The control of marine biofouling on xerogel surfaces with nanometer-scale topography

Mixtures of n-octadecyltrimethoxysilane (C18, 1-5 mole-%), n-octyltriethoxysilane (C8) and tetraethoxysilane (TEOS) gave xerogel surfaces of varying topography. The 1:49:50 C18/C8/TEOS xerogel formed 100-400-nm-wide, 2-7-nm deep pores by AFM while coatings with?≥3% C18 were free of such features. Segregation of the coating into alkane-rich and alkane-deficient regions in the 1:49:50 C18/C8/TEOS xerogel was observed by IR microscopy. Immersion in ASW for 48?h gave no statistical difference in surface energy for the 1:49:50 C18/C8/TEOS xerogel and a significant increase for the 50:50 C8/TEOS xerogel. Settlement of barnacle cyprids and removal of juvenile barnacles, settlement of zoospores of the alga Ulva linza, and strength of attachment of 7-day sporelings were compared amongst the xerogel formulations. Settlement of barnacle cyprids was significantly lower in comparison to glass and polystyrene standards. The 1:49:50 and 3:47:50 C18/C8/TEOS xerogels were comparable to PDMSE with respect to removal of juvenile barnacles and sporeling biomass, respectively.     

Hybrid xerogel films as novel coatings for antifouling and fouling release

Hybrid sol-gel-derived xerogel films prepared from 45/55 (mol ratio) n-propyltrimethoxysilane (C3-TMOS)/tetramethylorthosilane (TMOS), 2/98 (mol ratio) bis[3-(trimethoxysilyl)propyl]-ethylenediamine (enTMOS)/tetraethylorthosilane (TEOS), 50/50 (mol ratio) n-octyltriethoxysilane (C8-TEOS)/TMOS, and 50/50 (mol ratio) 3,3,3-trifluoropropyltrimethoxysilane (TFP-TMOS)/TMOS were found to inhibit settlement of zoospores of the marine fouling alga Ulva (syn. Enteromorpha) relative to settlement on acid-washed glass and give greater release of settled zoospores relative to glass upon exposure to pressure from a water jet. The more hydrophobic 50/50 C8-TEOS/TMOS xerogel films had the lowest critical surface tension by comprehensive contact angle analysis and gave significantly greater release of 8-day Ulva sporeling biomass after exposure to turbulent flow generated by a flow channel than the other xerogel surfaces or glass. The 50/50 C8-TEOS/TMOS xerogel was also a fouling release surface for juveniles of the tropical barnacle Balanus amphitrite. X-ray photon electron data indicated that the alkylsilyl residues of the C3-TMOS-, C8-TEOS-, and TFP-TMOS-containing xerogels were located on the surface of the xerogel films (in a vacuum), which contributes to the film hydrophobicity. Similarly, the amine-containing silyl residues of the enTMOS/TEOS films were located at the surface of the xerogel films, which contributes to the more hydrophilic character and increased critical surface tension of these films.     

The performance of hybrid titania/silica-derived xerogels as active antifouling/fouling-release surfaces against the marine alga Ulva linza: in situ generation of hypohalous acids

Mixed titania/silica xerogels were prepared using titanium tetraisopropoxide (TTIP) and tetraethoxy orthosilicate (TEOS). Xerogel properties were modified by incorporating n-octyltriethoxysilane (C8). The xerogels catalyze the oxidation of bromide and chloride with hydrogen peroxide (H₂O₂) to produce hypohalous acids at pH 7 and pH 8. The antifouling/ fouling-release performance of a TTIP/C8/TEOS xerogel in the presence and absence of H₂O₂ was evaluated for the settlement of zoospores of the marine alga Ulva linza and for the removal of sporelings (young plants). In the absence of H₂O₂, differences in the settlement of zoospores and removal of sporelings were not significant relative to a titanium-free C8/TEOS xerogel. Addition of H₂O₂ gave a significant reduction in zoospore settlement and sporeling removal relative to the C8/TEOS xerogel and relative to peroxide-free conditions. The impact of TTIP on xerogel characteristics was evaluated by comprehensive contact angle analysis, scanning electron microscopy, and X-ray photoelectron spectroscopy.     

