Antifouling potection at dfferent scales : multiple defence in Mytilus edulis and the global performance of mytilid microtopographies

Marine Organismen sind einem ständigen Besiedlungsdruck durch Mikroorganismen, Algensporen und Larvenstadien sessiler Invertebraten ausgesetzt. Weil die Gegenwart von Epibionten die Interaktionen zwischen Substratorganismus (Basibiont) und Umwelt stark beeinflusst, müssen Basibionten auf den Besiedlungsdruck reagieren. Natürliche Verteidigungssysteme mariner Organismen gegen Aufwuchs müssen auf die verschiedensten Taxa potentieller Besiedler, die eine große Bandbreite an Empfindlichkeiten besitzen, abzielen. Aus diesem Grunde sind diese Abwehrsysteme hoch komplex, und bestehen meist aus einer Kombination mechanischer, chemischer, physikalischer und ökologischer Mechanismen, die gleichzeitig oder abwechselnd die Anheftung und das Wachstum von Bakterien, Algen und Wirbellosen beeinflussen. Die vorliegende Arbeit untersucht die natürlichen Antifoulingmechanismen der Mytiliden generell und speziell von Mytilus edulis durch die Entkopplung potentieller mechanischer und chemischer Verteidigungsmechanismen. Des Weiteren wurde untersucht, ob eine dieser Verteidigungskomponenten, die Mikrotopographie, sich durch regionale Optimierung auszeichnet. Letztere wurde im Hinblick auf die hochinvasiven Mytiliden ausgewählt, da ein universeller, in den verschiedensten biogeographischen Regionen wirksamer Verteidigungsmechanismus die Invasion in neue Habitate erleichtern würde. Die Ergebnisse aller Experimente unterstützen nachdrücklich das Konzept eines multiplen Ver-teidigungssystems bei der Miesmuschel Mytilus edulis. Ich konnte zeigen, dass die Oberflächenmikrostruktur Besiedlung durch Makrofouler abwehrt, während die Oberflächenchemie die Besiedlung durch Mikrofouler behindert. Daher stehen mindestens zwei Verteidigungsmechanismen mit dem Periostracum in Zusammenhang. Diese beiden Mechanismen könnten durch kumulative Filtration im Muschelbett und säubernde Bewegungen des Muschelfußes an der Schalenoberfläche noch unterstützt werden. Eine regionale Optimierung der Oberflächenmikrostruktur konnte jedoch nicht nachgewiesen werden

