Antifouling Assays Research Articles

Three New Depsipeptides, Homiamides A–C, Isolated from Streptomyces sp., ROA-065

Three new depsipeptides, homiamides A–C (1–3), were isolated from a marine sediment-derived strain of Streptomyces sp., ROA-065. The planar structures of homiamides A–C (1–3) were elucidated using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic data. The absolute configurations of 1–3 were deduced from the application of the Marfey’s method and GC-MS analysis after formation of the O-trifluoroacetylated (S)-(+)-methyl-2-butyl ester derivatives of amino acids. Compounds 1–3 exhibited weak anti-bacterial activities against both Gram-positive bacteria and Gram-negative bacteria, with compound 1 showing MIC values of 32–64 μg/mL. In antifouling assays, compounds 1 and 2 displayed moderate activity against Micrococcus luteus KCTC 3063, while compound 3 exhibited weak activity against all tested bacteria.

In vitro and in vivo assessment of antimicrobial, enzymatic, and antifouling properties of self-potent lichen symbiotic bacteria.

This study aimed to comprehensively assess the antimicrobial, antifouling, and antibiofilm-forming potential of lichen symbiotic bacteria against marine fouling bacterial strains. A total of 50 lichen-associated bacteria (LAB) isolates were successfully characterized and evaluated for their effectiveness in mitigating biofouling caused by various marine biofoulers. Through a battery of biological assays encompassing enzymatic, antagonistic, antimicrobial, and antifouling assays, 15 LAB isolates were identified based on their antagonist activities. Notably, the strain LAB4 exhibited remarkable performance across all bioassays, demonstrating its proficiency as an antifouling agent. The production of crude LAB extracts was successfully scaled up using a large-scale fermentor and further optimized. Additionally, a phylogenetic analysis of the isolated strain Bacillus proteolyticus D65's 16S ribosomal RNA gene revealed a high query coverage and percentage identity of 92.62% (accession no. MK883171.1). In conclusion, the lichen bacterial symbiotic isolate B. proteolyticus exhibited significant in vitro and in vivo inhibition of foulants. This study highlights the potential of lichens as a valuable source of yet unexplored bacteria. The bacterial consortium associated with Parmotrema sp. holds promise in combatting biofouling, which poses a substantial threat to the maritime industries and their economic stability.

Mechanically robust antifouling coating with dual-functional antifouling strategy by infiltrating PDMS into plasma-sprayed porous Al2O3-Cu coating

Marine biofouling is a worldwide challenge that needs to be solved urgently. Poly(dimethylsiloxane) (PDMS)-based fouling release coatings with low surface free energy (SFE) could effectively inhibit biofouling. Nevertheless, their poor mechanical durability, adhesive strength, and antifouling performance under static conditions significantly limit their applications. Herein, a novel mechanically robust Al2O3-PDMS-Cu composite coating with strong adhesive strength and remarkable antifouling performance was developed. The Al2O3-PDMS-Cu coating loaded with a small amount of Cu was fabricated by infiltrating PDMS into plasma-sprayed micro/nano-scaled porous Al2O3-Cu coating. Results showed that the fabrication of this Al2O3-PDMS-Cu coating did not alter the surface hydrophobicity and SFE of PDMS significantly, thus presenting little influence on its inherent fouling release property. After rigorous abrasion test, the Al2O3-PDMS-Cu coating presented remarkably improved surface hydrophobicity due to the exposure of micro/nano structure, rather than falling off as that of PDMS coating. The combination of excellent abrasion resistance and one order of magnitude higher adhesive strength and hardness than PDMS coating contributed to the outstanding mechanical robustness of Al2O3-PDMS-Cu coating. Additionally, the antifouling assays against marine bacteria adhesion (95% reduction rate for Escherichia coli. (E. coli)) and algae attachment (96% and 94% reduction rates for Chlorella and Phaeodactylum tricornutum (P. tricornutum), respectively after 21 days of incubation) demonstrated the superior antifouling performance of the Al2O3-PDMS-Cu coating. Thus, a high-performance Al2O3-PDMS-Cu antifouling coating with excellent mechanical robustness and long-term antifouling performance was achieved via the combination of mechanical durability of Al2O3 skeleton and the dual-functional antifouling strategy, i.e., the fouling release property of PDMS and fouling resistance of Cu.

Exploring Antifouling Activity of Biosurfactants Producing Marine Bacteria Isolated from Gulf of California.