Spontaneous multiscale phase separation within fluorinated xerogel coatings for fouling-release surfaces

Four-component xerogel films consisting of 1 mole-% n-octadecyltrimethoxysilane (C18) and 50 mole-% tetraethoxysilane (TEOS) in combination with 1-24 mole-% tridecafluoro-1,1,2,2-tetrahydrooctyltriethoxysilane (TDF) and 25-48 mole-% n-octyltriethoxysilane (C8) and a 1:49:50 mole-% C18/TDF/TEOS were prepared. Settlement of barnacle cyprids and removal of juvenile barnacles, settlement of zoospores of the alga Ulva linza, and strength of attachment of 7-day sporelings (young plants) of Ulva were compared amongst the xerogel formulations. Several of the xerogel formulations were comparable to poly(dimethylsiloxane) elastomer with respect to removal of juvenile barnacles and removal of sporeling biomass. The 1:4:45:50 and 1:14:35:50 C18/TDF/C8/TEOS xerogels displayed some phase segregation by atomic force microscopy (AFM) pre- and post-immersion in water. Imaging reflectance infrared microscopy showed the formation of islands of alkane-rich and perfluoroalkane-rich regions in these same xerogels both pre- and post-immersion in water. Surface energies were unchanged upon immersion in water for 48?h amongst the TDF-containing xerogel coatings. AFM measurements demonstrated that surface roughness on the 1:4:45:50 and 1:14:35:50 C18/TDF/C8/TEOS xerogel coatings decreased upon immersion in water.     

Spontaneous multiscale phase separation within fluorinated xerogel coatings for fouling-release surfaces

Four-component xerogel films consisting of 1 mole-% n-octadecyltrimethoxysilane (C18) and 50 mole-% tetraethoxysilane (TEOS) in combination with 1–24 mole-% tridecafluoro-1,1,2,2-tetrahydrooctyltriethoxysilane (TDF) and 25–48 mole-% n-octyltriethoxysilane (C8) and a 1:49:50 mole-% C18/TDF/TEOS were prepared. Settlement of barnacle cyprids and removal of juvenile barnacles, settlement of zoospores of the alga Ulva linza, and strength of attachment of 7-day sporelings (young plants) of Ulva were compared amongst the xerogel formulations. Several of the xerogel formulations were comparable to poly(dimethylsiloxane) elastomer with respect to removal of juvenile barnacles and removal of sporeling biomass. The 1:4:45:50 and 1:14:35:50 C18/TDF/C8/TEOS xerogels displayed some phase segregation by atomic force microscopy (AFM) pre- and post-immersion in water. Imaging reflectance infrared microscopy showed the formation of islands of alkane-rich and perfluoroalkane-rich regions in these same xerogels both pre- and post-immersion in water. Surface energies were unchanged upon immersion in water for 48 h amongst the TDF-containing xerogel coatings. AFM measurements demonstrated that surface roughness on the 1:4:45:50 and 1:14:35:50 C18/TDF/C8/TEOS xerogel coatings decreased upon immersion in water.     

Correlative assays of barnacle cyprid behaviour for the laboratory evaluation of antifouling coatings: a study of surface energy components

Laboratory evaluation of antifouling coatings is underpinned by settlement studies with specific fouling organisms. Established methods provide insight into the likelihood of failure of a particular coating system, but can neglect the process of surface selection that often precedes attachment. The present approach for quantifying the exploratory behaviour of barnacle cypris larvae suggested that inspection behaviour can be a rapid and predictive proxy for settlement. Two series of xerogels with comparable total surface energy, but different dispersive and polar components, were evaluated. Settlement assays with three-day-old cyprids of Balanus improvisus demonstrated that while attachment was not linked directly to dispersive free energy, the composition of the xerogel was nevertheless significant. Behavioural analysis provided insight into the mechanism of surface rejection. In the case of a 50:50?PH/TEOS (phenyltriethoxysilane-based) xerogel vs a 50:50 TFP/TEOS (3,3,3-trifluoropropyltrimethoxysilane-based) xerogel, wide-searching behaviour was absent on the former.     

