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Biofouling
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The development of marine biofilms on two
commercial non-biocidal coatings: a comparison
between silicone and fluoropolymer technologies
Sergey Dobret sov
a
& Jeremy C. Thomason
b
a
Marine Science and Fisheries Depart ment , College of Agricult ural and Marine Sciences,
Sult an Qaboos Universit y, Al Khoud 123, PO Box 34, Oman
b
Marine Ecological Services, 10 Boulevard Barbes, Paris, 75018, France
Available online: 25 Aug 2011
To cite this article: Sergey Dobret sov & Jeremy C. Thomason (2011): The development of marine biof ilms on t wo commercial
non-biocidal coat ings: a comparison bet ween silicone and f luoropolymer t echnologies, Biof ouling, 27: 8, 869-880
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Biofouling
Vol. 27, No. 8, September 2011, 869–880
The development of marine biofilms on two commercial non-biocidal coatings: a comparison
between silicone and fluoropolymer technologies
Sergey Dobretsova* and Jeremy C. Thomasonb
a
Marine Science and Fisheries Department, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al Khoud 123 ,
PO Box 34, Oman; bMarine Ecological Services, 10 Boulevard Barbes, Paris, 75018, France
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(Received 2 May 2011; final version received 19 July 2011)
The antimicrobial performance of two fouling-release coating systems, Intersleek 7001 (IS700; silicone technology),
Intersleek 9001 (IS900; fluoropolymer technology) and a tie coat (TC, control surface) was investigated in a short
term (10 days) field experiment conducted at a depth of ca 0.5 m in the Marina Bandar Rawdha (Muscat, Oman).
Microfouling on coated glass slides was analyzed using epifluorescence microscopy and adenosine-50 -triphosphate
(ATP) luminometry. All the coatings developed biofilms composed of heterotrophic bacteria, cyanobacteria, seven
species of diatoms (2 species of Navicula, Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillaria
sp.) and algal spores (Ulva sp.). IS900 had significantly thinner biofilms with fewer diatom species, no algal spores
and the least number of bacteria in comparison with IS700 and the TC. The ATP readings did not correspond to the
numbers of bacteria and diatoms in the biofilms. The density of diatoms was negatively correlated with the density of
the bacteria in biofilms on the IS900 coating, and, conversely, diatom density was positively correlated in biofilms on
the TC. The higher antifouling efficacy of IS900 over IS700 may lead to lower roughness and thus lower fuel
consumption for those vessels that utilise the IS900 fouling-release coating.
Keywords: microfouling; bacteria; diatoms; microbial biofilms; antifouling coating; fouling-release; amphiphilic
coating
Introduction
Biofilms are sessile assemblages of microbes attached
to each other and to an interface, and are enclosed in
an exopolymeric matrix (Lewandowski 2000). Attachment of different biofilm-forming microorganisms
depends on the properties of the substratum (eg Dexter
et al. 1975; Cooksey and Wigglesworth-Cooksey 1995;
Becker et al. 1997; Jain and Boshle 2009; Mitik-Dineva
et al. 2009). Biofilms have distinctive architectures, the
exact form of which depends on both chemical (ie the
presence of particular ions and compounds) and
physical (ie the boundary layer properties or critical
surface tension) environmental parameters (Lewandowski 2000; Molino and Wetherbee 2008). Marine
biofilms on man made surfaces consist mainly of
numerous species of bacteria and diatoms that can
positively and/or negatively interact with each other
(Railkin 2003; Dobretsov 2010). It has been shown
that some biofilm-associated bacteria produce compounds that promote the growth of diatoms (Wigglesworth-Cooksey and Cooksey 2005), while others
inhibit growth of microorganisms (see reviews by
Dobretsov et al. 2006; Qian et al. 2010). Both bacteria
and diatoms may also have a significant impact on the
*Corresponding author. Email: sergey_dobretsov@yahoo.com
ISSN 0892-7014 print/ISSN 1029-2454 online
Ó 2011 Taylor & Francis
DOI: 10.1080/08927014.2011.607233
http://www.informaworld.com
recruitment of some invertebrate larvae and algal
spores by either enhancing or inhibiting their settlement (see reviews by Wieczorek and Todd 1998; Qian
et al. 2007; Prendergast 2010; Hadfield 2011). Biofilms
can change the physical and chemical properties of a
substratum and correspondingly modify macrofouling
attachment (Becker et al. 1997; Huggett et al. 2009).
Biofouling causes severe problems for marine
industries by increasing corrosion and hydrodynamic
drag which leads to elevated fuel consumption and
higher maintenance costs (Yebra et al. 2004; Schultz
2007; Schultz et al. 2011). Thus, marine installations
and vessels are protected against biofouling with
antifouling (AF) paints/coatings, which are still mostly
biocidal in nature (Yebra et al. 2004; Thomas and
Brooks 2010). Non-biocidal AF coatings have been
available since the 1950s, but economics (the relatively
low price of biocidal coatings) and the difficulty in
applying such coatings to hulls prevented their widespread use until relatively recently (see Finnie and
Williams 2010 for a recent review). Initially all the
commercially available non-biocidal coatings were
based on the silicone polymer polydimethylsiloxane
which minimizes adhesion of fouling organisms. These
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870
S. Dobretsov and J.C. Thomason
coatings work on the principle that loosely attached
organisms are removed by shear as the hull moves
through the water at high speed and are thus known as
foul release or fouling-release (FR) coatings.
