142 Chapter 5 Short-term sulfide batch experiments To determine preliminary sulfide toxic thresholds of the anaerobic consortium, we conducted 5-day long microcosm experiments using the culture described above. We assessed the methane oxidation potential of the biomass at 0 (control), 0.25 mM and 0.5 mM sulfide exposure. The medium´s pH was buffered with 20 mM HEPES and made anoxic by sparging with N2:CO2 (95:5) for approximately 2 h. The pH was then adjusted to 7.3 with 1 M KOH. A total volume of 60 ml biomass from the bioreactor was sampled per biological replicate (n=2-3) and immediately transferred to the anaerobic chamber in a capped lid with anoxic BD 60 ml Plastipak Becton Dickinson S.A. (Madrid, Spain). To remove residual nitrate and precipitates, the biomass was washed three times with mineral medium. For the incubations, 40 ml medium was placed in 120 ml serum bottles, leaving about 80 ml of headspace. The serum bottles were capped with aluminum crimp caps and red butyl rubber stoppers that were previously boiled twice for 5 to 10 min in 100 mM NaOH and washed twice in water. The bottles were subjected to an additional 5 min N2:CO2 (95:5) sparging to ensure full anoxic conditions. Serum bottles then received 25 ml 13C-CH4 and 2 mM NaNO3 resulting in an overpressure of 1.8 bars in all bottles. Batch incubations were kept in the dark, and shaken at 250 rpm at room temperature. Sulfide (Na2S x 3 H2O) was added at 0.25 mM and 0.5 mM approximately 2 h after the methane and nitrate addition. The sulfide source used for both batch and bioreactor incubations was sodium sulfide hydrate 60-64% (Na2S x 3 H2O) (Acros Organics, Thermo Fischer Scientific, The Hague, The Netherlands). Sulfide concentrations were measured immediately after addition using the methylene blue assay using the HACH 8131 method (1.5-50 µM) (HACH, Loveland, CO, USA). 12CO 2 and labeled 13CO 2 were measured in 50 μL headspace samples by gas chromatography-mass spectrometry (GC-MS), using an Agilent 8890 GC System and Agilent 5977B GC/MSD (Agilent Technologies, Santa Clara, CA, USA). A calibration gas mixture consisting of He/O2/N2/CH4/CO2/N2O with values of (%): balance/1.02/1.03/1.05/1.04/0.050 (Linde Gas Benelux BV, Schiedam, The Netherlands) was used to calculate concentrations. The chromatography data were analyzed using the Agilent OpenLab CDS Software. Nitrate and nitrite concentrations were monitored using MQuantTM colorimetric test strips (Merck, Darmstadt, Germany), same as for the bioreactor. When nitrate levels were nearly depleted (2 – 50 µM), 1 to 3 mM NaNO3 was added. Overpressure and a stable pH (7.2-7.5) were monitored before every injection.
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