Attachment of Marine Microorganisms to Surfaces

The attachment mechanisms of the main groups of marine microorganisms are similar. These similarities are particularly shown by the nature of the adhesive mucilages they produce. Marine bacteria, cyanobacteria and diatoms produce acidic polysaccharide mucilages. Macroalgal spores attach by the production of glycoprotein adhesives. The spores of several marine Ascomycete fungi attach by means of mucilaginous, fibrillar appendages, although little is known about their composition. Certain marine bacteria possess cell-surface structures such as stalks with holdfasts, flagella and spinae which may play an attachment role. Cyanobacteria possess pili and spinae which may serve the same purpose. The formation of proteinaceous conditioning films on substrata promotes bacterial adhesion. Polymeric polysaccharide materials and bacterial films also precondition surfaces for algal attachment. Substratum properties such as wettability and surface free energy can affect, and in some cases decrease, levels of bacterial and algal attachment. Marine microbial adhesion is of importance in disease pathogenesis, biotic interactions, physical interactions and certain economic aspects. In disease pathogenesis, the attachment of Aeromonas salmonicida to fish cells by the 'A'-protein layer is important in the initiation of furunculosis. Some invertebrate diseases are also caused by attached microorganisms. The bacterium Leucothrix mucor, along with diatoms, cyanobacteria and protozoa can accumulate on the gills of shrimps, crabs and lobsters. This heavy infestation causes death by suffocation. Vibrio cholerae attaches to the oral region of planktonic copepods and the hindgut of the blue crab Callinectes sapidus. These observations may be important for the epidemiology of cholera in aqueous environments. Microbial attachment may also be important in the establishment of symbiotic relationships with certain marine invertebrates. Biotic interactions include the formation of primary microbial films on any new surface immersed in seawater. Extensive microbial epiphytic layers form on seaweeds and estuarine salt marsh grasses. Bacteria which attach to the heterocysts of the cyanobacterium Anabaena spp. in the freshwater environment could be involved in nitrogen fixation. The parasitic marine bacterium Bdellovibrio bacteriovorus attaches to host cell membranes by cell-surface fibres, which ultimately leads to cell lysis. Attached bacteria are responsible for the aggregation of particulate detritus, which eventually disaggregates due to protozoal activity. Bacteria utilize dissolved organic carbon for the formation of adhesive materials which cause detrital aggregation. The attachment of bacteria to certain phytoplankton, including diatoms, ultimately results in the formation of amorphous detrital aggregates. Physical interactions include microbial attachment to sediments. Microbial attachment and adhesive production is important in the formation of microbial mats. Marine bacteria, cyanobacteria and diatoms are abundant in these mats. Microbial extracellular polymer material is responsible for lamination of mat layers and the structural integrity of the mats. Gelatinous mats are often formed in sediments from microalgal adhesive secretions. These mats are of importance in sediment stabilization. The adhesive mucilages of certain diatoms also reduce the resuspension of sediment particles. Microbial extracellular materials may also be utilized as a food source by benthic invertebrates. One economic aspect of marine microfouling is metal corrosion. Corrosion can result from the formation of differential aeration cells under a non-uniform film of attached microorganisms. Sulphate-reducing bacteria, which are often present in biofilms under anaerobic conditions corrode metals by cathodic depolarization. This process allows the formation of corrosive hydrogen sulphide and iron sulphide from sulphates. Some diatoms, such as Amphora spp., may inhibit corrosion by forming a uniform layer of adhesive mucilage over a metal surface. A further economic aspect is the affect of microbial attachment on the development of antifouling techniques. Bacterial and diatom slime films are easily formed on cuprous oxide and organometallic antifouling paints. This makes them less effective. The effects of surface free energy and wettability of substrata on bacterial and algal adhesion could provide a further antifouling technique. Conditioning film formation can alter these substratum properties towards a biocompatible range where lower rates of microfouling occur. The incorporation of silicone elastomers in substrata also decreases bacterial and diatom attachment. The use of metabolic inhibitors or calcium chelating agents to remove bacterial and diatom films could be a further antifouling development

Epoxy-based coatings with additions of titanium(IV) oxide and silver for marine antifouling applications