Intersleek 7001 and Intersleek 9001 are two nonbiocidal FR coating systems manufactured by International Paints Ltd. The Intersleek 700 (IS700) FR
system was patented in 1975 (Patent GB1470465) and
launched in 1999. The system is based on a silicone top
coat and it is primarily marketed for vessels such as
container ships, liquid natural gas carriers and cruise
vessels, whose speed and activity ensures adequate
shear for the removal of fouling organisms. Intersleek
900 (IS900) top coat is a fluoropolymer modified
polyorganosiloxane composition which was patented
in 2002 (Patent WO2002074870) and the product
launched in 2007. As a second generation FR system,
with improved performance against macrofouling
organisms, IS900 is marketed for slower or lower
activity vessels such tankers, bulkers and general cargo
ships, as well as those originally targeted by IS700.
The development of biofouling on FR and traditional AF coatings has been subject to several
investigations that have utilized destructive collection
and examination techniques (Robinson et al. 1985;
Callow 1986; Cassé and Swain 2006). These investigations demonstrate that FR coatings are very successful
against macrofouling organisms, such as barnacles
(Aldred and Clare 2008) and algae (Joshi et al. 2009),
but are still subject to biofilm colonisation. For
example, Molino et al. (2009b) found that the
percentage of bacterial cover on IS700 was 1.3–2 times
higher than on two biocidal coatings, Intersmooth
3601 and Super Yacht 8001. They also demonstrated
that within 2–4 days after immersion IS700 displayed
the quickest microbial colonization in comparison with
the biocidal coatings Intersmooth 360 and Super
Yacht 800 and microbial cover reached 470% after
16 days. Bacterial colonisation and growth on Super
Yacht and Intersmooth demonstrated an exponential
rate of growth, while bacterial development on IS700
revealed a logarithmic growth rate. Microfouling on
the new generation FR coatings, such as IS900, has not
yet been investigated.
Cassé and Swain (2006) found that while the
bacterial genera Micrococcus and Pseudomonas were
present on all coatings, Vibrio sp. was found specifically on FR coatings. Microbial biofilms on biocidal
and FR coatings typically contain diatoms belonging
to the genera Amphora, Navicula, Nitzschia, Licmophora, Navicula, and Achnanthes (Cassé and Swain
2006; Molino and Wetherbee 2008; Molino et al.
2009a). Species of the diatom Amphora are most
common on copper-based paints, Achnanthes and
Amphora on TBT paints (Callow 2000), while mixed
diatom communities have been found to dominate FR
coatings (Cassé and Swain 2006). The general differences in the bacterial and diatom assemblages between
biocidal paints and FR coatings are probably the result
of toxicity of the biocidal coatings.
The growth of biofilms on biocidal and FR
coatings increases shear stress and drag, leading to
increased fuel consumption (Yebra et al. 2006;
Edyvean 2010; Shultz et al. 2011). For example, a
1 mm thick biofilm developed on a 23 m ship caused
an 80% increase in friction drag and caused 15% loss
in ship speed (Lewthwaite et al. 1985). Formation of
‘heavy slime’ increases fuel consumption by 10.3%
which incurs an additional fuel cost of $1.15M per
naval vessel per year (Schultz et al. 2011).
Different microbial species have different dimensions and differential growth rates, thus potentially
affecting drag and shear stress of biofilmed coatings
to different extents (Howel 2009). Thus, the quantitative and qualitative description of biofilms (ie
number of species present, their thickness and
biomass) is important for better predictive systems
engineering. Traditionally, the densities of bacteria in
biofilms have been determined as colony forming
units (Cooksey and Wigglesworth-Cooksey 1995), but
this technique does not allow the determination of
uncultivable microbes that are revealed by direct
microscope counting using fluorescent dyes that stain
DNA, such as 40 ,6-diamidino-2-phenylindole (DAPI;
Kirchman et al. 1982). The densities of diatoms
determined by light or electron microscopy or
chlorophyll measurements can result in false results,
as discussed by Cooksey and Wigglesworth-Cooksey
(1995). Direct light microscopy and chlorophyll
measurement can be highly sensitive to low cells
numbers, while electron microscopy is not. However,
light microscopy has the advantage that a larger
surface area can be assessed for a given amount of
time. Biofilm thickness is an important parameter in
biofilm characterization as it will reflect the density
and the activity of the microbial assemblage in terms
of EPS formation. The biomass of a biofilm and the
number of cells determines the amount of adenosine5’-triphosphate (ATP) present. Therefore, determination of ATP using calibrated luciferase firefly
luminescence has been widely used for quick determination of bacterial contamination (Siragusa et al.
1995; Frundzhyan and Ugarova 2007) and bacterial
adhesion (Dexter et al. 2003) in medicine and
industry.