Biofouling causes major problems and economic losses to marine and shipping industries. In the search for new antifouling agents, marine bacteria with biosurfactants production capability can be an excellent option, due to the amphipathic surface-active characteristic that confers antimicrobial and antibiofilm activities. The aim of this study was to evaluate the antifouling activity of biosurfactants producing marine bacteria from the Gulf of California. The cell free culture supernatant (CFCS) of Bacillus niabensis (S-69), Ralstonia sp. (S-74) (isolated from marine sediment) and of B. niabensis (My-30) (bacteria associated to the sponge Mycale ramulosa) were screened for production of biosurfactants (using hemolysis and drop collapse test, oil displacement and emulsifying activity). The toxicity and antifouling activity were evaluated against biofoulers (bacteria forming biofilm and macrofoulers) both in laboratory and field assays. The results indicate that all bacteria were biosurfactant producers, but the higher capability was shown by B. niabensis (My-30) with high emulsifying properties (E24) of 71%. The CFCS showed moderate toxicity but were considered non-toxic against Artemia franciscana at low concentrations. In the antifouling assay, the CFCS of both strains of B. niabensis showed the best results for the reduction of the biofilm formation (up 50%) against all Gram-positive bacteria and most Gram-negative bacteria with low concentrations. In the field assay, the CFCS of B. niabensis (My-30) led to the reduction of 30% of biofouling compared to the control. The results indicate that the biosurfactant produced by B. niabensis (My-30) has promising antifouling activity.

Medusa polyps adherence inhibition: A novel experimental model for antifouling assays.

Although in the last decades significant advances have been made to improve antifouling formulations, the main current options continue to be highly toxic to marine environment, leading to an urgent need for new safer alternatives. For anti-adherence studies, barnacles and mussels are commonly the first choice for experimental purposes. However, the use of these organisms involves a series of laborious and time-consuming stages. In the present work, a new approach for testing antifouling formulations was developed under known formulations and novel proposed options. Due to their high resilience, ability of surviving in hostile environments and high abundance in different ecosystems, medusa polyps present themselves as prospect candidates for antifouling protocols. Thus, a complete protocol to test antifouling formulations using polyps is presented, while the antifouling properties of two invasive seaweeds, Asparagopsis armata and Sargassum muticum, were evaluated within this new test model framework. The use of medusa polyps as model to test antifouling substances revealed to be a reliable alternative to the conventional organisms, presenting several advantages since the protocol is less laborious, less time-consuming and reproductive. The results also show that the seaweeds A. armata and S. muticum produce compounds with anti-adherence properties being therefore potential candidates for the development of new greener antifouling formulations.

Extraction of Protease Produced by Sea Mud Bacteria and Evaluation of Antifouling Performance

Enzyme-based antifouling coatings are potential alternative to traditional tributyltin-based coatings in the marine bio-fouling control depended on its environmental friendliness. Proteases are usually the key antifouling active components in enzyme based antifouling coatings. In this work, based on biological antagonism effect, a marine proteolytic bacterial strain of Bacillus velezensis was isolated from the sea mud, and denoted as SM-1. The scanning electron microscope (SEM) revealed that the bacteria are rod-shaped with length 1-1.3 μm. The protease-producing conditions of SM-1 were investigated, and it was found that the culture solution displays higher proteolytic activity under the culture conditions of 35°C, 10‰-20‰ salinity, pH 6–9 and more than 7d culture time. Moreover, the 25kDa protein was confirmed to be the main active component in the crude protease, which was revealed via the experiment of SDS-PAGE. The antifouling assay indicated that the protease SM-1 has remarkable effect on the decomposition of barnacle cement and diatom secretion, and also can effectively inhibit the attachment of barnacle cyprids, diatom and mussel byssus. Therefore, this protease potentially can be used as environmental-friendly antifoulant of enzyme-based marine antifouling coatings, and this work also provides a new approach to obtain antifouling protease via isolating proteolytic bacteria from the sea mud surrounding representative fouling organisms.

