Ulva Zoospores Research Articles

A comparison of the biofouling potential of field-collected and laboratory-cultured Ulva

The marine algae Ulva spp. are commonly used as model biofouling organisms. As biofouling studies are primarily conducted using field-collected specimens, factors including species identity, seasonal availability, and physiological status can hinder the replicability of the results. To address these limitations, a protocol was developed for the on-demand laboratory culture and release of Ulva zoospores. The biofouling potential of laboratory-cultured and field-collected Ulva blades was compared using a waterjet. No significant differences were found between field and laboratory-cultured samples in either spore adhesion (before waterjet) or the proportion of spores retained after waterjet exposure. However, there was significant variability within each session type in pre- and post-waterjet exposures, indicating that spore adhesion and retention levels vary significantly among trial runs. In addition, all our laboratory cultures were Ulva Clade C (LPP complex). In contrast, our field samples contained a mix of Ulva Clade C, U. compressa clade I, and U. flexuosa Clade D. This protocol for on-demand production of Ulva spores can improve biofouling research approaches, enables comparison of results across laboratories and regions, and accelerate the development of anti-biofouling strategies.

Electric Potential Induced Prevention and Removal of an Algal Biofoulant from Planar SERS Substrates.

Ulva zoospores are widespread marine macroalgae and a common organism found in biofouling communities due to their strong adhesive properties and quick settlement times. Using Ulva as a model organism, a strategy is presented where direct-current (DC) electric potentials are applied in conjunction with surface-enhanced Raman spectroscopy (SERS) to characterize, remove, and prevent Ulva from forming a biofilm on gold-capped nanopillar SERS substrates. Experiments were conducted within a poly(tetrafluoroethylene) (PTFE) flow channel device where the SERS substrates were used as an electrode. Ulva density, determined in situ by SERS and ex situ by electron and fluorescence microscopy, decreased under successively increasing low negative potentials up to -1.0 V. The presence of damaged Ulva suggests that the applied potential led to spore rupture. At the highest negative applied potential (-1.0 V), microparticles containing copper, which is known for its antimicrobial properties, were associated with Ulva on the SERS substrate and the lowest Ulva density was observed. These findings indicate that (1) SERS can be employed to study biofilm formation on nanostructured metal surfaces and (2) applying low-voltage electric potentials may be used to control Ulva biofouling on SERS marine sensors.

Marine Macroalgae: Prospective Hitchhikers of Ship Ballast

Invasive alien species, on successful establishment, can displace native species. The threat of invasive species arises in view of their ability to outcompete and destabilize native biodiversity. Invasive species are found across all taxonomic groups of plants, animals and microorganisms. The green macroalga Ulva flexuosa has a potential to become invasive and this species was investigated for its hitchhiking potential under laboratory conditions. Zoospores of U. flexuosa were maintained at 4°C for nearly 10 months in the dark. Recruitment potential of zoospores after dark stress was tested in a modified Provasoli medium under optimal laboratoryconditions. The success rate of zoospore recruitment was 61%. The paper describes the transfer potential through shipping activities by correlating the Ulva zoospores recruitment potential and survivability.

A controlled release strategy of antifouling agent in coating based on intercalated layered double hydroxides

A controlled release method of antifouling agent was demonstrated via the model coating which was fabricated by sodium paeonolsilate (PAS) intercalated into Zn2Al Layered Double hydroxides (LDH) and then dispersed in the mixture of acrylic resin and colophony with weight ratio of 2:1. The powder X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) confirm the intercalation of PAS into the galleries of Zn2Al-LDH. Some larger particles of Zn2Al-PAS-LDH composite in the coating were observed owing to its aggregation, which was revealed by Laser scanning confocal microscope (LSCM). The release tests of the Zn2Al-PAS-LDH coating in 3.5% NaCl solution showed that no burst release phenomenon occurred at the beginning stage and a slower release rate of PAS (0.54ppm/d) was obtained compared with the pristine PAS coating (1.06ppm/d), exhibiting its preferable controlled release behavior. The adhesion experiment using Ulva zoospores indicated that Zn2Al-PAS-LDH composite obviously increases the utilization rate of PAS owing to its controlling release property. Therefore, the present work provided a useful strategy to decrease the waste of antifouling agent and prolong the lifetime of antifouling coatings.