Acyl-homoserine lactones modulate the settlement rate of zoospores of the marine alga Ulva intestinalis via a novel chemokinetic mechanism

Bacteria utilize quorum sensing to regulate the expression of cell density-dependant phenotypes such as biofilm formation and virulence. Zoospores of the marine alga Ulva intestinalis exploit the acyl-homoserine lactone (AHL) quorum sensing system to identify bacterial biofilms for preferential settlement. Here, we demonstrate that AHLs act as strong chemoattractants for Ulva zoospores. Chemoattraction does not involve a chemotactic orientation towards the AHL source. Instead, it occurs through a chemokinesis in which zoospore swimming speed is rapidly decreased in the presence of AHLs. The chemoresponse to AHLs was dependant on the nature of the acyl side chain, with N-(3-oxododecanoyl)-homoserine lactone (30-C12-HSL) being the most effective signal molecule. Mean zoospore swimming speed decreased more rapidly over wild-type biofilms of the marine bacteria Vibrio anguillarum relative to biofilms of the vanM mutant, in which AHL synthesis is disrupted. These data implicate a role for AHL-mediated chemokinesis in the location and preferential settlement of Ulva zoospores on marine bacterial assemblages. Exposure to AHLs did not inhibit the negative phototaxis of Ulva zoospores, indicating that chemoattraction to bacterial biofilms does not preclude the response to a light stimulus in substrate location.     

Poly(ethylene glycol)-containing hydrogel surfaces for antifouling applications in marine and freshwater environments

This work describes the fabrication, characterization, and biological evaluation of a thin protein-resistant poly(ethylene glycol) (PEG)-based hydrogel coating for antifouling applications. The coating was fabricated by free-radical polymerization on silanized glass and silicon and on polystyrene-covered silicon and gold. The physicochemical properties of the coating were characterized by infrared spectroscopy, ellipsometry, and contact angle measurements. In particular, the chemical stability of the coating in artificial seawater was evaluated over a six-month period. These measurements indicated that the degradation process was slow under the test conditions chosen, with the coating thickness and composition changing only marginally over the period. The settlement behavior of a broad and diverse group of marine and freshwater fouling organisms was evaluated. The tested organisms were barnacle larvae (Balanus amphitrite), algal zoospores (Ulva linza), diatoms (Navicula perminuta), and three bacteria species (Cobetia marina, Marinobacter hydrocarbonoclasticus, and Pseudomonas fluorescens). The biological results showed that the hydrogel coating exhibited excellent antifouling properties with respect to settlement and removal.     

Algal antifouling and fouling-release properties of metal surfaces coated with a polymer inspired by marine mussels

The marine antifouling and fouling-release performance of titanium surfaces coated with a bio-inspired polymer was investigated. The polymer consisted of methoxy-terminated poly(ethylene glycol) (mPEG) conjugated to the adhesive amino acid l-3,4-dihydroxyphenylalanine (DOPA) and was chosen based on its successful resistance to protein and mammalian cell fouling. Biofouling assays for the settlement and release of the diatom Navicula perminuta and settlement, growth and release of zoospores and sporelings (young plants) of the green alga Ulva linza were carried out. Results were compared to glass, a poly(dimethylsiloxane) elastomer (Silastic T2) and uncoated Ti. The mPEG-DOPA3 modified Ti surfaces exhibited a substantial decrease in attachment of both cells of N. perminuta and zoospores of U. linza as well as the highest detachment of attached cells under flow compared to control surfaces. The superior performance of this polymer over a standard silicone fouling-release coating in diatom assays and approximately equivalent performance in zoospore assays suggests that this bio-inspired polymer may be effective in marine antifouling and fouling-release applications.     

