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The development of marine biofilms on twocommercial non-biocidal coatings: a comparisonbetween silicone and fluoropolymer technologiesSergey Dobretsov a & Jeremy C. Thomason ba Marine Science and Fisheries Department, College of Agricultural and Marine Sciences,Sultan Qaboos University, Al Khoud 123, PO Box 34, Omanb Marine Ecological Services, 10 Boulevard Barbes, Paris, 75018, France

Available online: 25 Aug 2011

To cite this article: Sergey Dobretsov & Jeremy C. Thomason (2011): The development of marine biofilms on two commercialnon-biocidal coatings: a comparison between silicone and fluoropolymer technologies, Biofouling, 27:8, 869-880

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The development of marine biofilms on two commercial non-biocidal coatings: a comparison

between silicone and fluoropolymer technologies

Sergey Dobretsova* and Jeremy C. Thomasonb

aMarine 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

(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 shortterm (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, sevenspecies of diatoms (2 species of Navicula, Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillariasp.) and algal spores (Ulva sp.). IS900 had significantly thinner biofilms with fewer diatom species, no algal sporesand the least number of bacteria in comparison with IS700 and the TC. The ATP readings did not correspond to thenumbers of bacteria and diatoms in the biofilms. The density of diatoms was negatively correlated with the density ofthe bacteria in biofilms on the IS900 coating, and, conversely, diatom density was positively correlated in biofilms onthe TC. The higher antifouling efficacy of IS900 over IS700 may lead to lower roughness and thus lower fuelconsumption for those vessels that utilise the IS900 fouling-release coating.

Keywords: microfouling; bacteria; diatoms; microbial biofilms; antifouling coating; fouling-release; amphiphiliccoating

Introduction

Biofilms are sessile assemblages of microbes attachedto each other and to an interface, and are enclosed inan exopolymeric matrix (Lewandowski 2000). Attach-ment of different biofilm-forming microorganismsdepends on the properties of the substratum (eg Dexteret al. 1975; Cooksey and Wigglesworth-Cooksey 1995;Becker et al. 1997; Jain and Boshle 2009; Mitik-Dinevaet al. 2009). Biofilms have distinctive architectures, theexact form of which depends on both chemical (ie thepresence of particular ions and compounds) andphysical (ie the boundary layer properties or criticalsurface tension) environmental parameters (Lewan-dowski 2000; Molino and Wetherbee 2008). Marinebiofilms on man made surfaces consist mainly ofnumerous species of bacteria and diatoms that canpositively and/or negatively interact with each other(Railkin 2003; Dobretsov 2010). It has been shownthat some biofilm-associated bacteria produce com-pounds that promote the growth of diatoms (Wiggles-worth-Cooksey and Cooksey 2005), while othersinhibit growth of microorganisms (see reviews byDobretsov et al. 2006; Qian et al. 2010). Both bacteriaand diatoms may also have a significant impact on the

recruitment of some invertebrate larvae and algalspores by either enhancing or inhibiting their settle-ment (see reviews by Wieczorek and Todd 1998; Qianet al. 2007; Prendergast 2010; Hadfield 2011). Biofilmscan change the physical and chemical properties of asubstratum and correspondingly modify macrofoulingattachment (Becker et al. 1997; Huggett et al. 2009).

Biofouling causes severe problems for marineindustries by increasing corrosion and hydrodynamicdrag which leads to elevated fuel consumption andhigher maintenance costs (Yebra et al. 2004; Schultz2007; Schultz et al. 2011). Thus, marine installationsand vessels are protected against biofouling withantifouling (AF) paints/coatings, which are still mostlybiocidal in nature (Yebra et al. 2004; Thomas andBrooks 2010). Non-biocidal AF coatings have beenavailable since the 1950s, but economics (the relativelylow price of biocidal coatings) and the difficulty inapplying such coatings to hulls prevented their wide-spread use until relatively recently (see Finnie andWilliams 2010 for a recent review). Initially all thecommercially available non-biocidal coatings werebased on the silicone polymer polydimethylsiloxanewhich minimizes adhesion of fouling organisms. These

*Corresponding author. Email: sergey_dobretsov@yahoo.com

Biofouling

Vol. 27, No. 8, September 2011, 869–880

ISSN 0892-7014 print/ISSN 1029-2454 online

� 2011 Taylor & Francis

DOI: 10.1080/08927014.2011.607233

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coatings work on the principle that loosely attachedorganisms are removed by shear as the hull movesthrough the water at high speed and are thus known asfoul release or fouling-release (FR) coatings.

