Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R(2)=0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.     

Combinatorial materials research applied to the development of new surface coatings IX: an investigation of novel antifouling/fouling-release coatings containing quaternary ammonium salt groups

Polysiloxane coatings containing chemically-bound ("tethered") quaternary ammonium salt (QAS) moieties were investigated for potential application as environmental-friendly coatings to control marine biofouling. A combinatorial/high-throughput approach was applied to the investigation to enable multiple variables to be probed simultaneously and efficiently. The variables investigated for the moisture-curable coatings included QAS composition, ie alkyl chain length, and concentration as well as silanol-terminated polysiloxane molecular weight. A total of 75 compositionally unique coatings were prepared and characterized using surface characterization techniques and biological assays. Biological assays were based on two different marine microorganisms, a bacterium, Cellulophaga lytica and a diatom, Navicula incerta, as well as a macrofouling alga, Ulva. The results of the study showed that all three variables influenced coating surface properties as well as antifouling (AF) and fouling-release (FR) characteristics. The incorporation of QAS moieties into a polysiloxane matrix generally resulted in an increase in coating surface hydrophobicity. Characterization of coating surface morphology revealed a heterogeneous, two-phase morphology for many of the coatings investigated. A correlation was found between water contact angle and coating surface roughness, with the contact angle increasing with increasing surface roughness. Coatings based on the QAS moiety containing the longest alkyl chain (18 carbons) displayed the highest micro-roughness and, thus, the most hydrophobic surfaces. With regard to AF and FR properties, coatings based on the 18 carbon QAS moieties were very effective at inhibiting C. lytica biofilm formation and enabling easy removal of Ulva sporelings (young plants) while coatings based on the 14 carbon QAS moities were very effective at inhibiting biofilm growth of N. incerta.     

A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents

AIMS: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test. METHODS AND RESULTS: Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96-well polystyrene microplate. The 4'6-diamidino-2-phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 microm(2) and quantifying 2 x 10(7) to 2 x 10(8) bacteria adhered per cm(2). The antifouling properties of three commercial surface-active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose-response curve. CONCLUSIONS: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. Significance AND IMPACT OF THE STUDY: In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.     

A comparison of the antifouling/foul-release characteristics of non-biocidal xerogel and commercial coatings toward micro- and macrofouling organisms

Five non-biocidal xerogel coatings were compared to two commercial non-biocidal coatings and a silicone standard with respect to antifouling (AF)/fouling-release (FR) characteristics. The formation and release of biofilm of the marine bacterium Cellulophaga lytica, the attachment and release of the microalga Navicula incerta, and the fraction removal and critical removal stress of reattached adult barnacles of Amphibalanus amphitrite were evaluated in laboratory assays. Correlations of AF/FR performance with surface characteristics such as wettability, surface energy, elastic modulus, and surface roughness were examined. Several of the xerogel coating compositions performed well against both microfouling organisms while the commercial coatings performed less well toward the removal of microalgae. Reattached barnacle adhesion as measured by critical removal stress was significantly lower on the commercial coatings when compared to the xerogel coatings. However, two xerogel compositions showed release of 89-100% of reattached barnacles. These two formulations were also tested in the field and showed similar results.     

Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

Abstract

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R² = 0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.     

Selection of commercial hydrolytic enzymes with potential antifouling activity in marine environments

In this work, the marine antifouling potential of some commercially available hydrolytic enzymes acting on the main constituents of extracellular polymeric substances (EPS) involved in bacterial biofilm formation was determined. The selected protease (i.e., alpha-chymotrypsin from bovine pancreas), carbohydrase (i.e., alpha-amylase from porcine pancreas) and lipase (from porcine pancreas) exhibited remarkable hydrolytic activities towards target macromolecules typically composing EPS under a wide range of pHs (6.5-9.0 for alpha-chymotrysin and alpha-amylase; 7.0-8.5 for the lipase) and temperatures (from 10 °C to 30 °C), as well as relevant half-lives (from about 2 weeks to about 2 months), in a marine synthetic water. The activity displayed by each enzyme was poorly affected by the co-presence of the other enzymes, thus indicating their suitability to be employed in combination. None of the enzymes was able to inhibit the formation of biofilm by an actual site marine microbial community when applied singly. However, a mixture of the same enzymes reduced biofilm formation by about 90% without affecting planktonic growth of the same microbial community. This indicates that multiple hydrolytic activities are required to efficiently prevent biofilm formation by complex microbial communities, and that the mixture of enzymes selected in this study has the potential to be employed as an environmental friendly antifouling agent in marine antifouling coatings.     

