The development of marine biofilms on two commercial non-biocidal coatings: a comparison between silicone and fluoropolymer technologies

The antimicrobial performance of two fouling-release coating systems, Intersleek 700? (IS700; silicone technology), Intersleek 900? (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-5'-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.     

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon® and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.     

Colonisation and succession of marine biofilm-dwelling ciliate assemblages on biocidal antifouling and fouling-release coatings in temperate Australia

Ciliate assemblages are often overlooked, but ubiquitous components of microbial biofilms which require a better understanding. Ciliate, diatom and bacterial colonisation were evaluated on two fouling-release (FR) coatings, viz. Intersleek 970 and Hempasil X3, and two biocidal antifouling (AF) coatings, viz. Intersmooth 360 and Interspeed 5640, in Port Phillip Bay, Australia. A total of 15 genera were identified during the 10 week deployment. Intersleek 970 displayed the most rapid fouling by ciliates, reaching 63.3(± 5.9) cells cm^?2. After 10 weeks, all four coatings were extensively fouled. However, the toxicity of the AF coatings still significantly inhibited microbial fouling compared to the FR coatings. On all treatments, colonies of sessile peritrichs dominated the ciliate assemblage in the early stage of succession, but as the biofilm matured, vagile ciliates exerted more influence on the assemblage structure. The AF coatings showed selective toxic effects, causing significant differences in the ciliate species assemblages among the treatments.     

Interactions between microbial biofilms and marine fouling algae: a mini review

Natural and artificial substrata immersed in the marine environment are typically colonized by microorganisms, which may moderate the settlement/recruitment of algal spores and invertebrate larvae of macrofouling organisms. This mini-review summarizes the major interactions occurring between microbial biofilms and marine fouling algae, including their effects on the settlement, growth and morphology of the adult plants. The roles of chemical compounds that are produced by both bacteria and algae and which drive the interactions are reviewed. The possibility of using such bioactive compounds to control macrofouling will be discussed.     

Effect of bacterial biofilms formed on fouling-release coatings from natural seawater and Cobetia marina,on the adhesion of two marine algae

Previous studies have shown that bacterial biofilms formed from natural seawater (NSW) enhance the settlement of spores of the green alga Ulva linza, while single-species biofilms may enhance or reduce settlement, or have no effect at all. However, the effect of biofilms on the adhesion strength of algae, and how that may be influenced by coating/surface properties, is not known. In this study, the effect of biofilms formed from natural seawater and the marine bacterium Cobetia marina, on the settlement and the adhesion strength of spores and sporelings of the macroalga U. linza and the diatom Navicula incerta, was evaluated on Intersleek® 700, Intersleek® 900, poly(dimethylsiloxane) and glass. The settlement and adhesion strength of these algae were strongly influenced by biofilms and their nature. Biofilms formed from NSW enhanced the settlement (attachment) of both algae on all the surfaces while the effect of biofilms formed from C. marina varied with the coating type. The adhesion strength of spores and sporelings of U. linza and diatoms was reduced on all the surfaces biofilmed with C. marina, while adhesion strength on biofilms formed from NSW was dependent on the alga (and on its stage of development in the case of U. linza), and coating type. The results illustrate the complexity of the relationships between fouling algae and bacterial biofilms and suggest the need for caution to avoid over-generalisation.     

Investigation of the role of hydrophilic chain length in amphiphilic perfluoropolyether/poly(ethylene glycol) networks: towards high-performance antifouling coatings

