1 Department of Biomedical Engineering, University of Groningen, PO Box 196, 9700 AD Groningen, The Netherlands
2 Materials Technology Division Polymer Chemistry, TNO Industrial Technology, PO Box 6235 5600 HE, Eindhoven, The Netherlands
3 Antifouling Research Group, TNO Industrial Technology, PO Box 505, 1780 AM Den Helder, The Netherlands
Correspondence
Henny C. van der Mei
h.c.van.der.mei{at}med.rug.nl
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ABSTRACT |
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INTRODUCTION |
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Along with changes in the chemical composition of a substratum surface after conditioning film formation (Barth, 1989; Hogt et al., 1985
; Oga et al., 1988
), physico-chemical properties of the surface such as its hydrophobicity, roughness, charge and elasticity may alter (Bakker et al., 2003b
). Although many studies have shown relationships between substratum hydrophobicity, charge or roughness with bacterial adhesion, it is still hard to understand how the multiple changes brought about by the adsorption of a conditioning film affect bacterial adhesion. Conditioning films adsorbed from tear fluid on contact lenses changed the surface properties due to presence of proteinaceous material. Multiple linear regression analysis using the elemental surface composition, presence of proteins, surface hydrophobicity and surface roughness as input demonstrated that adhesion of Pseudomonas aeruginosa could be described by the hydrophobicity, surface roughness, percentage oxygen and nitrogen, O=C/OC ratio and the presence of proteins on the surface (Bruinsma et al., 2002
). The complexity of the changes brought about by the adsorption of a conditioning film and our lack of understanding of its impact on bacterial adhesion has hampered the development of antifouling coatings. This is especially important in the marine environment, as environmental restrictions have impeded the use of bacteriocidal coatings based on tributyltin and cuprous oxide.
The aim of this study was to determine the changes in physico-chemical surface properties of experimental polyurethane antifouling coatings after incubation in natural seawater and to relate these changes to the deposition of three marine bacterial strains in a stagnation-point flow chamber using multiple linear regression analysis.
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METHODS |
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Substrata, conditioning film formation and surface characterization.
Polyurethane coatings with different hydrophobicities (as shown in Table 1) and elastic moduli of 1·9 GPa and 1·5 GPa for hard 650 and soft 670 coatings, respectively, were applied to glass, as described before (Bakker et al., 2003a
). The hard 650 coating was obtained by the reaction of the branched, hydroxy-group-containing polyester Desmophen 650 MPA-65 (Bayer) and the aliphatic polyisocyanate, based on hexamethylenediisocyanate (HDI), Desmodur N75. The 670 coating, of lower elastic modulus, was obtained by the reaction of the weakly branched, hydroxy-group-containing polyester Desmophen 670 BA-80 and Desmodur N75. In order to change the hydrophobicity of the coatings, Fluowet EA 600 (Clariant) was added to the Desmodur N75 prior to addition of Desmophen, up to 5·4 mol% and 8·4 mol% in 670F and 650F, respectively. Conditioning films were allowed to form at room temperature by immersion of the plates in natural seawater, obtained from the Marsdiep near Den Helder, The Netherlands, in September 2002, with gentle shaking (30 r.p.m.) for 60 min. As a control, surfaces were also immersed in artificial seawater. After adsorption, conditioned surfaces were dipped once in demineralized water before further experimentation.
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X-ray photoelectron spectroscopy (XPS, S-Probe spectrometer, Surface Science Instruments) was performed on the polyurethane coatings prior to and after conditioning film formation to determine the chemical composition of the surfaces. The spectrometer was equipped with an aluminium anode (10 kV, 22 mA) and a quartz monochromator. The angle of the photoelectron collection was 55 degrees with the normal to the sample and the electron flood gun was set at 10 eV. A survey scan was made with a 1000x250 µm spot and a pass energy of 150 eV. Detailed scans of the C1s, O1s and N1s electron-binding energy peaks were obtained using a pass energy of 50 eV. Binding energies were determined by setting the binding energy of the C1s component due to the carboncarbon bond at 284·8 eV. The experimental peaks were integrated after nonlinear background subtraction and the peaks were decomposed assuming a Gaussian/Lorentzian ratio of 85/15 by using the SSI PC software package. All elemental surface concentrations presented are means of experiments on two polyurethane coatings.
Atomic force microscopy (Nanoscope III, Digital Instruments) was performed on conditioned and unconditioned polyurethane coatings with a silicon nitride cantilever (Veeco) in the contact mode and with a spring-constant of 0·06 N m1 in order to determine the mean surface roughness (Ra). Ra indicates the mean distance of the roughness profile to the centre plane of the profile and was determined on three randomly selected sites per surface. Furthermore, the elastic modulus of the coatings was assessed by measuring their indentation hardness with the atomic force microscope, as described in detail before (Bakker et al., 2003a; Tomasetti et al., 1998
).
Flow chamber experiments.
A stagnation-point flow chamber was used in this study, as described before in detail (Bakker et al., 2003a; Dabros & Van de Ven, 1983
). The flow chamber was incorporated between two communicating vessels placed at different heights and containing the bacterial suspension, to create a pulse-free flow by hydrostatic pressure. Fluid was recirculated with a roller pump between the vessels. In the stagnation-point flow chamber, adhesion in an area 1·1 mm away from the stagnation point was observed with a CCD-LDH camera (Philips) mounted on a dark-field microscope (Leitz) equipped with a 50x objective (Leitz). Live images were acquired with a PC-Vision+ frame grabber (Imaging Technology) and Sharp filtered. Deposited bacteria were discriminated from the background by single grey value thresholding. Experiments were carried out at a flow rate of 0·0088 ml s1, corresponding to a shear rate at the point of observation equal to 22·5 s1. The total number of adhering bacteria per unit area of coating was recorded as a function of time by image sequence analysis. All experiments were performed at 20 °C in triplicate, with separately cultured strains.
