170 Chapter 6 Bioreactor setup and operation A 2 L bioreactor (Applikon, Delft, The Netherlands) was anoxically inoculated with 1 L biomass of a “Ca. Methanoperedens Vercelli Strain 1” enrichment originating from freshwater Italian rice paddy fields (Vaksmaa et al., 2017). The bioreactor experiment was run for 12 weeks. It was operated as a sequencing fed-batch reactor (SBR) resulting in granular biomass; the sequences consisted of 22 h 45 min of receiving medium (250-300 ml/day), 15 min of settling and 45 min of supernatant removal, with approximately 4 days hydraulic retention time (Supplementary Fig. 1B). The bioreactor contained two standard six-blade turbines at 96 rpm and was operated at room temperature. The pH was buffered with 100 g/l KHCO3 solution and controlled by a BL 931700 pH controller Black Stone (Hanna Instruments, Rhode Island, USA) (Supplementary Fig. 1B). The bioreactor was constantly fed with CH4 with a flow of 10 ml/min and sparged with Ar:CO2 (95/5). Sulfate-free artificial sea water (ASW)(Nguyen, 2018) was increased stepwise by 0.25% every two weeks until 1.5% (brackish) was reached at 12 weeks (Supplementary Fig. 1A). To further investigate potential biomass-acclimation, the bioreactor ran at 1.5% for over 2.5 months prior to increasing salinity to 3% (marine) in 0.5%-steps over the course of a month (Supplementary Fig. 1A). The ASW composition was adapted and excluded MgSO4 to avoid growth of sulfatereducing bacteria (Nguyen, 2018). Traces of potassium, chlorides, and sulfate from the medium - together with sodium from the NaNO3 substrate contributed between 0.01 to 0.1% to the baseline salinity, increasing or, decreasing, depending on the nitrate-demand (with an average 0.6 mmol NaNO3/day). Physicochemical analysis Liquid nitrate consumption and/or nitrite toxicity were controlled daily in the bioreactor via MQuantTM colorimetric test strips (Merck, Darmstadt, Germany). From 0% to 1.5% salinity increase, ammonium was followed biweekly whereas during the 1-month 1.5%-3% salinity increase, it was done weekly. For this analysis, ammonium was determined either using a low or high sensitivity protocol (range from 40-400 μM or 0.5-5 mM) after reaction with 10% orthophthaldialdehyde as previously described(Taylor et al., 1974). For the low sensitivity assay, fluorescence was read with the Spark 10 M Plate Reader (Tecan, Grodig, Austria) whereas for the
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