Black freshwater sediment was retrieved, in June 2019 (TS1) and October 2020 (TS2), from a pond at Aarhus University campus (Vennelyst Park), Denmark (56.164672, 10.207908) at a water depth of 0.5–1 m. The sediment was stored with overlying water at 15°C for 2 weeks.
Before inoculation, sediment was homogenized, sieved (pore size: 0.5 mm) and autoclaved in 2 L bottles for 20 min as described in Thorup et al. (2021) (link) and Marzocchi et al. (2022) (link). Cooled down sediment (15°C) was distributed into 20 and 10 ethanol-cleaned Plexiglas core liners that were closed with a rubber stopper at the bottom. After 24 h settling time, the stoppers were pushed upwards to align the sediment surface with the core liner edge. The cores were inoculated by transferring a clump of sediment from a two-week-old, pre-grown single-strain enrichment culture of Ca. Electronema aureum GS (Thorup et al., 2021 (link)) and submerged in an aquarium with autoclaved tap water. The aquarium was covered with aluminum foil to prevent algae formation, equipped with aeration and a lid to prevent excessive evaporation, and kept at 15°C. Overlying water was replenished and refreshed several times during the incubation periods.
During the first time series (TS1), O2, pH, and EP profiles were measured combined with 16S rRNA sequencing and microscopy observations over 80 days, due to technical issues, the videos could not be used for flocking observations. The sequencing and microscopy observations were repeated in the second time series (TS2) with O2 and EP measurements over 81 days, and additionally the pH was measured on day 81. Following microsensor measurements (of both TS1 and TS2), 1–2 of the measured cores were sliced based on geochemical zone: the oxic layer (determined by the oxygen penetration depth) and the anoxic layer (below the oxygen penetration depth). Samples for light microscopy and 16S rRNA amplicon sequencing were taken from the anoxic layer of each core, of which the latter were frozen at −80°C until RNA extraction.
Before inoculation, sediment was homogenized, sieved (pore size: 0.5 mm) and autoclaved in 2 L bottles for 20 min as described in Thorup et al. (2021) (link) and Marzocchi et al. (2022) (link). Cooled down sediment (15°C) was distributed into 20 and 10 ethanol-cleaned Plexiglas core liners that were closed with a rubber stopper at the bottom. After 24 h settling time, the stoppers were pushed upwards to align the sediment surface with the core liner edge. The cores were inoculated by transferring a clump of sediment from a two-week-old, pre-grown single-strain enrichment culture of Ca. Electronema aureum GS (Thorup et al., 2021 (link)) and submerged in an aquarium with autoclaved tap water. The aquarium was covered with aluminum foil to prevent algae formation, equipped with aeration and a lid to prevent excessive evaporation, and kept at 15°C. Overlying water was replenished and refreshed several times during the incubation periods.
During the first time series (TS1), O2, pH, and EP profiles were measured combined with 16S rRNA sequencing and microscopy observations over 80 days, due to technical issues, the videos could not be used for flocking observations. The sequencing and microscopy observations were repeated in the second time series (TS2) with O2 and EP measurements over 81 days, and additionally the pH was measured on day 81. Following microsensor measurements (of both TS1 and TS2), 1–2 of the measured cores were sliced based on geochemical zone: the oxic layer (determined by the oxygen penetration depth) and the anoxic layer (below the oxygen penetration depth). Samples for light microscopy and 16S rRNA amplicon sequencing were taken from the anoxic layer of each core, of which the latter were frozen at −80°C until RNA extraction.
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