The facile preparation of amphiphilic network coatings having a hydrophobic dimethacryloxy-functionalized perfluoropolyether (PFPE-DMA; M _w = 1500 g mol^?1) crosslinked with hydrophilic monomethacryloxy functionalized poly(ethylene glycol) macromonomers (PEG-MA; M _w = 300, 475, 1100 g mol^?1), intended as non-toxic high-performance marine coatings exhibiting antifouling characteristics is demonstrated. The PFPE-DMA was found to be miscible with the PEG-MA. Photo-cured blends of these materials containing 10 wt% of PEG-MA oligomers did not swell significantly in water. PFPE-DMA crosslinked with the highest molecular weight PEG oligomer (ie PEG1100) deterred settlement (attachment) of algal cells and cypris larvae of barnacles compared to a PFPE control coating. Dynamic mechanical analysis of these networks revealed a flexible material. Preferential segregation of the PEG segments at the polymer/air interface resulted in enhanced antifouling performance. The cured amphiphilic PFPE/PEG films showed decreased advancing and receding contact angles with increasing PEG chain length. In particular, the PFPE/PEG1100 network had a much lower advancing contact angle than static contact angle, suggesting that the PEG1100 segments diffuse to the polymer/water interface quickly. The preferential interfacial aggregation of the larger PEG segments enables the coating surface to have a substantially enhanced resistance to settlement of spores of the green seaweed Ulva, cells of the diatom Navicula and cypris larvae of the barnacle Balanus amphitrite as well as low adhesion of sporelings (young plants) of Ulva, adhesion being lower than to a polydimethyl elastomer, Silastic T2.     

Quantitative exploration of the contribution of settlement,growth, dispersal and grazing to the accumulation of natural marine biofilms on antifouling and fouling-release coatings

The accumulation of microbial biofilms on ships’ hulls negatively affects ship performance and efficiency while also playing a role in the establishment of even more detrimental hard-fouling communities. However, there is little quantitative information on how the accumulation rate of microbial biofilms is impacted by the balance of the rates of cell settlement, in situ production (ie growth), dispersal to surrounding waters and mortality induced by grazers. These rates were quantified on test panels coated with copper-based antifouling (AF) or polymer-based fouling-release (FR) coatings by using phospholipids as molecular proxies for microbial biomass. The results confirmed the accepted modes of efficacy of these two types of coatings. In a more extensive set of experiments with only the FR coatings, it was found that seasonally averaged cellular production rates were 1.5 ± 0.5 times greater than settlement and the dispersal rates were 2.7 ± 0.8 greater than grazing. The results of this study quantitatively describe the dynamic balance of processes leading to the accumulation of microbial biofilm on coatings designed for ships’ hulls.     

Combinatorial materials research applied to the development of new surface coatings XVI: fouling-release properties of amphiphilic polysiloxane coatings

High-throughput methods were used to prepare and characterize the fouling-release (FR) properties of an array of amphiphilic polysiloxane-based coatings possessing systematic variations in composition. The coatings were derived from a silanol-terminated polydimethylsiloxane, a silanol-terminated polytrifluorpropylmethylsiloxane (CF₃-PDMS), 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (TMS-PEG), methyltriacetoxysilane and hexamethyldisilazane-treated fumed silica. The variables investigated were the concentration of TMS-PEG and the concentration of CF₃-PDMS. In general, it was found that the TMS-PEG and the CF₃-PDMS had a synergist effect on FR properties with these properties being enhanced by combining both compounds into the coating formulations. In addition, reattached adult barnacles removed from coatings possessing both TMS-PEG and relatively high levels of CF₃-PDMS displayed atypical base-plate morphologies. The majority of the barnacles removed from these coatings exhibited a cupped or domed base-plate as compared to the flat base-plate observed for the control coating that did not contain TMS-PEG or CF₃-PDMS. Coating surface analysis using water contact angle measurements indicated that the presence of CF₃-PDMS facilitated migration of TMS-PEG to the coating/air interface during the film formation/curing process. In general, coatings containing both TMS-PEG and relatively high levels of CF₃-PDMS possessed excellent FR properties.     

