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Methanoloxidation in Oxischen Böden Und Umweltparameter Assoziierter Methylotropher Mikroorganismen- Gemeinschaften

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Methanoloxidation in Oxischen Böden Und Umweltparameter Assoziierter Methylotropher Mikroorganismen- Gemeinschaften Methanoloxidation in oxischen Böden und Umweltparameter assoziierter methylotropher Mikroorganismen- Gemeinschaften Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften Dr. rer. nat. der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth vorgelegt von Astrid Stacheter Bayreuth, Juni 2013 Die vorliegende Arbeit wurde von Oktober 2008 bis Juni 2013 am Lehrstuhl Ökologische Mikrobiologie (Universität Bayreuth) unter der Anleitung von PD Dr. Steffen Kolb angefertigt. Die Arbeit wurde aus Mitteln der Deutschen Forschungsgemeinschaft (Fördernummer: DFG Dr310/5-1) und der Universität Bayreuth finanziert. Teile der Ergebnisse dieser Arbeit wurden als Artikel in einer wissenschaftlichen Zeitschrift veröffentlich: Stacheter, A., Noll, M., Lee, C. K., Selzer, M., Glowik, B., Ebertsch, L., Mertel, R., Schulz, D., Lampert, N., Drake, H. L., Kolb, S. (2012) Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs. ISME J. Online verfügbar. doi: 10.1038/ismej.2012.167. Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.). Dissertation eingereicht am: 04.06.2013 Zulassung durch die Prüfungskommission: 12.06.2013 Wissenschaftliches Kolloquium: 09.12.2013 Amtierender Dekan: Prof. Dr. Rhett Kempe Prüfungsausschuss: PD Dr. Steffen Kolb (Erstgutachter) Prof. Dr. Ortwin Meyer (Zweitgutachter) Prof. Dr. Bettina Engelbrecht (Vorsitz) Prof. Dr. Heike Feldhaar PD Dr. Werner Borken Inhaltsverzeichnis I INHALTSVERZEICHNIS ABBILDUNGSVERZEICHNIS ............................................................................................... V TABELLENVERZEICHNIS .................................................................................................. VII VERZEICHNIS DER GLEICHUNGEN ................................................................................... X VERZEICHNIS DER BEISPIELRECHNUNGEN .................................................................... X ABKÜRZUNGSVERZEICHNIS ............................................................................................. XI 1 EINLEITUNG ...................................................................................................................... 1 1.1 Die „Virtuosität" der Methylotrophen ............................................................................. 1 1.2 Die Bedeutung von Methanol für die globale Atmosphärenchemie .............................. 2 1.3 Der globale Methanolhaushalt...................................................................................... 4 1.4 Methanoloxidation im Boden ........................................................................................ 7 1.5 Die Vielfalt der Methylotrophen .................................................................................... 9 1.6 Methanol-abhängiger Metabolismus methylotropher Bacteria .....................................14 1.6.1 Oxidation von Methanol zu Formaldehyd ..............................................................15 1.6.2 Oxidation von Formaldehyd zu CO2 ......................................................................16 1.6.3 Kohlenstoffassimilation .........................................................................................19 1.7 Ökologische Nischen methylotropher Prokaryoten ......................................................19 1.7.1 Substrate ..............................................................................................................20 1.7.2 Sauerstoff .............................................................................................................21 1.7.3 Stickstoff ...............................................................................................................21 1.7.4 pH-Wert ................................................................................................................22 1.7.5 Temperatur ...........................................................................................................23 1.7.6 Salzkonzentration .................................................................................................23 1.7.7 Prädatoren ...........................................................................................................24 1.7.8 Vegetationstyp und Landnutzung .........................................................................24 1.8 Funktionelle Genmarker der Methylotrophen ..............................................................25 1.9 Ziele und Hypothesen .................................................................................................28 2 MATERIAL UND METHODEN ..........................................................................................29 2.1 Standortbeschreibung .................................................................................................29 2.2 Probenahme ...............................................................................................................32 2.3 Bestimmen von Bodenparametern ..............................................................................33 2.4 Verwendete Chemikalien und Gase ............................................................................34 2.5 Sterilisation .................................................................................................................34 II Inhaltsverzeichnis 2.6 Methanoloxidation .......................................................................................................34 2.6.1 Messung der Oxidation von Methanol zu CO2 ......................................................34 2.6.2 Methanoloxidation in Bodenaufschlämmungen und durch Pflanzenmaterial .........37 2.6.3 Anzucht von Arabidopsis thaliana .........................................................................37 2.6.4 Apparente Michaelis-Menten-Kinetiken ................................................................39 2.6.5 Wiederfindung der Radioaktivität ..........................................................................41 2.7 Kultivierung von Isolaten und Bestimmung der Zellzahlen ..........................................42 2.7.1 Kultivierungsmedien .............................................................................................42 2.7.2 Isolierung von Reinkulturen ..................................................................................46 2.7.3 Bestimmung der Zellzahlen mittels MPN-Methode ...............................................46 2.8 Molekularbiologische Methoden ..................................................................................48 2.8.1 DNA-Extraktion nach Stralis-Pavese ....................................................................49 2.8.2 Nukleinsäure-Extraktion nach Griffiths ..................................................................50 2.8.3 Bestimmung der Nukleinsäurekonzentration .........................................................52 2.8.3.1 Fluoreszenzbasierte Bestimmung der DNA-Konzentration .............................52 2.8.3.2 Spektrophotometrische Bestimmung der Nukleinsäurekonzentration .............52 2.8.4 Polymerase-Kettenreaktion ..................................................................................52 2.8.4.1 PCR-Protokolle ..............................................................................................53 2.8.4.2 Optimierung der Amplifikation von mxaF ........................................................56 2.8.5 Agarose-Gelelektrophorese ..................................................................................59 2.8.6 Reinigung der PCR-Produkte von mxaF, mch und fae .........................................59 2.8.7 TRFLP-Analyse zur Identifizierung von Methanol-induzierten Genotypen ............60 2.8.8 Taxonomische Einordnung der Isolate ..................................................................61 2.8.9 Analyse der funktionellen Genmarker mxaF, mch und fae mittels Amplikon- Pyrosequenzierung ...............................................................................................62 2.8.9.1 Amplikon-Pyrosequenzierung ........................................................................62 2.8.9.2 Bereinigung des Sequenzdatensatzes ...........................................................64 2.8.9.3 Berechnung von Cut-Off-Werten zur Differenzierung von OTUs ....................65 2.8.9.3.1 Berechnung eines Cut-Off-Wertes zur Differenzierung von OTUs auf Speziesebene ..........................................................................................65 2.8.9.3.2 Berechnung eines Datensatz-basierten Cut-Off-Wertes zur Differenzierung von OTUs .......................................................................68 2.9 Erstellen von Stammbäumen ......................................................................................68 2.10 Deskriptive Statistik ...................................................................................................68 2.10.1 Mittelwert und Standardabweichung ...................................................................68 2.10.2 Abschätzung des Stichprobenumfangs ...............................................................69 2.10.3 Abschätzung der Anzahl der OTUs ....................................................................69 2.10.4 Artendiversität ....................................................................................................70 2.11
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