UV-C LED-induced cyclobutane pyrimidine dimer formation, lesion repair and mutagenesis in the biofilm-forming diatom, Navicula incerta.

The use of ultraviolet-C (UV-C) irradiation in marine biofouling control is a relatively new and potentially disruptive technology. This study examined effects of UV-C exposure on the biofilm-forming diatom, Navicula incerta. UV-C-induced mutations were identified via Illumina HiSeq. A de novo genome was assembled from control sequences and reads from UV-C-exposed treatments were mapped to this genome, with a quantitative estimate of mutagenesis then derived from the frequency of single nucleotide polymorphisms. UV-C exposure increased cyclobutane pyrimidine dimer (CPD) abundance with a direct correlation between lesion formation and fluency. Cellular repair mechanisms gradually reduced CPDs over time, with the highest UV-C fluence treatments having the fastest repair rates. Mutation abundances were, however, negatively correlated with CPD abundance suggesting that UV-C exposure may influence lesion repair. The threshold fluence for CPD formation exceeding CPD repair was >1.27 J cm-2. Fluences >2.54 J cm-2 were predicted to inhibit repair mechanisms. While UV-C holds considerable promise for marine antifouling, diatoms are just one, albeit an important, component of marine biofouling communities. Determining fluence thresholds for other representative taxa, highlighting the most resistant, would allow UV-C treatments to be specifically tuned to target biofouling organisms, whilst limiting environmental effects and the power requirement.

Amphiphilic Balance: Effect of the Hydrophilic-Hydrophobic Ratio on Fouling-Release Surfaces.

This study demonstrated the importance of identifying the optimal balance of hydrophilic and hydrophobic moieties in amphiphilic coatings to achieve fouling-release (FR) performance that surpasses that of traditional hydrophobic marine coatings. While there have been many reports on fouling-release properties of amphiphilic surfaces, the offered understanding is often limited. Hence, this work is focused on further understanding of the amphiphilic surfaces. Poly(ethylene glycol) (PEG) and polydimethylsiloxane (PDMS) were used to create a series of noncross-linked amphiphilic additives that were then added to a hydrophobic-designed siloxane-polyurethane (SiPU) FR system. After being characterized by ATR-FTIR, XPS, contact angle analysis, and AFM, the FR performance was evaluated by using different marine organisms. The assessments showed that the closer the hydrophilic and hydrophobic moieties in a system reached a relatively equalized level, the more desirable the FR performance of the coating system became. A balanced ratio of hydrophilicity-hydrophobicity in the system at around 10-15 wt % of each component had the best FR performance and was comparable to or better than commercial FR coatings.

Diffusely Charged Polymeric Zwitterions as Loosely Hydrated Marine Antifouling Coatings.

Polymeric zwitterions exhibit exceptional fouling resistance through the formation of a strongly hydrated surface of immobilized water molecules. While being extensively tested for their performance in biomedical, membrane, and, to a lesser extent, marine environments, few studies have investigated how the molecular design of the zwitterion may enhance its performance. Furthermore, while theories of zwitterion antifouling mechanisms exist for molecular-scale foulant species (e.g., proteins and small molecules), it remains unclear how molecular-scale mechanisms influence the micro- and macroscopic interactions of relevance for marine applications. The present study addresses these gaps through the use of a modular zwitterion chemistry platform, which is characterized by a combination of surface-sensitive sum frequency generation (SFG) vibrational spectroscopy and marine assays. Zwitterions with increasingly delocalized cations demonstrate improved fouling resistance against the green alga Ulva linza. SFG spectra correlate well with the assay results, suggesting that the more diffuse charges exhibit greater surface hydration with more bound water molecules. Hence, the number of bound interfacial water molecules appears to be more influential in determining the marine antifouling activities of zwitterionic polymers than the binding strength of individual water molecules at the interface.

Nitroxide-Containing Amphiphilic Random Terpolymers for Marine Antifouling and Fouling-Release Coatings.

