Resistance of Amphiphilic Polysaccharides against Marine Fouling Organisms.

Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.

Conditioning of self-assembled monolayers at two static immersion test sites along the east coast of Florida and its effect on early fouling development.

Among the first events after immersion of surfaces in the ocean is surface 'conditioning'. Here, the accumulation and composition of the conditioning films formed after immersion in the ocean are analyzed. In order to account for different surface chemistries, five self-assembled monolayers that differ in resistance to microfouling and wettability were used. Water samples from two static immersion test sites along the east coast of Florida were collected at two different times of the year and used for experiments. Spectral ellipsometry revealed that conditioning films were formed within the first 24 h and contact angle goniometry showed that these films changed the wettability and rendered hydrophobic surfaces more hydrophilic and vice versa. Infrared reflection adsorption spectroscopy showed that the composition of the conditioning film depended on both the wettability and immersion site. Laboratory and field assays showed that the presence of a conditioning film did not markedly influence settlement of microorganisms.

A comparison between different fouling-release elastomer coatings containing surface-active polymers.

Surface-active polymers derived from styrene monomers containing siloxane (S), fluoroalkyl (F) and/or ethoxylated (E) side chains were blended with an elastomer matrix, either poly(dimethyl siloxane) (PDMS) or poly(styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS), and spray-coated on top of PDMS or SEBS preformed films. By contact angle and X-ray photoelectron spectroscopy measurements, it was found that the surface-active polymer preferentially populated the outermost layers of the coating, despite its low content in the blend. However, the self-segregation process and the response to the external environment strongly depended on both the chemistry of the polymer and the type of matrix used for the blend. Additionally, mechanical testing showed that the elastic modulus of SEBS-based coatings was one order of magnitude higher than that of the corresponding PDMS-based coatings. The coatings were subjected to laboratory bioassays with the marine alga Ulva linza. PDMS-based coatings had superior fouling-release properties compared to the SEBS-based coatings.

Settlement behavior of zoospores of Ulva linza during surface selection studied by digital holographic microscopy.

Settlement of the planktonic dispersal stages of marine organisms is the crucial step for the development of marine biofouling. Four-dimensional holographic tracking reveals the mechanism by which algal spores select surfaces suitable for colonization. Quantitative analysis of the three dimensional swimming trajectories of motile spores of a macroalga (Ulva linza) in the vicinity of surfaces functionalized with different chemistries reveals that their search strategy and swimming behavior is correlated to the number of settled spores found in spore settlement bioassays conducted over 45 min. The spore motility and exploration behavior can be classified into different motion patterns, with their relative occurrence changing with the surface chemistry. Based on the detailed motility analysis we derived a model for the surface selection and settlement process of Ulva zoospores.

Controlling the adhesion of the diatom Navicula perminuta using poly(N-isopropylacrylamide-co-N-(1-phenylethyl) acrylamide) films.

Poly(N-isopropylacrylamide)-co-N-(1-phenylethyl) acrylamide [P(NIPAAm-co-PEAAm)] thermo-responsive thin films with a lower critical solution temperature (LCST) adjusted to fit marine applications were used to investigate the effect of changes in the wetting properties of a surface on the adhesion of the diatom Navicula perminuta, an organism which forms slime films on surfaces immersed in an aquatic environment. Although the strength of attachment of cells was affected by whether the film was collapsed or expanded, no significant decrease in adhesion strength occurred upon temperature decrease. The effects were attributed to possible strong interactions between the hydrophobic segments of the responsive film when collapsed with components in the adhesive complex.

Interactions of zoospores of Ulva linza with arginine-rich oligopeptide monolayers.