Facile Synthesis of a Phosphorylcholine-Based Zwitterionic Amphiphilic Copolymer for Anti-Biofouling Coatings

An antibiofouling polymer coating, combined with both zwitterionic and amphiphilic features, is engineered by a two-step modification of a commodity polymer. The surface properties of the resultant polymer coating can be easily tuned by varying the extent of cross-linking in the network. Higher antibiofouling efficiency was observed for these surfaces vs. an elastomeric polydimethylsiloxane standard (Sylgard 184) against the adsorption of biomacromolecules and a marine fouling organism (Ulva zoospores) has been demonstrated. This design establishes a platform for the achievement of functionalized amphiphilic zwitterionic copolymers from relatively inexpensive starting materials via simple chemical manipulations.

Interference with the germination and growth ofUlvazoospores by quorum‐sensing molecules fromUlva‐associated epiphytic bacteria

Ulva zoospores preferentially settle on N-acylhomoserine lactone (AHL) producing marine bacterial biofilms. To investigate whether AHL signal molecules also affect the success and rate of zoospore germination in addition to zoospore attraction, the epiphytic bacteria associated with mature Ulva linza were characterized and bacterial isolates representative of this community tested for the ability to produce AHLs. Two of these AHL-producing isolates, Sulfitobacter spp. 376 and Shewanella spp. 79, were transformed with plasmids expressing the Bacillus spp. AHL lactonase gene aiiA to generate AHL-deficient variants. The germination and growth of U. linza zoospores was studied in the presence of these AHL-deficient strains and their AHL-producing counterparts. This revealed that the AHLs produced by Sulfitobacter spp. and Shewanella spp. or the bacterial products they regulate have a negative impact on both zoospore germination and the early growth of the Ulva germling. Further experiments with Escherichia coli biofilms expressing recombinant AHL synthases and synthetic AHLs provide data to demonstrate that zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. These results reveal an additional role for AHLs per se in the interactive relationships between marine bacteria and Ulva zoospores.

Thematic Issue: Protists

Ulva zoospores preferentially settle on N-acylhomoserine lactone (AHL) producing marine bacterial biofilms. To investigate whether AHL signal molecules also affect the success and rate of zoospore germination in addition to zoospore attraction, the epiphytic bacteria associated with mature Ulva linza were characterized and bacterial isolates representative of this community tested for the ability to produce AHLs. Two of these AHL-producing isolates, Sulfitobacter spp. 376 and Shewanella spp. 79, were transformed with plasmids expressing the Bacillus spp. AHL lactonase gene aiiA to generate AHL-deficient variants. The germination and growth of U. linza zoospores was studied in the presence of these AHL-deficient strains and their AHL-producing counterparts. This revealed that the AHLs produced by Sulfitobacter spp. and Shewanella spp. or the bacterial products they regulate have a negative impact on both zoospore germination and the early growth of the Ulva germling. Further experiments with Escherichia coli biofilms expressing recombinant AHL synthases and synthetic AHLs provide data to demonstrate that zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. These results reveal an additional role for AHLs per se in the interactive relationships between marine bacteria and Ulva zoospores.

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.

Engineered antifouling microtopographies: kinetic analysis of the attachment of zoospores of the green alga Ulva to silicone elastomers

Microtopography has been demonstrated as an effective deterrent to biofouling. The majority of published studies are fixed-time assays that raise questions regarding the kinetics of the attachment process. This study investigated the time-dependent attachment density of zoospores of Ulva, in a laboratory assay, on a micropatterned and smooth silicone elastomer. The attachment density of zoospores was reduced on average 70–80% by the microtopography relative to smooth surfaces over a 4 h exposure. Mapping the zoospore locations on the topography revealed that they settled preferentially in specific, recessed areas of the pattern. The kinetic data fit, with high correlation (r 2 > 0.9), models commonly used to describe the adhesion of bacteria to surfaces. The grouping of spores on the microtopography indicated that the pattern inhibited the ability of attached spores to recruit neighbors. This study demonstrates that the antifouling mechanism of topographies may involve disruption of the cooperative effects exhibited by fouling organisms such as Ulva.

