Bacterial biofilms on microplastics in the Baltic Sea – Composition, influences, and interactions with their environment kumulative Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Rostock vorgelegt von Katharina Kesy, geb. am 06.11.1985 in Berlin aus Rostock Rostock, 17.09.2019 https://doi.org/10.18453/rosdok_id00002636 Gutachter: Prof. Dr. Matthias Labrenz, Sektion Biologische Meereskunde, Leibniz-Institut für Ostseeforschug Warnemünde Assist. Prof. Dr. Melissa Duhaime, Department of Computational Medicine and Bioinformatics, University of Michigan, USA Jahr der Einreichung: 2019 Jahr der Verteidigung: 2020 Table of contents i Table of contents Summary/Zusammenfassung ............................................................................................. 1 General introduction ........................................................................................................... 6 Biofilms, their formation, and influential factors ............................................................. 6 The ecological importance of biofilms in aquatic systems ............................................... 8 Microplastics in aquatic environments: a newly available habitat for surface associated microorganisms and possible vector for potential pathogens ........................................... 9 Description of research aims ............................................................................................ 15 Summary of published papers .......................................................................................... 18 General discussion ............................................................................................................. 19 The vector potential of microplastics for putative pathogenic bacteria .......................... 19 Microplastics provide a habitat for opportunistic biofilm-forming bacteria .................. 28 Microplastic-specific colonisation is limited to bacteria specialised in the attachment to, and potential degradation of, organic surfaces ........................................ 32 Microplastics have the potential to alter pelagic bacterial communities ........................ 33 Conclusion & outlook ........................................................................................................ 34 Chapter I: Polystyrene influences bacterial assemblages in Arenicola marina-populated aquatic environments in vitro ......................................................................................................... 37 Abstract ........................................................................................................................... 38 1.1. Introduction ........................................................................................................... 38 1.1.1. Marine plastic pollution and its environmental implications ............................ 38 1.1.2. Microplastics as a substrate for marine microbial assemblages........................ 39 1.2. Material & methods .............................................................................................. 41 1.2.1. Collection of A. marina and natural sediment .................................................. 41 1.2.2. Experimental set-up and sample collection ...................................................... 41 1.2.3. Molecular analysis of bacterial assemblages .................................................... 43 1.2.3.1. DNA extraction, polymerase chain reaction (PCR) and 16S rRNA gene- fingerprinting .................................................................................................. 43 1.2.3.2. Digital processing of fingerprints and statistical analysis .............................. 44 1.2.3.3. Phylogenetic analysis ..................................................................................... 44 1.2.3.4. Quantitative PCR ............................................................................................ 45 Table of contents ii 1.3. Results ................................................................................................................... 45 1.3.1. Experimental settings ........................................................................................ 45 1.3.2. Substrate-specific bacterial assemblages in sediment and faeces at t0 .............. 45 1.3.3. Substrate-specific bacterial assemblages at t24 .................................................. 48 1.4. Discussion ............................................................................................................. 50 1.4.1. Microbial assemblages in sediment and faecal samples and the impact of A. marina .......................................................................................................... 50 1.4.1.1. High similarity of bacterial assemblages after passage through the A. marina gut .................................................................................................. 50 1.4.1.2. Low vector potential of PS for pathogens after gut passage .......................... 51 1.4.1.3. Substrate-related differences in assembly patterns occur only within sediment samples ............................................................................................ 52 1.4.2. Substrate-specific enrichment of potentially symbiotic bacteria on PS ............ 52 1.4.2.1. Amphritea sp. enrichment on PS occurs independently of A. marina ............ 52 1.4.2.2. Potential impact of PS on microbial assemblages in pelagic systems ........... 53 Chapter II: Fate and stability of polyamide-associated bacterial assemblages after their passage through the digestive tract of the blue mussel Mytilus edulis ........................................ 55 Abstract ........................................................................................................................... 56 2.1. Introduction ........................................................................................................... 57 2.1.1. Impact of M. edulis on biofilm formation on microplastics.............................. 58 2.2. Material & methods ............................................................................................... 59 2.2.1. Experimental set-up and sampling procedure ................................................... 59 2.2.2. Molecular analysis ............................................................................................ 61 2.2.2.1. DNA extraction and polymerase chain reaction (PCR) ................................. 61 2.2.2.2. 16S rRNA gene fingerprinting and diversity estimates .................................. 62 2.2.2.3. Phylogenetic 16S rRNA gene analysis and relative abundances ................... 63 2.3. Results ................................................................................................................... 63 2.3.1. Diversity and composition of the bacterial assemblages after passage through the digestive tract ................................................................................ 64 2.3.2. Stability of the microbial assemblages after particle egestion and subsequent incubation in seawater ...................................................................................... 66 2.4. Discussion ............................................................................................................. 67 2.4.1. Highly similar bacterial assemblages on egested polyamide and chitin particles ................................................................................................... 68 2.4.2. Limited vector function of polyamide for bacterial biofilms ............................ 69 Table of contents iii 2.5. Conclusions ........................................................................................................... 71 Chapter III: Spatial environmental heterogeneity determines young biofilm assemblages on microplastics in Baltic Sea mesocosms ............................................................................ 72 Abstract ........................................................................................................................... 73 3.1. Introduction ........................................................................................................... 74 3.2. Material and methods............................................................................................ 77 3.2.1. Sampling campaign and incubation experiments .............................................. 77 3.2.2. DNA extraction and 16S rRNA-gene amplicon sequencing ............................ 79 3.2.3. Sequence processing ......................................................................................... 79 3.2.4. Chao1 richness and species turnover ................................................................ 80 3.2.5. Relative abundances of the most abundant bacterial classes ............................ 81 3.2.6. Plastic-specific bacteria ..................................................................................... 81 3.2.7. Vibrio spp. relative abundances ........................................................................ 81 3.2.8. Multiple regression analysis of factors influencing bacterial assemblages ...... 82 3.3. Results ................................................................................................................... 83 3.3.1. Physico-chemical parameters of the stations and inorganic nutrient concentrations over