Effects of surface texture and interrelated properties on marine biofouling: a systematic review

This systematic review examines effects of surface texture on marine biofouling and characterizes key research methodologies. Seventy-five published articles met selection criteria for qualitative analysis; experimental data from 36 underwent quantitative meta-analysis. Most studies investigated fouling mechanisms and antifouling performance only in laboratory assays with one to several test species. Textures were almost exclusively a single layer of regularly arranged geometric features rather than complex hierarchical or irregular designs. Textures in general had no effect or an inconclusive effect on fouling in 46% of cases. However, effective textures more often decreased (35%) rather than increased (19%) fouling. Complex designs were more effective against fouling (51%) than were regular geometric features (32%). Ratios of feature height, width, or pitch to organism body length were significant influences. The authors recommend further research on promising complex and hierarchical texture designs with more test species, as well as field studies to ground-truth laboratory results.     

The effect of surface colour on the formation of marine micro and macrofouling communities

The effect of substratum colour on the formation of micro- and macro fouling communities was investigated. Acrylic tiles, painted either black or white were covered with transparent sheets in order to ensure similar surface properties. All substrata were exposed to biofouling at 1?m depth for 40?d in the Marina Bandar al Rowdha (Muscat, Sea of Oman). Studies were conducted in 2010 over a time course of 5, 10 and 20?d, and in 2012 samples were collected at 7, 14 and 21?d. The densities of bacteria on the black and white substrata were similar with the exception of day 10, when the black substrata had a higher abundance than white ones. Pyrosequencing via 454 of 16S rRNA genes of bacteria from white and black substrata revealed that Alphaproteobacteria and Firmicutes were the dominant groups. SIMPER analysis demonstrated that bacterial phylotypes (uncultured Gammaproteobacteria, Actibacter, Gaetbulicola, Thalassobius and Silicibacter) and the diatoms (Navicula directa, Navicula sp. and Nitzschia sp.) contributed to the dissimilarities between communities developed on white and black substrata. At day 20, the highest amount of chlorophyll a was recorded in biofilms developed on black substrata. SIMPER analysis showed that Folliculina sp., Ulva sp. and Balanus amphitrite were the major macro fouling species that contributed to the dissimilarities between the communities formed on white and black substrata. Higher densities of these species were observed on black tiles. The results emphasise the effect of substratum colour on the formation of micro and macro fouling communities; substratum colour should to be taken into account in future studies.     

Effects of marine paints on microbial biofilm development on three materials

The development of biofilms of Pseudomonas aeruginosa PAO-1 was studied using modified Robbins devices. Biofilm development was measured using viable counts, acridine orange direct counts (AODC), and a colorimetric method for exopolysaccharide (EPS). Biofilms reached their maximum population 24–72 h after inoculation on coupons with no paint or on coupons coated with marine paint VC-18 without additives. Biofilms on stainless steel contained higher numbers of total cells and of viable cells than biofilms on fiberglass or aluminum. Coating the surfaces with marine paint VC-18 resulted in decreased numbers of cells on stainless steel but had little effect on numbers of cells on fiberglass or aluminum. Addition to the paint of Cu or tributyltin (TBT), the active components in two types of antifouling paints, inhibited the initial development of biofilms. However, by 72–96 h, most biofilms contained the same number of cells as surfaces without additives as shown by both viable counts and AODC. Biofilms that formed on surfaces coated with Cu- or TBT-containing paint did not synthesize more EPS, suggesting that P. aeruginosa PAO-1 does not respond to these compounds by synthesizing more EPS, which could bind the metal and protect the cells. Rather, these biofilms may contain Cu- or TBT-resistant cells. TBT-resistant cells made up 1–10% of the viable counts in biofilms on uncoated stainless steel, but in biofilms on stainless steel coated with marine paint containing TBT, TBT-resistant cells made up as much as 50% of the population. For non-coated stainless steel surfaces, Cu-resistant cells initially made up the majority of the population, but after 48 h they made up less than 1% of the population. On Cu-coated stainless steel, Cu-resistant cells predominated through 48 h, but after 48 h they comprised less than 10% of the population. These results suggest that the growth of TBT-resistant and Cu-resistant cells contributes to biofilms of P. aeruginosa PAO-1 at early stages of development but not at later stages. Received 16 December 1997/ Accepted in revised form 9 March 1998     

