Fluorescent beads

Bulk chlorophyll fluorescence (FL) (ex440; em680) was measured almost daily using a Fluorescence Spectrophotometer (Cary Eclipse, Varian). Samples for flow cytometry were taken after 60, 100, and 140 days of experiment E1, fixed with glutaraldehyde (0.125% final concentration), incubated in the dark for 10 min and stored in −80 °C until analysis. Fluorescent beads (2 μm diameter, Polysciences, Warminster, PA, USA) were added as an internal standard. Data was acquired and processed with FlowJo software. Flow cytometry was performed unstained to count Prochlorococcus cells followed by staining with SYBR Green I (Molecular Probes/ ThermoFisher) to count Alteromonas cells.

The three culturable bacterial strains, Roseobacter sp., Marinobacter sp., and Bacillus sp., were cultured in 500 mL pre-combusted (450°C, 5 h) transparent conical flasks. The abundance of bacterial cells was measured using an Accuri C6 flow cytometer (BD Biosciences, Erembodegem, Belgium), as described previously (Moens et al., 2016 (link)). The sample was stained with 0.01% SYBR Green I for 30 min in the dark at 37°C (Marie et al., 1997 (link)). As an internal standard, 1 μm fluorescent beads (Polyscience, Warrington, PA, United States) were injected into each sample. The samples were measured at a flow rate of 0.25 μL s^–1 for 1 min.

For prokaryoplankton cell abundance determination, 5 mL samples were fixed with 2% paraformaldehyde and stored in liquid nitrogen until analysis with flow cytometry. Before counting, samples were thawed at room temperature and nucleic acids were stained with SYBRGreen-I and incubated in the dark for 15 min. Stained bacterial cells were enumerated according to their right-angle light scatter (RALS) and green fluorescence (FL1) collected at 530/30 nm. Counts were performed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with an air-cooled 488 nm argon laser. In order to calibrate the flow cytometer, fluorescent beads (1.002 μm; Polysciences Inc., Warrington, PA, USA) were systematically added to each sample. Sampling for prokaryoplankton enumeration was performed at one to two days interval. In total, 11 samples were taken between d0 and the end of the experiment (d21). Bacterial growth rates (μ in d⁻¹) were measured considering bacterial abundance (N) at the beginning (t_in) and the end (t_final) of the exponential growth in each microcosm [μ= ln(N_final/N_in)/(t_final − t_in)].