Enrichment and characterization of methanogenic hydrocarbon-degrading microbial communities in different ecosystems Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades DOKTORIN DER NATURWISSENSCHAFTEN Dr. rer. nat. genehmigte Dissertation von Diplom-Biologin Friederike Marie Luise Gründger geboren am 27. Februar 1982 in Weimar 2014 Referent: Prof. Dr. Axel Schippers Korreferent: Prof. Dr. Thomas Brüser Tag der Promotion: 4. April 2014 Schlagworte: mikrobielle Diversität, anaerober Kohlenwasserstoffabbau, Methanogenese Key words: microbial diversity, anaerobic hydrocarbon degradation, methanogenesis Contents Zusammenfassung .................................................................................................................... 7 Summary .................................................................................................................................. 8 List of abbreviations ................................................................................................................. 9 1. Introduction .................................................................................................................... 12 1.1. Hydrocarbons........................................................................................................... 12 1.1.1. Structure and natural occurrence ...................................................................... 12 1.1.2. The formation of crude oil and natural gas ...................................................... 14 1.1.3. The formation of coal ....................................................................................... 16 1.1.4. Methane – the simplest hydrocarbon ............................................................... 17 1.1.5. Isotope composition of methane and carbon dioxide and fractionation processes .......................................................................................................................... 19 1.2. The biodegradation of hydrocarbons ....................................................................... 21 1.2.1. Aerobic hydrocarbon degradation .................................................................... 21 1.2.2. Anaerobic hydrocarbon degradation ................................................................ 22 1.2.3. Microbiological anaerobic hydrocarbon activation .......................................... 25 1.2.4. Syntrophic interactions and in situ hydrocarbon biodegradation ..................... 29 1.2.5. Hydrocarbon biodegradation in petroleum reservoirs ...................................... 30 1.2.6. Hydrocarbon biodegradation in coal deposits .................................................. 35 1.3. Methanogenic processes .......................................................................................... 38 1.3.1. Hydrogenotrophic methanogenesis .................................................................. 38 1.3.2. Acetoclastic methanogenesis ............................................................................ 39 1.3.3. Methylotrophic methanogenesis ...................................................................... 39 1.3.4. The seventh order of methanogens ................................................................... 40 1.3.5. Enzymatic reactions during methanogenesis ................................................... 40 1.3.6. Cultivation methods and phylogenetic analysis of anaerobic microbes .......... 44 1.4. Motivation and main objectives of this work .......................................................... 46 1.5. Overview of publications and author contributions ................................................ 47 2. Evidence for in situ methanogenic oil degradation in the Dagang oil field ................... 52 2.1. Introduction ............................................................................................................. 54 2.2. Materials and methods/experimental details ........................................................... 55 2.2.1. Site description ................................................................................................. 55 2.2.2. Sampling and establishment of microcosms .................................................... 56 2.2.3. Analytical methods (isotopes and headspace gases) ........................................ 58 2.2.4. Gas-chromatography mass-spectrometry of oil ............................................... 58 2.2.5. Assessment of oil degradation .......................................................................... 59 2.2.6. Molecular methods ........................................................................................... 60 2.3. Results ..................................................................................................................... 60 2.3.1. Oil chemical analysis ....................................................................................... 60 2.3.2. Gas analysis ...................................................................................................... 64 2.3.3. Microbial abundance ........................................................................................ 66 2.3.4. In vitro methane production from labeled hydrocarbons ................................. 68 2.4. Discussion ................................................................................................................ 69 2.4.1. The microbiological potential of the Dagang oilfield for in situ methane production ......................................................................................................... 69 2.4.2. Chemical evidence for in situ oil biodegradation ............................................. 72 2.5. Appendix ................................................................................................................. 74 3. Microbial methane formation in deep aquifers of a coal-bearing sedimentary basin, Germany ......................................................................................................................... 78 3 3.1. Introduction ............................................................................................................. 80 3.2. Materials and methods ............................................................................................. 81 3.2.1. Sampling and sample preparation .................................................................... 81 3.2.2. Cultivation methods ......................................................................................... 82 3.2.3. Analytical methods ........................................................................................... 83 3.2.4. Molecular biological methods .......................................................................... 83 3.3. Results ..................................................................................................................... 85 3.3.1. Geochemical characterization of groundwater samples ................................... 85 3.3.2. Isotopic signatures of gasses from coal-rich sediments ................................... 86 3.3.3. Methanogenic and hydrocarbon degradation activity ...................................... 88 3.3.4. Quantification of microbial groups .................................................................. 88 3.3.5. Phylogenetic analysis of the microbial community composition ..................... 90 3.4. Discussion ................................................................................................................ 97 3.4.1. Geochemical links between groundwater and coal-bearing sediments ............ 97 3.4.2. The bacterial community in groundwater ........................................................ 99 3.4.3. The archaeal community in groundwater ....................................................... 101 3.4.4. The bacterial community in the coal-rich sediments and derived enrichment cultures ........................................................................................................... 102 3.4.5. The archaeal community in coal-rich sediments and derived enrichment cultures ........................................................................................................................ 104 3.5. Conclusion ............................................................................................................. 105 4. Similar features of microbial consortia from different ecosystems degrading hexadecane under methanogenic conditions .................................................................................... 108 4.1. Introduction ........................................................................................................... 110 4.2. Experimental procedures ....................................................................................... 112 4.2.1. Sample description and experimental set-up .................................................. 112 4.2.2. Analytical methods ......................................................................................... 113 4.2.3. Molecular biological methods ........................................................................ 115 4.3. Results ................................................................................................................... 117 4.3.1. Microbial activities ......................................................................................... 117 4.3.2. Physiological aspects .....................................................................................