Species-specific engineered antifouling topographies: correlations between the settlement of algal zoospores and barnacle cyprids

Novel, non-toxic antifouling technologies are focused on the manipulation of surface topography to deter settlement of the dispersal stages of fouling organisms. This study investigated the effect of the aspect ratio (feature height/feature width) of topographical features engineered in polydimethylsiloxane, on the settlement of cyprids of Balanus amphitrite and zoospores of Ulva linza. The correlation of relative aspect ratios to antifouling efficacy was proven to be significant. An increase in aspect ratio resulted in an increase of fouling deterrence for both zoospores and cyprids. The spore density of Ulva was reduced 42% with each unit increase in aspect ratio of the Ulva-specific Sharklet AF topography. Similarly, the number of settled cyprids was reduced 45% with each unit increase in aspect ratio. The newly described barnacle-specific Sharklet AF topography (40 microm feature height, aspect ratio of 2) reduced cyprid settled by 97%. Techniques have been developed to superimpose the smaller Ulva-specific topographies onto the barnacle-specific surfaces into a hierarchical structure to repel both organisms simultaneously. The results for spore settlement on first-generation hierarchical surfaces provide insight for the efficacious design of such structures when targeting multiple settling species.     

The performance of aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel coatings against the marine alga Ectocarpus crouaniorum: relative roles of surface energy and charge

The effect of a series of xerogel coatings modified with aminoalkyl/fluorocarbon/hydrocarbon groups on the adhesion of a new test species, the filamentous brown alga Ectocarpus crouaniorum, has been explored, and compared with the green alga Ulva linza. The results showed that E. crouaniorum adhered weakly to the less polar, low wettability coatings in the series, but stronger adhesion was shown on polar, higher surface energy coatings containing aminoalkyl groups. The results from a separate series of coatings tuned to have similar surface energies and polarities after immersion in artificial seawater (ASW), but widely different surface charges, demonstrated that surface charge was more important than surface energy and polarity in determining the adhesion strength of both E. crouaniorum and U. linza on xerogel coatings. No correlation was found between adhesion and contact angle hysteresis. X-ray photoelectron spectroscopy analysis of samples after immersion in ASW confirmed the presence of charged ammonium groups on the surface of the aminoalkylated coatings.     

Turnover of quorum sensing signal molecules modulates cross-kingdom signalling

N -acylhomoserine lactone (AHL) quorum-sensing molecules modulate the swimming behaviour of zoospores of the macroalga Ulva to facilitate the location of bacterial biofilms. Here we show that the intertidal surfaces colonized by Ulva are dominated by Alphaproteobacteria , particularly the Rhodobacteraceae family, and the Bacteroidetes family Flavobacteriaceae , and that this diverse assemblage both produces and degrades AHLs. N -acylhomoserine lactones could also be extracted from the surfaces of pebbles recovered from intertidal rock-pools. Bacteria representative of this assemblage were isolated and tested for the production and degradation of AHLs, and for their ability to modulate zoospore settlement at different biofilm densities. Of particular interest was a Shewanella sp. This strain produced three major AHLs (OC4, OC10 and OC12) in the late exponential phase, but the longer-chain AHLs were rapidly degraded in the stationary phase. Degradation occurred via both lactonase and amidase activity. A close relationship was found between AHL synthesis and Ulva zoospore settlement. The Shewanella isolate also interfered with AHL production by a Sulfitobacter isolate and its ability to enhance zoospore settlement in a polymicrobial biofilm. This influence on the attachment of Ulva zoospores suggests that AHL-degrading strains can affect bacterial community behaviour by interfering with quorum sensing between neighbouring bacteria. More importantly, these interactions may exert wider ecological effects across different kingdoms.     

Phospholipase A2 activation by hydrogen peroxide during in vitro capacitation of buffalo spermatozoa

Progressively motile, washed buffalo spermatozoa (50 x 10(6) cells in 0.5 ml) were in vitro capacitated in HEPES containing Bovine Gamete Medium 3 (BGM3) in presence of heparin (10 microg/ml), and different concentrations of hydrogen peroxide (10 to 100 microM). Spermatozoa (60%) were capacitated in presence of heparin compared to 56% in presence of 25 microM H2O2 (optimally found suitable for capacitation). The extent of capacitation was measured in terms of acrosome reaction (AR) induced by lysophosphatidyl choline (100 microg/ml). The acrosome reacted cells were counted after triple staining. Catalase (100 microg/ml) significantly reduced the sperm capacitation to 16-18% when added with H2O2, or alone in the capacitation medium. Phospholipase A2 activity of spermatozoa increased linearly up to 50 microM H2O2 concentration included in the assay system. Moreover, significant increase in phospholipase A2 activity was observed after capacitation by both, the heparin and 25 microM H2O2. The activity was always higher in acrosome reacted cells.     