Given the importance of biofilms in contributing to
hull fouling and the consequent increase in drag and
hence fuel penalty, the aim of this study was to
compare the initial development of marine biofilms on
IS700 and IS900 in a challenging tropical marine
Biofouling
environment to see if the general anti-macrofouling
efficacy improvement of IS900 over IS700 extends to
improved efficacy against biofilms.
Materials and methods
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Field experiment
Coating preparation
Three coating treatments were applied to standard
glass microscope slides (Fisher UK, 76 6 26 6 0.8
mm) using normal commercial application protocols
for each of the coatings (Intersleek 970 . . . 2009;
Intersleek 731 . . . 2010a; Intersleek 757 . . . 2010b; Intershield 300 . . . 2010c), at International Paint Ltd, Felling, UK. The coating treatments were the standard FR
anticorrosive primer and tie coat (TC) finished with
either the top coat for IS700 (silicone) or IS900
(fluoropolymer) coating system, or left unfinished
leaving the TC exposed. The TC was considered to
be the control and was used to provide a reference
surface on which biofilms would be expected to be
highly adherent. The primer used was Intershield
3001, the TC used was Intersleek 7311, the IS700
top coat was Intersleek 7571, and the IS900 topcoat
was Intersleek 9701. A complete system of primer, TC
and Intersleek 970 is known as the IS900 coating
scheme. Likewise a complete scheme with a top coat of
Intersleek 757 is known as the IS700 coating scheme.
The Intershield 300 primer and Intersleek 731 TC are
both two-pack products while Intersleek 757 and
Intersleek 970 are three-pack top coats. The mixing
ratio for the primer was 2.5:1, and TCs 1:1 by volume
and the mixing ratios for Intersleek 757 and Intersleek
970 was 15:4:1 and 16:3:1 by volume, respectively.
Both the top coats were standard commercial red and
the TC was pink. These schemes are the conventional
schemes as used on commercial vessels; the primer was
only included to ensure that a complete commercial
scheme was used and was not strictly necessary for
inclusion of its anticorrosive properties in this
experiment.
All the applications were carried out by airless
spray at 208C using a 0.48 mm tip at a pressure of
26.5 6 106 Pa to give a 42 cm standard fan width at
30 cm from the surface. Coated slides were dried at
358C for 5 h between anti-corrosive primer and TC
applications and at the same temperature for 24 h
before the application of the top coats. Nominal
thicknesses were 150 mm for the primer, 100 mm for
the TC and 150 mm for the top coats (Intersleek
970 . . . 2009; Intersleek 731 . . . 2010a; Intersleek
757 . . . 2010b; Intershield 300 . . . 2010c). After the
whole scheme was applied, coated slides were dried
at ambient temperature for 5 days prior to dispatch
to Oman for deployment.
871
Replication
Microscope slides are convenient for sending by
courier and immediately usable in microscopy, without
the need for sampling or reduction in size, and thus the
biofilms could be examined intact and undamaged.
However, since microscope slides present a small
surface for study and, as biofilms are often spatially
heterogeneous (Augspurger et al. 2010; Wagner et al.
2010), a high level of replication was used, consisting
of 84 slides of each treatment (ie 252 slides in total).
For each coating tested, the area for biofilm development was 0.16 m2, giving a total area for biofilm
growth in the experiment of 0.48 m2. The limits on the
number of slides deployed were imposed by the
necessity of some of the analytical methods having to
be completed in a relatively short time.
Deployment
The 252 coated slides were deployed in a replicated
randomised block design, with three blocks, such that
28 replicates of each treatment were randomly
allocated to a position in each block. Each block
corresponded to a single deployment frame that was
made up of a set of slide holders. The slide holders
were constructed from PVC. Each holder was made
from a PVC plate (986 6 151 mm) with two narrow
PVC battens (986 6 50 mm), one each side, used to
hold the slides to the PVC plate. The battens were held
in place by bolts and washers and a strip of neoprene
was placed under them to help trap the slides in place
without recourse to undue tightening of the bolts.
There were 21 equally spaced slides in each holder.
Four slide holders were attached lengthwise, next to
each other, to a 4 m long PVC pipe. The four slide
holders with slides attached to one pipe were considered to be the deployment frame and thus also an
experimental block. Three frames were made to give
three blocks. Each frame was deployed by ropes
attached at each end such that the whole frame was
kept parallel to the surface of the water. Each slide
holder on the frame was arranged such that the slides
in the holders were kept vertical with respect to the
surface as this limited amount of detritus on the slides.
Slides were deployed for 10 days at a depth of
*0.5 m in the semi-enclosed Marina Bandar Rawdha
(Muscat, Oman 238340 5500 N 588360 2700 E) in April 2010
and the distance between the blocks was *1 m.
Previous experiments had shown that longer deployment of the slides resulted in development of very thick
three dimensional biofilms, as well settlement of larvae,
which would have made clear visualization of the
substratum difficult. During the experiment the average water temperature was 28.88C and salinity was
872
S. Dobretsov and J.C. Thomason
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35.5 ppt. At the end of the experiment the slides were
retrieved from the water and placed into slide boxes
(Fisher Scientific, USA) with fresh filtered (0.22 mm)
seawater from the experimental site. ATP in each
biofilm was detected (see below) before each slide was
fixed with 4% formalin solution in artificial seawater.
