How to identify biofouling species in marine and freshwater.

The identification and management of biofouling remain pressing challenges in marine and freshwater ecosystems, with significant implications for environmental sustainability and industrial operations. This comprehensive review synthesizes the current state-of-the-art in biofouling identification technologies, examining eight prominent methodologies: Microscopy Examination, Molecular Biology, Remote Sensing, Community Involvement, Ecological Methods, Artificial Intelligence, Chemical Analysis, and Macro Photography. Each method is evaluated for its respective advantages and disadvantages, considering factors such as precision, scalability, cost, and data quality. Furthermore, the review identifies current obstacles that inhibit the optimal utilization of these technologies, ranging from technical limitations and high operational costs to issues of data inconsistency and subjectivity. Finally, the review posits a future outlook, advocating for the development of integrated, standardized systems that amalgamate the strengths of individual approaches. Such advancement will pave the way for more effective and sustainable strategies for biofouling identification and management.

Proof of Concept of Natural and Synthetic Antifouling Agents in Coatings.

Marine biofouling, caused by the deposition and accumulation of marine organisms on submerged surfaces, represents a huge concern for the maritime industries and also contributes to environmental pollution and health concerns. The most effective way to prevent this phenomenon is the use of biocide-based coatings which have proven to cause serious damage to marine ecosystems. Several research groups have focused on the search for new environmentally friendly antifoulants, including marine and terrestrial natural products and synthetic analogues. Some of these compounds have been incorporated into marine coatings and display interesting antifouling activities caused by the interference with the biofilm-forming species as well as by the inhibition of the settlement of macroorganisms. This review highlights the proof-of-concept studies of emerging natural or synthetic antifouling compounds in coatings, from lab-made to commercial ones, performed between 2019 and 2023 and their results in the field or in in vivo laboratorial tests.

Rapid discovery of a new antifoulant: From in silico studies targeting barnacle chitin synthase to efficacy against barnacle settlement.

Due to the adverse environmental impacts of toxic heavy metal-based antifoulants, the screening of environmentally friendly antifoulants has become important for the development of marine antifouling technology. Compared with the traditional lengthy and costly screening method, computer-aided drug design (CADD) offers a promising and efficient solution that can accelerate the screening process of green antifoulants. In this study, we selected barnacle chitin synthase (CHS, an important enzyme for barnacle settlement and development) as the target protein for docking screening. Three CHS genes were identified in the barnacle Amphibalanus amphitrite, and their encoded proteins were found to share a conserved glycosyltransferase domain. Molecular docking of 31,561 marine natural products with AaCHSs revealed that zoanthamine alkaloids had the best binding affinity (-11.8 to -12.6 kcal/mol) to AaCHSs. Considering that the low abundance of zoanthamine alkaloids in marine organisms would limit their application as antifoulants, a marine fungal-derived natural product, mycoepoxydiene (MED), which has a similar chemical structure to zoanthamine alkaloids and the potential for large-scale production by fermentation, was selected and validated for stable binding to AaCHS2L2 using molecular docking and molecular dynamics simulations. Finally, the efficacy of MED in inhibiting cyprid settlement of A. amphitrite was confirmed by a bioassay that demonstrated an EC50 of 1.97 µg/mL, suggesting its potential as an antifoulant candidate. Our research confirmed the reliability of using AaCHSs as antifouling targets and has provided insights for the efficient discovery of green antifoulants by CADD.

Ultrasonic-Assisted Marine Antifouling Strategy on Gel-like Epoxy Primer.

Ultrasonic technology has drawn extensive interests for its great potential in marine antifouling applications. However, its effects on the adhesion behavior of marine fouling organisms on marine structures remain underexplored. This work investigated how ultrasonic treatment impacted the adhesion of Pseudoalteromonas on a gel-like marine epoxy primer. And the process parameters for ultrasonic treatment were optimized using response surface analysis with Design-Expert software 11. The results revealed that ultrasonic treatment disrupted the cellular structure of Pseudoalteromonas, causing the deformation and fragmentation of the cell membrane, leading to bacterial death. Additionally, ultrasonic treatment reduced the particle size and Zeta potential value of Pseudoalteromonas, which disrupted the stability of bacterial suspensions. It also increased the relative surface hydrophobicity of Pseudoalteromonas cells, resulting in a reduction in adhesion to the gel-like marine epoxy primer. This study demonstrated that ultrasonic treatment significantly disturbed the adhesion behavior of microorganisms like Pseudoalteromonas on the gel-like marine epoxy primer, which provided an effective approach for controlling marine biofouling.

