Bacterial H2S oxidation coupled to the reduction of MnO2 studied on new isolated species of the genus Sulfurimonas Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Der Mathematisch-Naturwissenschaftlichen Fakultät Der Universität Rostock Vorgelegt von: Jan Henkel, geb. am 28. Juni 1989 in Fulda aus Rostock Rostock, den ____________________________________ https://doi.org/10.18453/rosdok_id00002580 Gutachter: Prof. Dr. Heide Schulz-Vogt, Leibniz- Institut für Ostseeforschung Warnemünde (IOW), Geomikrobiologie Prof. Dr. Axel Schippers, Bundesanstalt für Geowissenschaften und Rohstoffe (BGR), Geochemie der Rohstoffe Jun.-Prof. Dr. Mirko Basen, Universität Rostock, Mikrobielle Physiologie Jahr der Einreichung: 2019 Jahr der Verteidigung: 2019 Content Summary .................................................................................................................................................. I Zusammenfassung ................................................................................................................................. III 1 Introduction ...................................................................................................................................... 1 2 Material and methods ..................................................................................................................... 11 2.1 Preparation of anoxic cultivation medium ............................................................................ 11 2.2 Preparation of stock solutions ............................................................................................... 12 2.2.1 Manganese oxide (MnO2) .................................................................................................. 12 2.2.2 Elemental sulfur (S0) ......................................................................................................... 13 2.2.3 Iron oxides ......................................................................................................................... 13 2.2.4 Sulfide (Na2S) .................................................................................................................... 13 2.3 Origin of the three Sulfurimonas species ............................................................................... 13 2.3.1 S. marisnigri ...................................................................................................................... 13 2.3.2 S. baltica ............................................................................................................................ 14 2.3.3 S. gotlandica ...................................................................................................................... 14 2.4 Culture purity of S. marisnigri and S. baltica........................................................................ 15 2.4.1 16S rRNA sequencing and 16S rRNA clone libraries ....................................................... 15 2.5 Genome sequencing of S. marisnigri .................................................................................... 16 2.5.1 DNA isolation .................................................................................................................... 16 2.5.2 DNA sequencing ............................................................................................................... 17 2.5.3 Genome assembly and annotation ..................................................................................... 17 2.6 Analysis of cell counts .......................................................................................................... 17 2.6.1 DAPI stained epifluorescence microscopy ........................................................................ 17 2.6.2 Flow-cytometry ................................................................................................................. 18 2.7 Chemical analyses ................................................................................................................. 20 2.7.1 Sulfur species .................................................................................................................... 20 2.7.2 Metals and calcium (Ca) .................................................................................................... 21 2.7.3 Total inorganic carbon (TIC) ............................................................................................. 23 2.7.4 Cellular polyphosphate (polyP) ......................................................................................... 23 - - 2.7.5 Nitrate (NO3 ) and nitrite (NO2 ) ........................................................................................ 24 2.8 Analysis of O2, pH and H2S with microsensors .................................................................... 24 2.9 Combined imaging and elemental quantification .................................................................. 24 2.9.1 Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analysis ........................................................................................................................................... 24 2.9.2 Nanoscale secondary ion mass spectrometry (NanoSIMS) ............................................... 25 2.9.3 He- ion microscopy and secondary ion mass spectrometry (HIM-SIMS) ........................ 26 2.10 Rate calculations with exponential curve fitting ................................................................... 27 2.11 Numerical modeling .............................................................................................................. 27 2.12 Experimental design, cultivation conditions and specific supplementation .......................... 29 2.12.1 Particle dynamics analyzed with NanoSIMS ................................................................ 29 2.12.2 Dynamic of Mn and S species during growth of S. marisnigri ..................................... 29 - 2.12.3 S. marisnigri and S. baltica grown with NO3 and H2S additions.................................. 31 2.12.4 S. marisnigri grown with MnO2 and additions of H2S or H2S pre-reacted with MnO2 . 31 2.12.5 Determination of H2S oxidation rates of S. marisnigri .................................................. 32 2.12.6 Dissimilatory metabolism – electron acceptors ............................................................. 33 2.12.7 Dissimilatory metabolism – electron donors ................................................................. 34 0 2- 2.12.8 Disproportionation of S and S2O3 .............................................................................. 35 2- 2.12.9 SO4 - reduction ............................................................................................................ 35 2.12.10 Fermentation .................................................................................................................. 35 2.12.11 Assimilatory metabolism ............................................................................................... 36 2.12.12 Abiotic factors influencing growth ................................................................................ 36 3 Results ............................................................................................................................................ 38 2- 3.1 Chapter I: Reduction of MnO2 coupled to the oxidation of S2O3 and H2S .......................... 38 3.2 Chapter II: Influence of a biological H2S oxidation on the geochemical structure of the Black Sea suboxic zone. ............................................................................................................................... 48 3.2.1 Estimation of a biological H2S oxidation rate in the presence of MnO2 ........................... 48 3.2.2 Numerical modeling .......................................................................................................... 50 3.3 Chapter III: Ecological niches of the newly isolated Sulfurimonas strains in comparison to S. gotlandica. ......................................................................................................................................... 53 3.3.1 Dissimilatory metabolism - electron acceptors ................................................................. 53 3.3.2 Dissimilatory metabolism - electron donors ...................................................................... 57 0 2- 3.3.3 Disproportionation of S and S2O3 .................................................................................. 59 2- 3.3.4 Sulfate (SO4 ) - reduction ................................................................................................. 60 3.3.5 Fermentation ...................................................................................................................... 60 3.3.6 Assimilatory metabolism ................................................................................................... 61 3.3.7 Abiotic factors influencing growth .................................................................................... 63 4 Discussion ...................................................................................................................................... 70 4.1 Bacterial sulfur oxidation coupled to Mn reduction and a general view onto pelagic redoxclines with focus on the Black Sea ........................................................................................... 70 4.2 Mechanism of MnO2 reduction and potential implications for Mn(Ca)CO3