Intersleek 7001 and Intersleek 9001 are two non-biocidal FR coating systems manufactured by Inter-national Paints Ltd. The Intersleek 700 (IS700) FRsystem was patented in 1975 (Patent GB1470465) andlaunched in 1999. The system is based on a silicone topcoat and it is primarily marketed for vessels such ascontainer ships, liquid natural gas carriers and cruisevessels, whose speed and activity ensures adequateshear for the removal of fouling organisms. Intersleek900 (IS900) top coat is a fluoropolymer modifiedpolyorganosiloxane composition which was patentedin 2002 (Patent WO2002074870) and the productlaunched in 2007. As a second generation FR system,with improved performance against macrofoulingorganisms, IS900 is marketed for slower or loweractivity vessels such tankers, bulkers and general cargoships, as well as those originally targeted by IS700.

The development of biofouling on FR and tradi-tional AF coatings has been subject to severalinvestigations that have utilized destructive collectionand examination techniques (Robinson et al. 1985;Callow 1986; Casse and Swain 2006). These investiga-tions demonstrate that FR coatings are very successfulagainst macrofouling organisms, such as barnacles(Aldred and Clare 2008) and algae (Joshi et al. 2009),but are still subject to biofilm colonisation. Forexample, Molino et al. (2009b) found that thepercentage of bacterial cover on IS700 was 1.3–2 timeshigher than on two biocidal coatings, Intersmooth3601 and Super Yacht 8001. They also demonstratedthat within 2–4 days after immersion IS700 displayedthe quickest microbial colonization in comparison withthe biocidal coatings Intersmooth 360 and SuperYacht 800 and microbial cover reached 470% after16 days. Bacterial colonisation and growth on SuperYacht and Intersmooth demonstrated an exponentialrate of growth, while bacterial development on IS700revealed a logarithmic growth rate. Microfouling onthe new generation FR coatings, such as IS900, has notyet been investigated.

Casse and Swain (2006) found that while thebacterial genera Micrococcus and Pseudomonas werepresent on all coatings, Vibrio sp. was found specifi-cally on FR coatings. Microbial biofilms on biocidaland FR coatings typically contain diatoms belongingto the genera Amphora, Navicula, Nitzschia, Licmo-phora, Navicula, and Achnanthes (Casse and Swain2006; Molino and Wetherbee 2008; Molino et al.2009a). Species of the diatom Amphora are mostcommon on copper-based paints, Achnanthes andAmphora on TBT paints (Callow 2000), while mixed

diatom communities have been found to dominate FRcoatings (Casse and Swain 2006). The general differ-ences in the bacterial and diatom assemblages betweenbiocidal paints and FR coatings are probably the resultof toxicity of the biocidal coatings.

The growth of biofilms on biocidal and FRcoatings increases shear stress and drag, leading toincreased fuel consumption (Yebra et al. 2006;Edyvean 2010; Shultz et al. 2011). For example, a1 mm thick biofilm developed on a 23 m ship causedan 80% increase in friction drag and caused 15% lossin 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 pernaval vessel per year (Schultz et al. 2011).

Different microbial species have different dimen-sions and differential growth rates, thus potentiallyaffecting drag and shear stress of biofilmed coatingsto different extents (Howel 2009). Thus, the quanti-tative and qualitative description of biofilms (ienumber of species present, their thickness andbiomass) is important for better predictive systemsengineering. Traditionally, the densities of bacteria inbiofilms have been determined as colony formingunits (Cooksey and Wigglesworth-Cooksey 1995), butthis technique does not allow the determination ofuncultivable microbes that are revealed by directmicroscope counting using fluorescent dyes that stainDNA, such as 40,6-diamidino-2-phenylindole (DAPI;Kirchman et al. 1982). The densities of diatomsdetermined by light or electron microscopy orchlorophyll measurements can result in false results,as discussed by Cooksey and Wigglesworth-Cooksey(1995). Direct light microscopy and chlorophyllmeasurement can be highly sensitive to low cellsnumbers, while electron microscopy is not. However,light microscopy has the advantage that a largersurface area can be assessed for a given amount oftime. Biofilm thickness is an important parameter inbiofilm characterization as it will reflect the densityand the activity of the microbial assemblage in termsof EPS formation. The biomass of a biofilm and thenumber of cells determines the amount of adenosine-5’-triphosphate (ATP) present. Therefore, determina-tion of ATP using calibrated luciferase fireflyluminescence has been widely used for quick determi-nation of bacterial contamination (Siragusa et al.1995; Frundzhyan and Ugarova 2007) and bacterialadhesion (Dexter et al. 2003) in medicine andindustry.

Given the importance of biofilms in contributing tohull fouling and the consequent increase in drag andhence fuel penalty, the aim of this study was tocompare the initial development of marine biofilms onIS700 and IS900 in a challenging tropical marine

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environment to see if the general anti-macrofoulingefficacy improvement of IS900 over IS700 extends toimproved efficacy against biofilms.