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.

Reduction of biofilm accumulation by constant and alternating potentials in static and dynamic field experiments.

The application of electric fields to conductive coatings is an environmentally friendly way to reduce biofilm formation. In particular alternating potentials (APs) have received increasing attention in recent studies. Here, an electrochemical rotating disk setup for dynamic field exposure experiments was developed to study how APs alter the attachment of fouling organisms in a multispecies ocean environment. A specific focus of the device design was proper integration of the potentiostat in the strongly corroding saltwater environment. The effect of APs on the accumulation of fouling organisms in short term field exposures was studied. Potentials on conductive gold surfaces were periodically switched between -0.3 V and 0.3 V or between -0.8 V and 0.6 V at a frequency of 0.5 Hz. APs were capable of significantly reducing the attachment of marine fouling organisms compared with the conductive samples immersed at open circuit potentials.

Influence of coating type, colour, and deployment timing on biofouling by native and non-native species in a marine renewable energy context.

Biofouling on marine renewable energy devices presents engineering challenges for this developing sector, and has implications for the spread of marine non-native species (NNS) in coastal waters. This is particularly true at sites with abundant energy resource, little existing infrastructure, and few established NNS. Device coatings, such as antifouling paints, could reduce the risk of NNS spread. Settlement on coatings of various types and colours, representing those likely to be used on renewable energy devices, was assessed in the Orkney Islands, northern Scotland. Assemblage composition, but not overall biofouling cover, varied initially among different coloured surfaces, although differences decreased over time. Different coating types (an anticorrosive paint, a biocidal paint and a fouling-release coating) differed in biofouling abundance and composition for the full duration of the experiment. NNS were mostly, but not completely, absent from antifouling surfaces. These results can help informing antifouling strategies for the marine renewable energy industry.

Single Ultraviolet-C light treatment of early stage marine biofouling delays subsequent community development.

Past studies of Ultraviolet-C (UV-C) radiation as a marine antifoulant have focused on repeated doses. However, single or very low frequency exposures of UV-C could create more plausible applications for certain marine structures. The objective of the study reported here was to apply a single treatment of UV-C radiation to an early stage marine biofouling community to observe subsequent effects on biofouling development. Biofouling formed over a 2-week field immersion received UV-C treatments of 0 (control), 4, 20, or 120 min, and subsequent progression was then monitored weekly for 16 weeks. Analysis of acute effects and later macrofouling development suggested direct toxicity of UV-C illumination to invertebrate recruits caused reduction of subsequent biofouling (compared to controls) that persisted for up to 16 weeks following the longest UV-C treatment. Thus, UV-C treatments spaced by days or even weeks could be an option for some applications of UV-C radiation as an antifoulant.

Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments.

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic.

Quantitative proteomic analysis of marine biofilms formed by filamentous cyanobacterium.

Cyanobacterial molecular biology can identify pathways that affect the adhesion and settlement of biofouling organisms and, consequently, obtain novel antifouling strategies for marine applications. Proteomic analyses can provide an essential understanding of how cyanobacteria adapt to different environmental settings. However, only a few qualitative studies have been performed in some cyanobacterial strains. Considering the limited knowledge about protein expression in cyanobacteria in different growing conditions, a quantitative proteomic analysis by LC-MS/MS of biofilm cells from a filamentous strain was performed. Biofilms were also analysed through standard methodologies for following cyanobacterial biofilm development. Biofilms were formed on glass and perspex at two relevant hydrodynamic conditions for marine environments (average shear rates of 4 s-1 and 40 s-1). Biofilm development was higher at 4 s-1 and no significant differences were found between surfaces. Proteomic analysis identified 546 proteins and 41 were differentially expressed. Differences in protein expression were more noticeable between biofilms formed on glass and perspex at 4 s-1. When comparing biofilms formed on different surfaces, results suggest that biofilm development may be related to the expression of several proteins like a beta-propeller domain-containing protein, chaperone DnaK, SLH domain-containing proteins, an OMF family outer membrane protein, and/or additional uncharacterized proteins. Regarding the hydrodynamic effect, biofilm development can be related to SOD enzyme expression, to proteins related to photosynthetic processes and to a set of uncharacterized proteins with calcium binding domains, disordered proteins, and others involved in electron transfer activity. Studies that combine distinct approaches are essential for finding new targets for antibiofilm agents. The characterisation performed in this work provides new insights into how shear rate and surface affect cyanobacterial biofilm development and how cyanobacteria adapt to these different environmental settings from a macroscopic standpoint to a proteomics context.

