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Molekularbiologische Untersuchung Der Diversität Von Mikroorganismen in Gefluteten Und Ungefluteten Pappelmikrokosmen

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Molekularbiologische Untersuchung Der Diversität Von Mikroorganismen in Gefluteten Und Ungefluteten Pappelmikrokosmen Molekularbiologische Untersuchung der Diversität von Mikroorganismen in gefluteten und ungefluteten Pappelmikrokosmen Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Andrea Graff aus Stade Marburg / Lahn 2005 Die Untersuchungen zur vorliegenden Arbeit wurden von November 2000 bis November 2005 am Max-Planck-Institut für terrestrische Mikrobiologie in Marburg/ Lahn unter der Leitung von Prof. Dr. Ralf Conrad durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: 09. November 2005 Erstgutachter: Prof. Dr. R. Conrad Zweitgutachter: Prof. Dr. E. Bremer Tag der Disputation:16. Dezember 2005 Ein Großteil der in dieser Dissertation beschriebenen Ergebnisse ist in der folgenden Publikation veröffentlicht: Graff, A. und R. Conrad. 2005. Impact of flooding on soil bacterial communities associated with poplar (Populus sp.) trees. FEMS Microbiol. Ecol. 53:401-415. „Es ist des Lernens kein Ende“. Robert Schumann Inhaltsverzeichnis Inhaltsverzeichnis ABKÜRZUNGEN................................................................................................................ 1 ZUSAMMENFASSUNG..................................................................................................... 3 I. EINLEITUNG ............................................................................................................... 5 1. Boden und Pappelpflanze als mikrobieller Lebensraum........................................... 5 2. Mikrobielle Diversität ................................................................................................... 8 3. Molekularbiologische Methoden zur Charakterisierung eines Habitats ............... 11 4. Ziele der Arbeit............................................................................................................ 13 II. MATERIAL UND METHODEN............................................................................... 14 1. Materialien ................................................................................................................... 14 1.1. Chemikalien und Gase......................................................................................... 14 1.2. Bodenmaterial und Pappelpflanzen..................................................................... 15 1.2.1. Boden........................................................................................................... 15 1.2.2. Herstellung der Pappelmikrokosmen........................................................... 16 1.2.3. Definition der Habitate und Probennahme .................................................. 17 2. Inkubationsexperiment............................................................................................... 19 2.1. Ansatz der Bodenaufschlämmungen ................................................................... 19 2.2. Probenentnahme für chemische und physikalische Analysen............................. 19 2.2.1. Gasproben.................................................................................................... 19 2.2.2. Flüssigproben .............................................................................................. 19 3. Chemische und physikalische Methoden .................................................................. 20 3.1. Gaschromatographische Analysen ...................................................................... 20 3.1.1. Messung von Methan und Kohlendioxid .................................................... 20 3.1.2. Messung von Wasserstoff............................................................................ 20 3.2. Berechnungen ...................................................................................................... 21 3.3. Analyse organischer Säuren und Alkohole ......................................................... 22 3.4. Analyse anorganischer Ionen............................................................................... 23 3.5. Kolorimetrische Bestimmung von Eisen (II) ...................................................... 24 3.6. Bestimmung des pH-Wertes................................................................................ 24 3.7. Bestimmung des C/N-Gehaltes ........................................................................... 25 Inhaltsverzeichnis 3.8. Bestimmung des Trockengewichts...................................................................... 25 4. Molekularbiologische Methoden................................................................................ 26 4.1. Extraktion von DNA............................................................................................ 26 4.1.1. Extraktion von Umwelt-DNA aus Boden und Pflanzenmaterial................. 26 4.1.2. Extraktion von DNA aus E.coli–Klonen ..................................................... 28 4.2. Reinigung der Umwelt-DNA von Huminsäuren................................................. 28 4.3. Agarosegelelektrophorese ................................................................................... 30 4.4. Konzentrationsbestimmung von DNA-Lösungen ............................................... 31 4.5. PCR (Polymerase Chain Reaction – Polymerase Kettenreaktion) ...................... 31 4.5.1. Primer .......................................................................................................... 31 4.5.2. Amplifikation der 16S rDNA ...................................................................... 32 4.6. Terminale-Restriktionsfragment-Längen-Polymorphismus-Analyse (T-RFLP-Analyse)............................................................................................... 35 4.6.1. Restriktion der 16S rDNA-Amplifikate ...................................................... 35 4.6.2. Elektrophorese............................................................................................. 36 4.6.3. Auswertung der T-RFLP-Analyse............................................................... 37 4.7. Erstellung von 16S rDNA–Klonbibliotheken ..................................................... 38 4.8. Sequenzanalyse der klonierten 16S rDNA-Fragmente........................................ 40 4.8.1. Sequenzierreaktion ...................................................................................... 40 4.8.2. Reinigung der Sequenzierreaktions-Produkte ............................................. 42 4.8.3. Analyse der Sequenzierreaktions-Produkte................................................. 42 4.9. Phylogenetische Analyse..................................................................................... 44 4.9.1. Sequenzverarbeitung ................................................................................... 44 4.9.2. Phylogenetische Einordnung der partiellen und der vollständigen 16S rRNA-Gensequenzen ........................................................................... 44 5. Statistische Methoden ................................................................................................. 46 5.1. Abschätzung des Artenreichtums ........................................................................ 46 5.2. Statistische Auswertung der T-RFLP-Ergebnisse ............................................... 47 III. ERGEBNISSE ............................................................................................................. 49 1. Molekulare Analyse der mikrobiellen Lebensgemeinschaften in gefluteten und ungefluteten Pappelmikrokosmen...................................................................... 49 1.1. T-RFLP-Analyse der bakteriellen Lebensgemeinschaften.................................. 49 Inhaltsverzeichnis 1.2. Phylogenetische Identifikation einzelner bakterieller Gruppen .......................... 54 1.3. Abschätzung des bakteriellen Artenreichtums .................................................... 62 1.4. Struktur und phylogenetische Diversität der Archaea......................................... 63 2. Inkubationsexperiment aus Pappelmikrokosmen und feldfrischen Bodenproben70 2.1. Zeitlicher Verlauf geochemischer Parameter in Bodenaufschlämmungen ......... 70 2.1.1. Elekronenakzeptor-Konzentrationen........................................................... 70 2.1.2. Methan, Kohlendioxid und Wasserstoff...................................................... 71 2.1.3. Organische Säuren und pH-Wert................................................................. 73 2.2. Mikrobielle Gemeinschaften der Bodenaufschlämmungen ................................ 75 2.2.1. Struktur und phylogenetische Diversität der Bacteria ................................ 75 2.2.2. Struktur und phylogenetische Diversität der Archaea................................. 82 IV. DISKUSSION .............................................................................................................. 89 1. Pappel und Boden als mikrobieller Lebensraum ..................................................... 89 2. Biogeochemische Prozesse in Bodenaufschlämmungen........................................... 91 3. Bakterielle Diversität .................................................................................................. 94 3.1. Bodenaufschlämmungen ..................................................................................... 94 3.2. Pappelmikrokosmen ............................................................................................ 97 4. Archaeelle Diversität in Bodenaufschlämmungen und Pappelmikrokosmen ..... 104 5.
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