Cross-kingdom signalling: exploitation of bacterial quorum sensing molecules by the green seaweed Ulva

The green seaweed Ulva has been shown to detect signal molecules produced by bacteria. Biofilms that release N-acylhomoserine lactones (AHLs) attract zoospores--the motile reproductive stages of Ulva. The evidence for AHL involvement is based on several independent lines of evidence, including the observation that zoospores are attracted to wild-type bacteria that produce AHLs but are not attracted to mutants that do not produce signal molecules. Synthetic AHL also attracts zoospores and the attraction is lost in the presence of autoinducer inactivation (AiiA) protein. The mechanism of attraction is not chemotactic but involves chemokinesis. When zoospores detect AHLs, the swimming rate is reduced and this results in accumulation of cells at the source of the AHL. It has been demonstrated that the detection of AHLs results in calcium influx into the zoospore. This is the first example of a calcium signalling event in a eukaryote in response to bacterial quorum sensing molecules. The role of AHLs in the ecology of Ulva is discussed. It is probable that AHLs act as cues for the settlement of zoospores, rather than being directly involved as a signalling mechanism.     

Engineered antifouling microtopographies - effect of feature size, geometry, and roughness on settlement of zoospores of the green alga Ulva

The effect of feature size, geometry, and roughness on the settlement of zoospores of the ship fouling alga Ulva was evaluated using engineered microtopographies in polydimethylsiloxane elastomer. The topographies studied were designed at a feature spacing of 2 microm and all significantly reduced spore settlement compared to a smooth surface. An indirect correlation between spore settlement and a newly described engineered roughness index (ERI) was identified. ERI is a dimensionless ratio based on Wenzel's roughness factor, depressed surface fraction, and the degree of freedom of spore movement. Uniform surfaces of either 2 mum diameter circular pillars (ERI=5.0) or 2 microm wide ridges (ERI=6.1) reduced settlement by 36% and 31%, respectively. A novel multi-feature topography consisting of 2 mum diameter circular pillars and 10 microm equilateral triangles (ERI=8.7) reduced spore settlement by 58%. The largest reduction in spore settlement, 77%, was obtained with the Sharklet AF topography (ERI=9.5).     

Membrane recycling and calcium dynamics during settlement and adhesion of zoospores of the green alga Ulva linza

Recruitment of individuals of the marine alga Ulva linza on to a suitable habitat involves the settlement of motile zoospores on to a substratum during which a preformed adhesive is secreted by vesicular exocytosis. The fluorescent styryl dye FM 1-43 and fluorescent Ca(2+) indicators were used to follow membrane cycling and changes in cytosolic Ca(2+) ([Ca(2+)](cyt)) associated with settlement. When swimming zoospores were exposed continuously to FM 1-43, the plasma membrane was preferentially labelled. During settlement, FM 1-43-labelled plasma membrane was rapidly internalized reflecting high membrane turnover. The internalized membrane was focused into a discrete region indicating targeting of membrane to an endosome-like compartment. Acetoxymethyl (AM)-ester derivatives were found to be unsuitable for monitoring [Ca(2+)](cyt) because the dyes were rapidly sequestered from the cytoplasm into sub-cellular compartments. [Ca(2+)](cyt) was, however, reliably measured using dextran-conjugated calcium indicators delivered into cells using a biolistic technique. Cells loaded with Oregon Green BAPTA-1 dextran (Invitrogen, Paisley, UK) showed diffuse cytosolic loading and reliably responded to imposed changes in [Ca(2+)](cyt). During settlement, zoospores exhibited both localized and diffuse increases in [Ca(2+)](cyt) implying a role for [Ca(2+)](cyt) in exocytosis of the adhesive.     