Antifouling performance of copper-containing fused filament fabrication (FFF) 3-D printing polymer filaments for marine applications.

3-D printing allows for rapid prototyping and manufacture of components in a cost effective and timely manner, becoming a popular technology for oceanographic and maritime engineering applications. However, 3-D printed components are at risk of accumulating marine biofouling. This study examined the antifouling (AF) performance of three Cu-containing 3-D printing polymer filaments for potential marine use. The amount of Cu present in each filament was the primary determinant of AF performance. The lowest Cu-content (30%) filament recording 29% biofouling cover after 1 month and near 100% cover after 3 months. These AF results were comparable to polylactide acid (PLA) and acrylonitrile butadiene styrene (ABS) polymer filament controls. The medium (50%) Cu-content filament remained largely free of biofouling until 3 months (where 48% coverage was observed), after which fouling cover increased to 100% by ∼12 months. The highest Cu-content (80%) filament remained free of all macrofouling for the entire 24 months of the experiment.

Characterization of planktonic and biofilm cells from two filamentous cyanobacteria using a shotgun proteomic approach.

Cyanobacteria promote marine biofouling with significant impacts. A qualitative proteomic analysis, by LC-MS/MS, of planktonic and biofilm cells from two cyanobacteria was performed. Biofilms were formed on glass and perspex at two relevant hydrodynamic conditions for marine environments (average shear rates of 4 s-1 and 40 s-1). For both strains and surfaces, biofilm development was higher at 4 s-1. Biofilm development of Nodosilinea sp. LEGE 06145 was substantially higher than Nodosilinea sp. LEGE 06119, but no significant differences were found between surfaces. Overall, 377 and 301 different proteins were identified for Nodosilinea sp. LEGE 06145 and Nodosilinea sp. LEGE 06119. Differences in protein composition were more noticeable in biofilms formed under different hydrodynamic conditions than in those formed on different surfaces. Ribosomal and photosynthetic proteins were identified in most conditions. The characterization performed gives new insights into how shear rate and surface affect the planktonic to biofilm transition, from a structural and proteomics perspective.

A data-driven approach to predicting the attachment density of biofouling organisms.

The attachment efficiency of biofouling organisms on solid surfaces depends on a variety of factors, including fouler species, nutrition abundance, flow rate, surface morphology and the stiffness of the solid to which attachment is to be made. So far, extensive research has been carried out to investigate the effects of these factors on the attachment of various fouling species. However, the results obtained are species-dependent and scattered. There is no universal rule that can be applied to predict the attachment efficiency of different species. To solve this problem, the authors carried out meta-analysis of the effects of ten selected factors on attachment efficiency, resulting in a universal correlation between the attachment density and the selected factors, which was validated by attachment tests of tubeworms on PDMS surfaces. The results provide a practical approach to predicting the attachment efficiency of fouling organisms and should be of great value in the design of anti-biofouling materials.

Sediment challenge to promising ultra-low fouling hydrophilic surfaces in the marine environment.

Hydrophilic coatings exhibit ultra-low fouling properties in numerous laboratory experiments. In stark contrast, the antifouling effect of such coatings in vitro failed when performing field tests in the marine environment. The fouling release performance of nonionic and zwitterionic hydrophilic polymers was substantially reduced compared to the controlled laboratory environment. Microscopy and spectroscopy revealed that a large proportion of the accumulated material in field tests contains inorganic compounds and diatomaceous soil. Diatoms adhered to the accumulated material on the coating, but not to the pristine polymer. Simulating field tests in the laboratory using sediment samples collected from the test sites showed that incorporated sand and diatomaceous earth impairs the fouling release characteristics of the coatings. When exposed to marine sediment from multiple locations, particulate matter accumulated on these coatings and served as attachment points for diatom adhesion and enhanced fouling. Future developments of hydrophilic coatings should consider accumulated sediment and its potential impact on the antifouling performance.

Iodine-infused aeration for hull fouling prevention: a vessel-scale study.

Biofouling is a significant economic and ecological problem, causing reduced vessel performance and increases in fuel consumption and emissions. Previous research has shown iodine vapor (I2)-infused aeration to be an environmentally friendly method for deterring the settlement of fouling organisms. An aeration system was deployed on a vessel with hull sections coated with two types of antifoulant coatings, Intersleek® 1100 (fouling-release) and Interspeed® BRA-640 (ablative copper biocide), as well as an inert epoxy barrier coating, to assess the effectiveness of aeration in conjunction with common marine coatings. I2-infused aeration resulted in consistent reductions of 80-90% in hard fouling across all three coatings. Additionally, aeration reduced the soft fouling rate by 45-70% when used in conjunction with both Intersleek® and Interspeed® BRA versus those coatings alone. The results of this study highlight the contribution of I2-infused aeration as a standalone mechanism for fouling prevention or as a complement to traditional antifouling coatings.

