only in the chlorinated treatments.
Chlorophyta chloroplasts were relatively abundantly detected in the bacterial 16S rRNA gene
sequence data, especially in the non-chlorinated systems, indicating the presence of eukaryotic green
algae. Chlorophyta are common inhabitants of fouling biofilms on different surfaces (e.g., [29,30]).
Hydrogenase-containing Chlorophyta are able to use cathodic hydrogen and may, under acidic
conditions, contribute to corrosion events [31]. Other non-fungal eukaryotes detected were the
Cercozoa protists, which were especially abundant in the chlorinated systems. These protozoans are
also common in biofilms and may serve as hosts for pathogenic bacteria, such as Legionella [32].
Besides biological fouling, also the extent and nature of organic and inorganic fouling changed
due to chlorination. Because of chlorination, an overall decrease in the extent of fouling was detected.
Carbon was detected in all analyses, indicating that the presence of biofilm also contributed to the
results, but it is clear that much of the information also originated from inorganic deposits on the
surface. The results indicated that in non-chlorinated systems, the biofilm development facilitated
inorganic scaling, particularly SiO2 . Indeed, e.g., SiO2 is a common inorganic constituent in scales
developed in seawater environments [33–35], with the biofilm formation known to further enhance its
deposition on the surfaces [33]. This is also the case here, because carbon and silicon were systematically
detected in the same analyses. In the chlorinated systems, the contribution of “softer” inorganic
constituents, such as Ca, Mg and Al, increased, and SiO2 that forms hard and tenacious deposits
correspondingly decreased, thus demonstrating the viability of chlorination in the scale modification.
This is an important aspect with respect to long-term performance and maintenance of seawater cooling
systems, because the removal of SiO2 scales may be challenging due to their high hardness [35].
The composition of the coating was studied prior to exposure and was detected to contain
heterogeneous composite structure, having abundant fillers. The most common fillers were oxides of
magnesium and silicon with niobium, tantalum, molybdenum and barium. It is possible that barium
is included in the coating as barium sulfate, a common anti-fouling agent used due to its favorable
price [36]. In EDS spectra, the peaks of molybdenum and sulfur overlap, hiding the sulfur in the results.
Furthermore quartz, SiO2 , is a frequently-used low-price filler that improves the wear resistance
of the coating. Another important practical finding is the enhanced cracking of the epoxy coating
Inerta160 in the chlorinated system. It is expected that the cracks did not reach the substrate during
the three-month exposure period, because no corrosion products of steel were detected in the epoxy
coating. However, the operation of epoxy coatings relies on the barrier effect, i.e., the non-conducting
layer physically insulating the substrate (here steel) from the environment, which was challenged by
chlorination. Earlier, UV radiation [37] and thermal oxidation [38] have been linked with the cracking
of epoxy-based coatings.
The overall experiment period was short, three months at the longest, and lasted for only
one warm period (summer season) when pulse chlorination is utilized to prevent biofouling.
The chlorination was ended for the colder winter season, the same time as the experiment period
ended. The rate of biofilm formation is known to correlate with sea water temperature, which changes
with the season [39]. This is especially important in sub-arctic areas, such as the Baltic Sea area, where
this study was conducted, where the temperatures change drastically between, e.g., summer and
winter periods. To obtain reliable results of the effects of the fouling and chlorination on materials’
performance, experiments lasting for several chlorination periods are needed. Nevertheless, during
Materials 2016, 9, 475 15 of 21

this three-month experiment period, it was already demonstrated that chlorination has a clear effect
on the fouling and species composition of the biofilms developing on the surfaces.