Polysulfone and polyacrylate-based zwitterionic coatings for the prevention and easy removal of marine biofouling

A series of polysulfone and polyacrylate-based zwitterionic coatings were prepared on epoxy-primed aluminum substrata and characterized for their antifouling (AF) and fouling-release (FR) properties towards marine bacteria, microalgae and barnacles. The zwitterionic polymer coatings provided minimal resistance against bacterial biofilm retention and microalgal cell attachment, but facilitated good removal of attached microbial biomass by exposure to water-jet apparatus generated hydrodynamic shearing forces. Increasing the ion content of the coatings improved the AF properties, but required a stronger adhesive bond to the epoxy-primed aluminum substratum to prevent coating swelling and dissolution. Grafted poly(sulfobetaine) (gpSBMA), the most promising zwitterionic coating identified from microfouling evaluations, enabled the removal of four out of five barnacles reattached to its surface without incurring damage to their baseplates. This significant result indicated that gpSBMA relied predominately on its surface chemistry for its FR properties since it was very thin (~1–2 µm) relative to commercial coating standards (>200 µm).     

Quantitative analysis of biofilm EPS uronic acid content

The uronic acids assay was evaluated for its ability to measure the amount of uronic acids contained within a biofilm exopolysaccharide matrix. Cytophaga lytica, a marine bacterium isolated from a naturally occurring biofilm, was used to form single-species biofilms for the method assessment. The assay was found to be simple, reproducible, and sensitive to 1 microg levels, suggesting its potential for application as a screening technique for compounds that inhibit the production of microbial biofilm exopolysaccharide containing uronic acids.     

Combinatorial materials research applied to the development of new surface coatings IX: An investigation of novel antifouling/fouling-release coatings containing quaternary ammonium salt groups

Polysiloxane coatings containing chemically-bound (“tethered”) quaternary ammonium salt (QAS) moieties were investigated for potential application as environmental-friendly coatings to control marine biofouling. A combinatorial/high-throughput approach was applied to the investigation to enable multiple variables to be probed simultaneously and efficiently. The variables investigated for the moisture-curable coatings included QAS composition, ie alkyl chain length, and concentration as well as silanol-terminated polysiloxane molecular weight. A total of 75 compositionally unique coatings were prepared and characterized using surface characterization techniques and biological assays. Biological assays were based on two different marine microorganisms, a bacterium, Cellulophaga lytica and a diatom, Navicula incerta, as well as a macrofouling alga, Ulva. The results of the study showed that all three variables influenced coating surface properties as well as antifouling (AF) and fouling-release (FR) characteristics. The incorporation of QAS moieties into a polysiloxane matrix generally resulted in an increase in coating surface hydrophobicity. Characterization of coating surface morphology revealed a heterogeneous, two-phase morphology for many of the coatings investigated. A correlation was found between water contact angle and coating surface roughness, with the contact angle increasing with increasing surface roughness. Coatings based on the QAS moiety containing the longest alkyl chain (18 carbons) displayed the highest micro-roughness and, thus, the most hydrophobic surfaces. With regard to AF and FR properties, coatings based on the 18 carbon QAS moieties were very effective at inhibiting C. lytica biofilm formation and enabling easy removal of Ulva sporelings (young plants) while coatings based on the 14 carbon QAS moities were very effective at inhibiting biofilm growth of N. incerta.     

Comparative study of biofilm formation on biocidal antifouling and fouling-release coatings using next-generation DNA sequencing

The bacterial and eukaryotic communities forming biofilms on six different antifouling coatings, three biocidal and three fouling-release, on boards statically submerged in a marine environment were studied using next-generation sequencing. Sequenced amplicons of bacterial 16S ribosomal DNA and eukaryotic ribosomal DNA internal transcribed spacer were assigned taxonomy by comparison to reference databases and relative abundances were calculated. Differences in species composition, bacterial and eukaryotic, and relative abundance were observed between the biofilms on the various coatings; the main difference was between coating type, biocidal compared to fouling-release. Species composition and relative abundance also changed through time. Thus, it was possible to group replicate samples by coating and time point, indicating that there are fundamental and reproducible differences in biofilms assemblages. The routine use of next-generation sequencing to assess biofilm formation will allow evaluation of the efficacy of various commercial coatings and the identification of targets for novel formulations.     

