2.3 sewage water collection, storage, and microbial community extraction
Sewage from urban areas was collected in late May 2018 from Ellinge (55°49’18.1”N, 13°18’10.3”E), Klagshamn (55°31’31.2”N, 12°55’55.4”E) and Sjölunda (55°37’59.4”N, 13°02’32.2”E) WWTPs in Southern Sweden. Sewage was kept at 4°C post sampling in plastic jerry cans. Sample preparation was as follows; for disruption of particles, the sewage was distributed into sterile 250 ml centrifuge bottles (Nalgene®, Sigma-Aldrich) containing metal beads and were horizontally attached to a shaker (IKA®KS 260 basic, Sigma-Aldrich), shaking at 500 RPM for 15 min and then followed by 1 min of sonication (1510, Branson). Bottles were then incubated on ice for 30 min for sedimentation of larger particles and the supernatant was filtered through a 10 μm coated cellulose acetate (CMF) filter (Advantec®), using a vacuum pump. Filtrates were centrifuged at 8000xG for 7 min at 4°C. Pellets were resuspended in 1 ml ice-cold 0.9 % NaCl solution and pooled together. The up-concentrated suspension was filtered through a 10 μm syringe filter (Frisenette) into a new 50 ml screw-cap tube (Sarstedt) and kept at 4°C until use. In order to enumerate bacteria in the sewage, cells were stained with SYBR green I 10000 X (Invitrogen, ThermoFisher Scientific) as described previously, with minor changes (Paerl et al. 2018). Briefly, 10 ul cell suspension were stained in a 990 ul 2 x SYBR green I solution with 1x TE buffer and 0.9% NaCl. The stained cells were incubated in the dark for 20 min and subsequently quantified by FACS.