This art icle was downloaded by: [ Sult an Qaboos Universit y] On: 31 August 2011, At : 05: 07 Publisher: Taylor & Francis I nform a Lt d Regist ered in England and Wales Regist ered Num ber: 1072954 Regist ered office: Mort im er House, 37- 41 Mort im er St reet , London W1T 3JH, UK Biofouling Publicat ion det ails, including inst ruct ions f or aut hors and subscript ion inf ormat ion: ht t p: / / www. t andf online. com/ loi/ gbif 20 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 To link to this article: ht t p: / / dx. doi. org/ 10. 1080/ 08927014. 2011. 607233 PLEASE SCROLL DOWN FOR ARTI CLE Full t erm s and condit ions of use: ht t p: / / www.t andfonline.com / page/ t erm s- and- condit ions This art icle m ay be used for research, t eaching and privat e st udy purposes. Any subst ant ial or syst em at ic reproduct ion, re- dist ribut ion, re- selling, loan, sub- licensing, syst em at ic supply or dist ribut ion in any form t o anyone is expressly forbidden. The publisher does not give any warrant y express or im plied or m ake any represent at ion t hat t he cont ent s will be com plet e or accurat e or up t o dat e. The accuracy of any inst ruct ions, form ulae and drug doses should be independent ly verified wit h prim ary sources. The publisher shall not be liable for any loss, act ions, claim s, proceedings, dem and or cost s or dam ages what soever or howsoever caused arising direct ly or indirect ly in connect ion wit h or arising out of t he use of t his m at erial. 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 Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 (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 Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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 Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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 Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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. Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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. Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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 Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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 Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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 Downloaded by [Sultan Qaboos University] at 05:07 31 August 2011 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. 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