Phytoplankton sample collection and preparation. Samples for taxon-specific elemental content were collected using 12 liter (L) Niskin bottles, usually from two depths at each station (see supplemental file for map of stations from which samples were collected and cell-sorted to quantify cellular macronutrient content; one within the upper mixed layer (10-20 meters) and one near the deep chlorophyll maximum. Due to timing of the cruises, samples were collected from a seasonal window limited to summer and early fall. Samples were prepared for sorting by concentrating cells from the ambient sample under darkened conditions using a Memteq High Volume Cell Trap (0.2 micrometer pore size), and then flushed from the Cell Trap using 0.2 micrometer (µm) filtered seawater and a luer lock syringe; roughly 3-4 liters (L) of ambient seawater was concentrated down to ~5 milliliters (mls). Samples were either gravity filtered through the Cell Trap directly from the Niskin bottle or pushed through the cell trap using a peristaltic pump at <50 milliliter per minute (ml min-1). Samples were then kept cool at 4 degrees C if they were going to be sorted live and immediately after collection or fixed with freshly filtered paraformaldehyde (0.5% v/v final concentration) if they were going to be stored for later sorting. Samples were allowed to fix at 4 degrees C for 1-2 hours and then flash frozen in liquid nitrogen and then moved to -80 degrees C for longer term storage.
Cell sorting and elemental analysis. The cell counting and sorting protocols used follow that of Baer et al. (2017). Cell counts and sorts were performed on either a Becton Dickinson Influx or FACSJazz flow cytometer, each utilizing a 200 milliwatt (mW) 488 namometer (nm) laser, with detectors for forward scatter, side scatter, 692 namometer (nm), and 530 namometer (nm), and operated with 8 grams of NaCL kg-1 solution for sheath fluid, which was filtered inline using a 0.22 micrometer (µm) Millipore SterivexTM filter. Instrument alignment was performed with 3.0 micrometer (μm) 6-peak rainbow beads, while roughly hourly checks on forward scatter response were performed with 0.53 micrometer (μm) Nile Red beads (Spherotech). Prochlorococcus populations were discriminated based on forward scatter and red fluorescence, and a gate in orange (530 nm) discriminated for Synechococcus. Eukaryotes were all larger autofluorescing cells that did not fit the cyanobacterial gating scheme. For sorting, sort control software was set to "1.0 drop pure" sort mode. In excess of 17 million, 8 million, and 500,000 cells for Prochlorococcus, Synechococcus, and eukaryotes were sorted for chemical analysis, respectively. Not all phytoplankton groups were sufficiently abundant at each station/depth for practical sorting and subsequent analysis. Post-sort purity tests were run with subsamples of each sorted population; sort purity always exceeded 94%. Sorted populations were collected in polystyrene Falcon tubes (BD Biosciences Inc.) and subsequently filtered on pre-combusted (450 degrees C for 4 hours) GF-75 filters (Ahlstrom; nominal pore size = 0.3 micrometer). To ensure complete capture of all of the cells, the Falcon tubes were rinsed multiple times with 0.2 micrometer (μm) filtered sheath fluid. Filter and sheath fluid blanks were produced each day samples were sorted for subsequent subtraction from the mass of each unknown sample. Following filtration, filters were placed in acid-washed cryovials and frozen (-20 degrees C) until analysis as described below. Average cellular elemental content was determined by dividing the elemental content of the sample (see next section) by the number of sorted cells as determined in a direct post-sort count analysis of the sorted sample.
Particulate nutrients. Particulate organic P (POP) was analyzed using the ash-hydrolysis method, with oxidation efficiency and standard recovery tested with each sample run using an ATP standard solution and a certified phosphate standard (OSIL Phosphate Nutrient Standard Solution). Method precision is 1-2% at 5 nanomoles per kilogram (nmol kg-1). Samples for POC and PON were acid fumed in a desiccator over concentrated HCl prior to analysis. After acid fuming, POC and PON were determined on a Costech 4010 elemental analyzer or a Control Equipment 440 elemental analyzer, depending on the samples. L-glutamic acid (USGS40) was used for standard curve generation and as a check standard (tolerance of <0.1 microgram) approximately every ten samples and at the end of every instrument run. Empty tin capsules (Costech Analytical Technologies) were cleaned with acetone and dried and run as instrument blanks.
Additional methodological details can be found in Lomas et al. 2021 (currently in revision).
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