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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Sexual recognition and fertilization in brown algae.

Fertilization in the brown marine algae known as fucoids, is oogamous. The naked egg cell (80 micron diam.) is fertilized by small biflagellate spermatozoids and both monoecious and dioecious species are found. Fertilization is highly species-specific and this appears to be controlled during plasmogamy. Following fusion of egg and sperm, a rapid (less than 1 min) release of polyuronide cell wall material takes place from cytoplasmic vesicles within the egg. This is easily visualized using the fluorescent brightener Calcofluor, which therefore provides the basis of a quantitative fertilization bioassay. It has not proved possible to measure direct sperm binding to eggs. In experiments to investigate the molecular basis of egg-sperm recognition, the effect of exogenous agents on the initial rate of fertilization was examined. Predigestion of eggs with low concentrations of two glycosidases, alpha-fucosidase and alpha-mannosidase, caused inhibition of fertilization. The lectins concanavalin A and RCA120 bound strongly to egg surfaces, as detected using fluorescent labels, but not to sperm. The binding to eggs inhibited fertilization. On the other hand, Fucose Binding Protein bound only weakly to eggs but strongly to sperm, again causing inhibition of fertilization. It has not proved possible to quantitate lectin binding since high levels of lectin-nonspecific binding were detected using iodinated lectins. These inhibition experiments suggest that specific sugar residues may be involved in egg-sperm recognition, but the effects of lectins must be treated with caution since a large amount of variability in the sensitivity of gametes was detected. Attempts to isolate receptor fractions from egg cells have been partially successful. Egg membrane preparations bind sperm and sodium dodecyl sulphate-solubilized fractions, purified by concanavalin A affinity chromatography, have yielded low levels of a soluble receptor-like fraction that has not yet been fully characterized. Antisera raised against surface antigens of Fucus serratus sperm flagella, cause inhibition of fertilization in a species-specific manner, possibly by binding directly to the sperm fertilization receptor. A number of flagellar antigens were detected and future attempts to pinpoint the sperm receptor will make use of monoclonal antibodies.

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'.

How suspension cultures of Catharanthus roseus respond to oxygen limitation: small-scale tests with applications to large-scale cultures.

The effect of oxygen supply on the growth of suspension cultures of Catharanthus roseus in Erlenmeyer flasks was investigated. Below a critical oxygen supply rate the culture could not survive. By increasing the oxygen supply, a point is reached where the culture survives but no growth is possible. At higher oxygen supply rates there is a regime where both growth rate and the maximum biomass concentration increase with oxygen supply. Eventually there comes a point where no further increase in biomass is achieved, probably due to the depletion of the sugars; however, the growth rate continues to increase with oxygen supply until a maximum growth rate is obtained. The ratio of fresh to dry weight at maximum fresh weight increased with shaker table speed of rotation accompanied by a greater rate of sugar depletion.

Monoclonal antibodies to sperm surface antigens of the brown alga Fucus serratus exhibit region-, gamete-, species- and genus-preferential binding.

A panel of twelve monoclonal antibodies (MAbs), designated FS1 to FS12, have been raised against surface antigens of Fucus serratus sperm. The antibodies were selected on the basis that they show region-, gamete-, species- or genus-preferential binding. Indirect immunofluorescence shows that the antigens bound by the MAbs are distributed non-randomly over the cell surface. Seven MAbs (FS1, FS3, FS4, FS6, FS8, FS9, FS10) bind antigens located primarily on the cell body, while the others (FS2, FS5, FS7, FS11, FS12) bind antigens located primarily on the anterior flagellum. Of the MAbs that label the anterior flagellum, FS2, FS5, FS7 and FS12 form a 'halo' at the perimeter of the flagellum. Electron microscopic-immunogold studies indicate that the 'halo' results from labelling of the mastigonemes, as opposed to the flagellar plasmamembrane. Gamete-preferential binding of antibodies was detected using an enzyme-linked immunosorbent assay with egg membrane vesicles. Eight of the MAbs bind sperm antigens not common to eggs, though FS2, FS4, FS5 and FS9 bind antigens present on both sperm and eggs. In studies of species- and genus-specificity FS2, FS3, FS5, FS6, FS7, FS8, FS10, FS11 and FS12 exhibit genus-preferential binding, labelling sperm of F. serratus and F. vesiculosus more intensely than that of Ascophyllum nodosum. Only FS10 showed marked species-preferential binding, labelling sperm of F. serratus much more intensely than that of F. vesiculosus.