Engineered antifouling microtopographies: the role of Reynolds number in a model that predicts attachment of zoospores of Ulva and cells of Cobetia marina

A correlation between the attachment density of cells from two phylogenetic groups (prokaryotic Bacteria and eukaryotic Plantae), with surface roughness is reported for the first time. The results represent a paradigm shift in the understanding of cell attachment, which is a critical step in the biofouling process. The model predicts that the attachment densities of zoospores of the green alga, Ulva, and cells of the marine bacterium, Cobetia marina, scale inversely with surface roughness. The size and motility of the bacterial cells and algal spores were incorporated into the attachment model by multiplying the engineered roughness index (ERIII), which is a representation of surface energy, by the Reynolds number (Re) of the cells. The results showed a negative linear correlation of normalized, transformed attachment density for both organisms with ERIII · Re (R 2 = 0.77). These studies demonstrate for the first time that organisms respond in a uniform manner to a model, which incorporates surface energy and the Reynolds number of the organism.

The role of surface energy and water wettability in aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel surfaces in the control of marine biofouling

Xerogel films with uniform surface topogrophy, as determined by scanning electron microscopy, atomic force microscopy (AFM), and time-of-flight secondary ion mass spectrometry, were prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl- containing precursors. Young's modulus was determined from AFM indentation measurements. The xerogel coatings gave reduced settlement of zoospores of the marine fouling alga Ulva compared to a poly(dimethylsiloxane) elastomer (PDMSE) standard. Increased settlement correlated with decreased water wettability as measured by the static water contact angle, θWs, or with decreased polar contribution (γP) to the surface free energy (γS) as measured by comprehensive contact angle analysis. The strength of attachment of 7-day sporelings (young plants) of Ulva on several of the xerogels was similar to that on PDMSE although no overall correlation was observed with either θWs or γS. For sporelings attached to the fluorocarbon/hydrocarbon-modified xerogels, the strength of attachment increased with increased water wettability. The aminopropyl-modified xerogels did not follow this trend.

ABC Triblock Surface Active Block Copolymer with Grafted Ethoxylated Fluoroalkyl Amphiphilic Side Chains for Marine Antifouling/Fouling-Release Applications

An amphiphilic triblock surface-active block copolymer (SABC) possessing ethoxylated fluoroalkyl side chains was synthesized through the chemical modification of a polystyrene-block-poly(ethylene-ran-butylene)-block-polyisoprene polymer precursor. Bilayer coatings on glass slides consisting of a thin layer of the amphiphilic SABC spray coated on a thick layer of a polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (SEBS) thermoplastic elastomer were prepared for biofouling assays with the green alga Ulva and the diatom Navicula. Dynamic water contact angle analysis and X-ray photoelectron spectroscopy (XPS) were used to characterize the surfaces. Additionally, the effect of the Young's modulus of the coating on the release properties of sporelings (young plants) of the green alga Ulva was examined through the use of two different SEBS thermoplastic elastomers possessing modulus values of an order of magnitude in difference. The amphiphilic SABC was found to reduce the settlement density of zoospores of Ulva as well as the strength of attachment of sporelings. The attachment strength of the sporelings was further reduced for the amphiphilic SABC on the "low"-modulus SEBS base layer. The weaker adhesion of diatoms, relative to a PDMS standard, further highlights the antifouling potential of this amphiphilic triblock hybrid copolymer.

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.

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.

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.

Antifouling character of ‘active’ hybrid xerogel coatings with sequestered catalysts for the activation of hydrogen peroxide

Halide-permeable xerogel films prepared from sols containing 50 mol% aminopropyltriethoxysilane (APTES)/50 mol% tetraethoxysilane (TEOS) or 10 mol% APTES/90 mol% TEOS and 0.015 M selenoxide or telluride catalyst in the sol gave reduced settlement of cypris larvae of the barnacle Balanus amphitrite and larvae of the tubeworm Hydroides elegans in the presence of artificial seawater (ASW) and hydrogen peroxide (5–100 μM) relative to glass controls. Settlement of Ulva zoospores was lower on both the 50 mol% APTES/50 mol% TEOS and 10 mol% APTES/90 mol% TEOS xerogel formulations in comparison with glass controls with or without the added catalyst. The 50 mol% APTES/50 mol%TEOS xerogel containing telluride catalyst gave reduced settlement of Ulva zoospores in the presence of 100 μM H2O2 in ASW compared with the same coating without added peroxide. Scanning electron microscopy and XPS data suggest that exposure to H2O2 does not lead to chemical or morphological changes on the xerogel surface.

