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Charakterisierung Des Nitritoxidanten Nitrospira in Unterschiedlichen Ökosystemen

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Charakterisierung Des Nitritoxidanten Nitrospira in Unterschiedlichen Ökosystemen Charakterisierung des Nitritoxidanten Nitrospira in unterschiedlichen Ökosystemen DISSERTATION Zur Erlangung des akademischen Grades eines Doctor rerum naturalium (Dr. rer. nat.) eingereicht am Fachbereich Biologie/Chemie der Universität Osnabrück von Myriam Kruse aus Bremen Osnabrück, Mai 2011 Dissertation vorgelegt dem Fachbereich Biologie/Chemie der Universität Osnabrück. Gutachter der Dissertation: Erstgutachter: Prof. Dr. André Lipski Zweitgutachter: Prof. Dr. Evert P. Bakker Gerade wenn du schon fast aufgeben willst, gerade wenn du glaubst, dass das Leben zu hart mit dir umspringt, dann denk daran, wer du bist. Denk an deinen Traum. Sergio Bambaren Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis ................................................................................................. I Zusammenfassung.............................................................................................. IV Abkürzungsverzeichnis ..................................................................................... VII Tabellenverzeichnis ............................................................................................ IX Abbildungsverzeichnis ........................................................................................ X 1. Einführung ........................................................................................................ 1 1.1 Der biologische Stickstoffkreislauf...................................................................... 1 1.2 Organismen der Nitrifikation................................................................................ 2 1.2.1 Nitrifizierende Organismen ............................................................................................... 2 1.2.2 Lebensweise nitrifizierender Organismen ........................................................................ 4 1.3 Nitrit-oxidierende Bakterien ................................................................................. 5 1.3.1 Gattungen der Nitrit-oxidierenden Bakterien .................................................................... 5 1.3.2 Stoffwechsel und Zellmorphologie Nitrit-oxidierender Bakterien...................................... 7 1.3.3 Gattung Nitrospira ........................................................................................................... 11 1.4 Untersuchungsgebiete ........................................................................................12 1.4.1 Kläranlage des Klärwerkverbundes in Hamburg ............................................................ 13 1.4.1.1 Abwasserbehandlungen im kommunalen Klärwerk .................................................... 13 1.4.1.2 Belebtschlammverfahren............................................................................................. 14 1.4.2 Aquakulturanlagen in Büsum und Strande..................................................................... 15 1.4.2.1 Aquakulturanlage Büsum ............................................................................................ 18 1.4.2.2 Aquakulturanlage Strande ........................................................................................... 18 1.5 Fettsäureanalytik..................................................................................................19 1.5.1 Nomenklatur der Fettsäuren........................................................................................... 19 1.5.2 Fettsäuretypen in der Zellmembran bei unterschiedlichen Temperaturen..................... 19 1.5.3 Fettsäuren und stabile Isotope als Marker in Ökosystemen .......................................... 20 1.6 Charakterisierung autotropher Organismen ......................................................22 1.6.1 Charakterisierung über Gesamtfettsäureprofile und FAME-SIP .................................... 23 1.6.2 Charakterisierung über 16S rRNA-Analysen.................................................................. 25 1.7 Dominierende Nitritoxidanten in Biofiltersystemen ..........................................25 1.7.1 Ziel dieser Arbeit............................................................................................................. 25 I Inhaltsverzeichnis 2. Material und Methoden .................................................................................. 27 2.1 Herkunft und Kultivierung der Mikroorganismen ..............................................27 2.1.1 Herkunft und Probenahmestandorte .............................................................................. 27 2.1.2 Kultivierung von autotrophen Mikroorganismen ............................................................. 29 2.2 Fettsäureanalysen................................................................................................31 2.2.1 Gesamtzell-Fettsäurepräparation der Reinkulturen und Anreicherungen...................... 31 2.2.2 Markierungsexperimente von Biofiltermaterial über FAME-SIP..................................... 32 2.2.2.1 Inkubationsbedingungen der Biofilterproben............................................................... 32 2.2.2.1.1 Inkubationsbedingungen der Proben aus dem Klärwerkverbund Hamburg ............ 32 2.2.2.1.2 Inkubationsbedingungen der Proben aus marinen Aquakulturanlagen ................... 33 2.2.2.2 Extraktion der Phospholipidfettsäuren aus Biofiltermaterial........................................ 34 2.2.2.3 Parameter der Gaschromatographen für die Fettsäureanalysen................................ 34 2.2.2.4 Auswertungen der Fettsäureprofile ............................................................................. 35 2.2.2.4.1 Identifizierung der FAMEs ........................................................................................ 35 2.2.2.4.2 Berechnung der 13 C-Inkorporation in Fettsäuren ..................................................... 36 2.3 Molekularbiologische Methoden.........................................................................38 2.3.1 Entwicklung von Primern und Sonden............................................................................ 38 2.3.2 DNA-Extraktion und Amplifikation der 16S rRNA........................................................... 40 2.3.3 Analysen der Umweltproben über Klonierung................................................................ 44 2.3.4 Identifizierung über Sequenzierung und Sequenzanalysen........................................... 45 2.3.5 Fluoreszenz in situ Hybridisierung.................................................................................. 46 3. Ergebnisse...................................................................................................... 50 3.1 Gesamtzell-Fettsäureanalysen der Reinkulturen und Anreicherungen ...........50 3.2 Vorbereitende Analysen für die Markierung von Umweltproben......................55 3.2.1 Einbau von 13 C-Bicarbonat in die Fettsäuren von Nitrospira .......................................... 55 3.2.2 Markierungen einer Umweltprobe mit 12 C- und 13 C-Bicarbonat ..................................... 56 3.3 Ergebnisse zum Ökosystem Belebtschlamm der kommunalen Kläranlage ....57 3.3.1 FAME-SIP mit Proben aus Belebtschlamm.................................................................... 57 3.3.2 Anreicherungen aus Belebtschlamm.............................................................................. 66 3.3.3 Sequenzanalysen der 16S rRNA aus Belebtschlamm................................................... 68 3.3.4 Fluoreszenz in situ Hybridisierung für Anreicherungen aus Belebtschlamm ................. 69 3.4 Ergebnisse zum Biofiltermaterial aus marinen Fischfarmen............................76 3.4.1 FAME-SIP Analysen mit Proben aus Büsum und Strande......................................... 77 3.4.2 FAME-SIP und 16S rRNA Sequenzanalysen der Biofilter aus Strande..................... 79 II Inhaltsverzeichnis 4. Diskussion ...................................................................................................... 86 4.1 Gesamtzell-Fettsäureanalysen der Reinkulturen und Anreicherungen ...........86 4.2 Vorbereitende Analysen für die Markierung von Umweltproben......................92 4.3 Analysen zum Ökosystem Belebtschlamm aus einer Kläranlage ....................94 4.4 Analysen zum Biofiltermaterial aus Fischfarmen............................................104 5. Ausblick ........................................................................................................ 111 6. Literatur......................................................................................................... 112 7. Anhang.......................................................................................................... 125 7.1 Markierungen Belebtschlamm ..........................................................................125 7.1.1 Markierung Juni 2008 ................................................................................................... 126 7.1.2 Markierung Oktober 2008............................................................................................. 129 7.1.3 Markierung Januar 2009............................................................................................... 130 7.1.4 Markierung Mai 2009.................................................................................................... 132 7.1.5 Markierung August 2009 .............................................................................................. 134 7.2 Markierungen Biofilter Fischfarm .....................................................................137
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