The slides in the formalin were brought to the
laboratory in a large container (volume ¼ 15 L) and
processed (see below) within several weeks.
Biofilm quantification
In the laboratory the following components were
measured to quantify the biofilms: ATP, the number
of diatoms in each identifiable genus, the number of
bacteria and the thickness of the biofilm. All quantification was performed blind.
ATP measurements
For the ATP measurements, the slides were swabbed
with sterile 3 Ultrasnap1 Hygiena swabs (Hygiena,
UK) and the ATP measured using a SystemSURE II1
handheld luminometer (Hygiena, UK) according to
the manufacturer’s protocol. Briefly, for each slide,
three random horizontal swabs (each *125 mm2) were
taken which left an area of approximately 1500 mm2
for the quantification of diatoms and bacteria (see
below). Each swab was individually mixed with the
solution provided and ATP was measured as relative
light units (RLU) within 60 s. As a control, a clean
Hygiena swab was used. All ATP measurements were
made at a shady location of the marina on fresh
(unfixed) slides.
Quantification of diatoms and bacteria
Bacteria and diatoms were quantified according to
Dobretsov et al. (2005) in the area of each slide that
was left undisturbed after swabs were taken for the
ATP measurements. Biofilms were stained with a
100 ng ml71 solution of 40 ,6-diamidino-2-phenylindole
(DAPI, Sigma, Germany) in filtered (0.2 mm) seawater
for 15 min in the dark. The number of bacteria in 10
randomly selected fields of view (area ¼ 0.001 mm2)
was counted with an epifluorescence microscope
(Axiophot, Zeiss, Germany; magnification 10006;
lEx ¼ 359 nm, lEm ¼ 441 nm). The values obtained
were transformed into number of individuals per mm2.
The number of diatoms in 10 randomly selected fields
of view (area ¼ 3.5 mm2) was counted with a microscope (Nikon Eclipse, USA; magnification 4006). The
main genera were determined and their densities per
mm2 calculated. The thickness of the biofilms was
determined by using the calibrated fine focus control to
focus on the air/film and film/substratum interfaces
(see Yuehuei and Friedman 2000) of the microscope in
10 randomly selected fields of view.
Statistical analysis
Hypothesis testing was undertaken with linear mixed
modelling (LMM) in SPSS v18 following the protocols
described by McCulloch and Searle (2000) and
Verbeke and Molenberghs (2000). This approach
allows for the effect of a fixed factor to be determined
whilst taking into account the effect of a random
factor, as well as being able to include repeated
measurements on the same subject (McCulloch and
Searle 2000). For the LMMs, the response variables
were biofilm thickness, ATP score, number of bacteria
and diatoms, and species and richness of diatoms. To
achieve normality prior to modelling, data for the
number of bacteria and diatom species richness were
transformed by taking the natural log and diatom
counts were transformed by using the fifth root;
biofilm thickness and ATP score were not transformed.
For the response variables determined using microscopy, field of view number (N ¼ 10) was used to
identify the repeated measurements and slide number
was used to identify the unique subject (N ¼ 252).
Swab number (N ¼ 3) was used to identify the
repeated ATP measurements. Each LMM was estimated with coating as the fixed factor and block as the
random factor with an interaction between block and
coating included. Type III sums of squares were used
for fixed factor effect calculations. Estimation of the
variance/covariance repeated measures matrix was
initiated as an ante-dependence first order structure
for all variables except diatom numbers and richness
which required a diagonal matrix structure. A scaled
identity structure was used to initiate estimation of the
random effects variance/covariance matrix. Model
convergence and Akaike’s information criterion
(AIC) were used to determine the optimal covariance
matrix structure. Given the large sample size the LMM
was estimated using the maximum likelihood method.
A pairwise contrast test with Bonferroni correction
was used to determine differences between the coatings. Full details about the above procedures can be
found in McCulloch and Searle (2000) and Verbeke
and Molenberghs (2000). The surfaces of 45 slides were
obscured by large numbers of macroalgal spores which
prevented accurate enumeration of the bacteria; these
were excluded from the above analyses.
To determine the magnitude of the association
between the constituents of the biofilms, non-parametric Spearman’s correlation coefficients (r) were
calculated for all pairwise associations between numbers of diatoms and bacteria, diatom species richness
and biofilm thickness. This analysis was deemed to be
largely descriptive and was an attempt to describe the
Biofouling
873
effect of the coating on the assemblage within the
biofilm, hence the only hypothesis tested was that the
correlation 6¼ 0 and no adjustments for multiple
correlations were made.
