Characterization of Biofouling Resistant Coatings for Marine Applications
Ocean immersion testing is generally considered the “gold standard” for evaluating the performance of marine coatings. Coatings are typically prepared on large raft panels and immersed in the ocean to evaluate the accumulation and adhesion strength of fouling organisms over time. Although effective, ocean immersion testing does not adequately accommodate the testing of coatings generated with a combinatorial approach adopted by NDSU. In this regard, ocean immersion testing requires large amounts of coating material to coat panels, is seasonal, requires several weeks to months of exposure, and is quite labor intensive. As a result, a high-throughput biological screening workflow has been developed at NDSU to rapidly screen combinatorial coating arrays for antifouling and fouling-release properties. The high-throughput screening workflow employs a suite of relevant marine fouling organisms to efficiently and effectively assess coating performance to facilitate the down-selection and identification of promising candidates.
Coating Substrates for Biological Characterization
Coating libraries are applied to 4”x8” panels for fouling-release characterization with live barnacles. This includes draw down (left picture above) and well casting (right picture above) techniques to generate arrays of thin and thick films, respectively.
Coating libraries are prepared in 24-well plates (left picture above) for a variety of analyses with microbial organisms and mammalian cell based assays. 24-well plates are typically modified with discs of various substrates to facilitate solvent compatibility and promote good adhesion of coating films. A custom made pick and place apparatus (right picture above) was constructed at NDSU to enable the rapid production of modified plates.
Prior to coating evaluations, array plates and panels are immersed in a custom-built, large capacity water tank for an appropriate period of time to facilitate removal or “leaching” of toxic impurities (i.e., residual solvent, catalyst, monomers etc.). The system consists of a 200 gallon tank of deionized water that is continually re-circulated through an activated carbon bed and a UV light source to remove organic impurities and prevent un-wanted microbial growth. Plates and panels (maximum capacity of 300-400) are secured in racks and logged into a computerized database. E-mail notifications are automatically generated by the computerized database and sent to coating researchers to alert them when their plates/panels are ready to be removed for biological analysis.
Marine Fouling Organisms
A suite of relevant marine fouling organisms are maintained year round at NDSU for use in high-throughput screening assays to characterize antifouling and fouling-release coating properties.
Bacteria are unicellular microorganisms that have a rigid cell wall structure composed of peptidoglycan. They typically reside in a multispecies biofilm or “slime layer” in nature that can contribute up to a 25% increase in hydrodynamic drag on a ship’s hull. Recent evidence also suggests that bacterial biofilms can cue the settlement of other marine organisms, such as macroalgae and tubeworms. Two marine bacterial species, Cellulophaga lytica and Halomonas pacifica, are routinely utilized in our laboratory to screen coating performance.
Algae consist of a wide variety of unicellular and multicellular plant-like organisms. At NDSU, we utilize unicellular microalgae, called diatoms, to characterize the performance of marine coatings. Similar to bacteria, diatoms reside in the slime layer on surfaces submerged in the marine environment. However, their cell wall structure and composition is drastically different than bacteria as it is predominantly composed of silica.
Barnacles are often the most commonly recognized shell fouling organism found on surfaces submerged in the marine environment. Barnacles start off as nauplii or “swimmers” and metamorphose into cypris larvae or “settlers”. Once they find a suitable place to settle, cypris larvae metamorphose into the juvenile barnacles that spend the remainder of their life attached to a surface. Barnacles contribute to the “macrofouling” layer on surfaces submerged in the marine environment, which has been shown to contribute substantially to the hydrodynamic drag penalty, up to 75%, when attached to a ship’s hull.
Mussels also contribute to the development of “macrofouling” communities on surfaces submerged in the marine environment. Similar to barnacles, mussels start off as larvae that drift in the water column before settling onto suitable surfaces and metamorphosing into juveniles. Mussels attach to surfaces by generating numerous adhesive threads (byssus) and can quickly translocate to new positions by attaching and detaching these adhesive threads through the use of a large, muscular organ known as a ‘foot’.
Evaluation of Leachate Toxicity
The marine coatings program at NDSU is primarily focused on the development of non-toxic or environmentally friendly technologies. In this regard, it is important to verify that newly developed coating candidates do not release or “leach” any toxic components upon immersion in sea water. After pre-conditioning, coating arrays are incubated in artificial sea water (ASW) and the resulting extracts are collected. The appropriate marine organisms are then introduced into the coating extracts and monitored for growth or swimming behavior. A reduction in growth or the observation of abnormal swimming behavior is determined to be a consequence of toxic components leaching into the ASW. Coatings that exhibit no adverse affect of their leachates on the marine organisms are then evaluated with the suite of antifouling and fouling-release characterization assays.
Characterization of Antifouling Properties
One of the primary approaches employed at NDSU to create non-toxic, environmentally friendly antifouling coatings is to chemically attach or “tether” organic biocides into a coating matrix to deter settlement and or growth of marine fouling organisms. This strategy relies on the direct contact between the targeted fouling organisms and the coating surface to impart and antifouling character. A variety of high-throughput screening assays have been developed to rapidly characterize coatings based on this non-leaching, contact-active approach.