4. Materials and Methods

4.1. Materials
Two of the materials were uncoated: super austenitic stainless steel 254SMO and titanium-based
alloy Ti-6Al-4V. The composition of stainless steel 254SMO was 0.01 wt% C, 0.20% N, 20.0% Cr,
18.0% Ni, 6.1% Mo, 0.7% Cu and Fe (balance). For Ti-6Al-4V, the composition was 6.0 wt% Al,
4.0% V, ď0.25% Fe, ď0.2% and Ti (balance). Test materials also included epoxy-coated carbon steel.
Red epoxy was an abrasion-resistant epoxy Intershield 163 Inerta160 (International Marine Coatings,
Gateshead, UK) intended for corrosion protection applications in the underwater areas of ice-going
vessels. The coatings were applied as recommended by the manufacturer. In each case, the coupon
size was 25 ˆ 75 mm.
Coupons were cleaned with FreeBact-20 (AquaFix, Saltsjöbaden, Sweden) and sterile MQ water
and air-dried. Each biofouling cell contained 8 coupons of each material. Three parallel coupons were
used for material analyses, three coupons for molecular biological analyses and two for microscopy
analyses. The coupons were photographed prior to the experiment.

4.2. Experiment Setup

The test materials were placed in a biofouling cell in the cooling water system of a nuclear power
plant located in the western coast of Finland. Two cells were installed in the cooling water cycle before
the chlorination point and two cells after the chlorination point. The water flow through the biofouling
cell was set at 0.01 m¨ s´1 using rotameters. The chlorination of the cooling water system was started
in July 2015 and lasted 106 days. For chlorination, 15% of Na-hypochlorite was used with a final target
concentration 3 mg¨ L´1 . During the first 30 days, chlorination was applied for two hours with 10 h
brakes between treatments and, after that, for one hour with 11 h breaks between. The experimental
period started a week after chlorination treatment was initiated. One cell before the chlorination point
and one cell after it were removed after 1 month (32 days) of exposure. The remaining two cells were
removed after 3 months (90 days) of exposure. After the removal of the biofouling cells from the water
cycle, the cells were sealed to retain the water inside them. The cells were dismantled aseptically in
the laboratory.

4.3. Surface Characterization

Coupons for corrosion studies were immersed in 96% ethanol, air-dried and placed in
glass desiccators. All coupons for corrosion analyses were photographed and examined with a
stereomicroscope to verify the surface condition. The surface deposits of selected coupons were
analyzed with a scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS).
Furthermore, the as-received Inerta160-coated steel specimens were subjected to investigations by
SEM-EDS to disclose the original coating structure in the surface and cross-section. To enable the
investigations, a thin layer of gold was sputtered on the coated specimens to make them electrically
conductive. Field-emission (FE) SEM Zeiss ULTRAplus equipped with a Noran EDS system was
employed for the investigations. Surface characteristics (hydrophobicity/hydrophilicity) of the
Inerta160 coating were studied using an unexposed specimen by contact angle measurements.
To visualize the biofilm formed on the surface, selected samples were fixed in phosphate (0.1 M,
pH 7.2), buffered with 2.5% glutaraldehyde for 2 h and dehydrated with ethanol series followed
by final drying in hexamethyldisilazane. The specimens were coated with Au/Pd (10–15 nm) and
examined with a Zeiss SIGMA VP field-emission scanning microscope (FESEM; Carl Zeiss SMT GmBH,
Oberkochen, Germany).
Materials 2016, 9, 475 16 of 21

4.4. DNA Extraction

Coupons for microbiological analyses were subjected to biofilm extraction immediately after
they were removed from the biofouling cell. The biomass from the coupon surfaces was removed
by sonicating the coupons for 10 min (Bransonic 2210-DTH, Branson Ultrasonics, Danbury, CT,
USA, 47 kHz, 70W) and thereafter vortexing for 1 min. The biomass suspensions were filtered
into Sterivex-GP 0.22-µm filter units (Millipore, Billerica, MA, USA) and stored frozen (´80 ˝ C) until
DNA was extracted. Non-chlorinated and chlorinated water samples were filtered at the beginning of
the experiment and at both sampling times as two 500-mL replicates to Corning PES (polyethersulfone)
0.22-µm filter units (Corning, New York, NY, USA), which were cut out with a sterile scalpel and
stored frozen (´80 ˝ C). Subsequently, the Sterivex filter units were aseptically broken with a hammer,
and the Sterivex and Corning filters were cut into pieces with a sterile scalpel and placed into the
lysing tube of the DNA extraction kit with sterile forceps. DNA was extracted using the Fast DNA
Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions,
with the modification that the cells were homogenized in a FastPrep-24 instrument (MP Biomedicals,
Santa Ana, CA, USA) at 6 m¨ s´1 for 3 min.