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Results
All the coatings developed biofilms during the 10 day
deployment. These biofilms were made up of heterotrophic bacteria (no morphotype data recorded),
cyanobacteria (two distinguishable morphotypes),
seven species of diatom (two species of Navicula,
Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillaria sp.) and algal spores (probably
Ulva sp.). There were highly significant differences
between the coatings for each of the response
variables, namely numbers of bacteria and diatoms,
diatom species richness, ATP reading, and biofilm
thickness (Figure 1 and Table 1). IS900 had
significantly thinner biofilms, lower mean diatom
species richness and the least number of bacteria
(Figure 1, Tables 2 and 3). All diatom species were
found on all coatings. There was no significant
difference in ATP readings (RLU) and the number
of diatoms between IS900 and TC, whereas both of
these parameters were significantly higher for IS700
coated slides (Table 2). Thus, a general overview is
that IS900 had less biofilm with lower microbial
diversity and activity than IS700.
The ATP results did not show the same a pattern
across the three coatings as that shown for the other
variables measured, ie a consistently higher ATP RLU
was expected for surfaces with higher bacterial and
diatom counts (Figure 1, Table 2). Indeed, a correlation analysis found that the only significant result was
between ATP and the number of diatoms, but that this
was very weak (Spearman’s r ¼ 0.15, p 5 0.001,
N ¼ 621). An ordinary least squares regression analysis of ATP vs transformed diatom counts was
significant (ATP ¼ 544.96 þ 432.47 6 Transformed
Diatom Count, p 5 0.001), but the very small coefficient of determination, r2 ¼ 0.029, shows that diatoms
only accounted for *3% of the variation in the ATP
readings, and thus these data for ATP, although
significantly different between coatings, clearly do not
reflect bacterial or microalgal density in the biofilms,
and thus should be treated with a strong degree of
caution.
Of the 45 slides that were excluded from the LMM
analysis due to the presence of large numbers of algal
spores, 21 slides were coated with IS700 and 24 with
the TC; no slides with a finish coating of IS900 had
attached algal spores. There were significantly more
(p 5 0.001, Mann–Whitney U test) algal spores on the
TC (
x ¼ 19.52+18.78SD spores mm72) than on IS700
(
x ¼ 7.43+9.48SD spores mm72).
Figure 1. Mean and 95% CI plots of (a) ATP reading
(RLU, Hygiena systemSURE II relative light unit, N ¼ 621);
(b) biofilm thickness (mm, N ¼ 2070); (c) number of bacteria
(No. mm72, N ¼ 2070); (d) number of diatoms (No. mm72,
N ¼ 2070); (e) diatom species richness (N ¼ 2070).
874
S. Dobretsov and J.C. Thomason
Table 1.
Summary of linear mixed modelling for the fixed factor, coating.
Response variable
Numerator DF
Denominator DF
F
P
2
2
2
2
2
206.01
207.00
203.61
202.87
160.11
16.51
10.50
36.16
29.93
8.804
50.001
50.001
50.001
50.001
50.001
Bacteria (No. mm72)
ATP (Hygiena systemSURE II relative light unit)
Biofilm thickness (mm)
Diatoms (No. mm72)
Diatom species richness
Note: DF ¼ degrees of freedom; F ¼ F statistic; P ¼ probability.
Table 2. Summary of Bonferroni-adjusted p values for a pairwise contrast test of the estimated marginal means for coating
treatment for all the linear mixed models.
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Response variable
Reference
coating
IS700
IS900
TC
Comparison
coating
Bacteria
(No. mm72)
Diatom
species
richness
Diatoms
(No. mm72)
ATP (Hygiena systemSURE II
relative light unit)
Biofilm
thickness (mm)
IS900
TC
IS700
TC
IS700
IS900
0.001
0.198
0.001
50.001
0.198
50.001
50.001
0.995
50.001
50.001
0.995
50.001
50.001
0.018
50.001
0.309
0.018
50.001
0.002
50.001
0.002
0.744
50.001
0.744
50.001
0.131
50.001
50.001
0.131
50.001
Note: IS700 ¼ Intersleek 7001; IS900 ¼ Intersleek 9001; TC ¼ tie coat control.
Table 3. Summary of mean (and SD) of physico-chemical surface and biofilm properties of Intersleek 7001 (IS700) and
Intersleek 9001 (IS900).
Coating
Biofilm properties
Biofilm thickness (mm)
Diatom species richness
Number of bacteria (No. mm72)
Number of diatoms (No. mm72)
Physico-chemical surface properties
Roughness (mm)
Hardness (N mm72)
Modulus (MPa)
Polar surface energy (mN m71)
Dispersive surface energy (mN m71)
Total surface energy (mN m71)
Water contact angle (8)
Difference
IS700
IS900
Absolute
Percent
12.82 (10.55 )
1.03 (1.26)
26196 (17934.80)
20.31 (35.17)
8.06 (6.61 )
0.186 (0.54)
19541 (12549.84)
4.54 (20.84)
74.76
70.844
76655
715.77
737.13
781.94
725.40
777.65
711.85
0
70.1
10.11
78.73
0.33
722.56
718.07
0
79.09
3392.31
726.01
0.97
722.79
65.59
0.30
1.10
0.26
33.56
33.91
99.01
(7.68)
(0.26)
(0.83)
(0.81)
(1.11)
53.74
0.30
1.00
9.08
24.83
34.24
76.45
(4.80)
(0.46)
(1.82)
(1.99)
(1.95)
Note: Also given are the absolute and percentage differences between the mean values of each property. The biofilm data are from this study; the
physico-chemical data were supplied by International Paint Ltd. The elastic moduli of free films were measured using a Perkin Elmer Pyris
Diamond dynamic mechanical analyser in tension mode with sinusoidal oscillation at 1 Hz and cooling at *108C min71. Contact angles were
measured using a First Ten Ångstroms’ FTA32 automated video goniometer in sessile drop mode, using water, and diiodomethane, and surface
energies were calculated using the Owens-Wendt formula. Hardness was measured using a Fischer H100C Microhardness Tester using a 1368
Vickers’ diamond pyramid indentation probe with controlled test loading up to 1000 mN. Roughness (arithmetic mean, Ra) was measured using
a Nanofocus mscan 3D laser profilometer with a CF4 Confocal Point Sensor and SC200 scan module.