Two types of amphiphilic random terpolymers, poly(ethylene glycol methyl ether methacrylate)-ran-poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate)-ran-poly(polydimethyl siloxane methacrylate) (PEGMEMA-r-PTMA-r-PDMSMA), were synthesized and evaluated for antifouling (AF) and fouling-release (FR) properties using diverse marine fouling organisms. In the first stage of production, the two respective precursor amine terpolymers containing (2,2,6,6-tetramethyl-4-piperidyl methacrylate) units (PEGMEMA-r-PTMPM-r-PDMSMA) were synthesized by atom transfer radical polymerization using various comonomer ratios and two initiators: alkyl halide and fluoroalkyl halide. In the second stage, these were selectively oxidized to introduce nitroxide radical functionalities. Finally, the terpolymers were incorporated into a PDMS host matrix to create coatings. AF and FR properties were examined using the alga Ulva linza, the barnacle Balanus improvisus, and the tubeworm Ficopomatus enigmaticus. The effects of comonomer ratios on surface properties and fouling assay results for each set of coatings are discussed in detail. There were marked differences in the effectiveness of these systems against the different fouling organisms. The terpolymers had distinct advantages over monopolymeric systems across the different organisms, and the nonfluorinated PEG and nitroxide combination was identified as the most effective formulation against B. improvisus and F. enigmaticus.

Studying the Effect of Pre-Polymer Composition and Incorporation of Surface-Modifying Amphiphilic Additives on the Fouling-Release Performance of Amphiphilic Siloxane-Polyurethane Coatings.

Combining amphiphilic fouling-release (FR) coatings with the surface-active nature of amphiphilic additives can improve the antifouling/fouling-release (AF/FR) properties needed to offer broad-spectrum resistance to marine biofoulants. This work is focused on further tuning the amphiphilic character of a previously developed amphiphilic siloxane-polyurethane (SiPU) coating by varying the amount of PDMS and PEG in the base system. Furthermore, surface-modifying amphiphilic additives (SMAAs) were incorporated into these amphiphilic FR SiPU coatings in varying amounts. ATR-FTIR, contact angle and surface energy measurements, and AFM were performed to assess changes in surface composition, wettability, and morphology. AF/FR properties were evaluated using laboratory biological assays involving Cellulophaga lytica, Navicula incerta, Ulva linza, Amphibalanus amphitrite, and Geukensia demissa. The surfaces of these coatings varied significantly upon changes in PDMS and PEG content in the coating matrix, as well as with changes in SMAA incorporation. AF/FR properties were also significantly changed, with formulations containing the highest amounts of SMAA showing very high removal properties compared to other experimental formulations, in some cases better than that of commercial standard FR coatings.

Investigation of N-Substituted Morpholine Structures in an Amphiphilic PDMS-Based Antifouling and Fouling-Release Coating.

Biofouling is a major disruptive process affecting the fuel efficiency and durability of maritime vessel coatings. Previous research has shown that amphiphilic coatings consisting of a siloxane backbone functionalized with hydrophilic moieties are effective marine antifouling and fouling-release materials. Poly(ethylene glycol) (PEG) has been the primary hydrophilic component used in such systems. Recently, the morpholine group has emerged as a promising compact alternative in antifouling membranes but is yet to be studied against marine foulants. In this work, the use of morpholine moieties to generate amphiphilicity in a poly(dimethylsiloxane) (PDMS)-based antifouling and fouling-release coating was explored. Two separate coating sets were investigated. The first set examined the incorporation of an N-substituted morpholine amine, and while these coatings showed promising fouling-release properties for Ulva linza, they had unusually high settlement of spores compared to controls. Based on those results, a second set of materials was synthesized using an N-substituted morpholine amide to probe the source of the high settlement and was found to significantly improve antifouling performance. Both coating sets included PEG controls with varying lengths to compare the viability of the morpholine structures as alternative hydrophilic groups. Surfaces were evaluated through a combination of bubble contact angle goniometry, profilometry, X-ray photoelectron spectroscopy (XPS), and marine bioassays against two soft fouling species, U. linza and Navicula incerta, known to have different adhesion characteristics.

Low Fouling Polysulfobetaines with Variable Hydrophobic Content.

