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Distributions of model microorganisms along an estuarine gradient By Franck Carbonero A thesis submitted for the degree of Doctor of Philosophy Department of Biological Sciences University of Warwick June 2010 Table of contents LIST OF FIGURES ...........................................................................................................................IV LIST OF TABLES .............................................................................................................................VI ACKNOWLEDGMENTS ............................................................................................................... VII ABSTRACT........................................................................................................................................IX ABBREVIATIONS ..............................................................................................................................X CHAPTER 1 INTRODUCTION ........................................................................................................ 1 1.1 MICROBIAL ECOLOGY EARLY HISTORY......................................................................................... 1 1.1.1. Microbiology history and development of microbial ecology............................................. 1 1.1.2. The discovery of new microbial metabolisms ..................................................................... 1 1.1.3. Examples of ecological principles illustrated by microorganisms ..................................... 3 1.2. RECENT ADVANCES AND DRIFT FROM “TRADITIONAL ECOLOGY”................................................ 4 1.2.1. The revolution of molecular ecology .................................................................................. 4 1.2.1.1. Basic methods............................................................................................................................. 5 1.2.1.2. Fingerprinting methods............................................................................................................... 5 1.2.1.3. Phylogenetic methods ................................................................................................................. 6 1.2.1.4. Function and activity based methods .......................................................................................... 8 1.2.2. Limits of molecular assessment in an ecological context ................................................... 9 1.3. INTEGRATING MICROBIAL ECOLOGY INTO GENERAL ECOLOGY?................................................ 10 1.3.1. Environmental microbiology or microbial ecology?........................................................ 10 1.3.2. General principles in ecology........................................................................................... 12 1.3.3. Microorganisms versus macroorganisms......................................................................... 15 1.3.4. A need to fully integrate microbial ecology to general ecology ....................................... 16 1.4. DISTRIBUTION PATTERNS AND BIOGEOGRAPHY IN THE MICROBIAL WORLD............................... 17 1.4.1. Marine microbial ecology................................................................................................. 17 1.4.2. Freshwater microbial ecology.......................................................................................... 20 1.4.3. Terrestrial microbial ecology ........................................................................................... 21 1.4.4. Other environments .......................................................................................................... 22 1.5. HYPOTHESES ON FUNCTIONAL AND GENETIC DIVERSITY ROLE .................................................. 24 1.6. CONTEXT OF THIS WORK ........................................................................................................... 25 1.6.1. Estuarine microbiology .................................................................................................... 25 1.6.2. The Colne estuary............................................................................................................. 26 1.6.3. Microbial activities on the Colne estuary......................................................................... 27 1.6.4. The principal model, Methanosaeta ................................................................................. 29 1.6.5. The other models, Desulfobulbus, Desulfobacter, Methanosarcina ................................. 31 1.6.5.1. Desulfobulbus ........................................................................................................................... 31 1.6.5.2. Desulfobacter............................................................................................................................ 32 1.6.5.3. Methanosarcina ........................................................................................................................ 33 1.6.6. Experimental design ......................................................................................................... 34 CHAPTER 2 MATERIAL AND METHODS ................................................................................. 36 2.1. SAMPLING ................................................................................................................................. 36 2.1.1. Sediment sampling and conditioning................................................................................ 36 2.1.2. Pore water analysis .......................................................................................................... 37 2.2. METHANOSAETA ENRICHMENT CULTURES .................................................................................. 38 2.2.1. Anaerobic media preparation and techniques.................................................................. 38 2.2.2. Other chemicals and solutions.......................................................................................... 38 2.2.3. Methanosaeta enrichment method.................................................................................... 39 2.2.4. Methanosaeta development check methods ...................................................................... 41 2.2.4.1. Fluorescent in situ Hybridisation .............................................................................................. 41 2.2.4.2. Gas chromatography ................................................................................................................. 41 2.2.4.3. PCR amplification..................................................................................................................... 41 PCR using protocols and primers (bacterial, archaeal and Methanosaeta-specific) described in section 2.5 have been attempted on cell suspensions from cultures, or DNA extracted using the DNeasy Blood & Tissue Kit........................................................................................................................................... 41 2.3. METHANOSAETA ISOLATION ....................................................................................................... 42 I 2.3.1. Solid media ....................................................................................................................... 42 2.3.2. Purity controls .................................................................................................................. 42 2.4. NUCLEIC ACIDS EXTRACTION .................................................................................................... 42 2.4.1. Separate extraction of DNA and RNA .............................................................................. 42 2.4.2. PEG 6000 purification...................................................................................................... 43 2.4.3. DNA and RNA analyses.................................................................................................... 43 2.4.3.1. Spectrophotometry analyses...................................................................................................... 43 2.4.3.2. Agarose gel electrophoresis ...................................................................................................... 43 2.5. NUCLEIC ACIDS AMPLIFICATION ................................................................................................ 44 2.5.1. Genera-specific primers ................................................................................................... 44 2.5.1.1. Methanosaeta............................................................................................................................ 44 2.5.1.2. Desulfobulbus and Desulfobacter ............................................................................................. 44 2.5.2. PCR protocols and nested approaches............................................................................. 45 2.5.2.1. PCR protocols........................................................................................................................... 45 2.5.2.2.