4.5. Quantitative PCR

The bacterial, archaeal and fungal biomass on the surface of the samples and in seawater was
evaluated with quantitative PCR (qPCR) using the LightCycler 480 instrument (Roche Diagnostics,
Basel, Switzerland). The DNA concentration of all samples was adjusted to ď10 ng¨ µL´1 prior to
qPCR in order to avoid PCR inhibition. For bacteria, an approximately 200-bp fragment of the 16S
rRNA gene was amplified with primers 358F (5’-CCT ACG GGA GGC AGC AG-3’) and 534R (5’-ATT
ACC GCG GCT GCT GG-3’) [40] and detected with SYBR green-based detection for double-stranded
DNA. The amplification was done in a 10-µL reaction volume with KAPA Sybr Fast qPCR Master
mix optimized for Roche LightCycler 480 (KAPA Biosystems, Wilmington, MA, USA), 150 nM of each
primer and 1 µL of sample DNA. The amplification reaction consisted of initial denaturation at 95 ˝ C
for 15 min, 45 cycles with 10 s at 95 ˝ C, 35 s at 57 ˝ C and 30 s at 72 ˝ C, a final elongation of 3 min at 72 ˝ C
and a melting curve analysis. As an external standard, a dilution series (100–108 cfu/µL) of Escherichia
coli VTT E-90418 genomic DNA was used. For archaea, an approximately 400-bp fragment of the
16S rRNA gene was amplified with primers A344F (5’-ACG GGG TGC AGC AGG CGC GA-3’) [41]
and A744R (5’-CCC GGG TAT CTA ATC C-3’) modified from 744RA [42] and detected with SYBR
green. The amplification was done in a 10-µL reaction volume with KAPA Sybr Fast qPCR Master mix
optimized for Roche LightCycler 480 (KAPA Biosystems, Woburn, MA, USA), 300 nM of each primer
and 1 µL of template DNA. The amplification reaction consisted of initial denaturation at 95 ˝ C for
15 min, 45 cycles with 10 s at 95 ˝ C, 35 s at 56 ˝ C and 30 s at 72 ˝ C, final elongation of 3 min at 72 ˝ C
and a melting curve analysis. As an external standard, a dilution series of Halobacterium salinarum VTT
E-103154T genomic DNA was used (100–107 cells/µL). Fungal biomass was detected with TaqMan
probe assay using ITS (internal transcribed spacer) as a target. Primers 5.8F1 (5’-AAC TTT CAA
CAA CGG ATC TCT TGG-3’) and 5.8R1 (5’-GCG TTC AAA GAC TCG ATG ATT CAC-3’) and probe
5.8P1 (5’-CAT CGA TGA AGA ACG CAG CGA AAT GC-3’) were used [43]. The amplification was
done in 10 µL reaction volume with KAPA PROBE FAST qPCR Kit (KAPA Biosystems, Woburn, MA,
USA), 500 nM of each primer, 200 nM of probe, and 1 µL of template DNA. The amplification reaction
consisted of enzyme activation at 95 ˝ C for 3 min and 40 cycles with 10 s at 95 ˝ C (denaturation),
30 s at 62 ˝ C (annealing) and 1 s at 72 ˝ C (elongation). As an external standard, a dilution series of
Aspergillus versicolor VTT D-96667 genomic DNA was used (10–105 spores/µL).