Amphiphilic polymer coatings combining hydrophilic elements, in particular zwitterionic groups, and hydrophobic elements comprise a promising strategy to decrease biofouling. However, the influence of the content of the hydrophobic component in zwitterionic coatings on the interfacial molecular reorganization dynamics and the anti-fouling performance is not well understood. Therefore, coatings of amphiphilic copolymers of sulfobetaine methacrylate 3-[N-2'-(methacryloyloxy)ethyl-N,N-dimethyl]-ammonio propane-1-sulfonate (SPE) are prepared which contain increasing amounts of hydrophobic n-butyl methacrylate (BMA). Their fouling resistance is compared to that of their homopolymers PSPE and PBMA. The photo-crosslinked coatings form hydrogel films with a hydrophilic surface. Fouling by the proteins fibrinogen and lysozyme as well as by the diatom Navicula perminuta and the green algae Ulva linza is assessed in laboratory assays. While biofouling is strongly reduced by all zwitterionic coatings, the best fouling resistance is obtained for the amphiphilic copolymers. Also in preliminary field tests, the anti-fouling performance of the amphiphilic copolymer films is superior to that of both homopolymers. When the coatings are exposed to a marine environment, the reduced susceptibility to silt incorporation, in particular compared to the most hydrophilic polyzwitterion PSPE, likely contributes to the improved fouling resistance.

Amphiphilic Nitroxide-Bearing Siloxane-Based Block Copolymer Coatings for Enhanced Marine Fouling Release.

The buildup of organic matter and organisms on surfaces exposed to marine environments, known as biofouling, is a disruptive and costly process affecting maritime operations. Previous research has identified some of the surface characteristics particularly suited to the creation of antifouling and fouling-release surfaces, but there remains room for improvement against both macrofouling and microfouling organisms. Characterization of their adhesives has shown that many rely on oxidative chemistries. In this work, we explore the incorporation of the stable radical 2,2,6,6-tetramethylpipiderin-1-oxyl (TEMPO) as a component in an amphiphilic block copolymer system to act as an inhibitor for marine cements, disrupting adhesion of macrofouling organisms. Using polystyrene-b-poly(dimethylsiloxane-r-vinylmethysiloxane) block copolymers, pendent vinyl groups were functionalized with TEMPO and poly(ethylene glycol) to construct an amphiphilic material with redox active character. The antifouling and fouling-release performance of these materials was investigated through settlement and removal assays of three model fouling organisms and correlated to surface structure and chemistry. Surfaces showed significant antifouling character and fouling-release performance was increased substantially toward barnacles by the incorporation of stable radicals, indicating their potential for marine antifouling applications.

Effect of Multilayer Termination on Nonspecific Protein Adsorption and Antifouling Activity of Alginate-Based Layer-by-Layer Coatings.

Layer-by-layer (LbL) assembly is a versatile platform for applying coatings and studying the properties of promising compounds for antifouling applications. Here, alginate-based LbL coatings were fabricated by alternating the deposition of alginic acid and chitosan or polyethylenimine to form multilayer coatings. Films were prepared with either odd or even bilayer numbers to investigate if the termination of the LbL coatings affects the physicochemical properties, resistance against the nonspecific adsorption (NSA) of proteins, and antifouling efficacy. The hydrophilic films, which were characterized using spectroscopic ellipsometry, water contact angle goniometry, ATR-FTIR spectroscopy, AFM, XPS, and SPR spectroscopy, revealed high swelling in water and strongly reduced the NSA of proteins compared to the hydrophobic reference. While the choice of the polycation was important for the protein resistance of the LbL coatings, the termination mattered less. The attachment of diatoms and settling of barnacle cypris larvae revealed good antifouling properties that were controlled by the termination and the charge density of the LbL films.

Surface modifying amphiphilic additives and their effect on the fouling-release performance of siloxane-polyurethane coatings.

