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.     

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.     

The effects of nitric oxide in settlement and adhesion of zoospores of the green alga Ulva

Previous studies have shown that elevated nitric oxide (NO) reduces adhesion in diatom, bacterial and animal cells. This article reports experiments designed to investigate whether elevated NO reduces the adhesion of zoospores of the green alga Ulva, an important fouling species. Surface-normalised values of NO were measured using the fluorescent indicator DAF-FM DA and parallel hydrodynamic measurements of adhesion strength were made. Elevated levels of NO caused by the addition of the exogenous NO donor SNAP reduced spore settlement by 20% and resulted in lower adhesion strength. Addition of the NO scavenger cPTIO abolished the effects of SNAP on adhesion. The strength of attachment and NO production by spores in response to four coatings (Silastic® T2; Intersleek® 700; Intersleek® 900 and polyurethane) shows that reduced adhesion is correlated with an increase in NO production. It is proposed that in spores of Ulva, NO is used as an intracellular signalling molecule to detect how conducive a surface is for settlement and adhesion. The effect of NO on the adhesion of a range of organisms suggests that NO-releasing coatings could have the potential to control fouling.     

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.     

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.     

Drag-reducing riblets with fouling-release properties: development and testing

The manufacture and preliminary testing of a drag-reducing riblet texture with fouling-control properties is presented. The commercial fouling-release product Intersleek® 1100SR was modified to manufacture riblet-textured coatings with an embossing technology. Hydrodynamic drag measurements in a Taylor–Couette set-up showed that the modified Intersleek® riblets reduced drag by up to 6% compared to a smooth surface. Barnacle settlement assays demonstrated that the riblets did not substantially reduce the ability of Intersleek® 1100SR to prevent fouling by cyprids of Balanus amphitrite. Diatom adhesion tests revealed significantly higher diatom attachment on the riblet surface compared to smooth Intersleek® 1100SR. However, after exposure to flow, the final cell density was similar to the smooth surface. Statically immersed panels in natural seawater showed an increase of biofilm cover due to the riblets. However, the release of semi-natural biofilms grown in a multi-species biofilm culturing reactor was largely unaffected by the presence of a riblet texture.     

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.     

Antifouling potential of Subtilisin A immobilized onto maleic anhydride copolymer thin films

The proteinaceous nature of the adhesives used by most fouling organisms to attach to surfaces suggests that coatings incorporating proteolytic enzymes may provide a technology for the control of biofouling. In the present article, the antifouling (AF) and fouling release potential of model coatings incorporating the surface-immobilized protease, Subtilisin A, have been investigated. The enzyme was covalently attached to maleic anhydride copolymer thin films; the characteristics of the bioactive coatings obtained were adjusted through variation of the type of copolymer and the concentration of the enzyme solution used for immobilization. The bioactive coatings were tested for their effect on the settlement and adhesion strength of two major fouling species: the green alga Ulva linza and the diatom Navicula perminuta. The results show that the immobilized enzyme effectively reduced the settlement and adhesion strength of zoospores of Ulva and the adhesion strength of Navicula cells. The AF efficacy of the bioactive coatings increased with increasing enzyme surface concentration and activity, and was found to be superior to the equivalent amount of enzyme in solution. The results provide a rigorous analysis of one approach to the use of immobilized proteases to reduce the adhesion of marine fouling organisms and are of interest to those investigating enzyme-containing coating technologies for practical biofouling control.     

Topographic cues guide the attachment of diatom cells and algal zoospores

Surface topography plays a key role in the colonization of substrata by the colonizing stages of marine fouling organisms. For the innovation of marine antifouling coatings, it is essential to understand how topographic cues affect the settlement of these organisms. In this study, tapered, spiked microstructures and discrete honeycombs of varying feature dimensions were designed and fabricated in order to examine the influence of topography on the attachment of zoospores of the green macroalga Ulva linza and cells of the diatom (microalga) Navicula incerta. Contrasting results were obtained with these two species of algae. Indeed, the preferred location of cells of N. incerta was dominated by attachment point theory, which suggested a positive correlation between the density of cells adhering and the amount of available attachment points, while the settlement of spores of U. linza was mainly regulated by both Wenzel roughness and local binding geometry.     

Antifouling properties of oligo(lactose)-based self-assembled monolayers

The antifouling (AF) properties of oligo(lactose)-based self-assembled monolayers (SAMs), using four different proteins, zoospores of the green alga Ulva linza and cells of the diatom Navicula incerta, were investigated. The SAM-forming alkylthiols, which contained 1, 2 or 3 lactose units, showed significant variation in AF properties, with no differences in wettability. Non-specific adsorption of albumin and pepsin was low on all surfaces. Adsorption of lysozyme and fibrinogen decreased with increasing number of lactose units in the SAM, in agreement with the generally observed phenomenon that thicker hydrated layers provide higher barriers to protein adsorption. Settlement of spores of U. linza followed an opposite trend, being greater on the bulkier, more hydrated SAMs. These SAMs are more ordered for the larger saccharide units, and it is therefore hypothesized that the degree of order, and differences in crystallinity or stiffness between the surfaces, is an important parameter regulating spore settlement on these surfaces.     