Investigating the effect of temporal variation on fouling settlement using a rapid assay method

Biological fouling in the tropical marine environment is ubiquitous and occurs all year round. However, the fouling composition and settlement rates may differ, particularly due to hydrological factors. This may present many challenges in evaluating field performance of antifouling coatings. For short duration field testing, i.e., rapid field assessment, it may be difficult to reconcile differences in field activity for novel antifouling assays carried out at different immersion periods in a year, especially when there is little knowledge of temporal variation in fouling settlement. To understand how fouling settlement may vary over different periods, this study used a rapid field assessment method to examine fouling settlement on four types of control coatings at five different immersion periods over a year. Results suggested that antifouling assessment of coatings, using short duration assays (2–4 weeks), can be carried out all year round without being confounded by temporal variation. This study demonstrates, for practitioners, the significance of gaining insights on the effect of temporal variation in fouling settlement within a tropical marine environment in Singapore.

Mussel-Inspired Surface Functionalization of PET with Zwitterions and Silver Nanoparticles for the Dual-Enhanced Antifouling and Antibacterial Properties.

Herein, we designed and constructed a dual functional surface with antimicrobial and antifouling abilities to prevent protein and bacterial attachment that are significant challenges in biomedical devices. Primary amino-group-capped sulfobetaine of DMMSA was synthesized and then grafted onto polydopamine pretreated PET sheets via click chemistry. The sheets were subsequently immersed into silver ion solution, in which the absorbed silver ions were reduced to silver nanoparticles (AgNPs) in situ by a polydopamine layer. The antifouling assays demonstrated that the resultant PET/DMMSA/AgNPs sheets exhibited great antifouling performances against bovine serum albumin (BSA), bovine fibrinogen (BFG), platelets, and bacteria, the critical proteins/microorganisms leading to implant failure. The antibacterial data suggested that the sheets had dual functions as inhibitors of bacterial growth and bactericide and could efficiently delay the biofilm formation. This repelling and killing approach is green and simple, with potential biomedical applications.

Reproducible Superhydrophobic PVC Coatings; Investigating the Use of Plasticizers for Early Stage Biofouling Control

Here we show an easy to synthesize superhydrophobic material using a solvent phase–separation process of poly vinyl chloride. It is found that solvents mixed in different ratios increase the dielectric value of the solvent and can be tuned to produce superhydrophobic PVC. The PVC solution is then spin‐coated onto glass slides for characterization using scanning electron microscopy. Plasticizers are doped into the 70% (v/v) PVC to determine their overall effects; it is found that plasticizers reduce the water contact angle value. The final coatings were tested in a series of antifouling assays in a marine environment lab study; it is found that the superhydrophobic PVC material reduced marine biofouling.

Comparative effects of indole derivatives as antifouling agents on the growth of two marine diatom species

Antifouling agents, used to prevent biofouling, need to be assessed for their impacts on marine organisms and environment before the application. Diatoms are one of the main components of fouling biofilms, which play important roles in the formation of biofouling. Particularly, diatoms are also the important ingredients of primary production and present interest as ecotoxicological models in marine environment. In this study, two benthic diatoms Nitzschia closterium f. minutissima and Navicula climacospheniae, widely distributed in fouling biofilm, were used as models for screening the activities of potential antifoulants. Nine indole derivatives were tested and CuSO4 was used as a reference. Indole derivatives showed significant anti-algal activities and the EC50 values of most indole derivatives were lower than that of CuSO4. Halogen substituent enhanced the anti-algal activities of compounds, and the most efficient compounds for N. closterium f. minutissima were gramine and 7-chloroindole with the EC50 values of 1.94 and 2.1 mg/L, while for N. climacospheniae, 7-chloroindole and 6-bromoindole were the most efficient and the EC50 values were 3.91 and 4.25 mg/L, respectively. In conclusion, indole derivatives would be one of the promising candidates as antifoulants and our results strengthened the need to perform antifouling activity assays and environment-friendly evaluations.

Antifouling and Fungicidal Resorcylic Acid Lactones from the Sea Anemone-Derived FungusCochliobolus lunatus

Three new 14-membered resorcylic acid lactones, cochliomycins D-F, 1-3, and eight known analogues, 4-11, were isolated from the sea anemone-derived fungus Cochliobolus lunatus. Compounds 1-4 are diastereomers differing from each other by the absolute configurations of the 4',5'-diol chiral centers. The absolute configurations of 1-4 were established by the CD exciton chirality method and TDDFT ECD calculations. In antifouling assays, 1, 3-6, and 6a exhibited potent antifouling activities against the larval settlement of the barnacle Balanus amphitrite at nontoxic concentrations, with EC50 values ranging from 1.82 to 22.5 μg/mL. Noticeably, fungicide whole-plant assays indicated that 6 showed excellent activity on the Plasmopara viticola preventative test at 6 ppm and concentration-dependent activity on the Phytophthora infestans preventative application at 200, 60, and 20 ppm. Preliminary structure-activity relationships are also discussed.