Begroing defineres som avsetning eller opphopning av uønsket biologisk materiale på overflater under vann. Dette fenomenet har vært kjent lenge, og det kan potensielt ha alvorlige konsekvenser for miljø, økonomi og helse, særlig i forbindelse med marine næringer. Derfor brukes det ofte belegg med gro-hindrende/bunnstoffhindrende egenskaper på eksponerte overflater for å begrense effekten av begroing. Bruken av tradisjonelle bunnstoff har vært forbundet med utslipp av giftige forbindelser som kan påvirke arter som ikke er målarter, utgjøre en risiko for menneskers helse og skade det marine miljøet. Den økende bevisstheten om miljøpåvirkningen fra disse giftige bunnstoffene har fått forskere til å utforske mer bærekraftige alternativer til de tradisjonelle bunnstoffene. Epoksybaserte komposittbelegg med tilsetning av uorganiske partikler har vist lovende bunnstoffhindrende egenskaper. Det overordnede målet med denne masteroppgaven var å utvikle epoksybaserte belegg med enten titan(IV)oksid (TiO2)-pulver eller metalliske sølvpartikler (Ag) og undersøke deres bunnstoff-, overflate- og mekaniske egenskaper som egner seg for marine bruksområder. Sølvpartiklene ble syntetisert ved kjemisk reduksjon, og partiklene ble undersøkt for å finne deres renhet, størrelse og morfologi. Overflateegenskapene til Ag-beleggene ble undersøkt ved å måle beleggets fuktbarhet og beregne den frie overflateenergien i forhold til Ag-innholdet. Nanoindentasjonsmålinger ble utført på beleggene for å estimere deres ripebestandighet, hardhet og elastisitetsmodul. Deretter ble beleggene senket ned i et biofilmreaktorexperiment som sannsynligvis inneholdt en monokultur av kiselalger i to uker. Deretter ble algeveksten kvantifisert ved hjelp av optisk mikroskopi, ved å estimere den prosentvise algeveksten og ved en direkte tellemetode. De syntetiserte sølvpartiklene hadde høy krystallinitet og renhet, med partikkelstørrelser fra nanometer til mikrometer. Sol-gel-prosessen ble brukt til å syntetisere epoksy sol og slurries som inneholdt varierende konsentrasjoner av TiO2 eller Ag, nemlig 0%, 0.125 vekt%, 0.250 vekt% og 0.500 vekt%. Ag-slurryene viste seg å være stabile i opptil 30 minutter etter tilberedning, og ble spraybelagt på polyetylensubstrater før varmebehandling. Algene festet seg til beleggene hovedsakelig som homogent fordelte kiselalger, men det ble også observert noen klynger. Kvantifiseringen viste at Ag-beleggene hadde et betydelig potensial for bruk som bunnstoffhindrende middel, og at de bunnstoffhindrende egenskapene ble bedre med økende Ag-innhold. Dette ble ikke observert for TiO2-beleggene, noe som sannsynligvis skyldes den utilstrekkelige eksponeringen for UV-lys som kreves for å aktivere de fotokatalytiske egenskapene til TiO2. Alle de belagte prøvene hadde hydrofile egenskaper og viste bedre bunnstoffhindrende egenskaper enn de ubelagte prøvene. Tilsetningen av sølv hadde likevel ingen vesentlig innvirkning på fuktbarheten, til tross for de observerte endringene i bunnstoffegenskapene. På samme måte endret ikke inkorporeringen av sølv i beleggene ripebestandigheten i vesentlig grad. Hardhet og elastisitetsmodul ble målt for alle beleggene, og beleggene med økende innhold av Ag viste økt hardhet og stivhet. I tillegg til å ha overlegne bunnstoffhindrende egenskaper, viste Ag-beleggene større hardhet og stivhet enn epoksyol- og TiO2-beleggene.Biofouling is defined as the deposition or accumulation of undesirable biological matter on submerged surfaces. This phenomenon has been known for a considerable time and has the potential to cause severe consequences related to the environment, economy, and health, particularly in the context of marine industries. Consequently, coatings with antifouling properties are frequently employed on exposed surfaces to limit the effects of biofouling. The use of traditional antifouling coatings has been associated with the release of toxic compounds that can affect non-target species, pose risks to human health, and harm the marine environment. The growing awareness of the environmental impact of these toxic antifouling paints has prompted researchers to explore more sustainable alternatives to traditional antifouling agents. Epoxy-based composite coatings with the addition of inorganic particles have shown promising antifouling properties. The overall aim of this master's thesis was to develop epoxy-based coatings incorporating either titanium(IV) oxide (TiO2) powder or metallic silver (Ag) particles and investigate their antifouling, surface, and mechanical properties suitable for marine applications. Silver particles were synthesized by chemical reduction, and the particles were investigated to find their purity, size, and morphology. The surface properties of Ag coatings were investigated by measuring the coating's wettability and calculating the surface free energy with respect to the Ag content. Nanoindentation measurements were conducted on the coatings in order to estimate their scratch resistance, hardness, and Young's modulus. The coatings were subsequently submerged in a biofilm reactor experiment likely containing a monoculture of diatoms for two weeks. Following this, the algal growth was quantified using optical microscopy, with the percentage of algal growth estimated and a direct count method employed. The synthesized silver particles exhibited high crystallinity and purity, with particle sizes ranging from nanometers to micrometers. The sol-gel process was employed to synthesize epoxy sol and slurries containing varying concentrations of TiO2 or Ag, namely 0%, 0.125 wt%, 0.250 wt%, and 0.500 wt%. The Ag slurries were found stable up to 30 minutes after preparation and were spray-coated onto polyethylene substrates before heat treatment. Algae adhered to the coatings mainly as homogeneously distributed diatoms, but some clusters were also observed. The quantification demonstrated that the Ag coatings exhibited significant potential for antifouling applications, with the antifouling properties improving with increasing Ag content. This was not observed for the TiO2 coatings, likely due to the insufficient exposure to UV light required to activate the photocatalytic properties of TiO2. All coated samples exhibited hydrophilic properties and demonstrated superior antifouling characteristics in comparison to uncoated samples. Nevertheless, the addition of silver did not significantly impact wettability, despite the observed alterations in antifouling properties. Similarly, the incorporation of silver into the coatings did not significantly alter the scratch resistance. Hardness and Young's modulus were measured for all coatings, with the coatings containing increasing Ag content exhibiting increased hardness and stiffness. In addition to exhibiting superior antifouling properties, the Ag coatings demonstrated greater hardness and stiffness than the epoxy sol and TiO2 coatings