In vivo and in situ synchrotron radiation-based µ-XRF reveals elemental distributions during the early attachment phase of barnacle larvae and juvenile barnacles.

Barnacles are able to establish stable surface contacts and adhere underwater. While the composition of adult barnacle cement has been intensively studied, far less is known about the composition of the cement of the settlement-stage cypris larva. The main challenge in studying the adhesives used by these larvae is the small quantity of material available for analysis, being on the order of nanograms. In this work, we applied, for the first time, synchrotron radiation-based µ-X-ray fluorescence analysis (SR-µ-XRF) for in vivo and in situ analysis of young barnacles and barnacle cyprids. To obtain biologically relevant information relating to the body tissues, adhesives, and shell of the organisms, an in situ sample environment was developed to allow direct microprobe investigation of hydrated specimens without pretreatment of the samples. In 8-day-old juvenile barnacles (Balanus improvisus), the junctions between the six plates forming the shell wall showed elevated concentrations of calcium, potassium, bromine, strontium, and manganese. Confocal measurements allowed elemental characterization of the adhesive interface of recently attached cyprids (Balanus amphitrite), and substantiated the accumulation of bromine both at the point of initial attachment as well as within the cyprid carapace. In situ measurements of the cyprid cement established the presence of bromine, chlorine, iodine, sulfur, copper, iron, zinc, selenium, and nickel for both species. The previously unrecognized presence of bromine, iron, and selenium in the cyprid permanent adhesive will hopefully inspire further biochemical investigations of the function of these substances.

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.

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.

Understanding the flow behavior around marine biofilms.

In vitro platforms capable of mimicking the hydrodynamic conditions prevailing in natural aquatic environments have been previously validated and used to predict the fouling behavior on different surfaces. Computational Fluid Dynamics (CFD) has been used to predict the shear forces occurring in these platforms. In general, these predictions are made for the initial stages of biofilm formation, where the amount of biofilm does not affect the flow behavior, enabling the estimation of the shear forces that initial adhering organisms have to withstand. In this work, we go a step further in understanding the flow behavior when a mature biofilm is present in such platforms to better understand the shear rate distribution affecting marine biofilms. Using 3D images obtained by Optical Coherence Tomography, a mesh was produced and used in CFD simulations. Biofilms of two different marine cyanobacteria were developed in agitated microtiter plates incubated at two different shaking frequencies for 7 weeks. The biofilm-flow interactions were characterized in terms of the velocity field and shear rate distribution. Results show that global hydrodynamics imposed by the different shaking frequencies affect biofilm architecture and also that this architecture affects local hydrodynamics, causing a large heterogeneity in the shear rate field. Biofilm cells located in the streamers of the biofilm are subjected to much higher shear values than those located on the bottom of the streamers and this dispersion in shear rate values increases at lower bulk fluid velocities. This heterogeneity in the shear force field may be a contributing factor for the heterogeneous behavior in metabolic activity, growth status, gene expression pattern, and antibiotic resistance often associated with nutrient availability within the biofilm.

Development of Antifouling Strategies for Marine Applications.

Marine biofouling is an undeniable challenge for aquatic systems since it is responsible for several environmental and ecological problems and economic losses. Several strategies have been developed to mitigate fouling-related issues in marine environments, including developing marine coatings using nanotechnology and biomimetic models, and incorporating natural compounds, peptides, bacteriophages, or specific enzymes on surfaces. The advantages and limitations of these strategies are discussed in this review, and the development of novel surfaces and coatings is highlighted. The performance of these novel antibiofilm coatings is currently tested by in vitro experiments, which should try to mimic real conditions in the best way, and/or by in situ tests through the immersion of surfaces in marine environments. Both forms present their advantages and limitations, and these factors should be considered when the performance of a novel marine coating requires evaluation and validation. Despite all the advances and improvements against marine biofouling, progress toward an ideal operational strategy has been slow given the increasingly demanding regulatory requirements. Recent developments in self-polishing copolymers and fouling-release coatings have yielded promising results which set the basis for the development of more efficient and eco-friendly antifouling strategies.