Bacterial Biofilm Growth
The primary workhorse of the high-throughput screening workflow for antifouling characterization is based on the utilization of marine bacteria. In particular, the marine bacteria employed are allowed to attach and colonize the coating surfaces for the appropriate period of time needed to generate a biofilm (i.e., 24-48 hours). One of the defining characteristics of bacterial biofilm formation is the generation of an extracellular polymeric substance (EPS). The EPS is predominantly composed on polysaccharides and protein and serves as effective matrix to not only adhere bacteria to surfaces or interfaces, but protect them from environmental adversity (i.e., predation, antifouling agents etc.).
Marine bacteria are suspended in artificial sea water (ASW), supplemented with nutrients, and dispensed into wells of the coating array plates. The plates are then incubated at the appropriate temperature and duration required to facilitate attachment and subsequent biofilm growth. Coating plates are then rinsed with ASW, to remove loosely bound or planktonic growth, dried at ambient conditions, and stained with the biomass indicator dye, crystal violet. The crystal violet stained biofilms (purple color) are extracted with acetic acid and the resulting eluates are measured for absorbance at 600nm. The absorbance values obtained are directly proportional to the amount of biofilm growth retained on the coating surfaces.
Algal Biofilm Growth
Cultures of marine fouling diatoms are maintained year-round at NDSU for use in our high-throughput screening workflow. The rapid screening assays employing diatoms were developed in collaboration with researchers at the University of Birmingham, UK. Diatoms are utilized in conjunction with bacteria to rapidly assess the antifouling properties of coating arrays for broad spectrum activity. Diatoms also live in multispecies biofilms, often with bacteria, to facilitate adherence to surfaces and protection from the environment.
Diatoms are suspended in ASW, supplemented with nutrients, and dispensed into wells of the coating array plates. The plates are then incubated at the appropriate temperature, duration and lighting conditions to facilitate attachment and subsequent biofilm growth. The ASW growth medium is removed and the plates are immediately treated with dimethyl sulfoxide (DMSO) to extract chlorophyll. The resulting eluates are then measured for chlorophyll fluorescence (Excitation: 360 nm; Emission: 670 nm). The fluorescence values obtained are directly proportional to the amount of biofilm growth obtained on the coating surfaces.
Characterization of Fouling-Release Properties
In addition to non-leaching, contact-active antifouling coatings, considerable effort has been dedicated to the development of fouling-release technologies. Fouling-release coatings function by minimizing the strength of adhesion of the adhesives or “biological glues” produced by the various fouling organisms. Current fouling-release technologies are based on silicone elastomers or fluoropolymers. Researchers at NDSU are focusing on new strategies to improve their performance and durability while extending the active lifetime of these coating systems. As with the antifouling coatings, a number of high-throughput screening assays have been developed at NDSU to rapidly evaluate the adhesion strength of several relevant marine fouling organisms.
Bacterial Biofilm Adhesion
Two primary screening assays are utilized to rapidly determine the adhesion strength of marine bacterial biofilms to coating cast in multi-well plates.
Biofilm Retention and Retraction
A bacterial biofilm retention and retraction assay was developed to quickly obtain a first approximation of fouling-release performance of coating arrays. The high-throughput assay relies on the measurement of percent surface coverage from plate images, after crystal violet staining, as means to compare coating performance. The difference in percent surface coverage among fouling-release coating candidates is attributed to a “biofilm retraction” phenomenon that can occur as the coating surfaces dry after rinsing. The biofilm retraction process is theorized to be a consequence of removal and redistribution of adherent biofilms by the de-wetting of water droplets on the hydrophobic coating surfaces. In this regard, coatings that promote a weak biofilm adhesion exhibit a high degree of biofilm retraction (i.e., low surface coverage) while coatings that facilitate strong biofilm adhesion would show little or no biofilm retraction (i.e., high surface coverage) on the coating surface.
An additional screening assay for biofilm adhesion was adapted from a water-jet methodology commonly used at ocean immersion testing sites to quickly evaluate the adhesion strength of various fouling organisms in the field. An automated and miniaturized version of the water–jet apparatus used in the field was built in-house at NDSU. The system consists of a robotic arm that is mounted on a stainless steel deck which can access coating array plates from one of three plate stacking hotels. The robotic arm inverts each well of an array plate over an offset, rotating nozzle that delivers a jet of water perpendicular to the coating surface. The duration and pressure of the water-jet are precisely controlled through a customized software program. Coating array plates are subjected to water-jet treatments after bacterial incubation to facilitate removal of adherent biofilms. The difference in biomass, before and after water jetting treatments, is determined using the same crystal violet quantification assay used in the characterization of antifouling coatings. Experiments with Halomonas pacifica (right) demonstrated the difference in fouling-release performance observed between a silicone elastomer (Silastic T2) and polyurethane coating, with respect to biofilm adhesion.
