79 Contrasting methane, sulfide and nitrogen regimes in coastal sediment bioreactors samples of depths 9-16 cm from the 2019 Stockholm Archipelago’s coastal anoxic sediments campaign (Site 3, Sandöfjärden) (Dalcin Martins et al., 2024, Chapter 2), where a high methanotrophic activity was observed. The biomass was stored in fully anoxic conditions at 4ºC in sealed aluminum bags (Gruber-Folien GmbH & Co. KG, Germany) until April 4th, 2021. On that date, the samples were mixed with a mineral medium containing (per 1 L): 1.25 mL of CaCl₂·2H₂O (192 mg/L), 0.6 mL of KH₂PO₄ (100 g/L), 1.25 mL of MgSO₄·7H₂O (288 mg/L), 0.5 mL of DAMO trace elements, 0.6 mL of anammox trace elements, and 0.6 mL of FeSO₄, as specified in Arshad et al. (2017) and Chapter 4. The mixture was prepared under brackish salinity conditions (1% Red Sea, Red Sea, Israel) and subsequently used for bioreactor inoculation and monitoring. Nitrate, ammonium, and sulfide loading were slowly built up using a 2:1:1 molar ratio, starting with lower concentrations to adapt the microbial community to the nutrient concentrations and ensure complete sulfide conversion (Supplementary Figure 3). By the end of the 10-month monitoring, the eutrophic bioreactor was in steady state and received around 8 mmol nitrate, 4 mmol ammonium, and 4 mmol sulfide, while the oligotrophic bioreactor was supplied with approximately 0.4 mmol nitrate, 0.2 mmol ammonium and 0.2 mmol sulfide (Supplementary Figure 1, Supplementary Figure 3). The sulfide source employed was sodium sulfide hydrate 60-64% (Na2S x 3H2O) (Acros Organics, Thermo Fischer Scientific, The Hague, The Netherlands); an anoxic stock solution was prepared that was refreshed approximately every 10 days. Nitrate and ammonium were supplemented in the form of NaNO3 (VWR, Radnor, Pennsylvania, USA) and NH4Cl (Fischer Scientific, Gell, Belgium). Medium supply (1 L) and sulfide (0.2 L dissolved in mineral medium) were always provided from separate bottles. The SBR cycle consisted of: 23 h of medium influent inflow, 10 min of settling and 50 min of reactor liquid removal. The hydraulic retention time was approximately 4 days (1.2 L/day reactor liquid removal). Both bioreactors were operated at 150 rpm with stirrers that contained one standard six-blade and a double blade spiral turbine. The reactors were saturated with methane by receiving CH4:CO2 (95:5) at 10 mL/min. The bioreactors were flushed with Ar/CO2 (95:5) inflow during reactor settling to ensure anoxic conditions at all times. The pH of the bioreactor was monitored with a pH electrode (Applisense, Applikon, Delft, The Netherlands), maintained at pH 7 with 1 M KHCO3 solution controlled with a pH pump by an ADI 1010 biocontroller (Applikon, Delft, The Netherlands). To subject the microbiomes 3
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