171 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress high ammonium sensitivity assay, samples were read at 420 nm absorbance with a spectrophotometer Spectra Max 190 (VWR, Boxmeer, The Netherlands). Bioreactor batch activity assays To determine the methane oxidation rate of the bioreactor microbial community, the bioreactor was run as batch with 20% 13C-CH4 and additional N2 in the headspace to obtain an overpressure of 1.2-1.3 bar. The headspace was first flushed for 2-3 h with Ar:CO2 (95/5) to remove residual methane traces. The starting nitrate concentration was about 1.5 mM. Over a period of 4-5 days, labelled 13C-CO 2 and 12C-CO 2 were measured and nitrate limitation, overpressure and a stable pH were controlled. 50 μL of bioreactor headspace samples were analyzed as described above. As normalizers for activity assays, dry weight (n=2, 15 ml) and percentages of archaeal and bacterial 16S rRNA gene copy numbers (qPCR) were used. Our biomass grew in the form of granules and, for this growth-type, dry weight was preferred over archaeal cell counts. This way, the variation derived from granule heterogeneity in between conditions is minimized. Nucleic acid extractions and cDNA synthesis DNA for in-depth metagenomics was extracted at 0% and 1.5% salinity (n=1). We added a third DNA sample at 3% salinity for a less deep (2 Gbp/sample) metagenome sequencing (n=1). To obtain a detailed look at the microbial community shift via 16S rRNA gene amplicon analysis of bacteria and archaea, DNA was extracted biweekly. Additional DNA extractions for bacterial and archaeal abundance estimations via quantitative PCR (qPCR) were employed at (in %): 0,0.75,1.5,1.5-acclimated, and 3. RNA was extracted at 0% and 1.5% salinities (in four biological replicates, n=4). With the aim of achieving a targeted gene expression profile through reverse transcriptase qPCR, additional RNA extractions were performed at different salinities (in %): 0, 0.5, 1,1.5,1.5-acclimated, 3 (in three biological replicates per condition, n=3). DNA extractions were performed from 2 mL biomass using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) and eluted DNA was stored at -20ºC. 2 mL biomass for RNA extractions were anoxically sampled with four biological replicates (n=4) per time point, freeze dried overnight and stored at -70ºC. RNA was extracted 6
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