Dataset: Pico- and nanoplankton abundance: FCM: EN532 and EN538
Deployment: EN538

The cell abundance of pico- and nanophytoplankton (<~14 micrometers (µm) cell diameter) was determined by flow cytometric analysis of 1500 microliters (µl) of glutaraldehyde-preserved (1% v/v) (Marie et al. 1997) samples using a BD Accuri C6 flow cytometer equipped with a blue laser (488 nanometers (nm)), at a flow rate of 100 µl per minute, and a core diameter of 22 µm. Standard fluorescent bead solutions were prepared daily and used as an internal standard to assess instrument performance, to standardize scatter and fluorescence measurements (Rainbow Calibration Particles (8 peaks), BD Biosciences), and to validate the flow rate (TruCount, BD Biosciences) for quantitative applications. Each sample was run with fluorescent beads (YG beads, 0.94 µm Fluoresbrite® Yellow Green Microspheres, Polysciences, Inc.) as an internal standard for forward scatter measurements.

We distinguished several phytoplankton groups based on their forward (FSC) and side scatter (SSC), Chla, and phycoerythrin (PE) fluorescence signals: the picophytoplankton (<~2.5 µm) group comprised PE-containing Synechococcus and non-PE-containing picoeukaryotes (picoEuk), the nanophytoplankton groups (nanoEuk, >~2.5 - 14 µm) included PE-containing nanophytoplankton (PE_Euk), non-PE-containing phytoplankton, and coccolithophores (Cocco) (the latter group was identified based on their enhanced side scatter signal). The total concentration of nanoeukaryote phytoplankton (totnanoEuk) is the sum of PE_Euk, Cocco, and non-PE-containing phytoplankton other than Cocco.

The picoplanktonic Prochlorococcus cells were counted in SYBR Green I-stained samples (Marie et al. 1997), according to (Heywood et al. 2006), because of the difficulty of discriminating unstained cells from background noise. The concentration of heterotrophic bacterial cells was determined by flow cytometric analysis of 250 µl of glutaraldehyde-preserved (1% v/v) and SYBR Green I-stained (1:7500) samples according to Marie et al. (1997) and Gasol and Del Giorgio (2000). The Prochlorococcus and heterotrophic bacteria samples were analysed using a BD Accuri C6 flow cytometer, at a flow rate of 35 µl per minute, and a core diameter of 16 µm. All plankton groups were gated and their abundance quantified using FlowJo software (Tree Star, Inc., www.flowjo.com).

The biovolume of phytoplankton cells analyzed by flow cytometry was derived from forward scatter measurements of individual cells based on the polynomial relationship between the log10 of measured biovolumes of pico- and nanophytoplankton cells and the log10 of the peak area of their forward scatter signal (FSC-A) (Laney & Sosik 2014). A calibration procedure, using bead stocks and an unidentified cultured picoeukaryote from the Sosik Lab at Woods Hole Oceanographic Institution, confirmed the inter-lab agreement of flow cytometry-derived biovolume estimates. Since the largest phytoplankton cell in the empirical relationship of Laney & Sosik (2014) had a cell diameter of 14 µm and the number of cells larger than this in our samples was negligible, only cells up to ~14 µm in diameter were included in the cell abundance and biovolume calculations. Cellular biomass was estimated according to the relationship between cellular biovolume (cubic micrometers per cell (µm3 cell-1)) and carbon content (picomoles per cell (pmol cell-1)) for glutaraldehyde preserved pico- and nanophytoplankton cells from (Verity et al. 1992): C = (0.433 ⁄ 12) × biovolume^0.863.

Although Synechococcus cells could readily be counted based on their size and their characteristic PE fluorescence the high signal-to-noise ratio in the FSC-A channel of the Accuri precluded a reliable cell size estimate for particles smaller than 1 µm. Therefore, the biomass of Synechococcus was estimated using a conversion factor of 140 femtograms carbon per cell (fg C cell-1) assuming a cell diameter of 1 µm and 270 fg C µm-3 (Bertilsson et al. 2003). The biomass of Prochlorococcus cells was calculated by using an average cellular carbon content of 53.5 fg C cell-1 (Bertilsson et al. 2003), which is very similar to the range of cellular carbon content determined by Casey et al. (2013) for Prochlorococcus in the euphotic zone.