Anaerobic degradation of steroid hormones by novel denitrifying bacteria Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch- Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologe Michael Fahrbach aus Bad Mergentheim (Baden-Württemberg) Berichter: Professor Dr. Juliane Hollender Professor Dr. Andreas Schäffer Tag der mündlichen Prüfung: 12. Dezember 2006 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Table of Contents 1 Introduction.....................................................................................................................1 1.1 General information on steroids ...............................................................................1 1.2 Steroid hormones in the environment.......................................................................2 1.2.1 Natural and anthropogenic sources and deposits ............................................2 1.2.2 Potential impact on the environment ................................................................3 1.2.3 Fate of steroid hormones..................................................................................4 1.3 Microbial degradation of steroid hormones and sterols............................................5 1.3.1 Aerobic degradation..........................................................................................5 1.3.2 Anaerobic degradation......................................................................................7 1.4 Aim of dissertation ....................................................................................................9 2 Materials and Methods.................................................................................................11 2.1 Microbiological methods .........................................................................................11 2.1.1 Sources of bacteria.........................................................................................11 2.1.2 Media and growth conditions ..........................................................................11 2.1.3 Anoxic cultivation techniques..........................................................................13 2.1.4 Isolation, purity control and maintenance .......................................................14 2.1.5 Characterization of isolated bacteria ..............................................................14 2.1.6 Phylogenetic analysis .....................................................................................15 2.1.7 MPN dilution series.........................................................................................15 2.1.8 Quantification of steroid hormone oxidation and denitrification ......................16 2.1.9 Degradation time course.................................................................................16 2.2 Biochemical methods .............................................................................................17 2.2.1 Preparation of cell-free extracts for proteomics ..............................................17 2.2.2 Two-dimensional gel electrophoresis .............................................................18 2.2.3 In-gel digestion ...............................................................................................19 2.3 Analytical methods .................................................................................................19 2.3.1 Analysis of nitrite and nitrate by ion chromatography .....................................19 2.3.2 Analysis of dinitrogen monoxide and dinitrogen by gas chromatography.......20 2.3.3 Analysis of steroid hormones by HPLC ..........................................................21 2.3.4 Proteomic analysis..........................................................................................21 2.3.5 Protein determination......................................................................................22 2.3.6 Chemotaxonomy.............................................................................................23 2.4 Chemicals...............................................................................................................23 3 Results...........................................................................................................................25 3.1 Characterization of strain AcBE2-1T .......................................................................25 3.1.1 Enrichment and isolation ................................................................................25 Table of Contents 3.1.2 Physiological, cytological, and morphological properties................................26 3.1.3 Environmental significance .............................................................................28 3.1.4 Quantification of steroid hormone degradation and denitrification..................29 3.1.5 Phylogenetic analysis and chemotaxonomy...................................................30 3.2 Proteomic analysis of strain AcBE2-1T ...................................................................32 3.3 Characterization of strain FST.................................................................................40 3.3.1 Enrichment and isolation ................................................................................40 3.3.2 Physiological, cytological, and morphological properties................................41 3.3.3 Quantification of steroid hormone degradation and denitrification..................43 3.3.4 Phylogenetic analysis and chemotaxonomy...................................................46 4 Discussion ....................................................................................................................49 4.1 Description of strain AcBE2-1T as Denitratisoma oestradiolicum gen. nov., sp. nov. ……………………………………………………………………………………………..49 4.1.1 Physiology ......................................................................................................49 4.1.2 Taxonomy .......................................................................................................50 4.2 Proteomic analysis of Denitratisoma oestradiolicum AcBE2-1T..............................53 4.3 Description of strain FST as Steroidobacter denitrificans gen. nov., sp. nov. .........60 4.3.1 Physiology ......................................................................................................60 4.3.2 Taxonomy .......................................................................................................63 4.4 General discussion .................................................................................................67 5 Summary .......................................................................................................................71 6 Zusammenfassung.......................................................................................................73 7 References ....................................................................................................................75 8 Appendix .......................................................................................................................87 8.1 Phylogenetic analysis of Denitratisoma oestradiolicum..........................................87 8.2 Assimilation equations of estradiol and testosterone..............................................88 8.3 Quantification of steroid hormone degradation and denitrification..........................89 8.4 Complete datasets of time course experiments......................................................90 8.5 Enrichment cultures................................................................................................97 8.6 Proteomic analysis of Denitratisoma oestradiolicum AcBE2-1T..............................98 8.7 Table of Figures....................................................................................................111 8.8 List of Tables ........................................................................................................114 9 Acknowledgements....................................................................................................115 List of Abbreviations % (v/v) % volume per volume % (w/v) % weight per volume ARB Software for sequence data analysis ATCC American Type Culture Collection BLAST Basic Local Alignment Search Tool Casein-peptone soymeal-peptone agar. The medium is identical CASO agar with trypticase soy agar (TSA) CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate Da Dalton (molecular weight) DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DNase I Endonuclease that nonspecifically cleaves DNA Deutsche Sammlung von Mikroorganismen DSMZ und Zellkulturen GmbH DTT Dithiotreithol E1 Estrone E2 Estradiol EE2 Ethinyl estradiol ESI Electrospray ionization FAD Flavin-adenine dinucleotide FISH Fluorescence in situ hybridization G+C content of the DNA Guanine+Cytosine content of the DNA GC Gas chromatography HEPES N-[2-hydroxyethyl] piperazine-N’-[ethansulfonic acid] HPLC High performance liquid chromatography IEF Isoelectric focussing IMG system Integrated Microbial Genomes system IPG strip Immobilized pH gradient strip JCM Japan Collection of Microorganisms Log KOW Log octanol-water partition coefficient MPN Most probable number MS Mass spectrometry MS/MS Tandem mass spectrometry NA No data available NAD(P) Nicotinamide-adenine dinucleotide (phosphate) ND Not determined