In this work, surface-modifying amphiphilic additives (SMAAs) were synthesized via hydrosilylation using various polymethylhydrosiloxanes (PMHS) and allyl-terminated polyethylene glycol monomethyl ethers (APEG) of varying molecular weights. The additives synthesized were incorporated into a hydrophobic, self-stratifying siloxane-polyurethane (SiPU) coating system to produce an amphiphilic surface. Contact angle experiments and atomic force microscopy (AFM), in a dry and hydrated state, were performed to assess changes in surface wettability and morphology. The antifouling and fouling-release (AF/FR) performances were evaluated by performing laboratory biological assays using the marine bacterium Cellulophaga lytica, the microalga Navicula incerta, the macroalga Ulva linza, the barnacle Amphibalanus amphitrite, and the marine mussel, Geukensia demissa. Several of the formulations showed improved AF/FR performance vs the base SiPU and performed better than some of the commercial standard marine coatings. Formulations containing SMAAs with a low grafting density of relatively high molecular weight PEG chains showed the best performance overall.

Critical Amphiphilic Concentration: Effect of the Extent of Amphiphilicity on Marine Fouling-Release Performance.

Amphiphilic surfaces, containing both hydrophilic and hydrophobic domains, offer desirable performance for many applications such as marine coatings or anti-icing purposes. This work explores the effect of the concentration of amphiphilic moieties on converting a polyurethane (PU) system to a coating having fouling-release properties. A novel amphiphilic compound is synthesized and added at increasing amounts to a PU system, where the amount of the additive is the only variable in the study. The additive-modified surfaces are characterized by a variety of techniques including ATR-FTIR, XPS, contact angle measurements, and AFM. Surface characterizations indicate the presence of amphiphilic domains on the surface due to the introduction of the self-stratifying amphiphilic additive. The fouling-release properties of the surfaces are assessed with three biological assays using Ulva linza, Cellulophaga lytica, and Navicula Incerta as the test organisms. A change in the fouling-release performance is observed and plateaued once a certain amount of amphiphilicity is attained in the coating system, which we call the critical amphiphilic concentration (CAC).

Anti-biofouling properties of poly(dimethyl siloxane) with RAFT photopolymerized acrylate/methacrylate surface grafts against model marine organisms.

Biofouling of man-made surfaces by marine organisms is a global problem with both financial and environmental consequences. However, the development of non-toxic anti-biofouling coatings is challenged by the diversity of fouling organisms. One possible solution leverages coatings composed of diverse chemical constituents. Reversible addition-fragmentation chain-transfer (RAFT) photopolymerization was used to modify poly(dimethylsiloxane) (PDMSe) surfaces with polymeric grafts composed of three successive combinations of acrylamide, acrylic acid, and hydroxyethyl methacrylate. RAFT limited conflicting variables and allowed for the effect of graft chemistry to be isolated. While all compositions enhanced the anti-biofouling performance compared with the PDMSe control, the ternary, amphiphilic copolymer was the most effective with 98% inhibition of the attachment of zoospores of the green alga Ulva linza, 94% removal of cells of the diatom Navicula incerta, and 62% removal of cells of the bacterium Cellulophaga lytica. However, none of the graft compositions tested were able to mitigate reattachment of adult barnacles, Amphibalanus amphitrite.

Layer-by-Layer Deposited Hybrid Polymer Coatings Based on Polysaccharides and Zwitterionic Silanes with Marine Antifouling Properties.

Polyelectrolyte multilayer (PEM) assembly is a versatile tool to construct low-fouling coatings. For application in the marine environment, their structure needs to be stabilized by covalent linkage. Here, we introduce an approach for spin coating of silane-based sol-gel chemistries using layer-by-layer assembly of polysaccharide-based hybrid polymer coatings (LBLHPs). The silane sol-gel chemistry allows the films to be cross-linked under water-based and mild reaction conditions. Two different silanes were used for this purpose, a conventional triethoxymethyl silane and a de novo synthesized zwitterionic silane. The polysaccharide-silane hybrid polymer coatings were thoroughly characterized with spectroscopic ellipsometry, water contact angle (WCA) goniometry, attenuated total reflection-Fourier transform infrared spectroscopy, and atomic force microscopy. The coatings showed good stability in seawater, smooth surfaces, a high degree of hydration, and WCAs below or close to the Berg limit. LBLHPs showed low-fouling properties in biological assays against nonspecific protein adsorption, attachment of the diatom Navicula perminuta, and settlement of zoospores of the macroalga Ulva linza.

Degradable hyaluronic acid/chitosan polyelectrolyte multilayers with marine fouling-release properties.

