Carbon Metabolism of Methylotrophic Methanogens and Asgard Archaea in Marine Sediments DISSERTATION zur Erlangung des Grades eines Doktors der Naturwissenschaften −Dr. rer. nat.− dem Fachbereich Biologie/Chemie der Universität Bremen vorgelegt von Xiuran Yin Bremen 2019 This PhD thesis was conducted in the frame work of the International Max Planck Research School of Marine Microbiology (MarMic) and supported by the China Scholarship Council. Gutachter: Prof. Dr. Michael W. Friedrich (Universität Bremen) Gutachter: Prof. Dr. Jens Harder (Max-Planck-Institut für Marine Mikrobiologie) Prüfer: Dr. Marcus Elvert (Marum) Prüfer: Tilmann Harder (Universität Bremen) Contents Summary ........................................................................................................................................ ……1 Zusammenfassung................................................................................................................................. 3 Chapter 1 ............................................................................................................................................... 5 Introduction ....................................................................................................................................... 5 1.1. Carbon utilization in marine sediments.................................................................................... 5 1.2. Carbon metabolisms in methanogenesis .................................................................................. 9 1.2.1. Hydrogenotrophic methanogens ....................................................................................... 9 1.2.2. Methylotrophic methanogens .......................................................................................... 10 1.2.3. Acetoclastic methanogens ............................................................................................... 11 1.3. Carbon metabolisms in other uncultured archaea .................................................................. 12 1.3.1. Archaeal diversity in the Helgoland Mud sediments ...................................................... 13 1.3.2. Carbon fixation ............................................................................................................... 13 1.3.3. Alkane and fatty acids degradation ................................................................................. 14 1.3.4. Fermentation and organic polymer degradation ............................................................. 15 1.4. Identification of active microorganisms by stable isotope probing ....................................... 17 1.4.1. Nucleic acid-SIP ............................................................................................................. 17 1.4.2. Lipid-SIP ......................................................................................................................... 18 1.5. Objectives and structure of the thesis .................................................................................... 18 1.6. Reference ............................................................................................................................... 21 Chapter 2 ............................................................................................................................................. 33 DNA and RNA stable isotope probing of methylotrophic methanogenic archaea .................... 33 Abstract ......................................................................................................................................... 34 2.1. Introduction ............................................................................................................................ 35 2.2. Materials ................................................................................................................................ 36 2.2.1. Sediment slurry incubations ............................................................................................ 36 2.2.2. Media .............................................................................................................................. 36 2.2.3. Nucleic acid extraction.................................................................................................... 37 2.2.4. DNA- and RNA-SIP ....................................................................................................... 37 2.3. Methods .................................................................................................................................. 38 2.3.1. SIP incubations ............................................................................................................... 38 2.3.2. Nucleic acids extraction .................................................................................................. 39 2.3.3. Density separation of isotopically labeled nucleic acids ................................................. 40 2.3.4. Anticipated results........................................................................................................... 42 2.3.5. Experimental run time ..................................................................................................... 43 2.4. Notes ...................................................................................................................................... 43 2.5. Figures .................................................................................................................................... 49 2.6. Table ...................................................................................................................................... 51 2.7. References .............................................................................................................................. 52 Chapter 3 ............................................................................................................................................. 55 CO2 conversion to methane and biomass in obligate methylotrophic methanogens in marine sediments .......................................................................................................................................... 55 Abstract ......................................................................................................................................... 57 3.1. Introduction ............................................................................................................................ 58 3.2. Materials and Methods ........................................................................................................... 59 3.2.1. Sediment incubation setup for SIP .................................................................................. 59 3.2.2. Pure culture setup ............................................................................................................ 60 3.2.3. Slurry incubations inoculated with M. methylutens ....................................................... 60 3.2.4. Gas analysis .................................................................................................................... 61 3.2.5. Nucleic acids extraction, quantification and DNase treatment ....................................... 61 3.2.6. Isopycnic centrifugation, gradient fractionation and reverse transcription ..................... 61 3.2.7. Quantitative PCR (qPCR) ............................................................................................... 62 3.2.8. Sequencing and bioinformatics analysis ......................................................................... 62 3.2.9. Lipid analysis .................................................................................................................. 62 3.2.10. δ13C calculation ............................................................................................................. 62 3.3. Results .................................................................................................................................... 63 3.3.1. Methylotrophic methanogenesis and increase in methanogenic archaea ........................ 63 3.3.2. Carbon assimilation into RNA and identification of metabolically active archaea ........ 64 3.3.3. Methane formation from DIC during methylotrophic methanogenesis .......................... 65 3.4. Discussion .............................................................................................................................. 66 3.4.1. Carbon assimilation by methylotrophic methanogens in sediment incubations ............. 66 3.4.2. CO2 reduction to methane by obligate methylotrophic methanogens ............................. 68 3.5. Acknowledgments .................................................................................................................. 70 3.6. Conflict of interest ................................................................................................................. 70 3.7. Data availability ..................................................................................................................... 70 3.8. Table ...................................................................................................................................... 70 3.9. Figures .................................................................................................................................... 72 3.10. Supplementary figures ........................................................................................................