Data analysis and statistical evaluation.
The initial deposition rate, j0, describing the affinity of a bacterium for a substratum surface, was calculated by linear regression analysis from the initial increase of the numbers of adhering bacteria as a function of time. To analyse the differences in initial deposition rates, a one-way ANOVA was performed with SPSS for Windows (SPSS Inc.), using a significance level of 0·05. Backward multiple linear regression was performed in order to identify the surface properties most predictive for the difference in bacterial deposition with and without a conditioning film. The difference in initial deposition rate was used as a dependent variable, while the differences in water contact angle, mean surface roughness, and the percentage surface composition (%C, %O, %N), were taken as independent variables. Variables were excluded when equal in both situations or when correlating significantly with other variables, as determined by Pearson's correlation test.
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RESULTS |
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The mean surface roughness of the substratum surfaces, as determined by atomic force microscopy, was on average 2·7 nm for the hard coatings (650 and 650F) and 5·9 nm for the soft coatings (670 and 670F). After conditioning film formation in natural seawater, surface roughness increased to 4·7 nm for the hard coatings (650 and 650F) and to 12·9 nm for the soft coatings (670 and 670F).
Bacterial deposition
Table 2 shows the initial deposition rates for the three strains on substrata after incubation in artificial or natural seawater. In the presence of a conditioning film from natural seawater no significant differences in initial deposition rates between the hydrophobic (650F and 670F) and more hydrophilic (650 and 670) polyurethane coatings were observed in contrast to the bare polyurethane coatings. Psychrobacter sp. adhered preferentially to the soft fluoridated coating, whereas in the absence of a conditioning film highest initial deposition rates were observed for the hard non-fluoridated coating. The most hydrophilic strain, H. pacifica, showed the highest initial deposition rate to the most hydrophobic coating with a conditioning film of natural seawater.
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Table 3 shows that the difference in water contact angle upon conditioning film formation influences the deposition of all three strains, although for Psychrobacter sp. (the intermediately hydrophobic strain) the influence of the water contact angle difference was not significant. Conditioning film formation also affected the initial deposition rates of Psychrobacter sp. and H. pacifica through changes of the mean surface roughness, although here too the influence of mean surface roughness on deposition of Psychrobacter sp. was not significant. Note that in the case of M. hydrocarbonoclasticus ATCC 27132, a small and almost significant influence of the presence of adsorbed nitrogen-rich components on initial deposition was seen.
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DISCUSSION |
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Even though only small amounts of organic carbon (part of it being proteins) are present in the natural seawater used, the results obtained clearly demonstrate that a conditioning film strongly affects the adhesion of bacteria, with a residual influence of the original surface properties. Longer exposure to natural seawaters would possibly have diminished the effects of the underlying surface, as others have reported that the time scale in which the composition of the conditioning film becomes independent of the substratum surface properties ranges from 4 h (Maki et al., 1990) up to 3 days (Little & Zsolnay, 1985
).
Multiple linear regression analysis indicated that hydrophobicity was the main determinant for bacterial deposition, as evidenced by the relatively high standardized coefficients and the fact that hydrophobicity was predictive for all strains. Coatings that became more hydrophobic appeared to be more attractive for bacteria that were hydrophobic as well, and vice versa, which is in agreement with the observation on the coatings without a conditioning film (Bakker et al., 2003a) and the general notion that bacteria with hydrophobic surface properties prefer hydrophobic material surfaces and those with hydrophilic surface properties prefer hydrophilic surfaces (Hogt et al., 1985
; Satou et al., 1988
). Furthermore, hydrophobic bacteria adhere to a greater extent than hydrophilic bacteria (Van Loosdrecht et al., 1987
), as evidenced by the higher mean initial deposition rate for M. hydrocarbonoclasticus (1582 cm2 s1) as compared to H. pacifica (825 cm2 s1).
A positive relation was found between mean surface roughness (which ranged from 2·3 nm up to 12·9 nm) and the deposition of micron-sized bacteria. An influence of nanometer-scale roughness on the deposition of P. aeruginosa to contact lenses was also observed by Bruinsma et al. (2003). This influence of nanometer-scale roughness is surprising and indicates that adhesion of bacteria is mediated by structures much smaller than the bacterium itself, like fimbriae, flagella or polymeric substances excreted by bacteria. Although no significant relation was shown between elemental surface composition and roughness, adsorbed macromolecules are inevitably involved in the changes of roughness observed.
Summarizing, this study shows the rapid change of the surface properties of surfaces incubated in natural seawater, with subsequent changes in deposition of marine bacteria, which were due to changes in multiple surface properties: hydrophobicity, roughness and the amount of nitrogen on the surface. Attention should be paid to ensure that antifouling coatings based on optimal non-adhesive physico-chemical properties maintain their optimal properties after exposure to natural waters, which requires that conditioning film formation should be a first target in the development of antifouling coatings.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 December 2003;
revised 2 February 2004;
accepted 5 February 2004.
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