Fertilization in brown algae. III. Preliminary characterization of putative gamete receptors from eggs and sperm of Fucus serratus.

Membrane fractions have been isolated from eggs of Fucus serrratus which inhibit fertilization in a species-specific manner. This activity is destroyed by alpha-fucosidase and alpha-mannosidase. Some 6% of the protein of this membrane fraction binds to Con A-agarose, following SDS solubilization, and when eluted with alpha-methyl mannoside inhibits fertilization when preincubated with sperm, but not eggs. This inhibitory activity is species-specific and destroyed by alpha-fucosidase but not by trypsin. SDS-gel electrophoresis reveals 1 band staining strongly with Coomassie Brilliant Blue G and weakly with the PAS reagent. This major band represents a glycoprotein with an approximate molecular weight of 30 000 Daltons. Membrane fractions from sperm of Fucus serratus solubilized in KC1 yielded a protein-containing fraction, after affinity chromatography on desulphated focoidan-Sepharose. This fraction is 100-fold more effective in inhibiting fertilization after preincubation with eggs than either Con A or fucose-binding protein. It is species-specific and inhibition is reversed when pretreated eggs are washed with fucoidan. Activity is destroyed by heat and trypsin and only one diffuse band is apparent on SDS gels. This stains positively with Coomassie Brilliant Blue G but not with PAS and has a molecular weight of approximately 60 000 Daltons. Tentative calculation of the numbers of putative receptor molecules gives a figure of 2.5 X 10(9) receptors per egg and 1.8 X 10(6) receptors per sperm.

Product enhancement and recovery from transformed root cultures of Nicotiana glauca.

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.

The molecular nature of Fucus serratus sperm surface antigens recognised by monoclonal antibodies FS1 to FS12.

Sperm of the brown alga Fucus serratus are highly differentiated, biflagellate, naked cells. Immunolocalisation studies, employing monoclonal antibodies (MAbs - designated FS1 to FS12) raised against antigens of these sperm cells, have revealed that some sperm surface components are distributed over the entire cell, whereas others are restricted to, or occur preferentially on, the surface of the anterior flagellum or cell body. This report describes the use of these MAbs in Western-blot procedures and antigen-modification binding assays to determine the nature of these sperm surface components. Monoclonal antibodies which bind to antigens found on the cell body and both flagella (FS3, FS4, FS6, FS8, FS10) recognise carbohydrate epitopes of a high-molecular-weight glycoprotein (Mr=205 kDa). These MAbs were initially chosen at random from a much larger number of antibodies which bound to sperm in a similar fashion, indicating that this glycoprotein is an immunodominant antigen. Though these MAbs compete under conditions of limited antigen availability, differences in the effects of periodate on antibody binding and differences in other binding data indicate that the MAbs recognise epitopes of this glycoprotein which are neighbouring or overlapping, rather than common. The MAb FS9, which has a similar binding pattern to the above antibodies, also seems to bind to carbohydrate epitopes, but the antigen recognised by this antibody could not be identified in Western-blotting procedures. The MAbs FS7 and FS12, which bind to the mastigonemes on the anterior flagellum and to the cell body and posterior flagellum, recognise a set of glycoproteins in the molecular-weight range 40-250 kDa. The evidence indicates that the antibodies are binding to N-linked carbohydrate side chains of these glycoproteins. Three MAbs that bind to the anterior flagellum (FS2, FS5 and FS11) recognise protein antigens in the molecular-weight range 90-250 kDa; it is not known whether these antigens are glycosylated. The MAb FS1, which binds primarily to the sperm cell body, could not be used in enzyme-linked immunosorbent assays or Western-blotting procedures and the antigen recognised by this antibody is so far uncharacterised.