Growth ofEnteromorpha linza in sewage effluent and sewage effluent-seawater mixtures

Enteromorpha linza (L.) J. Ag. was grown in full strength sewage effluent, various combinations of sewage effluent and seawater, and in natural seawater. It was found that full strength sewage effluent with a salinity of 14 supported best growth of the alga. After a 12 day cultivation period, growth ofE. linza in full strength sewage effluent and 75% sewage effluent- seawater mixture showed 3.5-fold and 2-fold increase in fresh weight over that grown in natural seawater; respectively. Uptake of PO _inf4 ^sup3– -P, NH₃-N and NO _inf3 ^sup– -N by cells ofE. linza was extremely efficient in all tested media. Data obtained from the experiments indicated that inorganic nitrogen rather than phosphorus was the limiting nutrient factor for growth ofE. linza in full strength sewage effluent and in other sewage effluent- seawater mixtures. NH₃-N at concentrations above 4.5 ppm was found to inhibit uptake of NO _inf3 ^sup– -N in the same culture medium by the algal cells. The fact that sewage grownE. linza contained comparatively much higher protein content (30.2% dry weight) than that grown in natural seawater (12.5% dry weight) leads to the conclusion that sewage grownE. linza could serve as an economically feasible feed for livestock in Hong Kong where the sewage is characterized by having a salinity of approximately 14. It is proposed that this multicellular green alga is a suitable algal species to serve the dual function of wastewater purification through the production of algal protein from sewage effluent having high salinities.     

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.     

SIZE INCREMENTS DUE TO INTERINDIVIDUAL FUSIONS: HOW MUCH AND FOR HOW LONG?1

Size increments following interindividual fusions appear as a general benefit for organisms, such as coalescing seaweeds and modular invertebrates, with the capacity to fuse with conspecifics. Using sporelings of the red algae Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira and Mazzaella laminarioides (Bory) Fredericq, we measured the growth patterns of sporelings built with different numbers of spores, and the magnitude and persistence of the size increments gained by fusions. Then we studied three morphological processes that could help explain the observed growth patterns. Results indicate that in these algae, coalescence is followed by immediate increase in total size of the coalesced individual and that the increment is proportional to the number of individuals fusing. However, the size increments in sporelings of both species do not last >60 d. Increasing reductions of marginal meristematic cells and increasing abundance of necrotic cells in sporelings built with increasing numbers of initial spores are partial explanations for the above growth patterns. Since sporelings formed by many spores differentiate erect axes earlier and in larger quantities than sporelings formed by one or only a few spores, differentiation, emergence, and growth of erect axes appear as a more likely explanation for the slow radial growth of the multisporic sporelings. Erect axis differentiation involves significant morphological and physiological changes and a shift from radial to axial growth. It is concluded that the growth pattern exhibited by these macroalgae after fusion differs from equivalent processes described for other organisms with the capacity to fuse, such as modular invertebrates.     

Effects of Macroalgal Chemical Extracts on Spore Behavior of the Antarctic Epiphyte Elachista antarctica Phaeophyceae

Most macroalgal species along the Western Antarctic Peninsula (WAP) are defended against predation, many using chemical defenses. These subtidal communities are also mostly devoid of free living filamentous algae. However, one endo/epiphyte, Elachista antarctica, is found growing exclusively out of the palatable rhodophyte Palmaria decipiens. To understand this unusual and exclusive epiphytization, we tested whether macroalgal secondary metabolites such as those responsible for deterring sympatric grazers, affect the behaviors of the epiphyte's spores. Settlement, germination, and swimming behaviors of the epiphyte's motile spores were quantified in the presence of fractionated lipophilic and hydrophilic extracts of host P. decipiens and other rhodophytes from the shallow subtidal. Host P. decipiens was the only alga tested that did not inhibit spore settlement or germination. We also examined whether extracts from these chemically rich algae affect spore swimming behaviors and found spores to be chemotactically attracted to seawater soluble extract fractions of host P. decipiens. These results indicate that chemosensory behaviors of the epiphyte's spores to metabolites associated with these chemically defended macrophytes can explain this exclusive epiphyte–host interaction.     

Poly(dimethyl siloxane) (PDMS) network blends of amphiphilic acrylic copolymers with poly(ethylene glycol)-fluoroalkyl side chains for fouling-release coatings. II. Laboratory assays and field immersion trials

Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700?. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance.     

