DIPLOMA THESIS Characterisation of biogeochemical processes in methane-containing sediments of the Beaufort Sea (Arctic Ocean) by Johanna Schweers Supervisor Prof. Dr. Tina Treude (IFM-GEOMAR, Kiel) Faculty of Mathematics and Natural Sciences at the Christian-Albrechts-University, Kiel Leibniz Institute of Marine Sciences/ IFM-GEOMAR, Kiel January 2011 Contents List of Abbreviations ........................................................................................................................ IV Abstract .............................................................................................................................................1 1. Introduction ...............................................................................................................................3 1.1 Microbial processes in marine sediments .................................................................................... 3 1.2 Microbial processes in sediments of continental margins ........................................................... 4 1.3 Climate change .............................................................................................................................. 7 1.4 Temperature dependence of microbial processes ....................................................................... 8 1.5 Climate change in the Arctic ......................................................................................................... 9 1.6 Aims of this study ........................................................................................................................ 11 2. Material and Methods .............................................................................................................. 12 2.1 Study site ..................................................................................................................................... 12 2.2 Sampling sites ............................................................................................................................. 12 2.3 Core sampling ............................................................................................................................. 13 2.4 In-vitro experiments ................................................................................................................... 16 2.5 Molecular methods ..................................................................................................................... 26 3. Results ..................................................................................................................................... 30 3.1 Temperature experiment ............................................................................................................ 30 3.2 Inhibition experiments ................................................................................................................ 39 3.3 Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) ......................... 42 4. Discussion ................................................................................................................................ 45 4.1 Microbial processes in sediments from the Beaufort Sea .......................................................... 46 4.2 Proportion of Bacteria and Archaea in the sediment ................................................................. 58 4.3 Conclusion and Outlook .............................................................................................................. 58 5. Literature ................................................................................................................................. 60 6. Annex....................................................................................................................................... 68 I List of Figures Figure 1.1: Redox cascade of oxidants and electron carriers in the sediment 3 Figure 1.2: Division of a continental margin 4 Figure 1.3: Stability of hydrate in the ocean 10 Figure 2.1: Sampling sites at the Arctic Shelf in the Beaufort Sea 12 Figure 2.2: Profiles of methane and sulfate concentration and ex situ rates of AOM and SR 17 Figure 2.3: Profiles of methane and sulfate concentration, ex situ rates of AOM and SR and concentration of barium 25 Figure 3.1: Sulfide development in sample MC10 from the SR zone 31 Figure 3.2: SR rates determined by radiotracer techniques in samples from the SR zone 32 Figure 3.3: Sulfide development in sample PC13 S6 from the AOM zone 33 Figure 3.4: Sulfate reduction rates determined by radiotracer measurements in the samples from the AOM zone 34 Figure 3.5: AOM determined by radiotracer measurements in the samples from the AOM zone 35 Figure 3.6: Methane development over time in the samples from the MG zone 37 Figure 3.7: Comparison of MG rates determined by time series and radiotracer measurements 38 Figure 3.8: SR (A) and AOM (B) rates determined by radiotracer measurements in samples from the MG zone with and without addition of molybdate (22mM) 40 Figure 3.9: SR (A) and AOM (B) rates determined by radiotracer measurements in samples from the MG zonewith and without addition of BES (60 mM) 41 Figure 3.10: Total cells stained with Sybr Green I 43 Figure 3.11: Bacteria stained with Alexa 546 (A) and Sybr Green I(B) 43 Figure 3.12: Archaea stained with Alexa 546 (A) and Sybr Green (B) I 43 Figure 3.13: Cell counts per ml sample (total, bacterial, and archaeal cell numbers) 44 II List of Tables Table 2.1: Geographic coordinates 13 Table 2.2: Sampling of PC13 14 Table 2.3: Sampling of PC12 15 Table 2.4: Cooled, anoxic sediment for in-vitro studies of PC06 and MC10 16 Table 2.5: Division of sediment samples 18 Table 2.6: Temperature scale of the used temperatures in the experiment 18 Table 2.7: Main salts of artificial seawater medium 19 Table 2.8: Mass of added acetate 20 Table 2.9: Modified masses of magnesium sulfate and magnesium chloride 20 Table 2.10: Different filter sets for cell detection 29 III List of Abbreviations °C degree Celsius % percent α alpha µ- mikro (10-6) ANME anaerobic methanotrophs AOM anaerobic oxidation of methane Bq Becquerel CARD-FISH catalyzed reporter deposition fluorescence in situ hybridization CH4 methane cmbsf cm before seafloor cm-3 cubic centimeter =milliliter cpm counts per minute CO2 carbon dioxide CoM coenzyme-M CTD conductivity-temperature-depth-logger Cu copper d day e.g. for example, Latin: exempli gratia et al et alteri etc. et cetera: and so on Fe iron Fig. figure g gram or gravitational acceleration GHG greenhouse gas H2 molecular hydrogen - HCO3 bicarbonate H2Odist distilled water H2S hydrogen sulfide i.e. id est: that means k kilo L liter IV m- milli (1 x 10-3) m meter M molarity MC multi corer MG methanogenesis min minute Mn manganese n- nano (1 x 10-9) N2 molecular nitrogen - NO3 nitrate O2 molecular oxygen PC piston corer + pH negative logarithm of the molar concentration of dissolved hydronium ions (H3O ) ppm parts per million ppmv parts per million by volume RT room temperature sec second SMTZ sulfate-methane-stability-zone 2- SO4 sulfate SR sulfate reduction SRB sulfate reducing bacteria SRR sulfate reduction rate t time UV ultra violet v/v volume/volume w/v mass/volume w/w mass/mass V Abstract Abstract Polar Regions are experiencing and will experience the greatest impact from global change, especially in the Arctic, which has displayed the highest increase in temperature over the last decades. However, the microbial processes in arctic sediments, and how they could be impacted by temperature changes, are still poorly understood. To investigate the microbial processes in arctic sediments and to see if methane releases from sediments are changing with the projected temperature rise, samples were taken from the continental shelf and slope of the Beaufort Sea. Sediment cores from different water depths (ranging from 30 - 2000 m) were collected and the microbial processes of sulfate reduction (SR), anaerobic oxidation of methane (AOM) and methanogenesis (MG) were examined to a maximum sediment depth of ~5 m. First, temperature experiments were conducted to determine in-vitro rates of SR, AOM and MG at a temperature range from -5 to 37 °C. In-vitro rates were measured applying both direct measurements of sulfide and methane development, as well as radiotracer techniques. The ex-situ rates of AOM and SR, which were made on board, displayed additional peaks in the sediment layer where normally only MG would be expected. Second, to examine if, and to what extent, these three processes coexisted in this layer, inhibition experiments with different inhibitors were conducted. The first experiment was carried out with the chemical sodium molybdate, which inhibits SR. The second experiment was performed with 2-bromoethanesulfonate (BES), a substance that inhibits the metabolism of archaea mediating MG and AOM. Lastly, catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was applied to determine the ratio of bacteria to archaea in the sediment. For this analysis, one sample was examined from three different sediment layers where SR, AOM and MG dominated, respectively. In the sediments of the Beaufort Sea, the SR microbial community showed a psychrophilic to mesophilic metabolism in the surface sediment depth (2-32 cmbsf), with optima around 0.5-13 °C and 25 °C, respectively, and most likely only psychrophilic metabolism at a depth of 290 cmbsf, with activity only below 25 °C and optima at -1 and 13 °C. For AOM, optima were