Method development for enhanced antifouling testing using novel natural products against marine biofilms

Abstract

Marine biofouling is the accumulation of organisms on underwater surfaces, causing increased ship hydrodynamic drag, which results in higher fuel consumption and decreased speed and range. Biofilms constitute a major component of the overall biofouling and may lead to a 14 % increase in ship fuel costs. Past solutions to antifouling (AF) have used toxic coatings which have subsequently been shown to severely affect marine life. The prohibited use of these antifoulants has led to the search for bio-inspired AF strategies. Current approaches towards the production of alternative coatings include the incorporation of natural AF compounds into paints.

Screening assays for novel AF compounds are often separated into two categories; toxicity and AF assays. Increasingly there is evidence that active compounds affect organisms at non-toxic concentrations, hence, the necessity for more insightful AF testing, such as bacterial and diatom attachment. This study assessed natural product (NP) antifouling performance of two marine seaweeds (Chondrus crispus and Bifurcaria bifurcata) and two isolated pure compounds from terrestrial sources (usnic acid and juglone) against two marine biofilm bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Overall it was found that all NPs affected bacterial attachment, however, juglone demonstrated the best AF performance against both bacterial species at a concentration range between 5 - 20 ppm.

Biofilm colonisation is a surface related phenomenon, thus novel bioassays have been developed to directly test biofilm attachment and growth on NP-containing coatings for both static and hydrodynamic conditions. This study has incorporated NPs into a model coating system, using two formulations in order to assess their effect on biofilm growth. Laboratory screening of NP-containing coatings is often largely unexplored mainly due to difficulties in assessing their activity over short experimental time scales (typically only a maximum of a few days). To date there are only a limited number of reports on laboratory assessment for antifouling paints and their effect on biofilm growth and/or attachment. In this study, NP-containing model paints were applied on to coupons, placed in 24-well plates and then inoculated with the marine biofilm forming bacteria. This has been achieved by the development of a novel bioassay protocol that has allowed the in situ observation of biofilm formation and growth, by corroborating different techniques such as a multidetection microplate reader and confocal laser scanning microscopy (through nucleic acid staining). There was good correlation between the two techniques which showed that the NP containing coatings significantly inhibited biofilm growth and also revealed marked differences in biofilm structure (e.g. bio-volume, morphology and thickness). The goal of this study was to develop a new protocol to allow assessment of biofilm formation on coatings in a high throughput non-invasive manner.

New protocols and methods using microfluidic devices were developed for the assessment of bacterial attachments and initial biofilm formation in the presence and absence of a NP under hydrodynamic conditions. This led to the development and fabrication of a novel lab-on-a-chip device for the investigation of the biofilm response to different hydrodynamic conditions. The microfluidic flow channels were designed using computational fluid dynamic simulations so as to have a pre-defined, homogeneous wall shear stress in the channels, ranging from 0.03 to 4.30 Pa, which are relevant to in-service conditions on a ship hull.

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Identifiers

ORCID for J.A. Wharton: orcid.org/0000-0002-3439-017X

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