The adhesion strength of algae on coatings prepared in multi-well plates is determined with the same water-jet apparatus utilized for the rapid assessment of bacterial biofilm adhesion. However, as with the quantification of antifouling performance, a DMSO extraction and fluorescence measurement of chlorophyll is utilized to determine the biomass before and after water-jet treatments. Two species of marine algae are utilized to evaluate fouling-release performance, the macrofouling green algae, Ulva linza, and the microfouling diatom,N. incerta. Evaluations with N. incerta are carried out year-round at NDSU while evaluations with Ulva are carried out seasonally at the University of Birmingham, UK.
Barnacle Attachment and Adhesion
An adult barnacle reattachment and adhesion assay was developed in collaboration with researchers at the Duke University Marine Laboratory in Beaufort, NC. Adult barnacles are shipped monthly to NDSU on glass panels coated with a silicone elastomer and maintained year-round in an automated aquarium system. Adult barnacles are pushed off the silicone elastomer and placed on experimental coating patches prepared on array panels (right picture above). The array panels are then placed in an aquarium system for two weeks to facilitate reattachment and subsequently removed for force gauge measurements and calculation of adhesion strengths. The barnacle reattachment assay provides valuable information regarding the ease of removal of shell fouling from fouling-release coatings and serves as a nice complement to the bacterial and algal adhesion screening assays.
Mussel Attachment and Adhesion
An adult mussel attachment and adhesion assay was developed in collaboration with researchers at the Duke University Marine Laboratory in Beaufort, NC. Adult mussels are shipped monthly to NDSU and maintained year round in an automated aquarium system. Adult mussels are affixed with a threaded ‘stud’ and immobilized onto experimental coating panels using a customized template. The panels are then placed in an aquarium system for three days to facilitate attachment and subsequently removed for pull-off adhesion measurements using an fully automated device. As with the barnacle reattachment assay, the mussel attachment assay provides valuable information regarding the ease of removal of shell fouling from fouling-release coatings and serves as a nice complement to the bacterial and algal adhesion screening assays.
Examples of High-Throughput Coating Analysis
Evaluation of Silicone Coatings Containing Tethered Quaternary Ammonium Salts (QAS)
A coating library consisting of 24 unique coating compositions was prepared from silanol terminated polydimethylsiloxane (PDMS). The library was comprised of four different quaternary ammonium salts (QASs figure) with different alkyl chains attached with nitrogen (top). The QASs varied from a low to high concentration in the PDMS matrix to generate six unique coatings for each of the four QAS (bottom figure).
The rapid marine bacterial biofilm retention (C. lytica) and algal biofilm growth (N. incerta) assays were able to adequately discern differences in antifouling performance of this coating library. Leachate toxicity evaluations with each organism were utilized to determine whether the antifouling character observed for a particular coating was a consequence of surface contact activity or a result of toxic components leaching from the coating matrix. For the marine algal species, coatings that contained the QAS with an alkyl chain length of C14 (series B) showed an increase in the antifouling activity as the concentration of the QAS was increased in the PDMS matrix. In regards to the marine bacterium, the coatings that contained the QAS with a C18 alkyl chain length (series D) showed excellent antifouling activity at relatively low amounts of QAS in the PDMS matrix.
Correlations to Ocean Immersion Testing
The ability to accurately down select and identify the most promising coating candidates is essential to the overall success of a combinatorial materials research program. In regards to marine coating development, it is imperative that the high-throughput biological screening workflow demonstrates some degree of correlation to results obtained from ocean immersion testing. The establishment of such correlations enables a powerful, “real world” predictive capability that coating researchers can leverage to expedite the discovery of new marine coating technologies.
Bacterial Biofilm Retraction (NDSU) vs. Fouling and Barnacle Adhesion (FIT)
A series of nine polysiloxane fouling-release coatings was evaluated with the bacterial biofilm retraction assay at NDSU and ocean immersion testing at the Florida Institute of Technology (FIT) field testing site in Melbourne, FL. A strong correlation (r = 0.90) was established between the bacterial biofilm retraction data and the barnacle adhesion results. Good agreement was also observed (r = 0.77) between the bacterial biofilm retraction assay and mean fouling rating (i.e., total accumulation of fouling organisms). The bacterial biofilm retraction assay accurately identified the best (LMLC Oil) and worst (HMHC No Oil) performing coatings determined at FIT.
Algal Biofilm Growth (NDSU) vs. Fouling (FIT)
A series of nine antifouling silicone coatings, containing a chemically tethered biocide, was evaluated with the algal biofilm growth assay at NDSU and ocean immersion testing at FIT. A high correlation (r = 0.99) was established between the algal biofilm growth assay and the mean fouling rating. The two coating compositions that showed good antifouling performance at FIT, 3A3P and 3A6P, also exhibited good reduction in algal biofilm growth at NDSU.
Barnacle Adhesion (NDSU) vs. Barnacle Adhesion (FIT)
The reattached barnacle adhesion assay was also utilized to evaluate the series of nine polysiloxane fouling-release coatings evaluated at the FIT ocean immersion testing site. A strong correlation (r = 0.87) was established between the reattached barnacle adhesion data in the laboratory and the barnacle adhesion results obtained in the field. As with the bacterial biofilm retraction assay, the reattached barnacle adhesion assay accurately identified the best and worst performing coatings determined at FIT.