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Distributions of Model Microorganisms Along an Estuarine Gradient

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Distributions of Model Microorganisms Along an Estuarine Gradient University of Warwick institutional repository: http://go.warwick.ac.uk/wrap A Thesis Submitted for the Degree of PhD at the University of Warwick http://go.warwick.ac.uk/wrap/4511 This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. 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.
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