Biofilm formation by Pseudoalteromonas ruthenica and its removal by chlorine

The distribution of a recently described marine bacterium, SBT 033 GenBank Accession No. AY723742), Pseudoalteromonas ruthenica, at the seawater intake point, outfall and mixing point of an atomic power plant is described, and its ability to form biofilm was investigated. The effectiveness of the antifouling biocide chlorine in the inactivation of planktonic as well as biofilm cells of P. ruthenica was studied in the laboratory. The results show that the planktonic cells were more readily inactivated than the cells enclosed in a biofilm matrix. Viable counting showed that P. ruthenica cells in biofilms were up to 10 times more resistant to chlorine than those in liquid suspension. Using confocal laser scanning microscopy it was shown that significant detachment of P. ruthenica biofilm developed on a glass substratum could be accomplished by treatment with a dose of 1 mg l-1 chlorine. Chlorine-induced detachment led to a significant reduction in biofilm thickness (up to 69%) and substratum coverage (up to 61%), after 5-min contact time. The results show that P. ruthenica has a remarkable ability to form biofilms but chlorine, a common biocide, can be used to effectively kill and detach these biofilms.     

Effectiveness of sodium benzoate as a freshwater low toxicity antifoulant when dispersed in solution and entrapped in silicone coatings

The traditional solution for preventing organisms from attaching to submerged surfaces is to apply antifouling coatings or biocides. Based on the varied defence mechanisms exhibited by biofilms, the antifoulant needs to prevent bacterial attachment during the early stages of biofilm formation. The potential of benzoic acid and sodium benzoate (NaB) as antifoulants for deterring freshwater bacterial attachment was evaluated with the antifoulants dispersed in solution or entrapped in silicone coatings. Effectiveness was based on the decrease in microbial attachment, limited toxicity, and minimum alteration of the properties of the coatings. The optimal NaB concentration when dispersed in solution, 700 mg l-1, resulted in a biofilm surface coverage of only 3.34% after four weeks. The model silicone, Sylgard 184, demonstrated a better overall performance than the commercial coating, RTV11. Sylgard 184 containing sodium benzoate had 41-52% less biofilm in comparison to the control Sylgard 184, whereas both the control and NaB-entrapped RTV11 coatings had significant biofilm coverage.     

On-line monitoring of antifouling and fouling-release surfaces using bioluminescence and fluorescence measurements during laminar flow

A laminar flow biofilm-monitoring system was used to determine the efficacies of three antifouling (AF) coatings and five fouling-release (FR) coatings againstVibrio harveyi attachment. On-line measurements of tryptophan fluorescence and bioluminescence from each coating, normalized to an upstream stainless steel coupon, were used to determine the effects of AF and FR surfaces on biofilm formation. The AF coatings consisted of 5, 10, and 35 wt% Sea Nine 211 (C9211) incorporated into a vinyl copolymer. Both the 10 and 35 wt% coatings significantly inhibited biofilm biomass development measured by tryptophan fluorescence compared to the stainless steel control.V. harveyi bioluminescence was significantly greater than tryptophan fluorescence in cells attached to these coatings, suggesting that bioluminescence expression may be a marker for cellular stress or toxicity in biofilms. Five different polydimethylsiloxane (PDMS) FR coatings did not inhibit biofilm formation under low flow conditions. However, four PDMS coatings demonstrated decreased biomass levels compared to stainless steel after exposure to a shear stress of 330 dynes cm^–2. There was no toxic additive in these coatings; bioluminescence and tryptophan fluorescence were proportional.     

Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

新型海洋防污剂应用组分的简述

海洋防污涂料是通过海洋防污剂的可控释放,与海洋污损生物发生作用,从而阻止海生物在物体表面附着。海洋防污涂料中最重要的组成部分是基体树脂和海洋防污剂。海洋防污剂主要是从农药、杀虫剂、杀菌剂、防霉剂以及带有生物活性的聚合物中筛选出来的,随着海洋环境保护日益严格,制备和筛选高效、广谱的海洋防污剂对海洋防污涂料的发展日显重要,本文对现有的防污剂材料作了简述。     

Molecular approaches to nontoxic antifouling

Summary

A consequence of environmental and human health concerns arising from the use of toxic metals in marine antifouling coatings has been to recognise the need for a nontoxic alternative to fouling control. Recent research has focused on two approaches to this problem: the development of (a) foul-release coatings that work on the principle of either low surface free energy or coating ablation, and (b) coatings that incorporate a compound(s) that is nontoxic, or at least environmentally benign, that will deter fouling. Here we discuss the nature of the fouling problem and a new technology that is emerging to address it. The use of natural marine products and of analogues to these compounds holds considerable promise and is an area of intense research. It is recognized, however, that a melding of the technologies of foul-release and foul-deterrence may be required to develop broad spectrum, nontoxic antifouling coatings. This approach may more closely reflect antifouling strategies adopted by marine organisms that maintain a foul-free surface.     