4.6. Amplicon Library

The amplification libraries for high throughput sequencing with Ion Torrent PGM were
prepared by PCR from the DNA samples. Bacterial 16S genes were amplified with primers
Materials 2016, 9, 475 17 of 21

S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21 [44], targeting the variable region V3-V4 of the 16S

rDNA gene, S-D-Arch-0349-a-S-17/S-D-Arch-0787-a-A-20 [44], archaeal 16S genes with primers
S-D-Arch-0349-a-S-17/S-D-Arch-0787-a-A-20 [44], targeting the V4 region of the gene and fungal
internal transcribed spacer (ITS) gene markers with primer pair ITS1 and 58A2R targeting the
fungal ITS1 region [45,46]. PCR amplification was performed in parallel 25-µL reactions for every
sample containing 1ˆ MyTaq™ Red Mix (Bioline, London, UK), 20 pmol of each primer, up to 25 µL
molecular-biology-grade water (Sigma, St. Louis, MO, USA) and 2 µL of template. The PCR program
consisted of an initial denaturation step at 95 ˝ C for 3 min, 35 cycles for bacteria and fungi and 40 cycles
for archaea of 15 s at 95 ˝ C, 15 s at 50 ˝ C and 15 s at 72 ˝ C. A final elongation step of 30 s was performed
at 72 ˝ C. The PCR products were verified with agarose gel electrophoresis. Amplicons were sent to Ion
Torrent sequencing with PGM equipment (Bioser, Oulu, Finland), and amplicons were purified before
sequencing at Bioser.

4.7. Sequence Processing and Analysis

The sequence reads obtained from Ion Torrent sequencing were subjected to quality control
using the QIIME-software Version 1.8 [47] using a minimum quality score of 20, minimum and
maximum sequence length of 200 bp and 600 bp, respectively, maximum primer mismatch of
2 nucleotides (nt) and maximum homopolymer stretches of 8 nt. Adapters, barcodes and primers
were removed from the sequence reads, and chimeric sequence reads were removed from the dataset
with the USEARCH-algorithm [48] by de novo detection and through similarity searches against
the Greengenes reference dataset (Version gg_13_8) [49] with bacterial and archaeal sequences, as
well as the UNITE (User-friendly Nordic ITS Ectomycorrhiza Database) reference dataset (Version
sh_taxonomy_qiime_ver7_97_s_31.01.2016) [50] with fungal sequences.
The sequences were grouped into Operational Taxonomic Units (OTUs), following the
open-reference OTU-picking protocol of QIIME-software. First, all reads that failed to hit the
Greengenes or UNITE reference database with a minimum of a 60% identity threshold were discarded
as sequencing errors. Subsequently, closed-reference OTUs were picked at 97% clustering identity
against the Greengenes or UNITE database, and de novo OTUs were picked from a randomly
subsampled sequence subset that failed the closed-reference OTU-picking stage. Next, singleton OTUs,
i.e., OTUs that were represented by a single sequence, were filtered from the dataset. Finally, taxonomy
from the domain level to the species level was assigned to OTUs via representative OTU sequences
with the Ribosomal Database Project (RDP) classifier algorithm at a minimum of 80% confidence [51]
with bacterial and archaeal sequences. With fungal ITS sequences, taxonomic assignments were
made by the Basic Local Alignment Search Tool (BLAST) algorithm with a maximum E-value of
0.001 [52]. Unassigned and unidentified sequences were filtered from the datasets before alpha
diversity estimations. Alpha diversity indices were calculated on randomly subsampled and filtered
datasets containing 1243, 1674 and 1624 sequences for ITS, bacteria and archaea, respectively.
The sequences were deposited in the European Nucleotide Archive (ENA, https://www.ebi.ac.
uk/) under accession number PRJEB14388.