The effect of surface colour on the adhesion strength of Elminius modestus Darwin on a commercial non-biocidal antifouling coating at two locations in the UK

A number of factors affect the adhesion strength of organisms to fouling-release coatings, and except for a few studies focussing on black or white surfaces none have dealt specifically with the effect of coating colour. The aim was to test the effect of colour on the adhesion strength of the barnacle Elminius modestus. Panels coated in six commercial colours of Intersleek 700® were submerged at two field sites and barnacles were pushed-off using a standard assay procedure. The strength of adhesion (SOA) varied between and within sites for colour and by barnacle basal area, SOA per unit area being higher for smaller barnacles. Higher SOA with a small basal area may be because of size-specific predation, differential hydrodynamic effects or adhesive failure with age. The complex effect of colour on barnacle adhesion may be because of physico-chemical surface characteristics varying with pigments, and their interactions with local environmental conditions, as well as interactions with the settling barnacle larvae.     

Presence and effects of marine microbial biofilms on biocide-based antifouling paints

Abstract

Marine microorganisms are capable of successfully colonizing toxic surfaces through the formation of biofilm structures. In this article, most of the literature reporting the presence of marine biofilms on chemically-active antifouling paints is briefly reviewed. Of special concern is the influence of the dense extracellular polymeric substances (EPS) matrix on the release rate of the compounds involved in antifouling paint performance (i.e. active compounds and controlled-release binder molecules). A deeper understanding of these phenomena is of interest for both environmental legislators and paint formulators.     

The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms

The elucidation of the mechanisms by which diverse species survive and interact in drinking water (DW) biofilm communities may allow the identification of new biofilm control strategies. The purpose of the present study was to investigate the effects of metabolite molecules produced by bacteria isolated from DW on biofilm formation. Six opportunistic bacteria, viz. Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp. isolated from a drinking water distribution systems (DWDS) were used to form single and multispecies biofilms in the presence and absence of crude cell-free supernatants produced by the partner bacteria. Biofilms were characterized in terms of mass and metabolic activity. Additionally, several physiological aspects regulating interspecies interactions (sessile growth rates, antimicrobial activity of cell-free supernatants, and production of iron chelators) were studied to identify bacterial species with biocontrol potential in DWDS. Biofilms of Methylobacterium sp. had the highest growth rate and M. mucogenicum biofilms the lowest. Only B. cepacia was able to produce extracellular iron-chelating molecules. A. calcoaceticus, B. cepacia, Methylobacterium sp. and M. mucogenicum biofilms were strongly inhibited by crude cell-free supernatants from the other bacteria. The crude cell-free supernatants of M. mucogenicum and S. capsulata demonstrated a high potential for inhibiting the growth of counterpart biofilms. Multispecies biofilm formation was strongly inhibited in the absence of A. calcoaceticus. Only crude cell-free supernatants produced by B. cepacia and A. calcoaceticus had no inhibitory effects on multispecies biofilm formation, while metabolite molecules of M. mucogenicum showed the most significant biocontrol potential.     

The adhesive strategies of cyprids and development of barnacle-resistant marine coatings

Over the last decade, approaches to the development of surfaces that perturb settlement and/or adhesion by barnacles have diversified substantially. Although, previously, coatings research focussed almost exclusively on biocidal technologies and low modulus, low surface-free-energy ‘fouling-release’ materials, novel strategies to control surface colonisation are now receiving significant attention. It is timely, therefore, to review the current ‘state of knowledge’ regarding fouling-resistant surface characteristics and their mechanisms of action against settling larvae of barnacles. The role of the barnacle in marine fouling is discussed here in the context of its life cycle and the behavioural ecology of its cypris larva. The temporary and permanent adhesion mechanisms of cyprids are covered in detail and an overview of adult barnacle adhesion is presented. Recent legislation has directed academic research firmly towards environmentally inert marine coatings, so the actions of traditional biocides on barnacles are not described here. Instead, the discussion is restricted to those surface modifications that interfere with settlement-site selection and adhesion of barnacle cypris larvae; specifically, textural engineering of surfaces, development of inert ‘non-fouling’ surfaces and the use of enzymes in antifouling.     