Modeling biocide action against biofilms

A phenomenological model of biocide action against microbial biofilms was derived. Processes incorporated in the model include bulk flow in and out of a well-mixed reactor, transport of dissolved species into the biofilm, substrate consumption by bacterial metabolism, bacterial growth, advection of cell mass within the biofilm, cell detachment from the biofilm, cell death, and biocide concentration-dependent disinfection. Simulations were performed to analyze the general behavior of the model and to perform preliminary sensitivity analysis to identify key input parameters. The model captured several general features of antimicrobial agent action against biofilms that have been observed widely by experimenters and practitioners. These included (1) rapid disinfection followed by biofilm regrowth, (2) slower detachment than disinfection, and (3) reduced susceptibility of microorganisms in biofilms. The results support the plausibility of a mechanism of biofilm resistance in which the biocide is neutralized by reaction with biofilm constituents, leading to a reduction in the bulk biocide concentration and, more significantly, biocide concentration gradients within the biofilm. Sensitivity experiments and analyses identified which input parameters influence key response variables. Each of three response variables was sensitive to each of the five input parameters, but they were most sensitive to the initial biofilm thickness and next most sensitive to the biocide disinfection rate coefficient. Statistical regression modeling produced simple equations for approximating the response variables for situations within the range of conditions covered by the sensitivity experiment. The model should be useful as a tool for studying alternative biocide control strategies. For example, the simulations suggested that a good interval between pulses of biocide is the time to minimum thickness. (c) 1996 John Wiley & Sons, Inc.     

Fluorescence microscopy techniques for quantitative evaluation of organic biocide distribution in antifouling paint coatings: application to model antifouling coatings

A test matrix of antifouling (AF) coatings including pMMA, an erodible binder and a novel trityl copolymer incorporating Cu?O and a furan derivative (FD) natural product, were subjected to pontoon immersion and accelerated rotor tests. Fluorescence and optical microscopy techniques were applied to these coatings for quantification of organic biocide and pigment distribution. Total leaching of the biocide from the novel copolymer binder was observed within 6 months of rotor immersion, compared to 35% from the pMMA coating. In pontoon immersions, 61% of the additive was lost from the pMMA coating, and 53% from the erodible binder. Profiles of FD content in the binders revealed an accelerated loss of additive from the surface of the CDP resulting from rosin degradation, compared to even depletion from pMMA. In all samples, release of the biocide was inhibited beyond the Cu?O front, corresponding to the leached layer in samples where Cu?O release occurred.     

Biofilm retention on surfaces with variable roughness and hydrophobicity

Biofilms on food processing equipment cause food spoilage and pose a hazard to consumers. The bacterial community on steel surfaces in a butcher's shop was characterized, and bacteria representative of this community enriched from minced pork were used to study biofilm retention. Stainless steel (SS) was compared to two novel nanostructured sol-gel coatings with differing hydrophobicity. Surfaces were characterized with respect to roughness, hydrophobicity, protein adsorption, biofilm retention, and community composition of the retained bacteria. Fewer bacteria were retained on the sol-gel coated surfaces compared to the rougher SS. However, the two sol-gel coatings did not differ in either protein adsorption, biofilm retention, or microbial community composition. When polished to a roughness similar to sol-gel, the SS was colonized by the same amount of bacteria as the sol-gel, but the bacterial community contained fewer Pseudomonas cells. In conclusion, biofilm retention was affected more by surface roughness than chemical composition under the condition described in this study.     

The serine protease Esperase HPF inhibits the formation of multispecies biofilm

The antifouling (AF) potential of the serine protease Esperase HPF (subtilisin) was evaluated for the ability to prevent the formation of a four-species bacterial biofilm. The effects of enzyme activity, time and application of the enzyme were tested on the density and the oxidative metabolism of biofilm developed in microtiter wells. Esperase HPF did not inhibit the oxidative metabolism of the bacterial biofilm or planktonic growth, but the enzyme inhibited biofilm formation by its proteolytic activity as inactivated enzyme had no effect. The effective enzyme concentration was determined over a period of 72 h, as by then all the tested concentrations inhibited biofilm formation maximally. The effective concentrations of the enzymes in solution were the same regardless of time of application (ie before or after biofilm formation), but immobilisation of the enzymes caused a lower effective concentration. Esperase HPF is an attractive alternative to the biocidal compounds used in AF coatings today.