Multilayers of Fluorinated Amphiphilic Polyions for Marine Fouling Prevention

Sequential layer-by-layer (LbL) deposition of polyelectrolytes followed by chemical cross-linking was investigated as a method to fabricate functional amphiphilic surfaces for marine biofouling prevention applications. A novel polyanion, grafted with amphiphilic perfluoroalkyl polyethylene glycol (fPEG) side chains, was synthesized and subsequently used to introduce amphiphilic character to the LbL film. The structure of the polyanion was confirmed by FTIR and NMR. Amphiphilicity of the film assembly was demonstrated by both water and hexadecane static contact angles. XPS studies of the cross-linked and annealed amphiphilic LbL films revealed the increased concentration of fPEG content at the film interface. In antifouling assays, the amphiphilic LbL films effectively prevented the adhesion of the marine bacterium Pseudomonas (NCIMB 2021).

Bioactivity of the red algae Asparagopsis taxiformis collected from the Southwestern coast of India

Among the diverse variety of red algae, Asparagopsis taxiformis constitutes one of the abundant biomass in the Kollam coast (Southwest coast of India). Therefore, in the present study, A. taxiformis was collected, extracted and fractionated using column chromatography. The individual fractions were evaluated in vitro for their antifouling, anticyanobacterial, piscicidal and crustaceans toxicity assays. The fraction eluted with 2:8, petroleum ether and ethyl acetate exhibited strong and broad spectrum of bioactivity. In antifouling assay against Limnea truncatula, the active algal fraction produced 80% of foot repellency at 150 mg/L whereas in anticyanobacterial assay, the active fraction inhibited 100% growth of Trichodesmium sp. at 320 mg/L. The algal fraction showed higher piscicidal effect at the level of 60 mg/L. The crustacean toxicity of the active fraction was also evaluated to find compounds without toxicity in non target organisms, Penaeus monodon and Macrobrachium rosenbergii. It was found that column fraction showed less toxicity against the non target organisms. The chemical constituents of the active fraction were identified by means of chromatographic systems such as TLC, reverse phase HPLC and GC-MS. The overall activity profile envisages that the active column fraction of A. taxiformis might contain synergistic bioactive metabolites that could be utilized for the control of fouling organisms, algal bloom and herbivorous/predaceous fishes in aquaculture ponds.

Antifouling activity of seaweed extracts from Guarujá, São Paulo, Brazil

Marine biofouling historically constitutes one of the major constraints faced by mankind in its oceanic activities. The search for alternatives to TBT-based antifouling paints has led several researchers to focus efforts in the development of environmentally friendly natural compounds. This work has contributed with this search, testing the antifouling potential of crude organic extracts from four seaweed species collected at Praia Branca, Guarujá district, São Paulo, Brazil. Throughout laboratory antifouling assays in which the attachment of a common fouling organism, the brown mussel Perna perna, was employed, antifouling activity (p < 0.05) was detected in natural concentrations of the extracts of Jania rubens (Rhodophyta, Cryptonemiales) and Bryothamnion seaforthii (Rhodophyta, Ceramiales), while Dictyopteris delicatula (Phaeophyta, Dictyotales) and Heterosiphonia gibbesii (Rhodophyta, Ceramiales) did not exhibit fouling inhibition. From the algae that exhibited antifouling activity, J. rubens presented best performance when compared to that of B. seaforthii. Future field studies would be necessary to obtain results that can better reflect natural conditions, as well as to assess the activity spectrum of the antifouling activity presently recorded. Further bioassay-guided purification of the active extracts can lead to new alternatives to the metal-based antifouling paints currently in use.

Zebra Mussel Antifouling Activity of the Marine Natural Product Aaptamine and Analogs

Several aaptamine derivatives were selected as potential zebra mussel (Dreissena polymorpha) antifoulants because of the noteworthy absence of fouling observed on Aaptos sponges. Sponges of the genus Aaptos collected in Manado, Indonesia consistently produce aaptamine-type alkaloids. To date, aaptamine and its derivatives have not been carefully evaluated for their antifoulant properties. Structure-activity relationship studies were conducted using several aaptamine derivatives in a zebra mussel antifouling assay. From these data, three analogs have shown significant antifouling activity against zebra mussel attachment. Aaptamine, isoaaptamine, and the demethylated aaptamine compounds used in the zebra mussel assay produced EC(50) values of 24.2, 11.6, and 18.6 microM, respectively. In addition, neither aaptamine nor isoaaptamine produced a phytotoxic response (as high as 300 microM) toward a nontarget organism, Lemna pausicostata, in a 7-day exposure. The use of these aaptamine derivatives from Aaptos sp. as potential environmentally benign antifouling alternatives to metal-based paints and preservatives is significant, not only as a possible control of fouling organisms, but also to highlight the ecological importance of these and similar biochemical defenses.