Functional polymer materials for modern marine biofouling control

Marine biofouling is a well-known massive problem: within the shortest time, ship hulls and other man-made submerged surfaces are inevitably populated by various marine organisms. Marine biofouling causes severe economic and environmental problems. Thus, effective biofouling control on submerged surfaces is of utmost importance. Since the middle of the 20th century, scientists and engineers have developed antifouling coatings mainly based on the continuous release of toxic metal ions and accompanying booster biocides to repel or kill organisms approaching the surface. However, these coatings caused serious harm to non-target organisms and the ocean. Therefore, the development of environmentally friendly alternative coatings is an urgent need, and research in this field is growing rapidly. This review includes concise basic theory from biology, chemistry, and physics. It provides an introduction into the biofouling formation, as well as physicochemical surface properties that can be manipulated to achieve an effective biofouling control. Furthermore, a complete overview of the currently developed biofouling control coatings is presented and summarized. This overview includes coatings based on surface wettability, self-renewable coatings, coatings containing antifouling agents, switchable coatings, and biomimetic coatings.</p

Effect of cuprous oxide particles on the drag characteristics of marine coatings

Ph. D ThesisCu2O is an active antifouling substance which is commercially produced with different particle sizes before being formulated into antifouling products, and this consequently leads to different surface finish roughness conditions. The further effects of particle size on the drag performance of antifouling coatings and hence on ship hull resistance, as well as biofilm attachment, has not been explored and studied systematically. Accordingly, about the question of how to make an optimised selection of Cu2O size remains for the ship owners or paint developers. In this research, a number of different sized Cu2O (2μm ≤D50≤250μm) were applied to Newcastle University’s (UNEW) flat test panels. The boundary layer characteristics of the coated surfaces and the uncoated reference surface were measured using two-dimensional Laser Doppler Velocimetry (LDV) in the Emerson Cavitation Tunnel. Pressure drop measurements were carried out using a turbulent flow channel under dynamic flow conditions. The effect of biofilm on the drag characteristics of the Cu2O surfaces under “in-service” conditions was investigated by mounting the test panels the research vessel, The Princess Royal, and examining them every six weeks during a six-month dynamic/static immersion period. The subsequent streamwise pressure drop measurements were conducted on all of the test panels along with uncoated reference panels. In addition, roughness characteristics were analysed by using an optical surface profilometer and the microstructure was studied using Scanning Electron Microscopy (SEM). Based on the roughness function results obtained from the tests, the additional resistance diagrams for full-scale ships were developed according to the Granville similarity scaling law by employing an in-house programme. The research study has systematically explored the effect of different sized Cu2O on the drag and roughness characteristics of marine coatings for the first time in the open literature and hence demonstrated the significant potential impact of this effect on ship performance in-service. It was found that the lowest roughness surface was not desmonstrated by the smallest sized particles because of aggregations which caused an unexpectedly rougher surface and higher fricitonal drag. Apart from the aggregated particles, the overall fricitonal drag was found to increase with particle size and this can be expressed by an empirical linear equation. Details of the time-dependent influence of biofilms on the Cu2O surfaces have been presented. Based on this, the frictional drag changes on different size full-scale ships under similar surface conditions may become more predictable. It was also observed that significant fluctuation occurs on the roughness and frictional drag values for surfaces coated with larger sizes of Cu2O particles, e.g. D50=60μm and therefore, similar behaviours are expected for ship surfaces which have similar or rougher characteristics than this particle size. It has to be noted that the test coatings were purely experimental, having exceptionally high Cu2O contents in the dry film that do not correspond to any commercial antifoulings