Multiple allelopathic activity of the crustose coralline algaLithophyllum yessoenseagainst settlement and germination of seaweed spores

A study was made to investigate possible formation by the crustose coralline algaLithophyllum yessoenseof multiple allelopathic-related substances against the settlement and germination of spores of various seaweeds. Seven different solvents (n-hexane, diethyl ether, acetone, ethyl acetate, acetonitrile, methanol, distilled water) and seawater were used to obtain crude extracts and secretory exudates from the coralline alga. The extracts and the algal conditioned seawater were tested for inhibitory activity against the settlement and germination of spores from 17 species representing 15 genera. Spore settlement of 14 species was inhibited over 90% by one or more extracts of the six organic solvents and conditioned seawater. The germination of spores from 13 species was inhibited by one or more extracts of all seven solvents and conditioned seawater. The species where spore settlement was not significantly affected showed strong inhibition of germination, andvice versa.     

Effect of Substratum Surface Chemistry and Surface Energy on Attachment of Marine Bacteria and Algal Spores

Two series of self-assembled monolayers (SAMs) of ω-substituted alkanethiolates on gold were used to systematically examine the effects of varying substratum surface chemistry and energy on the attachment of two model organisms of interest to the study of marine biofouling, the bacterium Cobetia marina (formerly Halomonas marina) and zoospores of the alga Ulva linza (formerly Enteromorpha linza). SAMs were formed on gold-coated glass slides from solutions containing mixtures of methyl- and carboxylic acid-terminated alkanethiols and mixtures of methyl- and hydroxyl-terminated alkanethiols. C. marina attached in increasing numbers to SAMs with decreasing advancing water contact angles (θ_AW), in accordance with equation-of-state models of colloidal attachment. Previous studies of Ulva zoospore attachment to a series of mixed methyl- and hydroxyl-terminated SAMs showed a similar correlation between substratum θ_AW and zoospore attachment. When the hydrophilic component of the SAMs was changed to carboxylate, however, the profile of attachment of Ulva was significantly different, suggesting that a more complex model of interfacial energetics is required.     

Effects of temperature and irradiance on photosynthesis and growth of a green-tide-forming species (<Emphasis Type="Italic">Ulva linza</Emphasis>) in the Yellow Sea

Samples of the massive drifting green alga, Ulva linza, were collected from the coastal waters of the Yellow Sea, southwest of Korea, in early July 2009, and cultured under laboratory conditions. The effects of various temperature (10–30°C) and irradiance levels (0–1,000 μmol photons m⁻² s⁻¹) on photosynthesis, growth, and tissue nutrient content of U. linza were investigated in laboratory for both individuals of the late-stage vegetation (LSV) and the early-stage vegetation (ESV). After 1 h acclimation to various irradiance and temperature conditions, maximum gross photosynthetic rate of ESV was significantly higher than those of LSV. In the long-term (7-d) acclimation experiments to various irradiance and temperature levels, gross photosynthetic rates of ESV individuals were also significantly higher than those of LSV individuals. High photosynthetic rate of ESV individuals induced increase in mass of about 60% over the growth saturation irradiance (136 μmol photons m⁻² s⁻¹) and about 20% under low temperature conditions (10–15°C) during 7-d. The gross photosynthesis of LSV individuals was low when examined under temperature and irradiance conditions that were optimum for ESV growth. Consequently, free-floating U. linza exhibits cellular senescence beginning in early July in the Yellow Sea, and green tides formed by this species cannot be maintained beyond this time in the open sea. However, we expect that U. linza can proliferate quickly after settlement on new coastal habitats of the Yellow Sea because of the high tissue nitrogen utilization for photosynthesis in ESV, which is formed by germination of reproductive cells.     

Antialgal activity of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes against the marine alga Ulva

Marine biofouling has detrimental effects on the environment and economy, and current antifouling coatings research is aimed at environmentally benign, non-toxic materials. The possibility of using contact-active coatings is explored, by considering the antialgal activity of cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes. The antialgal activity was investigated via zoospore settlement and sporeling growth assays of the marine algae Ulva linza and U. lactuca. The assay results for PDMAEMA brushes were compared to those for anionic and neutral surfaces. It was found that only PDMAEMA could disrupt zoospores that come into contact with it, and that it also inhibits the subsequent growth of normally settled spores. Based on the spore membrane properties, and characterization of the PDMAEMA brushes over a wide pH range, it is hypothesized that the algicidal mechanisms are similar to the bactericidal mechanisms of cationic polymers, and that further development could lead to successful contact-active antialgal coatings.     

Bioactive volatile compounds from marine algae: feeding attractants

Chemical communications play an important role in plants, fungi, and algae. Volatile organic compounds in marine algae are released into the seawater. These compounds play a role as either pheromones or allelochemicals. We observed that the turbinid gastropod Lunella coronata coreensis inhabits the intertidal zone and often grazes the green alga Ulva pertusa. Feeding tests and feeding preference studies were performed with green, brown and red algae or by using the powdered freeze-dried seaweed in agar. The snails fed on U. pertusa preferentially compared to the other marine algae, and recognized chemoreception compounds from the alga but not their structural or morphological differences. From feeding tests using artificial foods, it is suggested that the feeding attractants are in the essential oil of the alga U. pertusa.