Materials and methods

Field experiment

Coating preparation

Three coating treatments were applied to standardglass microscope slides (Fisher UK, 766 266 0.8mm) using normal commercial application protocolsfor each of the coatings (Intersleek 970 . . . 2009;Intersleek 731 . . . 2010a; Intersleek 757 . . . 2010b; Inter-shield 300 . . . 2010c), at International Paint Ltd, Fell-ing, UK. The coating treatments were the standard FRanticorrosive primer and tie coat (TC) finished witheither the top coat for IS700 (silicone) or IS900(fluoropolymer) coating system, or left unfinishedleaving the TC exposed. The TC was considered tobe the control and was used to provide a referencesurface on which biofilms would be expected to behighly adherent. The primer used was Intershield3001, the TC used was Intersleek 7311, the IS700top coat was Intersleek 7571, and the IS900 topcoatwas Intersleek 9701. A complete system of primer, TCand Intersleek 970 is known as the IS900 coatingscheme. Likewise a complete scheme with a top coat ofIntersleek 757 is known as the IS700 coating scheme.The Intershield 300 primer and Intersleek 731 TC areboth two-pack products while Intersleek 757 andIntersleek 970 are three-pack top coats. The mixingratio for the primer was 2.5:1, and TCs 1:1 by volumeand the mixing ratios for Intersleek 757 and Intersleek970 was 15:4:1 and 16:3:1 by volume, respectively.Both the top coats were standard commercial red andthe TC was pink. These schemes are the conventionalschemes as used on commercial vessels; the primer wasonly included to ensure that a complete commercialscheme was used and was not strictly necessary forinclusion of its anticorrosive properties in thisexperiment.

All the applications were carried out by airlessspray at 208C using a 0.48 mm tip at a pressure of26.56 106 Pa to give a 42 cm standard fan width at30 cm from the surface. Coated slides were dried at358C for 5 h between anti-corrosive primer and TCapplications and at the same temperature for 24 hbefore the application of the top coats. Nominalthicknesses were 150 mm for the primer, 100 mm forthe TC and 150 mm for the top coats (Intersleek970 . . . 2009; Intersleek 731 . . . 2010a; Intersleek757 . . . 2010b; Intershield 300 . . . 2010c). After thewhole scheme was applied, coated slides were driedat ambient temperature for 5 days prior to dispatchto Oman for deployment.

Replication

Microscope slides are convenient for sending bycourier and immediately usable in microscopy, withoutthe need for sampling or reduction in size, and thus thebiofilms could be examined intact and undamaged.However, since microscope slides present a smallsurface for study and, as biofilms are often spatiallyheterogeneous (Augspurger et al. 2010; Wagner et al.2010), a high level of replication was used, consistingof 84 slides of each treatment (ie 252 slides in total).For each coating tested, the area for biofilm develop-ment was 0.16 m2, giving a total area for biofilmgrowth in the experiment of 0.48 m2. The limits on thenumber of slides deployed were imposed by thenecessity of some of the analytical methods having tobe completed in a relatively short time.

Deployment

The 252 coated slides were deployed in a replicatedrandomised block design, with three blocks, such that28 replicates of each treatment were randomlyallocated to a position in each block. Each blockcorresponded to a single deployment frame that wasmade up of a set of slide holders. The slide holderswere constructed from PVC. Each holder was madefrom a PVC plate (9866 151 mm) with two narrowPVC battens (9866 50 mm), one each side, used tohold the slides to the PVC plate. The battens were heldin place by bolts and washers and a strip of neoprenewas placed under them to help trap the slides in placewithout recourse to undue tightening of the bolts.There were 21 equally spaced slides in each holder.Four slide holders were attached lengthwise, next toeach other, to a 4 m long PVC pipe. The four slideholders with slides attached to one pipe were con-sidered to be the deployment frame and thus also anexperimental block. Three frames were made to givethree blocks. Each frame was deployed by ropesattached at each end such that the whole frame waskept parallel to the surface of the water. Each slideholder on the frame was arranged such that the slidesin the holders were kept vertical with respect to thesurface 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 2383405500N 5883602700E) in April 2010and the distance between the blocks was *1 m.Previous experiments had shown that longer deploy-ment of the slides resulted in development of very thickthree dimensional biofilms, as well settlement of larvae,which would have made clear visualization of thesubstratum difficult. During the experiment the aver-age water temperature was 28.88C and salinity was

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35.5 ppt. At the end of the experiment the slides wereretrieved from the water and placed into slide boxes(Fisher Scientific, USA) with fresh filtered (0.22 mm)seawater from the experimental site. ATP in eachbiofilm was detected (see below) before each slide wasfixed with 4% formalin solution in artificial seawater.The slides in the formalin were brought to thelaboratory in a large container (volume ¼ 15 L) andprocessed (see below) within several weeks.