Polysaccharide multilayers consisting of hyaluronic acid and chitosan were prepared by layer-by-layer assembly. To be used in seawater, the multilayers were crosslinked to a different degree using thermal or chemical methods. ATR-FTIR revealed different amide densities as a result of the crosslinking conditions. AFM showed that the crosslinking affected the roughness and swelling behavior of the coatings. The stability and degradability of the multilayers in aqueous environments were monitored with spectroscopic ellipsometry. The resistance of the coatings against non-specific protein adsorption was characterized by SPR spectroscopy. Settlement assays using Ulva linza zoospores and removal assays using the diatom Navicula incerta showed that the slowly degradable coatings were less prone to fouling than the strongly crosslinked ones. Thus, the coatings were a suitable model system to show that crosslinking the multilayers under mild conditions and equipping the coatings with controlled degradation rates enhances their antifouling and fouling-release properties against marine fouling organisms.

Sol-Gel-Based Hybrid Materials as Antifouling and Fouling-Release Coatings for Marine Applications.

Hybrid materials (HMs) offer unique properties as they combine inorganic and organic components into a single material. Here, we developed HM coatings for marine antifouling applications using sol-gel chemistry and naturally occurring polysaccharides. The coatings were characterized by spectroscopic ellipsometry, contact angle goniometry, AFM, and ATR-FTIR, and their stability was tested in saline media. Marine antifouling and fouling-release properties were tested in laboratory assays against the settlement of larvae of the barnacle Balanus improvisus and against the settlement and removal of the diatom Navicula incerta. Furthermore, laboratory data were confirmed in short-term dynamic field assays in Florida, USA. All hybrid coatings revealed a superior performance in the assays compared to a hydrophobic reference. Within the hybrids, those with the highest degree of hydrophilicity and negative net charge across the surface performed best. Alginate and heparin showed good performance, making these hybrid materials promising building blocks for fouling-resistant coatings.

Effect of Dipole Orientation in Mixed, Charge-Equilibrated Self-assembled Monolayers on Protein Adsorption and Marine Biofouling.

While zwitterionic interfaces are known for their excellent low-fouling properties, the underlying molecular principles are still under debate. In particular, the role of the zwitterion orientation at the interface has been discussed recently. For elucidation of the effect of this parameter, self-assembled monolayers (SAMs) on gold were prepared from stoichiometric mixtures of oppositely charged alkyl thiols bearing either a quaternary ammonium or a carboxylate moiety. The alkyl chain length of the cationic component (11-mercaptoundecyl)-N,N,N-trimethylammonium, which controls the distance of the positively charged end group from the substrate's surface, was kept constant. In contrast, the anionic component and, correspondingly, the distance of the negatively charged carboxylate groups from the surface was varied by changing the alkyl chain length in the thiol molecules from 7 (8-mercaptooctanoic acid) to 11 (12-mercaptododecanoic acid) to 15 (16-mercaptohexadecanoic acid). In this way, the charge neutrality of the coating was maintained, but the charged groups exposed at the interface to water were varied, and thus, the orientation of the dipoles in the SAMs was altered. In model biofouling studies, protein adsorption, diatom accumulation, and the settlement of zoospores were all affected by the altered charge distribution. This demonstrates the importance of the dipole orientation in mixed-charged SAMs for their inertness to nonspecific protein adsorption and the accumulation of marine organisms. Overall, biofouling was lowest when both the anionic and the cationic groups were placed at the same distance from the substrate's surface.

Effects of crosslink density in zwitterionic hydrogel coatings on their antifouling performance and susceptibility to silt uptake.

Hydrogel coatings effectively reduce the attachment of proteins and organisms in laboratory assays, in particular when made from zwitterionic monomers. In field experiments with multiple species and non-living material, such coatings suffer from adsorption of particulate matter. In this study, the zwitterionic monomer 3-[N-(2-methacryloyloxyethyl)-N,N-dimethylammonio] propanesulfonate (SPE) was copolymerized with increasing amounts of the photo-crosslinker benzophenon-4-yloxyethyl methacrylate (BPEMA) to systematically alter the density of crosslinks between the polymer chains. The effect of increasing crosslink density on the antifouling (AF) performance of the coatings was investigated in laboratory assays and fields tests. In both cases, the AF performance was improved by increasing the crosslinker content. The coatings reduced protein, diatom, and barnacle accumulation, and showed better resistance to biomass accumulation. The findings underline that the marine AF performance of hydrogel coatings does not only depend on the specific chemical structure of the polymers, but also on their physico-chemical properties such as rigidity and swelling.