A coulometric biosensor to determine hydrogen peroxide using a monomolecular layer of horseradish peroxidase immobilized on a glass surface

A biosensor to detect hydrogen peroxide, by coulometry, down to submicromolar concentration using a monomolecular layer of horseradish peroxidase was developed. In this device 0.3 pmol of the enzyme were covalently immobilized on the glass surface of the biosensor and the enzyme layer was characterized by atomic force microscopy and activity measurements. The glass surface bearing the peroxidase was faced to a carbon electrode in a cell of 1 microl of active volume. The polarization of the working electrode at -100 mV versus Ag/AgCl, in the presence of 1,4-hydroquinone as mediator, allowed the fast reduction of the injected hydrogen peroxide via the hydroquinone-peroxidase system. This device permitted to measure the total number of H(2)O(2) molecules present in the cell in the concentration range of 0.3-100 microM H(2)O(2), with a sensitivity of 196 nC/microM H(2)O(2), which is close to the theoretical value (193 nC/microM).     

Charged hydrophilic polymer brushes and their relevance for understanding marine biofouling

The resistance of charged polymers to biofouling was investigated by subjecting cationic (PDMAEMA), anionic (PSPMA), neutral (PHEMA-co-PEG₁₀MA), and zwitterionic (PSBMA) brushes to assays testing protein adsorption; attachment of the marine bacterium Cobetia marina; settlement and adhesion strength of zoospores of the green alga Ulva linza; settlement of barnacle (Balanus amphitrite and B. improvisus) cypris larvae; and field immersion tests. Several results go beyond the expected dependence on direct electrostatic attraction; PSPMA showed good resistance towards attachment of C. marina, low settlement and adhesion of U. linza zoospores, and significantly lower biofouling than on PHEMA-co-PEG₁₀MA or PSBMA after a field test for one week. PDMAEMA showed potential as a contact-active anti-algal coating due to its capacity to damage attached spores. However, after field testing for eight weeks, there were no significant differences in biofouling coverage among the surfaces. While charged polymers are unsuitable as antifouling coatings in the natural environment, they provide valuable insights into fouling processes, and are relevant for studies due to charging of nominally neutral surfaces.     

Light-evoked depolarizations in the retina ofStrombus: Role of calcium and other divalent cations

Previous studies indicate that overlapping inward sodium and outward potassium currents play a role in generating the waveform of light-evoked depolarizations (LEDs) in one type of retinal neuron in Strombus luhuanus, a marine gastropod [Chinn, K. S., and Gillary, H. L. (1985). Comp. Biochem. Physiol. 80A:233-245]. This paper concerns the effects of divalent cations on the LED. The LED can exhibit a distinct early phase of depolarization (DE). Increasing the [Ca2+] in the artificial seawater (ASW) bathing medium reduced the amplitude of the entire LED, and omitting Ca2+ increased it. Adding 10 mM Sr2+ or 10 mM Mn2+ to either normal ASW or 0-Ca2+ ASW decreased the LED amplitude. Adding 10 mM Ba2+ to 0-Ca2+ ASW also decreased the LED amplitude, but adding Ba2+ to normal ASW selectively increased DE. Cd2+ (100 microM) selectively reduced DE when added to normal ASW but not when added to 0-Ca2+ ASW. The results show that a variety of divalent cations can alter the currents that underlie the LED. They also suggest that an inward Ca2+ current occurs during DE.     

Conditioning of surfaces by macromolecules and its implication for the settlement of zoospores of the green alga Ulva linza

Conditioning, ie the adsorption of proteins and other macromolecules, is the first process that occurs in the natural environment once a surface is immersed in seawater, but no information is available either regarding the conditioning of surfaces by artificial seawater or whether conditioning affects data obtained from laboratory assays. A range of self-assembled monolayers (SAMs) with different chemical terminations was used to investigate the time-dependent formation of conditioning layers in commercial and self-prepared artificial seawaters. Subsequently, these results were compared with conditioning by solutions in which zoospores of the green alga Ulva linza had been swimming. Spectral ellipsometry and contact angle measurements as well as infrared reflection absorption spectroscopy (IRRAS) were used to reveal the thickness and chemical composition of the conditioning layers. The extent that surface preconditioning affected the settlement of zoospores of U. linza was also investigated. The results showed that in standard spore settlement bioassays (45-60 min), the influence of a molecular conditioning layer is likely to be small, although more substantial effects are possible at longer settlement times.