Self-decontaminating photocatalytic zinc oxide nanorod coatings for prevention of marine microfouling: a mesocosm study

The antifouling (AF) properties of zinc oxide (ZnO) nanorod coated glass substrata were investigated in an out-door mesocosm experiment under natural sunlight (14:10 light: dark photoperiod) over a period of five days. The total bacterial density (a six-fold reduction) and viability (a three-fold reduction) was significantly reduced by nanocoatings in the presence of sunlight. In the absence of sunlight, coated and control substrata were colonized equally by bacteria. MiSeq Illumina sequencing of 16S rRNA genes revealed distinct bacterial communities on the nanocoated and control substrata in the presence and absence of light. Diatom communities also varied on nanocoated substrata in the presence and the absence of light. The observed AF activity of the ZnO nanocoatings is attributed to the formation of reactive oxygen species (ROS) through photocatalysis in the presence of sunlight. These nanocoatings are a significant step towards the production of an environmentally friendly AF coating that utilizes a sustainable supply of sunlight.     

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.     

Bioassays and field immersion tests: a comparison of the antifouling activity of copper-free poly(methacrylic)-based coatings containing tertiary amines and ammonium salt groups

This paper focuses on the activity spectrum of three dimethylalkyl tertiary amines as potential active molecules and the corresponding ammonium salt-based antifouling (AF) paints. Bioassays (using marine bacteria, microalgae and barnacles) and field tests were combined to assess the AF activity of coatings. Bioassay results demonstrated that the ammonium salt-based paints did not inhibit the growth of microorganisms (except the dimethyldodecylammonium-based coatings) and that the tertiary amines were potent towards bacteria, diatoms, and barnacle larvae at non-toxic concentrations (therapeutic ratio, LC₅₀/EC₅₀, < 1). The results from field tests indicated that the ammonium salt-based coatings inhibited the settlement of macrofouling and the dimethylhexadecylammonium-based coatings provided protection against slime in comparison with PVC blank panels. Thus, results from laboratory assays did not fully concur with the AF activity of the paints in the field trial.     

The effect of quorum-sensing blockers on the formation of marine microbial communities and larval attachment

We studied the effect of the quorum-sensing (QS) blockers 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-2(5H)-one (FUR1), (5R)-3,4-dihydroxy-5-[(1S)-1,2-dihydroxyethyl]furan-2(5H)-one (FUR2) and triclosan (TRI) on the formation of bacterial biofilms, and the effect of these biofilms on the larval attachment of the polychaete Hydroides elegans and the bryozoan Bugula neritina. 14-day-old subtidal biofilms were harvested from artificial substrata and were allowed to develop in the laboratory with and without QS blockers. QS blockers inhibited the production of violacein by the QS reporter strain Chromobacterium violaceum CV026 and did not affect the metabolic activity of bacteria in multispecies biofilms. At a concentration of 10(-3) M all three tested compounds inhibited the establishment of microbial communities, but at one of 10(-4) M only FUR2 inhibited establishment. The tested QS blockers caused changes in bacterial density and bacterial community structure, as revealed by terminal restriction fragment length polymorphism and FISH. The groups most affected by QS blockers were Alphaproteobacteria, Gammaproteobacteria and the Cytophagales. Larvae of H. elegans and B. neritina avoided settling on biofilms that had developed in the presence of QS blockers. Our results suggest that QS blockers directly control the formation of multi-species biofilms, and indirectly - by means of biofilm properties - affect larval attachment on these modified biofilms.