Selected Papers from XVI MaNaPro and XI ECMNP

The oceans harbor the majority of the Earth´s biodiversity. Marine organisms/microorganisms provide a diverse array of natural products, which are important sources of biologically active agents with unique chemical structures and a broad range of medical and biotechnological applications. The XVI MaNaPro and XI ECMNP conferences aim to present advances and future perspectives on marine natural product research to the scientific community by gathering scientists who work in marine chemistry and related scientific fields from all over the world and at different seniority levels. This Special Issue was organized on the occasion of the 2nd joint XVI MaNaPro and XI ECMNP meeting (http://wmnp2019.ipleiria.pt/) held in Peniche, Portugal, in 2019. It comprises 12 original research articles that exemplify research performed in the scope of the conference topic

Laser Surface Texturing To Create Biomimetic Surface Topographies For Marine Antifouling Efficacy Testing

Biofouling is the unwanted colonisation of organisms on a living or artificial surface. Convergent evolution has led to the development of antifouling textures on many marine species. This thesis provides novel investigation into creating biomimetic antifouling surface directly onto marine grade stainless steel using laser micro machining. The investigation was split into three main research questions: (1) can laser surface texturing be used to create antifouling surfaces, and their effects on surface parameters (roughness / contact angle); (2) can biomimetic antifouling surfaces be created using laser surface texturing?; (3) can features of those successful surfaces be combined to create enhanced biomimetic antifouling surface?. All three experiments had similar methods, as laser processing was used to transfer the selected biomimetic micro-topography patterns onto marine grade stainless steel (316L). Samples were deployed in the field (Liverpool South Docks, UK) for 7 days. Abundance of biofilm was assessed using random systematic sampling. For the biomimetic surfaces, a fringe projection microscope (GFM) was used to investigate 3D scans of the surface topography of shells of bivalve and crab species, to provide bio-inspiration for the design of the surfaces created in this research. It was found that the micro-topography pattern limits the attachment of the biofilm to the surface. This thesis shows that (1) laser surface texturing can be used to create antifouling surfaces; (2) biomimetic antifouling surfaces can be created and enhance antifouling efficacy, and (3) that combining biomimetic features into multi-scale and multi-feature patterns have enhanced antifouling effects. This reinforces that biomimetic surfaces have the potential to be a non-toxic, eco-friendly antifouling technology that work directly on marine metal structures without the need for further coatings or chemicals

Influence of surface conditioning and morphology on biofouling

Biofouling, the undesired colonization of surfaces, is a major problem for marine-related industries. To prevent unwanted effects caused by biofouling, suitable non-toxic coatings for the marine environment are required. Conditioning, i.e. the adsorption of proteins and macromolecules influences, as surface chemistry and morphology do, the settlement of fouling oragnisms. Investigating the temporal dynamics of conditioning film formation on functionalized self assembled monolayers (SAMs), it was shown that the obtained film thickness of about 10Å to 20Å is independent of the surface chemistry but differences occur concerning the composition of these films. While on hydrophilic surfaces more proteinaceous compounds are detectable, the hydrophobic surfaces show a lower intensity of proteins. Furthermore, is was shown that in standard Ulva linza spore settlement assays the influence of a molecular conditioning layer is likely to be small, but by increasing pre-conditioning time this influence gains importance and should be considered in long term experiments. Preconditioning also resulted in detectable surface differences in field studies. It was found that preconditioned samples which contain more proteinaceous compounds seem to be more attractive for settlement. Experiments with matured laboratory biofilms formed by Pseudomonas aeruginosa demonstrated that using a protein-rich medium results in conditioning film formation. Conversely, surface conditioning is reduced when a media containing a smaller amount of proteins is utilized. Furthermore, it was observed that surface chemistry has no remarkable effect on the fraction of inoculated bacteria that adhere to a surface. Finally, inspired by the nanostructured skin of dolphins, electron-beam lithography was utilized to create a honeycomb topography made of poly(N-isopropylacrylamide) (PNIPAM). The evaluation showed that structures from 0.75 μm to 2.5 μm in diameter reduce Ulva settlement in comparison to a smooth PNIPAM surface. It should be noted, that wet polymer structure heights in the range of only 0.01 μm do have an effect on spores with a body size of around 5 μm

