169 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress Our investigation also shed light on the overall stress response, transcriptionallydetected morphology change, storage polymer usage and EPS production by freshwater “Ca. Methanoperedens” archaea. MATERIALS AND METHODS “Ca. Methanoperedens” microcosm experiment at increasing salinities Prior to the bioreactor experiment, we first performed a preliminary 18-day long salinity stress microcosm experiment on the same “Ca. Methanoperedens” enrichment. Here, we determined the methane oxidation potential of the enrichment at three different salinities: 1, 2, and 3%. Freshwater and sulfate-free ASW medium had otherwise the same composition as specified for the bioreactor experiment. For the microcosm experiment, pH was adjusted with NaOH pellets to 7.3 and buffered with 10mM HEPES to secure pH values between 7.2-7.5. Prior to inoculation with bioreactor material, medium was made anoxic by Ar:CO2 (95:5) sparging for 2 h. A total volume of 30 ml was sampled per biological replicate and immediately transferred to the anaerobic chamber in capped and anoxic 60 ml syringes. To remove precipitates, biomass was washed three times with the respective medium. Briefly, medium was dispensed in final volumes of 20 ml in 120 ml serum bottles. Overpressure (Ar:CO2, 95:5) was checked to be around 1.3 bars in all bottles. Serum bottles were capped with red butyl rubber stoppers that were previously boiled twice in 100 mM NaOH and twice washed in water. Aluminum crimps were used to ensure full attachment of the stoppers. Later, biomass samples were subjected to an extra 1 min Ar:CO2 (95:5) sparging to ensure full anoxic conditions. Serum bottles then received 10% 13C-CH4 and 2 mM NaNO3. 12C-CO 2 and labelled 13C-CO 2 were measured in 50 μL headspace samples by gas chromatography-mass spectrometry (GC-MS), Agilent 8890 GC System and Agilent 5977B GC/MSD (Agilent Technologies, Santa Clara, CA, USA). Calibration was performed with standard gas consisting of He/O2/N2/CH4/CO2/N2O with terminal values of (%): balance/1.02/1.03/1.05/1.04/0.050 (Linde Gas Benelux BV, Schiedam, The Netherlands). Nitrate consumption was followed by MQuantTM colorimetric test strips (Merck, Darmstadt, Germany). Overpressure and a stable pH (7.2-7.5) were monitored before every injection. Chromatography data was analyzed using the Agilent OpenLab CDS Software. 6
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