80 Chapter 3 to a second environmentally relevant coastal microbiome stressor, the salinity was increased to 2 % Red Sea Salt in the last month of operation (13.5-14.5 months) (Supplementary Figure 1). Physicochemical measurements The bioreactor was checked daily for nitrate and nitrite concentrations with MQuantTM colorimetric test strips (Merck, Darmstadt, Germany). Monthly sulfide was either measured in liquid samples with the methylene blue assay (Moest, 1975) (HACH, Loveland, CO, USA), or in the headspace as gas using a 7890B GC System (Agilent Technologies, Santa Clara, CA, USA) (Supplementary Figure 3). Liquid samples were collected every week for ammonium measurements (Supplementary Figure 1, 3). Ammonium was determined fluorescently using a high sensitivity protocol (range from 40-400 μM): 2-mercaptoethanol created reduced conditions throughout the entire mixture, allowing it to react with ortho-phthaldialdehyde (Taylor et al., 1974). Fluorescence was recorded via a Spark 10M Plate Reader (Tecan, Grodig, Austria). Nucleic acid extraction DNA samples for downstream 16S rRNA gene amplicon sequencing were obtained in months: 0 (inoculation day), 2, 3, 5-12, 14.5 (before salt increase to 2% NaCl) and a month afterwards, 15.5 (Supplementary Figure 1). A total volume of 40 mL of biomass was extracted from both bioreactors at each time point. Samples were then centrifuged for 5 min at maximum speed and the remaining pellet was used for DNA extraction. From month 6 onwards, DNA was also extracted from the wall-associated biofilm by using a sterile Sigma SIAL0010 Cell scraper (Sigma Aldrich, MO, USA) (Supplementary Figure 1, Supplementary Figure 2). The biofilms covered both bioreactors” heights between 3.8 L and 5 L, corresponding to the minimum and maximum volumes of the SBR cycle (Supplementary Figure 2). DNA extraction for metagenomic sequencing was performed in triplicates in bioreactors, planktonic and wall biofilm samples, on months: 7,14.5, and 15.5. DNA extractions were performed using the Power Soil Kit (Qiagen, Hilden, Germany), with a modified initial bead-beating step with solution C1 at 10 min 50 oscillations per second and PowerBead tubes on a TissueLyser LT (Qiagen,
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