Abstract
Biofilms are assemblages of microbial cells irreversibly attached to artificial or living surfaces, embedded in a self-produced matrix of hydrated extracellular polymeric substances (EPS) with beneficial or detrimental implications to anthropogenic activities. Marine biofilms, commonly dominated by marine bacteria and diatoms, are ubiquitous in the marine environment and constitute an essential component of biofouling on all immersed surfaces. Biofouling is the undesirable accumulation of marine organisms, such as invertebrates and algae, on submerged surfaces. Biofouling constitutes a considerable impediment in marine applications imposing serious environmental implications and economic penalties to human activities, especially in the shipping industry. In order to combat biofouling, technologies for antifouling control have been developed based on the application of protective paints on vessel hulls. Modern antifouling coatings are divided into biocidal and non-toxic fouling-release technologies. However, to date the development of a successful antifouling coating that is simultaneously effective against biofouling and environmentally friendly, is very challenging. Moreover, the successful performance of a coating is influenced by the activity of the pioneering surface colonisers, i.e. marine biofilms.The central focus of this PhD thesis is to provide better understanding of the marine biofilm components formed on artificial surfaces, specifically antifouling coatings. The first aim of this work was to investigate the in-situ marine biofilm community on different commercial antifouling coatings using 16S rRNA gene amplicon and metatranscriptomic total RNA sequencing. In addition, the second aim was to explore for the first time the gene expression profile of these communities in the marine environment and screen for metal resistance genes using metatranscriptomic (mRNA) sequencing. The last aim of this thesis was to establish a proof-of- concept for successful phototrophic biofilm quantification based on high-throughput, non- destructive and rapid chlorophyll fluorescence measurements using microplate reader technology.
The results showed that biocidal and fouling-release coatings exhibited distinct biofilm communities as demonstrated by both taxonomic approaches. The 16S rRNA gene-based taxonomic profiling demonstrated that biocidal coating had the lowest diversity, highest abundance and was characterised by abundant phyla belonging to the Proteobacteria, Bacteroidetes, Planctomycetes and Verrucomicrobia. The fouling release coating was characterised by the dominant phyla of Proteobacteria, Bacteroidetes, Actinobacteria and Planctomycetes. The total RNA-based taxonomic profiling demonstrated that biocidal coating had the lowest diversity and abundance and was characterised by the dominant phyla belonging to Ochrophyta, Cyanobacteria, Ciliophora and Phragmoplastophyta. The fouling release coating had the highest abundance characterised by the dominant phyla Cyanobacteria, Ochrophyta, Jakobida, Phragmoplastophyta and Ciliophora. The genera Cylindrotheca, Acineta and Nicotiana were abundant in all samples. Both taxonomic profiling analyses generated the bacterial abundant phyla of Proteobacteria, Bacteroidetes, Cyanobacteria and Planctomycetes. New taxa of protists, ciliates and fungi were recorded using RNA-Seq.
The functional profile generated with mRNA sequencing revealed distinct gene expression profiles between the biocidal and fouling release coatings. Biofilms recovered from the biocidal coating were characterised by upregulated genes for translocation, chloramphenicol resistance, stimulation of GTPase and genes regulating metal defense mechanisms, such as SodA, CueO, yfjQ, ymfF, Mco, actP, znuC, tldD, mdtC, copB. The fouling-release coating displayed genes associated with coper resistance (copA) and zinc-binding (fdh). Genes encoding biofilm regulation and formation were enriched across all samples, as well as MntH divalent metal cation transporter gene.
A novel method for phototrophic biofilm quantification was developed. A proof-of-concept method based on the intrinsic chlorophyll of autofluorescent phototrophic cells was successfully confirmed using in vitro monoculture biofilms in the lab and in situ mixed biofilms in the marine environment. The method introduces a non-destructive, rapid and reproducible quantification using microplate reader technology in lieu of labour-intensive and prone to bias microscopy.
The results of the present work are expected to have important implications on the design of novel and robust antifouling coatings. The new findings illuminated some essential mechanisms characterising the overwhelming diversity and unexplored functionality of marine biofilms, that allow them to contribute successfully to biofouling and adapt to toxic conditions created by biocidal surfaces. The insights and developed method provided here will inform the antifouling industry and support the optimization of the antimicrobial-antibiofilm performance of antifouling coatings with more environmentally sustainable solutions.
| Date of Award | 23 Nov 2020 |
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| Original language | English |
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| Supervisor | Maria Salta (Supervisor), Sam Robson (Supervisor), John Tsibouklis (Supervisor) & Joy Watts (Supervisor) |