Frictional drag measurements of large-scale plates in an enhanced plane channel flowcell.

This paper describes the design of an enhanced, plane channel, flowcell and its use for testing large-scale coated plates (0.6 m × 0.22 m) in fully developed flow, over a wide range of Reynolds numbers, with low uncertainty. Two identical, hydraulically smooth plates were experimentally tested. Uniform biofilms were grown on clean surfaces to test skin friction changes resulting from different biofilm thickness and densities. A velocity survey of the flowcell measurement section, using laser Doppler anemometry, showed a consistent velocity profile and low turbulence intensity in the central flow channel. The skin friction coefficient was experimentally determined using a pressure drop method. Results correlate closely to previously published regression data, particularly at higher speeds. Repeated measurements indicated very low uncertainty. This study demonstrates this flowcell's applicability for representing consistent frictional drag of ship hull surfaces, enabling comparability of hydrodynamic drag caused by surface roughness to the reference surface measurements.

Chitin is a functional component of the larval adhesive of barnacles.

Barnacles are the only sessile crustaceans, and their larva, the cyprid, is supremely adapted for attachment to surfaces. Barnacles have a universal requirement for strong adhesion at the point of larval attachment. Selective pressure on the cyprid adhesive has been intense and led to evolution of a tenacious and versatile natural glue. Here we provide evidence that carbohydrate polymers in the form of chitin provide stability to the cyprid adhesive of Balanus amphitrite. Chitin was identified surrounding lipid-rich vesicles in the cyprid cement glands. The functional role of chitin was demonstrated via removal of freshly attached cyprids from surfaces using a chitinase. Proteomic analysis identified a single cement gland-specific protein via its association with chitin and localized this protein to the same vesicles. The role of chitin in cyprid adhesion raises intriguing questions about the evolution of barnacle adhesion, as well as providing a new target for antifouling technologies.

Engineered Chemical Nanotopographies: Reversible Addition-Fragmentation Chain-Transfer Mediated Grafting of Anisotropic Poly(acrylamide) Patterns on Poly(dimethylsiloxane) To Modulate Marine Biofouling.

Effectively negating the deleterious impact of marine biofouling on the world's maritime fleet in an environmentally conscientious manner presents a difficult challenge due to a variety of factors including the complexity and diversity of fouling species and the differing surface adhesion strategies. Understanding how surface properties relate to biofouling can inform and guide the development of new antibiofouling coatings to address this challenge. Herein, we report on the development of a living photopolymerization strategy used to tailor the surface properties of silicone rubber using controlled anisotropic poly(acrylamide) patterns and the resulting antibiofouling efficacy of these surfaces against zoospores of the model marine fouling organism, Ulva linza. Chemical patterns were fabricated using reversible addition-fragmentation chain-transfer (RAFT) living polymerization in conjunction with photolithography. Pattern geometries were inspired by the physical (i.e., nonchemical) Sharklet engineered microtopography system that has been shown to be effective against the same model organism. Sharklet chemical patterns and analogous parallel channels were fabricated in sizes ranging from 2 to 10 µm in the lateral dimension with tailorable feature heights ranging from tens to hundreds of nanometers. Nonpatterned, chemically grafted poly(acrylamide) silicone surfaces inhibited algal spore attachment density by 59% compared to the silicone control; however, attachment density on chemical nanotopographies was not statistically different from the control. While these results indicate that the chemical nanotopographies chosen do not represent an effective antibiofouling coating, it was found that the Sharklet pattern geometry, when sized below the 5 µm critical attachment size of the spores, significantly reduced the algal spore density compared to the equally sized channel geometry. These results indicate that specific chemical geometry of the proper sizing can impact the behavior of the algal spores and could be used to further study the mechanistic behavior of biofouling organisms.