For the all coatings (IS700, IS900 and TC) strong
positive associations were found between the densities
of diatoms and their species richness (Table 4). This
was the only significant association between any of the
biofilm variables for IS700. Additional correlations
were also significant for IS900: both the number and
species richness of diatoms were negatively correlated
with the density of bacteria. These correlations,
although significant, were weak (both 0.35; Table
4). The lack of strength in these associations precluded
further breakdown of the correlation analyses by
diatom genera. On TC treated surfaces the association
Biofouling
875
Table 4. All pairwise Spearman’s correlations between densities of diatoms and bacteria, diatom richness and thickness of the
biofilms on Intersleek 7001 (IS700), Intersleek 9001 (IS900) and control (TC) coatings.
Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011
Number of diatoms
(No. mm72)
IS700
Number of diatoms (No.
Diatom species richness
Number of bacteria (No.
Biofilm thickness (mm)
IS900
Number of diatoms (No.
Diatom species richness
Number of bacteria (No.
Biofilm thickness (mm)
TC
Number of diatoms (No.
Diatom species richness
Number of bacteria (No.
Biofilm thickness (mm)
mm72)
mm72)
0.938 (50.001)
0.938 (50.001)
70.021 (0.870)
0.117 (0.360)
mm72)
mm
72
)
70.027 (0.836)
0.102 (0.426)
0.969 (50.001)
0.969 (50.001)
70.309 (0.004)
0.088 (0.427)
mm72)
mm72)
Diatom species
richness
70.353 (0.001)
0.058 (0.602)
0.983 (50.001)
0.983 (50.001)
0.275 (0.033)
0.099 (0.450)
0.275 (0.034)
0.125 (0.340)
Number of
bacteria (No. mm72)
Biofilm
thickness (mm)
70.021 (0.870)
70.027 (0.836)
0.117 (0.360)
0.102 (0.426)
70.143 (0.263)
70.143 (0.263)
70.309 (0.004)
70.353 (0.001)
0.088 (0.427)
0.058 (0.602)
70.002 (0.989)
70.002 (0.989)
0.275 (0.033)
0.275 (0.034)
0.099 (0.450)
0.125 (0.340)
70.114 (0.388)
70.114 (0.388)
Note: Values for r and its significance (in brackets) are presented. Ho: r 6¼ 0.
between the diatom and biofilm communities was also
weak (*28%) and significant, but conversely it was
positive.
Analysis of the diatom counts by species showed
that the most common species across all coatings was
Cylindrotheca sp. with *5 cells mm72, with Navicula
sp. 2 the second most common with *1 cell mm72 and
then Nitzschia sp. and, finally, Amphora sp. with
*0.7 cells mm72 (Figure 2a). The diatoms present at
the lowest density were Diploneis sp., Bacillaria sp. and
Navicula sp. 1 with *50.3 cells mm72 (Figure 2a).
Comparison of these diatom species data by
coating was only feasible for the four most common
species (Figure 2b), ie Cylindrotheca sp., Navicula sp. 2,
Nitzschia sp. and Amphora sp., due to the low densities
and patchiness of their distributions. Even so, with
these four species the planned LMM analysis was not
possible, as the algorithm was not able to converge to a
solution. Thus a somewhat cruder analysis was done
using generalized linear modelling using mean counts
(aggregated at the slide level). The linear models with
identity link functions were estimated using type III
sums of squares with both coating and block as fixed
factors with an interaction between block and coating
included. To detect significant differences in diatom
counts between coatings, estimated means from each
model were compared using all pair-wise contrast tests
with sequential Bonferroni corrections of the errorrate (Table 5). There was no significant difference
(p ¼ 1.000) in numbers of Nizschia sp. between the
coatings (Table 5, Figure 2b), but the numbers of the
other genera were significantly higher on IS700:
Cylindrotheca sp. (*46, p ¼ 0.001), Amphora sp.
(*66, p 5 0.001) and Navicula sp. 2 (*96,
p 5 0.001).
