Interactions between Diatoms and Bacteria from phototrophic Biofilms of the littoral Zone of Lake Constance Dissertation zur Erlangung des Doktorgrades der Mathematisch – Naturwissenschaftlichen Sektion, Fachbereich Biologie, der Universität Konstanz vorgelegt von Christian G. Bruckner, Konstanz 2008 Tag der mündlichen Prüfung: 19. Januar 2009 1. Referent: Prof. Dr. Peter Kroth 2. Referentin: Prof. Dr. Iwona Adamska “…we live in the Age of Bacteria. As it was in the beginning, is now and ever shall be, until the world ends…” Stephen Jay Gould in: “The Spread of Excellence from Plato to Darwin” 2 Contents General Introduction................................................................................................................5 1 Protocols for the Removal of Bacteria from benthic Diatom Cultures..........................................................................................16 Abstract.........................................................................................................................17 Introduction...................................................................................................................17 Methods to verify the axenic state of a culture.............................................................18 Generation of axenic diatom strains directly from natural biofilms.............................................................................................................19 Generation of axenic diatom strains from xenic cultures: ultrasound, heavy antibiotic treatment and “single cell care”.....................................................................................................21 Generation of axenic diatom strains from xenic cultures: antibiotic treatment and co-cultivation with Escherichia coli.............................................................................22 Generation of axenic diatom strains from xenic cultures: dissolution of EPS, differential centrifugation and moderate antibiotic treatment..........................................................25 Summary and Discussion..............................................................................................26 Acknowledgements.......................................................................................................28 2 Bacteria associated with benthic Diatoms from Lake Constance: Phylogeny and Influences on Diatom Growth and EPS Secretion..............................................................................29 Abstract.........................................................................................................................30 Introduction...................................................................................................................31 Materials and Methods..................................................................................................33 Results...........................................................................................................................38 Discussion.....................................................................................................................48 Acknowledgements.......................................................................................................53 3 3 Bacterial Influences on Growth and Carbohydrate Secretion of representative epilithic Diatoms and Correlations with dissolved free Amino Acids.................................................................54 Abstract.........................................................................................................................55 Introduction...................................................................................................................56 Materials and Methods..................................................................................................58 Results...........................................................................................................................60 Discussion.....................................................................................................................74 Acknowledgements.......................................................................................................80 4 Analysis of the extracellular Metaproteome of Diatoms and Bacteria in Co-Cultures indicate characteristic functional Interactions...............................................................................81 Abstract.........................................................................................................................82 Introduction...................................................................................................................82 Materials and Methods..................................................................................................85 Results...........................................................................................................................87 Discussion.....................................................................................................................92 Acknowledgements.......................................................................................................98 General Discussion..................................................................................................................99 Summary................................................................................................................................108 Zusammenfassung.................................................................................................................109 References..............................................................................................................................110 Contributions.........................................................................................................................127 General Acknowledgments...................................................................................................128 4 General Introduction General Introduction Biofilms “The importance of microorganisms in human health and disease, and the massive impact of the pure-culture approach devised by Robert Koch and others, has understandably led to a philosophy in microbiological research that emphasizes the study of microorganisms in pure liquid culture. This approach has so prominently pervaded microbiology that biofilm research was long neglected until microbiologists “re-discovered” these fascinating communities almost 40 years ago” (Battin et al., 2007). Exaggerated one could regard the planktonic phase of microorganisms just as a transport mechanism for translocation between surfaces (Watnick & Kolter, 2000) where they can settle to form biofilms. In fact, “most surfaces on this planet teem with microbial life, creating ecosystems of diverse organisms that flourish in slimy beds of their own making” (Kolter & Greenberg, 2006). Biofilms are very complex communities often exhibiting a high phenotypic plurality regarding substrate and nutrient utilization, production of extracellular polymeric substances (EPS) and cell/cell communication, resulting in surprisingly coordinated multicellular behavior, even perceived as “city of microbes” (Watnick & Kolter, 2000). Integrating opportunistic individuals in coordinated units is assumed to be mediated by quorum sensing (Dunlap, 1997; Fuqua et al., 1996; Ruby, 1996). Being an inhabitant of such “biofilm-cities” is often advantageous, e.g. biofilm cells are more resistant against changes in salinity and temperature, UV radiation, desiccation or toxins and detergents (Decho, 2000; Costeron et al., 1987). 6 General Introduction Epilithic phototrophic biofilms are based on interactions between the primary producers (algae and cyanobacteria) and bacteria, fungi, protozoa, insects, larvae, shellfish etc. (Makk et al., 2003). For Lake Constance, a large mesotrophic, warm-monomictic lake in central Europe, phototrophic organisms in biofilms on stones, sand, mud and other particles in the littoral zone, are dominated by diatoms (Bahulikar, unpublished data, Fig.1). A B C Fig 1: A: Diatom dominated biofilms in the littoral zone of Lake Constance; B: Such biofilms can become several millimeters thick; C: Microscopy reveals a high biodiversity in such biofilms; Single microbial strains or even individual cells are thought to fill distinct niches within the biofilm, possibly regulated by a complicated “conversation” based on many different soluble signals (Watnick & Kolter, 2000) forming a metacommunity (Battin et al., 2007). In Lake Constance biofilms (Fig.1) are widely distributed, occupying to our observations several square kilometers. These biofilms are exposed to a spatial heterogeneity regarding substrates like rocks, wood, sand, mud, macrophytes, changing water levels, atmospheric pressure, light intensities, nutrient availability etc., thus forming itself an entity that can be regarded as a landscape (Battin et al., 2007). 7 General Introduction Besides the living organisms, EPS and trapped water are main components of the biofilms, which are thought to act as a kind of glue sticking particles together, thus stabilizing whole sediments (Stal & Brouwer, 2003; Wigglesworth-Cooksey et al, 2001; Decho, 2000; Sutherland & Grant, 1998). It is assumed that biofilm formation is initiated by the adsorption of organic and inorganic ions to a surface, followed by the settlement of bacteria which again serve as a substrate for the attachment of eukaryotic algae