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Free University of Brussels FACULTEIT WETENSCHAPPEN VAKGROEP TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN DIENST MICROBIËLE INTERACTIES SCK•CEN DEPARTEMENT RADIOPROTECTIE LABORATORIUM VOOR MICROBIOLOGIE EN RADIOBIOLOGIE Academiejaar: 2004-2005 Microbial characterization of the nitrifying packed-bed pilot reactor in the Micro-Ecological Life Support System Alternative (MELiSSA) Promotor: Prof. Dr. P. Cornelis Scriptie voorgedragen tot het behalen van de graad Licentiaat in de Biologie Co-promotor: Dr. Ir. L. Hendrickx Benny PYCKE FACULTEIT WETENSCHAPPEN VAKGROEP TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN DIENST MICROBIËLE INTERACTIES SCK•CEN DEPARTEMENT RADIOPROTECTIE LABORATORIUM VOOR MICROBIOLOGIE EN RADIOBIOLOGIE Academiejaar: 2004-2005 Microbial characterization of the nitrifying packed-bed pilot reactor in the Micro-Ecological Life Support System Alternative (MELiSSA) Promotor: Prof. Dr. P. Cornelis Scriptie voorgedragen tot het behalen van de graad Licentiaat in de Biologie Co-promotor: Dr. Ir. L. Hendrickx Benny PYCKE 2 Acknowledgements First, I would like to thank my promoter, Prof. Dr. Pierre Cornelis, and Prof. Dr. Max Mergeay for allowing me to work at SCK•CEN, and for the advice that they have given me over the year. All my appreciation goes out to my mentor, Dr. Ir. Larissa Hendrickx, whom I would like to thank for all the assistance and advice during my stay at SCK•CEN, and for helping me master what there was to learn in a microbiological laboratory. I also owe many thanks to the researchers at the Free University of Barcelona for lending their support to this research. I would also like to thank everyone in the SCK•CEN Radiobiology and Microbiology laboratory (Annik, Fabienne, Felice, Greet, Hanane, Ilse, Iris, Jasmine, Joris, Joyce, Louis, Marcella, Mieke, Monica, Natalie, Nicolas, Patrick, Paul & Paul, Rafi, Sarah, Sébastien & Sébastien, and Werner) and the VITO Biology laboratory (An, Annemie, Leni, Karolien, and Winnie) for the pleasant work atmosphere and providing assistance when I was in need, and especially Arlette for all the help with and advice about several molecular techniques. In addition, I would like to thank everybody who lives at the dormitory and made my stay a pleasant one, Aurélie, James, Ruth, Sandor, etc. Finally, I owe my thanks to my parents and girlfriend for supporting me during this whole period. Benny. 3 Table of contents LIST OF FIGURES ............................................................................................................................................... 7 LIST OF TABLES.................................................................................................................................................. 9 ABSTRACT .......................................................................................................................................................... 10 SAMENVATTING ............................................................................................................................................... 11 ABBREVIATIONS............................................................................................................................................... 12 A. INTRODUCTION............................................................................................................................................ 14 B. STATE OF KNOWLEDGE............................................................................................................................. 16 PART I: LIFE SUPPORT .................................................................................................................................. 16 1. LIFE SUPPORT SYSTEMS ................................................................................................................................. 16 1.1. General introduction............................................................................................................................. 16 1.2. Micro-Ecological Life-Support System Alternative (MELiSSA) ........................................................... 16 PART II: NITROGEN RECYCLING IN NATURAL AND ENGINEERED SYSTEMS............................ 20 1. NITROGEN AND MICROORGANISMS................................................................................................................ 20 1.1. Introduction .......................................................................................................................................... 20 1.2. Nitrogen cycle....................................................................................................................................... 20 1.3. Aerobic nitrification.............................................................................................................................. 24 1.3.1. Autotrophic nitrification................................................................................................................ 24 1.3.2. Classification of autotrophic nitrifying bacteria............................................................................ 24 1.3.3. The sensitiveness of the AMO enzyme......................................................................................... 26 1.3.4. Heterotrophic nitrification............................................................................................................. 26 2. NITROGEN RECYCLING IN BIOREACTOR CIII ................................................................................................... 28 2.1. Nitrosomonas europaea ATCC 19718 .................................................................................................. 28 2.2. Nitrobacter winogradskyi Agilis ATCC 25391 ..................................................................................... 30 PART III: THE BACTERIAL COMMUNITY OF NITRIFYING REACTORS ......................................... 33 1. BACTERIAL COMMUNITIES IN ENGINEERED ENVIRONMENTS.......................................................................... 33 1.1. General introduction............................................................................................................................. 33 1.2. Bacterial communities in nitrifying reactors and wastewater treatment plants.................................... 34 1.2.1. Community composition in nitrifying reactors and wastewater treatment plants ......................... 34 1.2.2. Spatial patterns in nitryifying reactors .......................................................................................... 36 2. ANALYSIS OF BACTERIAL COMMUNITIES ....................................................................................................... 37 2.1. Approaches to bacterial community analysis........................................................................................ 37 2.2. Analysis by means of DGGE................................................................................................................. 38 PART IV: HORIZONTAL GENE TRANSFER .............................................................................................. 40 1. BACTERIAL CONJUGATION............................................................................................................................. 40 1.1. Mechanisms of Horizontal Gene Transfer (HGT)................................................................................. 40 1.2. Bacterial conjugation............................................................................................................................ 41 1.2.1. Properties and implications ........................................................................................................... 41 1.2.2. Requirements for bacterial conjugation......................................................................................... 42 1.2.3. Plasmids ........................................................................................................................................ 44 2. EXOGENOUS PLASMID ISOLATION .................................................................................................................. 46 C. OBJECTIVES.................................................................................................................................................. 48 D. MATERIAL AND METHODS ....................................................................................................................... 49 1. BACTERIAL STRAINS, CULTURE CONDITIONS, BACTERIAL MEDIA, AND ANTIBIOTICS ..................................... 49 1.1. Bacterial strains.................................................................................................................................... 49 1.2. Plasmids................................................................................................................................................ 49 4 1.3. Growth conditions................................................................................................................................. 50 1.4. Bacterial media..................................................................................................................................... 50 1.4.1. 1-2-3 medium................................................................................................................................ 50 1.4.2. 284-medium .................................................................................................................................. 51 1.4.3. 869-medium .................................................................................................................................
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