Discussion
This study has shown that the latest generation of nonbiocidal FR coatings based on fluoropolymer technology, namely Intersleek 900 (IS900), has significantly
reduced short-term biofilm accumulation when compared with a first generation silicone non-biocidal
fouling-release coating, Intersleek 700 (IS700). On
IS900, the biofilm was on average *1.58 times thinner
and contained only *75%, and *22% of the number
of bacteria and diatoms, respectively, and had only
*18% of the diatom species richness of IS700. The
densities of the diatoms Cylindrotheca sp., Amphora sp.
and Navicula sp. 2 on IS900 were at least 4-fold lower
than on IS700. The study also showed that during this
experiment IS900 was completely resistant to settlement by algal spores, whereas *8% of the IS700
coated slides and *9.5% of the TC slides (control) had
adhered spores. The number of spores was more than
2.5 times lower on the IS700 coating in comparison
with the control. Similarly, low adhesion of Ulva
spores has been observed on IS900 compared to with
IS700 (Thompson et al. 2010). These results suggest
that utilization of the new FR coating IS900 will result
in lower microfouling, which will lead to reduced shear
stress, drag and fuel consumption.
Diatom species of the genera Navicula, Cylindrotheca, Nitzschia, Amphora, Diploneis and Bacillaria were
found on IS700, IS900 and control TC coatings.
Diatoms belonging to the genera Amphora and
Navicula are well known cosmopolitan fouling species
and have been reported on both biocidal and nonbiocidal AF coatings (Callow 1986; Cassé and Swain
2006; Molino and Wetherbee 2008; Molino et al.
2009a; Pelletier et al. 2009). Production of strong
S. Dobretsov and J.C. Thomason
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876
Figure 2. Mean and 95% CI plot of (a) the number of each
species of diatom (No. mm72) across all coatings and (b) the
number of the four most common species of diatom (No.
mm72) on Intersleek 700 (IS700), Intersleek 900 (IS900) and
control (TC) coatings. Significant differences are given in
Table 5.
adhesives and high tolerance to biocides possibly
explain the wide distribution of these two species on
AF coatings (Molino and Wetherbee 2008). In the
present study, the genus Cylindrotheca dominated
the diatom assemblages found on all tested coatings.
In field experiments undertaken in Australia on IS700,
the genera Licmophora, Nitzschia, Cylindrotheca,
Amphora, Cocconeis and Toxarium were the most
abundant (Molino et al. 2009a), suggesting perhaps
that Cylindrotheca is characteristic of biofilms on FR
coatings. However, this contrasts with the diatom
assemblage recorded on the FR coating Biox L
exposed in Florida which comprised four dominant
genera, viz. Fragilaria, Synedra, Navicula and Amphora
(Cassé and Swain 2006). Thus these data suggest that
there is unlikely to be a single globally dominant
diatom genus on FR coatings. The presence of these
three different diatom assemblages on FR coatings
exposed in Oman, Florida and Australia could reflect
differential local diatom supply or some differences in
the experimental design between the three studies.
The difference between the fluoropolymer technology and the silicone technology used in the IS900 and
IS700 coating systems, respectively, results in different
surface parameters, with IS900 having lower roughness
and contact angle. Although the total surface energy is
similar, IS900 has a higher polar component of the
surface energy (Table 3) resulting in an amphiphilic
surface in comparison to surface if IS700, which is
hydrophobic. The hardness and modulus for both
coatings were similar. All these have previously been
shown to affect adhesion, settlement and development
of microfouling (Dexter et al. 1975; Becker 1998; Allion
et al. 2006; Schumacher et al. 2007; Jain and Boshle
2009; Mitik-Dineva et al. 2009; Bhushan and Jung 2010;
Myint et al. 2010) and any one of them (or a
combination thereof) might have resulted in the lower
abundance of microbial organisms on IS900. Further
field trials with manipulated and controlled combinations of surface properties are required for clarification.
According to the ‘Baier’ hypothesis, the adhesion
strength of foulers is expected to be minimal on
substrata with surface energies between 22 and 24 nM
m71 (Baier 1973). According to this hypothesis, the
minimum in bioadhesion at 22–24 nM m71 does not
occur at the lowest surface energy (Vladkova 2009).
Numerous field and laboratory experiments have indeed
demonstrated that adhesion of bacteria, larvae and algal
spores is low at surface energies between 20 and 30 nM
m71 (reviewed by Vladkova 2009). However, in a few
studies it has been shown that surface wettability alone
cannot predict attachment of biofouling organisms
(Youngblood et al. 2003), and Ulva spores (Bennett
et al. 2010) and the diatom Seminavis robusta (Thompson et al. 2008) were attached more strongly to
hydrophobic surfaces, such as silicone fouling-release
coatings, compared to hydrophilic surfaces. In the case
of polar or amphiphilic systems, such as IS900, the
percentage of the polar contribution to the total surface
energy is better correlated with spore attachment
(Bennett et al. 2010). The water contact angle of IS700
is higher than for IS900 and although the total surface
Biofouling
877
Table 5. Sequential Bonferroni adjusted results of contrast tests for all pair-wise comparisons of the number of each of the four
most common diatom species recorded on Intersleek 7001 (IS700), Intersleek 9001 (IS900) and control (TC) coatings.
Sequential Bonferroni significance
Comparison
IS700
IS900
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TC
IS900
TC
IS700
TC
IS700
IS 900
Amphora sp.
Cylindrotheca sp.
Navicula sp. 2
Nitzschia sp.