Settlement of Ulva Zoospores on Patterned Fluorinated and PEGylated Monolayer Surfaces

Various designs for coatings that resist the attachment of marine organisms are based on the concept of "ambiguous" surfaces that present both hydrophobic and hydrophilic functionalities as surface domains. In order to facilitate the optimal design of such surfaces, information is needed on the scale of the domains that the settling stages of marine organisms are able to distinguish. Previous experiments showed that Ulva zoospores settle (attach) in high numbers onto fluorinated monolayers compared to PEGylated monolayers. The main aim of the present study was to determine, when zoospores of the green alga Ulva are presented with a choice of fluorinated or PEGylated surfaces, what the minimum dimensions of the two types of surface are that zoospores can detect and consequently settle on. Silicon wafers were chemically modified to produce a pattern of squares containing alternating fluorinated and PEGylated stripes of different widths on either a uniform fluorinated or PEGylated background. Each 1 cm x 1 cm square contained stripes with widths of 500, 200, 100, 50, 20, 5, or 2 microm as well as an unpatterned square with a chemistry opposite that of the background. Spores were selective in choosing where to settle, settling at higher densities on fluorinated stripes compared to PEGylated stripes. However, the magnitude of response, and the consequences for settlement on patterned areas overall, was dependent on both the width of the stripes and the chemistry of the background. The data are discussed in relation to the ability of spores to "choose" favorable sites for settlement and the implications for the development of novel antifouling coatings.

Settlement and adhesion of algal cells to hexa(ethylene glycol)-containing self-assembled monolayers with systematically changed wetting properties

Protein resistance of self-assembled monolayers (SAMs) of hexa(ethylene glycols) (EG(6)) has previously been shown to be dependent on the alkoxyl end-group termination of the SAM, which determines wettability [S. Herrwerth, W. Eck, S. Reinhardt, and M. Grunze, J. Am. Chem. Soc. 125, 9359 (2003)]. In the present study, the same series of hexa(ethylene glycols) was used to examine the correlation between protein resistance and the settlement and adhesion of eukaryotic algal cells, viz., zoospores of the macroalga Ulva and cells of the diatom Navicula, which adhere to the substratum through the secretion of protein-containing glues. Results showed that the initial settlement of Ulva zoospores was highest on the hydrophilic EG(6)OH but that cells were only weakly adhered. The number of Ulva zoospores and Navicula cells firmly adhered to the SAMs systematically increased with decreasing wettability, as shown for the protein fibrinogen. The data are discussed in terms of hydration forces and surface charges in the SAMs.

Laboratory screening of coating libraries for algal adhesion

Coatings libraries achieved through a combinatorial chemistry approach, which may generate tens to hundreds of formulations, can be deposited in an array of 12 patches, each approximately 9 cm2, on 10 × 20 cm primed aluminum panels. However, existing methods to quantify algal biomass on coatings are unsuitable for this type of array format. This paper describes an algorithm modelled on a probability distribution that quantifies the area of surface covered by a green alga from digital images. The method allows coatings with potential fouling-release properties to be down-selected for further evaluation. The use of the algorithm is illustrated by a set of eight siloxane-polyurethane coatings made using organofunctional poly(dimethylsiloxane) (PDMS) and poly(ϵ-caprolactone)-PDMS-poly(ϵ-caprolactone) (PCL-PDMS-PCL) triblock copolymers along with four PDMS standards which were deposited on one panel. Six replicate panels were seeded with Ulva zoospores which grew into sporelings (small plants) that completely covered the surface. The ease of removal of the Ulva sporeling biofilms was determined by automated water jetting at six different impact pressures. The coverage of the biofilm on the twelve individual formulations after jet washing was quantified from the green colour of digital images. The data are discussed in relation to the composition of the coatings.

Effect of Marine Bacterial Isolates on the Growth and Morphology of Axenic Plantlets of the Green Alga Ulva linza

The green marine macroalga, Ulva linza, adopts an "atypical" form when grown in the absence of bacteria. Twenty unique strains of periphytic bacteria, isolated from three species of Ulva, were identified by 16S rDNA sequencing. These isolates were assessed for their effect on the growth and morphological development of axenic plantlets of U. linza. Results showed that the effect of bacterial strains was strain- but not taxon-specific. Thirteen isolates returned the aberrant morphology to normal and of these, five also significantly increased growth rate. One isolate increased growth, but had no effect on morphology. Biofilms of some of these isolates stimulated the settlement of Ulva zoospores but there was no correlation between bacterial isolates that stimulated zoospore settlement and those that initiated changes in morphology and/or growth of the cultured alga.