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Charakterisierung Der Anaeroben Gärung Verschiedener Ausgangssubstrate Mittels 16S Rrna Gen Amplikonsequenzierung Und Metaproteomanalytik

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Charakterisierung Der Anaeroben Gärung Verschiedener Ausgangssubstrate Mittels 16S Rrna Gen Amplikonsequenzierung Und Metaproteomanalytik Charakterisierung der anaeroben Gärung verschiedener Ausgangssubstrate mittels 16S rRNA Gen Amplikonsequenzierung und Metaproteomanalytik DISSERTATION zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) in der Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften (BayNAT) der Universität Bayreuth vorgelegt von M. Sc. Bioanalytik Christian Büttner aus Lichtenfels Bayreuth, 2020 Die vorliegende Arbeit wurde im Zeitraum von März 2016 bis August 2020 an der Fakultät für angewandte Naturwissenschaften der Hochschule für angewandte Wissenschaften Coburg unter Betreuung von Herrn Prof. Dr. Matthias Noll angefertigt. Vollständiger Abdruck der von der Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften (BayNAT) der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.). Dissertation eingereicht am: 17.08.2020 Zulassung durch das Leitungsgremium: 20.08.2020 Wissenschaftliches Kolloquium: 27.04.2021 Amtierender Direktor: Prof. Dr. Markus Lippitz Prüfungsausschuss: Prof. Dr. Matthias Noll (Gutachter) Prof. Dr. Dirk Schüler (Gutachter) PD. Dr. Alfons Weig (Vorsitz) Prof. Dr. Andreas Römpp (Weiterer Gutachter: Prof. Dr. Tillmann Lüders) Was wir wissen, ist ein Tropfen; was wir nicht wissen, ein Ozean. - Sir Isaac Newton - I Inhaltsverzeichnis Inhaltsverzeichnis ...................................................................................... I Zusammenfassung ................................................................................... V Abstract .................................................................................................. VIII Abkürzungsverzeichnis ......................................................................... XI Abbildungsverzeichnis ....................................................................... XIII Tabellenverzeichnis .............................................................................. XV 1 Einleitung und Theorie ...................................................................... 1 Biogas als Energieträger ................................................................................. 1 1.1.1 Nutzung von Biogas in Deutschland ......................................................... 1 1.1.2 Aufbau und Funktionsweise von anaeroben Fermentern ...................... 3 Die vier Phasen des anaeroben Abbaus organischen Materials ............ 5 1.2.1 Hydrolyse ....................................................................................................... 6 1.2.2 Acidogenese ................................................................................................... 6 1.2.3 Acetogenese ................................................................................................... 7 1.2.4 Methanogenese .............................................................................................. 9 Relevante Prozessparameter beim anaeroben Abbau ............................ 10 1.3.1 Substratbasis ................................................................................................ 10 1.3.2 Temperatur ................................................................................................... 11 1.3.3 pH-Wert ........................................................................................................ 12 1.3.4 Flüchtige organische Säuren und totale Alkalinität ............................... 12 Die Bedeutung mikrobieller Gemeinschaften für die Umwelt ........... 14 1.4.1 Molekularbiologische Methoden zur Charakterisierung mikrobieller Gemeinschaften ........................................................................................... 14 1.4.2 OMICS-Technologien im Kontext der Mikrobiomforschung ............... 16 Berechnung mikrobieller Netzwerke auf Basis von OMICS- Datensätzen .................................................................................................... 19 1.5.1 Einordnung von Netzwerken in den ökologischen Kontext ................ 19 1.5.2 Aufbau und Eigenschaften von mikrobiellen Netzwerken .................. 20 1.5.3 Die Berechnung mikrobieller Netzwerke ................................................ 21 1.5.4 Die topologische Rolle von Knoten .......................................................... 24 Motivation und Hypothesen ....................................................................... 26 II 2 Material und Methoden ................................................................... 29 Geräte und Materialien ................................................................................ 29 Verwendete Chemikalien ............................................................................ 30 Probennahme ................................................................................................. 33 Ermittlung der Anlagenparameter ............................................................. 34 2.4.1 Prozessparameter der Biogasanlagen ...................................................... 34 2.4.2 Prozessparameter von Kläranlagen .......................................................... 37 16S rRNA Gen Amplikonsequenzierung ................................................. 38 2.5.1 Nukleinsäureextraktion ............................................................................. 38 2.5.2 Quantitative und qualitative Überprüfung der Extrakte ...................... 39 2.5.3 Primerwahl und Überprüfung der Amplifizierbarkeit ......................... 40 2.5.4 Sequenzierung ............................................................................................. 41 2.5.5 Auswertung der Sequenzierung des 16S rRNA Gens ........................... 42 Metaproteomuntersuchungen ..................................................................... 46 2.6.1 Proteinextraktion ......................................................................................... 46 2.6.2 Proteinquantifizierung ............................................................................... 47 2.6.3 SDS-Page und tryptischer Proteinverdau................................................ 47 2.6.4 NanoLC-MS/MS-Messungen ................................................................... 49 2.6.5 Auswertung der Metaproteomdatensätze .............................................. 50 Verfügbarkeit der generierten Datensätze ............................................... 53 3 Ergebnisse ........................................................................................... 54 Prozessparameter der beprobten Anlagen ............................................... 54 3.1.1 Biogasanlagenparameter ............................................................................ 54 3.1.2 Kläranlagenparameter ................................................................................ 56 Mikrobielle Gemeinschaften in Biogas- und Kläranlagen ................... 57 3.2.1 Beschreibung der Biodiversität ................................................................. 57 3.2.2 Vergleich der mikrobiellen Gemeinschaften in Biogas- und Kläranlagen .................................................................................................. 58 3.2.3 Kernmikrobiome von Biogas- und Kläranlagen ..................................... 59 3.2.4 Die Varianz in der Zusammensetzung der mikrobiellen Gemeinschaften in BPs und STPs ............................................................. 64 3.2.5 Einfluss von Prozessparametern auf die mikrobielle Gemeinschaft ... 70 3.2.6 Mikrobielle Interaktionsmuster ................................................................ 72 III Funktionelle Charakterisierung der Biogasanlagen .............................. 78 3.3.1 Taxonomische und funktionelle Proteinzusammensetzung ................ 78 3.3.2 Einteilung der Anlagen auf Basis des vorherrschenden Methanogenesewegs ................................................................................... 81 3.3.3 Anlagencluster auf verschiedenen taxonomischen und funktionellen Ebenen........................................................................................................... 82 3.3.4 Funktionelle Charakterisierung der mikrobiellen Lebensgemeinschaften ............................................................................... 87 3.3.5 Zusammenhang zwischen relativer Abundanz und funktioneller Redundanz auf KO-Ebene ......................................................................... 91 3.3.6 Einfluss von Prozessparametern auf verschiedene taxonomische und funktionelle Ebenen .................................................................................... 92 3.3.7 Mikrobielle Interaktionsmuster in Abhängigkeit des Methanogenesewegs ................................................................................... 96 4 Diskussion der Ergebnisse ............................................................ 103 Mikrobielle Gemeinschaften der anaeroben Gärung .......................... 103 4.1.1 Unterschiede in der Diversität zwischen den Anlagentypen ............. 103 4.1.2 Unterschiedliche Kernmikrobiome in BPs und STPs .......................... 105 4.1.3 Einfluss der Umweltparameter auf die mikrobielle Zusammensetzung .................................................................................... 108 4.1.4 Identifikation mikrobieller Interaktionsmuster mittels Netzwerkanalysen .................................................................................... 111 Metaproteomuntersuchungen ..................................................................
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