50.001
0.002
50.001
0.269
0.002
0.269
0.001
0.006
0.001
0.713
0.006
0.713
50.001
0.110
50.001
0.110
0.110
0.110
1.000
1.000
1.000
1.000
1.000
1.000
energy is similar for both surfaces, the polar contribution of IS900 is higher than IS700 suggesting that the
polar component may be responsible for the higher
fouling resistance of IS900. Furthermore, a number of
recent papers have shown the benefits of amphiphilic
surfaces in laboratory assays with algae (eg Park et al.
2010; Martinelli et al. 2011; Sundaram et al. 2011).
The patterns of correlations between bacterial and
diatom numbers on the three substrata are perhaps
indicative of the effect of the coatings on the biofilm
assemblages. Negative correlations were only found
for IS900 between the density of bacteria and diatoms.
This might indicate that the physical properties of
IS900 are hostile to diatoms while still favourable for
particular species of bacteria. Additionally, the hypothesis that the bacterial species that colonized IS900
inhibited the subsequent attachment and growth of
diatoms cannot be ruled out. Conversely, there was no
correlation between diatoms and bacteria on IS700
and a positive one on the TC.
The presence of positive and negative correlations
between diatoms and bacteria on biocidal and experimental coatings with narcotizing and repellent compounds has been previously reported (Dobretsov and
Railkin 1994), though generally the causal relationship
between bacteria and diatoms present in biofilms
remains unclear (Molino and Wetherbee 2008). The
densities of bacteria and microalgae growing in
biofilms under light conditions are correlated and
organic compounds produced by photosynthetic microorganisms are used by bacterial heterotrophs (Ylla
et al. 2009). A significant positive linear relationship
between the number of diatoms and bacteria on the
glass slides exposed in the Clyde Sea was found (Head
et al. 2004). Beneficial interactions between the diatom
Navicula sp. and the bacterium Pseudoalteromonas sp.
have been reported (Wigglesworth-Cooksey and Cooksey 2005). These authors demonstrated that bacterial
waterborne metabolites enhanced agglutination and
attachment of diatoms in the laboratory. Other
bacterial species can produce antibiotic, anti-diatom
compounds and quorum sensing inhibitors that can
reduce attachment and growth of other microorganisms (reviewed by Dobretsov et al. 2006, 2009).
Bacterial composition and bacterial culture conditions
affected attachment of the diatom Achnanthes longipes
(Gawne et al. 1998). Additionally, the presence of
bacterial biofilms on substrata with different physical
properties either enhanced or inhibited attachment of
this diatom. These results demonstrate the complexity
of the relationship between substrata and diatom cells
as well as bacterial colonizers. It remains unclear
whether different physical properties or/and negative
and positive interactions between diatoms and bacteria
in the biofilms determine the formation of significantly
different microbial communities on the coatings that
were tested in the present study.
Usually the amount of ATP is determined by the
measurement of luciferase firefly bioluminescence using
a luminometer (De Luca 1976). Since the amount of
ATP is related to the biomass of cells, this method has
been widely used for quick determination of bacterial
contamination (Siragusa et al. 1995; Frundzhyan and
Ugarova 2007) and bacterial adhesion (Dexter et al.
2003) in the medical and food industries. The use of
ATP measurement has been recently proposed as an
indicator of biofilm accumulation in seawater desalination plants (Veza et al. 2008). In the highly replicated
experiment presented here (N ¼ 621), the amount of
ATP in biofilms determined by luciferase firefly
bioluminescence was not related to the quantity of
bacteria and diatoms in the biofilms. This is possibly
because of the presence of many salts and non-ionic
chemicals in the biofilms originating from the seawater,
which inhibited the luminescence reaction (Webster and
Leach 1980). Therefore, although the amount of ATP
was significantly higher on IS700 it is unclear what this
indicated as it certainly did not reflect biofilm accumulation and perhaps represented lower salt inhibition.
Thus, this potentially useful and very rapid technique is
not suitable for the quantification of marine biofilms.
In conclusion, this highly replicated experiment
clearly demonstrated that the latest generation of
fluoropolymer FR coatings, IS900, significantly resisted
878
S. Dobretsov and J.C. Thomason
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the initial development of microbial biofilms. While the
experiment was conducted for only a short time under
static conditions, and because a higher reduction in
biofilm can be expected under dynamic exposure (Cassé
and Swain 2006), it still reflects the comparative
performance of the coatings tested, although how long
this difference persists requires further work. When
compared to the older generation silicone-based IS700,
IS900 is also more resistant to settlement of algal spores.
As both biofilm and algal fouling have been shown to
significantly increase hull friction (Schultz 2007), it is
likely to lead to lower fuel consumption for those vessels
with the right operational profile that are capable of
utilizing the newest FR coatings.
Acknowledgements
The authors would like to thank Brent Tyson, David Stark,
Zhiyi Li and David Williams of International Paint Ltd,
Felling for their technical assistance. The work of SD and the
travel of JCT were supported by a Sultan Qaboos University
(SQU) internal grant IG/AGR/FISH/09/03 and by the HM
Sultan Qaboos Research Trust Fund SR/AGR/FISH/10/01.
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