Biofilm quantification

In the laboratory the following components weremeasured to quantify the biofilms: ATP, the numberof diatoms in each identifiable genus, the number ofbacteria and the thickness of the biofilm. All quanti-fication was performed blind.

ATP measurements

For the ATP measurements, the slides were swabbedwith sterile 3 Ultrasnap1 Hygiena swabs (Hygiena,UK) and the ATP measured using a SystemSURE II1

handheld luminometer (Hygiena, UK) according tothe manufacturer’s protocol. Briefly, for each slide,three random horizontal swabs (each *125 mm2) weretaken which left an area of approximately 1500 mm2

for the quantification of diatoms and bacteria (seebelow). Each swab was individually mixed with thesolution provided and ATP was measured as relativelight units (RLU) within 60 s. As a control, a cleanHygiena swab was used. All ATP measurements weremade at a shady location of the marina on fresh(unfixed) slides.

Quantification of diatoms and bacteria

Bacteria and diatoms were quantified according toDobretsov et al. (2005) in the area of each slide thatwas left undisturbed after swabs were taken for theATP measurements. Biofilms were stained with a100 ng ml71 solution of 40,6-diamidino-2-phenylindole(DAPI, Sigma, Germany) in filtered (0.2 mm) seawaterfor 15 min in the dark. The number of bacteria in 10randomly 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 obtainedwere transformed into number of individuals per mm2.The number of diatoms in 10 randomly selected fieldsof view (area ¼ 3.5 mm2) was counted with a micro-scope (Nikon Eclipse, USA; magnification 4006). Themain genera were determined and their densities permm2 calculated. The thickness of the biofilms wasdetermined by using the calibrated fine focus control tofocus on the air/film and film/substratum interfaces

(see Yuehuei and Friedman 2000) of the microscope in10 randomly selected fields of view.

Statistical analysis

Hypothesis testing was undertaken with linear mixedmodelling (LMM) in SPSS v18 following the protocolsdescribed by McCulloch and Searle (2000) andVerbeke and Molenberghs (2000). This approachallows for the effect of a fixed factor to be determinedwhilst taking into account the effect of a randomfactor, as well as being able to include repeatedmeasurements on the same subject (McCulloch andSearle 2000). For the LMMs, the response variableswere biofilm thickness, ATP score, number of bacteriaand diatoms, and species and richness of diatoms. Toachieve normality prior to modelling, data for thenumber of bacteria and diatom species richness weretransformed by taking the natural log and diatomcounts were transformed by using the fifth root;biofilm thickness and ATP score were not transformed.For the response variables determined using micro-scopy, field of view number (N ¼ 10) was used toidentify the repeated measurements and slide numberwas used to identify the unique subject (N ¼ 252).Swab number (N ¼ 3) was used to identify therepeated ATP measurements. Each LMM was esti-mated with coating as the fixed factor and block as therandom factor with an interaction between block andcoating included. Type III sums of squares were usedfor fixed factor effect calculations. Estimation of thevariance/covariance repeated measures matrix wasinitiated as an ante-dependence first order structurefor all variables except diatom numbers and richnesswhich required a diagonal matrix structure. A scaledidentity structure was used to initiate estimation of therandom effects variance/covariance matrix. Modelconvergence and Akaike’s information criterion(AIC) were used to determine the optimal covariancematrix structure. Given the large sample size the LMMwas estimated using the maximum likelihood method.A pairwise contrast test with Bonferroni correctionwas used to determine differences between the coat-ings. Full details about the above procedures can befound in McCulloch and Searle (2000) and Verbekeand Molenberghs (2000). The surfaces of 45 slides wereobscured by large numbers of macroalgal spores whichprevented accurate enumeration of the bacteria; thesewere excluded from the above analyses.

To determine the magnitude of the associationbetween the constituents of the biofilms, non-para-metric Spearman’s correlation coefficients (r) werecalculated for all pairwise associations between num-bers of diatoms and bacteria, diatom species richnessand biofilm thickness. This analysis was deemed to belargely descriptive and was an attempt to describe the

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effect of the coating on the assemblage within thebiofilm, hence the only hypothesis tested was that thecorrelation 6¼ 0 and no adjustments for multiplecorrelations were made.