PRELIMINARY RESULTS ON SECONDARY METABOLITES FROM ANTARCTIC BROWN ALGAE AND THEIR ECOLOGICAL RELEVANCE

Iken, K. 1, Amsler, C. D.1, Hubbard, J. M.2, McClintock, J. B.1, Baker, B. J.3 1University of Alabama at Birmingham, Dept. of Biology, Birmingham, AL 35294‐1170 USA 2Hanshew Middle School, 10121 Lake Otis Pkwy., Anchorage, AK 99507 USA; 3Dept. of Chemistry, Florida Institute of Technology, Melbourne, FL 32901 USABrown algae (Phaeophyceae) are often dominant members in abundance and biomass of Antarctic macroalgal communities. They are ecologically important in providing habitat structure and shelter to many invertebrates and fish. However, as live algae they often appear to be of little importance as food for herbivores. Palatability of thalli of eight abundant Antarctic brown algae was tested against two sympatric algal grazers, the fish Notothenia coriiceps and the sea star Odontaster validus. Polar and non‐polar organic extracts of six brown algae were also tested against these grazers and against the omnivorous amphipod Gondogeneia antarctica. Almost all thalli and at least one of the extract types per algal species were unpalatable to the grazers, suggesting that chemical defenses are probably present in Antarctic brown algae. We further tested biological activity of brown algal extracts in antimicrobial assays as well as antifouling assays with sympatric bacteria and diatoms, respectively. We chemically screened organic brown algal extracts for the occurrence of secondary metabolites. Acetogeno‐terpenes were detected in Desmarestia menziesii and D. anceps. Phlorotannins were present in all brown algal species analyzed, but at different concentrations between species as well as between different thallus parts within individuals. Phlorotannin extracts were size fractionated to examine distribution and abundance of various size classes between species as well as between different thallus parts within individuals.

Phenoloxidase (E.C. 1.14.18.1) from the byssus gland of Mytilus edulis: Purification, partial characterization and application for screening products with potential antifouling activities

Although a total ban on the use of TBT coatings is not expected in the short term, there is a growing need for environmentally safe antifouling systems. To assist in the rapid screening of a large number of potential antifouling substances, a method that is simple, efficient and inexpensive is required. The production of byssus threads by the blue mussel, Mytilus edulis, has often been studied for testing the antifouling efficacy of various compounds. The present study reports a new antifouling assay based on the inhibition of purified M. edulis phenoloxidase activity. The method has the advantage of being specific, reliable, sensitive and rapid.

A new antifouling assay method: results from field experiments using extracts of four marine organisms

Antifouling is one possible defensive function of marine natural products isolated from sessile benthic organisms, but there is little experimental evidence to support this claim. We developed a technique in which crude organic extracts of marine organisms are incorporated into hard, stable gels that serve as substrata for larval settlement in the field. These gels contain extracts at concentrations that are volumetrically equivalent to those in living tissues, and compounds diffuse from assay gels in a manner that may mimic their natural release from some organisms. After 21 days in flowing seawater, a mean of 56% of the mass of crude extract from the sponge Hymeniacidon heliophila (Parker) remained in gels. Extracts from two sponges, Aplysilla longispina (George & Wilson) and Hymeniacidon heliophila, an ascidian, Eudistoma hepaticum (VanName), and an alga, Codium decorticatum (Woodward & Howe), were incorporated into gels and deployed in the field over a period of 28 days; extracts from A. longispina deterred settlement of invertebrates and algae relative to control gels, while extracts from C. decorticatum enhanced settlement. Mean settlement on control gels was similar to that on plexiglas plates of the same size. This technique represents a more ecologically relevant method for assaying the antifouling properties of extracts of marine organisms because (1) assay gels are exposed to a natural population of settling propagules, (2) extracts are incorporated into gels at natural volumetric concentrations, and (3) extracts within the gel matrix do not alter the physical characteristics of the settlement surface.