We recently reported on the strong interactions of zoospores of the green alga, Ulva linza with an arginine-rich oligopeptide self-assembled monolayer (SAM) [Biofouling 2008, 24, 303-312], where the arginine-rich peptide induced not only high spore settlement, but also a form of abnormal settlement, or "pseudo-settlement", whereby a proportion of spores do not go through the normal process of surface exploration, adhesive exocytosis, and loss of flagella. Further, it was demonstrated that both the total number of settled spores and the fraction of pseudosettled spores were related to the surface density of the arginine-rich peptide. Here we present a further investigation of the interactions of zoospores of Ulva with a set of oligomeric, de novo designed, arginine-rich peptides, specifically aimed to test the effect of peptide primary structure on the interaction. Via variations in the peptide length and by permutations in the amino acid sequences, we gain further insight into the spore-surface interactions. The interpretation of the biological assays is supported by physicochemical characterization of the SAMs using infrared spectroscopy, ellipsometry, and contact angle measurements. Results confirm the importance of arginine residues for the anomalous pseudosettlement, and we found that settlement is modulated by variations in both the total length and peptide primary structure. To elucidate the causes of the anomalous settlement and the possible relation to peptide-membrane interactions, we also compared the settlement of the "naked" zoospores of Ulva (which present a lipoprotein membrane to the exterior without a discrete polysaccharide cell wall), with the settlement of diatoms (unicellular algae that are surrounded by a silica cell wall), onto the peptide SAMs. Cationic SAMs do not notably affect settlement (attachment), adhesion strength, or viability of diatom cells, suggesting that the effect of the peptides on zoospores of Ulva is mediated via specific peptide-membrane interactions.

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.

The influence of surface lubricity on the adhesion of Navicula perminuta and Ulva linza to alkanethiol self-assembled monolayers.

The settlement and adhesion of Navicula perminuta and Ulva linza to methyl-terminated alkanethiol self-assembled monolayers (SAMs) of increasing chain length has been investigated. Organisms were allowed to settle onto the monolayers and were subsequently exposed to hydrodynamic shear stress in order to determine their adhesion strength. Results show that as the SAM structure changes from amorphous to crystalline (C14), there is a marked change in the adhesion of N. perminuta and U. linza. Given that the SAMs in the series all exhibit similar contact angle behaviour and surface energy, it is hypothesized that the lubricity of the surface plays a role in determining the surface adhesion.

Mussel (Mytilus edulis) byssus deposition in response to variations in surface wettability.

Mussels (Mytilus edulis) are economically important in their role as an aquaculture species and also with regard to marine biofouling. They attach tenaciously to a wide variety of submerged surfaces by virtue of collagenous attachment threads termed 'byssi'. The aim of this study was to characterize the spreading of the byssal attachment plaque, which mediates attachment to the surface, on a range of surfaces in response to changes in wettability. To achieve this, well characterized self-assembled monolayers of omega-terminated alkanethiolates on gold were used, allowing correlation of byssal plaque spreading with a single surface characteristic--wettability. The present results were inconsistent with those from previous studies, in that there was a positive correlation between plaque size and surface wettability; a trend which is not explained by conventional wetting theory for a three-phase system. A recent extension to wetting theory with regard to hydrophilic proteins is discussed and the results of settlement assays are used to attempt reconciliation of these results with those of similar previous studies and, also, with recent data presented for the spreading of Ulva linza spore adhesive.

Biofilms.

Biofilms of bacteria, frequently in association with algae, protozoa and fungi, are found on all submerged structures in the marine environment. Although it is likely that for the majority of organisms a biofilmed surface is not a pre-requisite for settlement, in practice, colonization by spores and larvae of fouling organisms almost always takes place via a biofilmed surface. Therefore, the properties of the latter may be expected to influence colonization, positively or negatively. Biofilms are responsible for a range of surface-associated and diffusible signals, which may moderate the settling behaviour of cells, spores and larvae. However, there is no consensus view regarding either cause and effect or the mechanism(s) by which biofilms moderate settlement. Studies with mixed biofilms, especially field experiments, are difficult to interpret because of the conflicting signals produced by different members of the biofilm community as well as their spatial organisation. Molecular techniques highlight the deficiencies of culture methods in identifying biofilm bacteria; hence, the strains with the most impact on settlement of spores and larvae may not yet have been isolated and cultured. Furthermore, secondary products isolated from cultured organisms may not reflect the situation that pertains in nature. The evidence that bacterial quorum sensing signal molecules stimulate settlement of spores of the green macroalga, Ulva, is discussed in some detail. New molecular and analytical tools should provide the opportunity to improve our fundamental understanding of the interactions between fouling organisms and biofilms, which in turn may inform novel strategies to control biofouling.

Settlement behavior of swimming algal spores on gradient surfaces.