Results

All the coatings developed biofilms during the 10 daydeployment. These biofilms were made up of hetero-trophic bacteria (no morphotype data recorded),cyanobacteria (two distinguishable morphotypes),seven species of diatom (two species of Navicula,Cylindrotheca sp., Nitzschia sp., Amphora sp., Diplo-neis sp., and Bacillaria sp.) and algal spores (probablyUlva sp.). There were highly significant differencesbetween the coatings for each of the responsevariables, namely numbers of bacteria and diatoms,diatom species richness, ATP reading, and biofilmthickness (Figure 1 and Table 1). IS900 hadsignificantly thinner biofilms, lower mean diatomspecies richness and the least number of bacteria(Figure 1, Tables 2 and 3). All diatom species werefound on all coatings. There was no significantdifference in ATP readings (RLU) and the numberof diatoms between IS900 and TC, whereas both ofthese parameters were significantly higher for IS700coated slides (Table 2). Thus, a general overview isthat IS900 had less biofilm with lower microbialdiversity and activity than IS700.

The ATP results did not show the same a patternacross the three coatings as that shown for the othervariables measured, ie a consistently higher ATP RLUwas expected for surfaces with higher bacterial anddiatom counts (Figure 1, Table 2). Indeed, a correla-tion analysis found that the only significant result wasbetween ATP and the number of diatoms, but that thiswas very weak (Spearman’s r ¼ 0.15, p5 0.001,N ¼ 621). An ordinary least squares regression analy-sis of ATP vs transformed diatom counts wassignificant (ATP ¼ 544.96 þ 432.47 6 TransformedDiatom Count, p5 0.001), but the very small coeffi-cient of determination, r2 ¼ 0.029, shows that diatomsonly accounted for *3% of the variation in the ATPreadings, and thus these data for ATP, althoughsignificantly different between coatings, clearly do notreflect bacterial or microalgal density in the biofilms,and thus should be treated with a strong degree ofcaution.

Of the 45 slides that were excluded from the LMManalysis due to the presence of large numbers of algalspores, 21 slides were coated with IS700 and 24 withthe TC; no slides with a finish coating of IS900 hadattached algal spores. There were significantly more(p5 0.001, Mann–Whitney U test) algal spores on theTC (�x¼ 19.52+18.78SD spores mm72) than on IS700(�x¼ 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).

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For the all coatings (IS700, IS900 and TC) strongpositive associations were found between the densitiesof diatoms and their species richness (Table 4). Thiswas the only significant association between any of thebiofilm variables for IS700. Additional correlationswere also significant for IS900: both the number and

species richness of diatoms were negatively correlatedwith the density of bacteria. These correlations,although significant, were weak (both �0.35; Table4). The lack of strength in these associations precludedfurther breakdown of the correlation analyses bydiatom genera. On TC treated surfaces the association

Table 1. Summary of linear mixed modelling for the fixed factor, coating.

Response variable Numerator DF Denominator DF F P

Bacteria (No. mm72) 2 206.01 16.51 50.001ATP (Hygiena systemSURE II relative light unit) 2 207.00 10.50 50.001Biofilm thickness (mm) 2 203.61 36.16 50.001Diatoms (No. mm72) 2 202.87 29.93 50.001Diatom species richness 2 160.11 8.804 50.001

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 coatingtreatment for all the linear mixed models.

Referencecoating

Comparisoncoating

Response variable

Bacteria(No. mm72)

Diatomspeciesrichness

Diatoms(No. mm72)

ATP (Hygiena systemSURE IIrelative light unit)

Biofilmthickness (mm)

IS700 IS900 0.001 50.001 50.001 0.002 50.001TC 0.198 0.995 0.018 50.001 0.131

IS900 IS700 0.001 50.001 50.001 0.002 50.001TC 50.001 50.001 0.309 0.744 50.001

TC IS700 0.198 0.995 0.018 50.001 0.131IS900 50.001 50.001 50.001 0.744 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) andIntersleek 9001 (IS900).

Coating Difference

IS700 IS900 Absolute Percent

Biofilm propertiesBiofilm thickness (mm) 12.82 (10.55 ) 8.06 (6.61 ) 74.76 737.13Diatom species richness 1.03 (1.26) 0.186 (0.54) 70.844 781.94Number of bacteria (No. mm72) 26196 (17934.80) 19541 (12549.84) 76655 725.40Number of diatoms (No. mm72) 20.31 (35.17) 4.54 (20.84) 715.77 777.65

Physico-chemical surface propertiesRoughness (mm) 65.59 (7.68) 53.74 (4.80) 711.85 718.07Hardness (N mm72) 0.30 0.30 0 0Modulus (MPa) 1.10 1.00 70.1 79.09Polar surface energy (mN m71) 0.26 (0.26) 9.08 (0.46) 10.11 3392.31Dispersive surface energy (mN m71) 33.56 (0.83) 24.83 (1.82) 78.73 726.01Total surface energy (mN m71) 33.91 (0.81) 34.24 (1.99) 0.33 0.97Water contact angle (8) 99.01 (1.11) 76.45 (1.95) 722.56 722.79