4.8. Statistics Analyses

Significant differences of the qPCR means were tested with variance analysis using sampling
time, surface material and chlorination as fixed factors and the subsequent Tukey’s post hoc test using
IBM SPSS Statistics Version 21 software. The results were log-transformed to fulfil the assumptions of
variance analysis.
Differences in the number of OTUs detected from the different biofilm samples, the Chao1 richness
estimates and Shannon indices were tested based on normalized sequence data using One-way ANOVA
in the PAST3 program [52]. We tested significant differences of the mean between chlorinated and
non-chlorinated samples using Levine’s test for the homogeneity of variance, Kruskal–Wallis and
Tukey’s Q test. The difference in community composition between all samples was tested with principal
Materials 2016, 9, 475 18 of 21

component analysis of 100 bootstrap repeats on the taxonomic relative abundance data using the
PAST3 program.

5. Conclusions
The present work demonstrates the powerfulness of molecular biological tools in studying
biofouling, especially microfouling in cooling water environments. Biofilm formation in the
non-chlorinated system was intensive, but also in the chlorinated system, a biofilm consisting of
bacteria, archaea, fungi, algae and protozoa was detected. According to the results presented here, the
chlorination reduces microfouling in brackish water by 10–1000-fold, but also alters the structure of
the remaining community forming the biofilm on surfaces and the composition of inorganic scaling.
In addition, the choice of material had an effect on the biofilm-forming community in the beginning, but
prolonged exposure time unified the composition of the biofilm-forming community between different
materials. Multiple potentially corrosion-inducing species, e.g., manganese oxidizing, sulfate reducing
and iron oxidizing bacteria, were detected on the surfaces of all materials. In addition to having a
reducing and species selecting influence on biofilm formation, the chlorination also deteriorated the
epoxy coating already during one month of exposure. On the other hand, coating combined with
chlorination further reduced the microfouling on the surfaces compared to metallic surfaces.

Supplementary Materials: The following are available online at www.mdpi.com/1996-1944/9/6/475/s1.

Figure S1: EDS spectra for coupons. (a) 254SMO chlorinated; (b) Ti alloy chlorinated; (c) 254SMO non-chlorinated
system; (e) Ti alloy non-chlorinated system. Figure S2: SEM images. (a) Surface; (b) cross-section; (c–e) EDS
spectra for particles included in the Inerta160 coating. Figure S3: Temperature (˝ C) of the seawater during the
experiment. Table S1: Diversity and sequence stats. Table S2: Bacteria, relative abundance and genus level.
Table S3: Archaea, relative abundance and genus level. Table S4: Fungi, relative abundance and genus level.
Acknowledgments: Finnish Metals and Engineering Competence Cluster (FIMECC) as a part of Breakthrough
steels and applications program and Teollisuuden Voima Oyj (TVO) are thanked for funding this work.
Pekka Simula and Merja Levy of TVO Olkiluoto Nuclear Power Plant are thanked for their skillful
assistance during the sampling and setting up of the experiment. Irina Tsitko is thanked for FE-SEM
imaging. FE-SEM imaging made use of the Aalto University Nanomicroscopy Center (Aalto-NMC) premises.
Jarkko Metsäjoki is thanked for performing EDS analyses, and Mirva Pyrhönen, Taru Lehtikuusi and
Tuomo Kinnunen are thanked for technical assistance.
Author Contributions: P.R., M.B., E.H.-S., O.P., M.T. and L.C. conceived of, designed and performed the
experiments, analyzed the data and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript:
ANOVA Analysis of variance
BLAST Basic Local Alignment Search Tool
bp Base pair (in DNA)
DNA Deoxyribonucleic acid
FE-SEM Field emission scanning electron microscopy
EDS Energy-dispersive spectroscopy
ITS fungal internal transcribed spacer
MCG Miscellaneous crenarchaeotal group
OTU Operational taxonomic unit
PCA Principal component analysis
PCR Polymerase chain reaction
STD Standard deviation
Ti in this paper, Ti-6Al-4V
TOC Total organic carbon
Materials 2016, 9, 475 19 of 21

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