When surfaces possessing gradients of surface energy are incubated with motile spores from the green seaweed Ulva, the spores attach on the hydrophilic part of the gradient in larger numbers than they do on the hydrophobic part. This result is opposite to the behavior of the spores observed on the homogeneous hydrophobic and hydrophic surfaces. The data suggest that the gradients have a direct and active influence on the spores, which may be due to the biased migration of the spores during the initial stages of surface sensing.

The influence of surface energy on the wetting behaviour of the spore adhesive of the marine alga Ulva linza (synonym Enteromorpha linza).

The environmental scanning electron microscope has been used to image the adhesive pads secreted by zoospores of the marine alga Ulva linza as they settle on a range of self-assembled and grafted monolayers of different wettability, under natural, hydrated conditions. Results reveal that the diameter of the adhesive pad is strongly influenced by surface wettability, the adhesive spreading more (i.e. wetting the surface better) on the more hydrophilic surfaces. This is in direct contrast to previous observations on the spreading of marine bioadhesives and is in apparent contradiction to the predictions of the Young-Dupre equation for three-phase systems. In this paper, we attempt an explanation based upon thermodynamic analysis of the wetting properties of hydrophilic proteins.

Roughness-dependent removal of settled spores of the green alga Ulva (syn. Enteromorpha) exposed to hydrodynamic forces from a water jet.

Topographic features change the hydrodynamic regime over surfaces subjected to flow. Hydrodynamic microenvironments around topographic structures may have consequences for recruitment and removal of propagules of marine benthic organisms. The settlement and adhesion of zoospores from the green alga Ulva linza (syn. Enteromorpha linza) to defined topographies was investigated. A range of topographic size scales (Rz: 25-100 microm) was manufactured from plankton nets, creating patterns with ridges and depressions. The topographic scales span a roughness similar to that of natural substrata and antifouling coatings. Spores were removed from the surfaces by a calibrated water jet. Fewer spores were removed from the smallest topographic structure tested (Rz: 25 microm) compared to both the smooth (Rz: 1) and the roughest (Rz: 100 microm) structures. Zoospores that settled in depressions were less likely to be removed compared to spores on the ridges. The results in terms of the interaction between surface topography and hydrodynamic forces have implications for both natural substrata exposed to wave action and antifouling surfaces on ships' hulls. The possible effects of topography on increasing zoospore adhesion and offering a refuge from hydrodynamic forces are discussed.

Activity of commercial enzymes on settlement and adhesion of cypris larvae of the barnacle Balanus amphitrite, spores of the green alga Ulva linza, and the diatom Navicula perminuta.

Fouling species produce adhesive polymers during the settlement, adhesion and colonization of new surfaces in the marine environment. The present paper tests the hypothesis that enzymes of the appropriate specificity may prevent biofouling by hydrolysing these adhesive polymers. Seventeen commercially available enzyme preparations designed originally for bulk use in a range of end-use applications were tested for their effects on the settlement and/or adhesion of three major fouling species, viz. the green alga Ulva linza, the diatom Navicula perminuta and the barnacle Balanus amphitrite. The serine-proteases were found to have the broadest antifouling potential reducing the adhesion strength of spores and sporelings of U. linza, cells of N. perminuta and inhibiting settlement of cypris larvae of B. amphitrite. Mode-of-action studies on the serine-protease, Alcalase, indicated that this enzyme reduced adhesion of U. linza in a concentration-dependent manner, that spores of the species could recover their adhesive strength if the enzyme was removed and that the adhesive of U. linza and juvenile cement of B. amphitrite became progressively less sensitive to hydrolysis as they cured.

Adhesion and motility of fouling diatoms on a silicone elastomer.