Note: Also given are the absolute and percentage differences between the mean values of each property. The biofilm data are from this study; thephysico-chemical data were supplied by International Paint Ltd. The elastic moduli of free films were measured using a Perkin Elmer PyrisDiamond dynamic mechanical analyser in tension mode with sinusoidal oscillation at 1 Hz and cooling at *108C min71. Contact angles weremeasured using a First Ten Angstroms’ FTA32 automated video goniometer in sessile drop mode, using water, and diiodomethane, and surfaceenergies were calculated using the Owens-Wendt formula. Hardness was measured using a Fischer H100C Microhardness Tester using a 1368Vickers’ diamond pyramid indentation probe with controlled test loading up to 1000 mN. Roughness (arithmetic mean, Ra) was measured usinga Nanofocus mscan 3D laser profilometer with a CF4 Confocal Point Sensor and SC200 scan module.

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between the diatom and biofilm communities was alsoweak (*28%) and significant, but conversely it waspositive.

Analysis of the diatom counts by species showedthat the most common species across all coatings wasCylindrotheca sp. with *5 cells mm72, with Naviculasp. 2 the second most common with*1 cell mm72 andthen Nitzschia sp. and, finally, Amphora sp. with*0.7 cells mm72 (Figure 2a). The diatoms present atthe lowest density were Diploneis sp., Bacillaria sp. andNavicula sp. 1 with *50.3 cells mm72 (Figure 2a).

Comparison of these diatom species data bycoating was only feasible for the four most commonspecies (Figure 2b), ie Cylindrotheca sp., Navicula sp. 2,Nitzschia sp. and Amphora sp., due to the low densitiesand patchiness of their distributions. Even so, withthese four species the planned LMM analysis was notpossible, as the algorithm was not able to converge to asolution. Thus a somewhat cruder analysis was doneusing generalized linear modelling using mean counts(aggregated at the slide level). The linear models withidentity link functions were estimated using type IIIsums of squares with both coating and block as fixedfactors with an interaction between block and coatingincluded. To detect significant differences in diatomcounts between coatings, estimated means from eachmodel were compared using all pair-wise contrast testswith sequential Bonferroni corrections of the error-rate (Table 5). There was no significant difference(p ¼ 1.000) in numbers of Nizschia sp. between thecoatings (Table 5, Figure 2b), but the numbers of theother genera were significantly higher on IS700:Cylindrotheca sp. (*46, p ¼ 0.001), Amphora sp.(*66, p5 0.001) and Navicula sp. 2 (*96,p5 0.001).

Discussion

This study has shown that the latest generation of non-biocidal FR coatings based on fluoropolymer technol-ogy, namely Intersleek 900 (IS900), has significantlyreduced short-term biofilm accumulation when com-pared with a first generation silicone non-biocidalfouling-release coating, Intersleek 700 (IS700). OnIS900, the biofilm was on average *1.58 times thinnerand contained only *75%, and *22% of the numberof bacteria and diatoms, respectively, and had only*18% of the diatom species richness of IS700. Thedensities of the diatoms Cylindrotheca sp., Amphora sp.and Navicula sp. 2 on IS900 were at least 4-fold lowerthan on IS700. The study also showed that during thisexperiment IS900 was completely resistant to settle-ment by algal spores, whereas *8% of the IS700coated slides and*9.5% of the TC slides (control) hadadhered spores. The number of spores was more than2.5 times lower on the IS700 coating in comparisonwith the control. Similarly, low adhesion of Ulvaspores has been observed on IS900 compared to withIS700 (Thompson et al. 2010). These results suggestthat utilization of the new FR coating IS900 will resultin lower microfouling, which will lead to reduced shearstress, drag and fuel consumption.

Diatom species of the genera Navicula, Cylindrothe-ca, Nitzschia, Amphora, Diploneis and Bacillaria werefound on IS700, IS900 and control TC coatings.Diatoms belonging to the genera Amphora andNavicula are well known cosmopolitan fouling speciesand have been reported on both biocidal and non-biocidal AF coatings (Callow 1986; Casse and Swain2006; Molino and Wetherbee 2008; Molino et al.2009a; Pelletier et al. 2009). Production of strong

Table 4. All pairwise Spearman’s correlations between densities of diatoms and bacteria, diatom richness and thickness of thebiofilms on Intersleek 7001 (IS700), Intersleek 9001 (IS900) and control (TC) coatings.