Nano-pollutants – big impact? Investigating the ecotoxicological effects of nanomaterials in our oceans

Engineered nanomaterials (NMs) used for industrial and commercial applications, represent an emerging contaminant of environmental concern. Due to their widespread use, their entry into the environment is believed inevitable, where the ocean represents the sink for contaminants entered into the aquatic environment. In this thesis new insight is provided on the likely environment risk of engineered NMs towards marine microbial organisms which represent the base of the marine food web and play major roles in global climatic and biogeochemical processes. Three commonly used NMs with clear pathways into the aquatic environment were selected for study; silver nanoparticles (AgNPs), titanium dioxide (nTiO2) and cerium oxide (nCeO2). Preliminary investigation exposing a natural phytoplankton community to AgNPs identified the marine cyanobacterium Prochlorococcus as particularly sensitive to NMs, and hence the model Prochlorococcus sp. MED4 was selected for use in ecotoxicity assessments. Toxicity testing and subsequent investigation of toxic mechanisms revealed varying impacts of metal-based NMs such as AgNPs, and metal oxide NMs upon Prochlorococcus, largely determined by their respective fate and behaviour upon entry into saline media. In chapter 2, AgNPs were observed to exert significant cell declines upon Prochlorococcus via the release of toxic silver ions and superoxide. This perturbation was associated with a significant alteration to the cellular proteome and was irreversible in the longer-term. However, cell declines were mitigated by increasing cell density likely due to an increase in production of superoxide dismutase at higher cell numbers. Significant cell declines were observed at concentrations ≥10 μg L-1 under natural conditions, representing the upper limit of AgNPs predicted in the environment. Hence, it is believed that negative impacts of AgNPs upon marine microbial species is only likely in hotspots of contamination. In chapter 3, the potential impacts of metal oxide NMs (nTiO2 and nCeO2) were investigated. Here, little impact upon Prochlorococcus was recorded in extended exposure (240 h) except at extremely high concentrations (100 mg L-1). Temporary declines were observed in the short-term (≤72 h) and were associated with the heteroaggregation and co-precipitation with nanoparticles which were observed to aggregate rapidly upon entry into seawater, as confirmed by dynamic light scattering (DLS) and fluorescent microscopy. The key role of physical interactions in driving cell declines was further supported by shotgun proteomic analysis of Prochlorococcus exposed to nTiO2 (100 μg L-1), revealing no significant alteration to the cellular or exo- proteome. Effective protocols were established to extract and characterise nTiO2 utilised in consumer products such as cosmetics and sunscreen. Use of nTiO2 extracted from consumer sunscreen in exposures with natural marine microbial communities, found negligible impact upon the structure and diversity of prokaryotic or eukaryotic communities. Hence the current environmental risk of nTiO2 and nCeO2 towards marine microbial species is believed low. In the final experimental chapter 4, in collaboration with an industrial partner, the effectiveness of novel nCeO2 antifouling surface coatings was examined by use of amplicon sequencing, nutrient analysis and 3D microscopic imaging. Investigation revealed negligible impact of the addition of nCeO2 upon biofilm community structure, however nutrient analysis suggested a slight reduction in biofouling in the presence of nanoparticles