Recent demands for non-toxic antifouling technologies have led to increased interest in coatings based on silicone elastomers that 'release' macrofouling organisms when hydrodynamic conditions are sufficiently robust. However, these types of coatings accumulate diatom slimes, which are not released even from vessels operating at high speeds (>30 knots). In this study, adhesion strength and motility of three common fouling diatoms (Amphora coffeaeformis var. perpusilla (Grunow) Cleve, Craspedostauros australis Cox and Navicula perminuta Grunow) were measured on a poly-dimethylsiloxane elastomer (PDMSE) and acid-washed glass. Adhesion of the three species was stronger to PDMSE than to glass but the adhesion strengths varied. The wall shear stress required to remove 50% of cells from PDMSE was 17 Pa for Craspedostauros, 24 Pa for Amphora and >>53 Pa for Navicula; the corresponding values for glass were 3, 10 and 25 Pa. In contrast, the motility of the three species showed little or no correlation between the two surfaces. Craspedostauros moved equally well on glass and PDMSE, Amphora moved more on glass initially before movement ceased and Navicula moved more on PDMSE before movement ceased. The results show that fouling diatoms adhere more strongly to a hydrophobic PDMSE surface, and this feature may contribute to their successful colonization of low surface energy, foul-release coatings. The results also indicate that diatom motility is not related to adhesion strength, and motility does not appear to be a useful indicator of surface preference by diatoms.

Three models to relate detachment of low form fouling at laboratory and ship scale.

Since fouling-release coating systems do not prevent settlement, various methods to quantify the tenacity of adhesion of fouling organisms on these systems have been offered. One such method is the turbulent channel flow apparatus. The question remains how the results from laboratory scale tests relate to the self-cleaning of a ship coated with a fouling-release surface. This paper relates the detachment strength of low form fouling determined in the laboratory using a turbulent channel flow to the conditions necessary for detachment of these organisms in a turbulent boundary layer at ship scale. A power-law formula, the ITTC-57 formula, and a computational fluid dynamics (CFD) model are used to predict the skin-friction at ship scale. The results from all three methods show good agreement and are illustrated using turbulent channel flow data for sporelings of the green macrofouling alga Enteromorpha growing on a fouling-release coating.

Brefeldin A affects adhesion of zoospores of the green alga Enteromorpha.

Primary adhesion of zoospores of the green macroalga Enteromorpha to substrata involves a massive release of adhesive glycoproteins from Golgi-derived, membrane-bounded vesicles in the anterior region of the spore, followed by rapid curing. This process is sensitive to low concentrations (5-10 microg x ml(-1)) of the secretion-inhibiting antibiotic, brefeldin A (BFA). The proportion of cells that settled in BFA was reduced by approximately 50%, but the effect was fully reversed by washing in seawater to remove the BFA. Ultrastructural observations showed that BFA caused the breakdown of Golgi stacks in the majority of cells examined. When settled cells were subjected to shear stress, a greater proportion of those settled in the presence of BFA were detached, compared with controls, indicating reduced adhesion strength in the presence of the antibiotic. The most likely reason for this is that strong adhesion to substrata either requires the synthesis of extra adhesive materials beyond those present in the swimming spore, or the secretion of an additional component required for adhesive curing. The novel use of atomic force microscopy in force modulation mode demonstrated that the adhesive secreted by most spores in the presence of BFA did not undergo the rapid curing process typical of control spores. However, some variation between zoospores was observed, with some cells showing no ultrastructural changes and normal adhesive curing. These results are discussed in relation to variations observed in the propensity and competence of spores to settle, which may be reflected in differential requirements for de novo synthesis and secretion of materials needed for full adhesion.

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha.

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7-10 microm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 microm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 +/- 1.7 mN m(-1) (mean +/- SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m(-1). The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 x 10(6) +/- 0.05 x 10(6) N m-2 (mean +/- SE; n=30). Within minutes of release the adhesive underwent a progressive 'curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives.

Use of self-assembled monolayers of different wettabilities to study surface selection and primary adhesion processes of green algal (Enteromorpha) zoospores.

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH(3)) or hydroxyl (OH) groups or mixtures of CH(3)- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and "searching" above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.

Substratum location and zoospore behaviour in the fouling alga Enteromorpha.

The green alga Enteromorpha is the most important macroalga that fouls ships, submarines and underwater structures. Major factors in its success in colonising new substrata are the production of enormous numbers of swimming spores and their ability to locate surfaces on which to settle. Factors facilitating the settlement and adhesion of asexual zoospores are examined in this article. Settlement and adhesion may be regulated by topographical, biological, chemical and physico-chemical cues, all of which are modified by the presence of microbial biofilm. The level of gregarious zoospore settlement is related to spore density and may be mediated by a number of external cues including fatty acids and 'detritus'.