Number of diatoms(No. mm72)

Diatom speciesrichness

Number ofbacteria (No. mm72)

Biofilmthickness (mm)

IS700Number of diatoms (No. mm72) 0.938 (50.001) 70.021 (0.870) 0.117 (0.360)Diatom species richness 0.938 (50.001) 70.027 (0.836) 0.102 (0.426)Number of bacteria (No. mm72) 70.021 (0.870) 70.027 (0.836) 70.143 (0.263)Biofilm thickness (mm) 0.117 (0.360) 0.102 (0.426) 70.143 (0.263)

IS900Number of diatoms (No. mm72) 0.969 (50.001) 70.309 (0.004) 0.088 (0.427)Diatom species richness 0.969 (50.001) 70.353 (0.001) 0.058 (0.602)Number of bacteria (No. mm72) 70.309 (0.004) 70.353 (0.001) 70.002 (0.989)Biofilm thickness (mm) 0.088 (0.427) 0.058 (0.602) 70.002 (0.989)

TCNumber of diatoms (No. mm72) 0.983 (50.001) 0.275 (0.033) 0.099 (0.450)Diatom species richness 0.983 (50.001) 0.275 (0.034) 0.125 (0.340)Number of bacteria (No. mm72) 0.275 (0.033) 0.275 (0.034) 70.114 (0.388)Biofilm thickness (mm) 0.099 (0.450) 0.125 (0.340) 70.114 (0.388)

Note: Values for r and its significance (in brackets) are presented. Ho: r 6¼ 0.

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adhesives and high tolerance to biocides possiblyexplain the wide distribution of these two species onAF coatings (Molino and Wetherbee 2008). In thepresent study, the genus Cylindrotheca dominatedthe 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 mostabundant (Molino et al. 2009a), suggesting perhapsthat Cylindrotheca is characteristic of biofilms on FRcoatings. However, this contrasts with the diatomassemblage recorded on the FR coating Biox Lexposed in Florida which comprised four dominantgenera, viz. Fragilaria, Synedra, Navicula and Amphora(Casse and Swain 2006). Thus these data suggest thatthere is unlikely to be a single globally dominantdiatom genus on FR coatings. The presence of thesethree different diatom assemblages on FR coatingsexposed in Oman, Florida and Australia could reflectdifferential local diatom supply or some differences inthe experimental design between the three studies.

The difference between the fluoropolymer technol-ogy and the silicone technology used in the IS900 andIS700 coating systems, respectively, results in differentsurface parameters, with IS900 having lower roughnessand contact angle. Although the total surface energy issimilar, IS900 has a higher polar component of thesurface energy (Table 3) resulting in an amphiphilicsurface in comparison to surface if IS700, which ishydrophobic. The hardness and modulus for bothcoatings were similar. All these have previously beenshown to affect adhesion, settlement and developmentof microfouling (Dexter et al. 1975; Becker 1998; Allionet al. 2006; Schumacher et al. 2007; Jain and Boshle2009; Mitik-Dineva et al. 2009; Bhushan and Jung 2010;Myint et al. 2010) and any one of them (or acombination thereof) might have resulted in the lowerabundance of microbial organisms on IS900. Furtherfield trials with manipulated and controlled combina-tions of surface properties are required for clarification.According to the ‘Baier’ hypothesis, the adhesionstrength of foulers is expected to be minimal onsubstrata with surface energies between 22 and 24 nMm71 (Baier 1973). According to this hypothesis, theminimum in bioadhesion at 22–24 nM m71 does notoccur at the lowest surface energy (Vladkova 2009).Numerous field and laboratory experiments have indeeddemonstrated that adhesion of bacteria, larvae and algalspores is low at surface energies between 20 and 30 nMm71 (reviewed by Vladkova 2009). However, in a fewstudies it has been shown that surface wettability alonecannot predict attachment of biofouling organisms(Youngblood et al. 2003), and Ulva spores (Bennettet al. 2010) and the diatom Seminavis robusta (Thomp-son et al. 2008) were attached more strongly tohydrophobic surfaces, such as silicone fouling-releasecoatings, compared to hydrophilic surfaces. In the caseof polar or amphiphilic systems, such as IS900, thepercentage of the polar contribution to the total surfaceenergy is better correlated with spore attachment(Bennett et al. 2010). The water contact angle of IS700is higher than for IS900 and although the total surface

Figure 2. Mean and 95% CI plot of (a) the number of eachspecies of diatom (No. mm72) across all coatings and (b) thenumber of the four most common species of diatom (No.mm72) on Intersleek 700 (IS700), Intersleek 900 (IS900) andcontrol (TC) coatings. Significant differences are given inTable 5.

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energy is similar for both surfaces, the polar contribu-tion of IS900 is higher than IS700 suggesting that thepolar component may be responsible for the higherfouling resistance of IS900. Furthermore, a number ofrecent papers have shown the benefits of amphiphilicsurfaces in laboratory assays with algae (eg Park et al.2010; Martinelli et al. 2011; Sundaram et al. 2011).

The patterns of correlations between bacterial anddiatom numbers on the three substrata are perhapsindicative of the effect of the coatings on the biofilmassemblages. Negative correlations were only foundfor IS900 between the density of bacteria and diatoms.This might indicate that the physical properties ofIS900 are hostile to diatoms while still favourable forparticular species of bacteria. Additionally, the hy-pothesis that the bacterial species that colonized IS900inhibited the subsequent attachment and growth ofdiatoms cannot be ruled out. Conversely, there was nocorrelation between diatoms and bacteria on IS700and a positive one on the TC.

The presence of positive and negative correlationsbetween diatoms and bacteria on biocidal and experi-mental coatings with narcotizing and repellent com-pounds has been previously reported (Dobretsov andRailkin 1994), though generally the causal relationshipbetween bacteria and diatoms present in biofilmsremains unclear (Molino and Wetherbee 2008). Thedensities of bacteria and microalgae growing inbiofilms under light conditions are correlated andorganic compounds produced by photosynthetic mi-croorganisms are used by bacterial heterotrophs (Yllaet al. 2009). A significant positive linear relationshipbetween the number of diatoms and bacteria on theglass slides exposed in the Clyde Sea was found (Headet al. 2004). Beneficial interactions between the diatomNavicula sp. and the bacterium Pseudoalteromonas sp.have been reported (Wigglesworth-Cooksey and Cook-sey 2005). These authors demonstrated that bacterialwaterborne metabolites enhanced agglutination andattachment of diatoms in the laboratory. Otherbacterial species can produce antibiotic, anti-diatomcompounds and quorum sensing inhibitors that can

reduce attachment and growth of other microorgan-isms (reviewed by Dobretsov et al. 2006, 2009).Bacterial composition and bacterial culture conditionsaffected attachment of the diatom Achnanthes longipes(Gawne et al. 1998). Additionally, the presence ofbacterial biofilms on substrata with different physicalproperties either enhanced or inhibited attachment ofthis diatom. These results demonstrate the complexityof the relationship between substrata and diatom cellsas well as bacterial colonizers. It remains unclearwhether different physical properties or/and negativeand positive interactions between diatoms and bacteriain the biofilms determine the formation of significantlydifferent microbial communities on the coatings thatwere tested in the present study.

Usually the amount of ATP is determined by themeasurement of luciferase firefly bioluminescence usinga luminometer (De Luca 1976). Since the amount ofATP is related to the biomass of cells, this method hasbeen widely used for quick determination of bacterialcontamination (Siragusa et al. 1995; Frundzhyan andUgarova 2007) and bacterial adhesion (Dexter et al.2003) in the medical and food industries. The use ofATP measurement has been recently proposed as anindicator of biofilm accumulation in seawater desalina-tion plants (Veza et al. 2008). In the highly replicatedexperiment presented here (N ¼ 621), the amount ofATP in biofilms determined by luciferase fireflybioluminescence was not related to the quantity ofbacteria and diatoms in the biofilms. This is possiblybecause of the presence of many salts and non-ionicchemicals in the biofilms originating from the seawater,which inhibited the luminescence reaction (Webster andLeach 1980). Therefore, although the amount of ATPwas significantly higher on IS700 it is unclear what thisindicated as it certainly did not reflect biofilm accumu-lation and perhaps represented lower salt inhibition.Thus, this potentially useful and very rapid technique isnot suitable for the quantification of marine biofilms.

In conclusion, this highly replicated experimentclearly demonstrated that the latest generation offluoropolymer FR coatings, IS900, significantly resisted

Table 5. Sequential Bonferroni adjusted results of contrast tests for all pair-wise comparisons of the number of each of the fourmost common diatom species recorded on Intersleek 7001 (IS700), Intersleek 9001 (IS900) and control (TC) coatings.

Sequential Bonferroni significance

Comparison Amphora sp. Cylindrotheca sp. Navicula sp. 2 Nitzschia sp.

IS700 IS900 50.001 0.001 50.001 1.000TC 0.002 0.006 0.110 1.000

IS900 IS700 50.001 0.001 50.001 1.000TC 0.269 0.713 0.110 1.000

TC IS700 0.002 0.006 0.110 1.000IS 900 0.269 0.713 0.110 1.000

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the initial development of microbial biofilms. While theexperiment was conducted for only a short time understatic conditions, and because a higher reduction inbiofilm can be expected under dynamic exposure (Casseand Swain 2006), it still reflects the comparativeperformance of the coatings tested, although how longthis difference persists requires further work. Whencompared 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 tosignificantly increase hull friction (Schultz 2007), it islikely to lead to lower fuel consumption for those vesselswith the right operational profile that are capable ofutilizing 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 thetravel of JCT were supported by a Sultan Qaboos University(SQU) internal grant IG/AGR/FISH/09/03 and by the HMSultan Qaboos Research Trust Fund SR/AGR/FISH/10/01.

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