Dataset: Nutrients and Flow Cytometry 2017

Cruises and sample collection
A cruise was carried out on August 7, 2017 on R/V Mediterranean Explorer to study the photic zone in the Eastern Mediterranean at high depth resolution. Genetic data and nano-SIMS measurements were used to infer and quantify mixotrophy by Prochlorococcus at the base of the photic zone.

Water samples were collected using a 12-bottle rosette with 8 L Niskin bottles. Sampling depths were selected based on real-time data from a Conductivity, Temperature, Depth (CTD) profiler (Seabird 19 Plus) from the down-cast before each sample collection in the up-cast. The continuous data were processed using custom Excel files taking into account the location of each sensor and the sensor delay, and binned over 1-meter intervals.

Nutrients
Nutrient samples were collected in acid-washed 500 ml plastic containers and immediately filtered through 0.22 μm Nalgene rapid-flow filter units. The first two filtrates were discarded, and the final sub-samples were collected in new 50 ml falcon tubes and stored in the dark at 4 degrees Celsius in racks. The samples were transported to the analytical lab at the marine station at Sdot-Yam within 12–15 h of sampling and dissolved nutrients were determined using a SEAL AA-3 autoanalyzer system (SEAL, 2011). To minimize cross-contamination in the laboratory, ammonium was analyzed first alone using an automated ophthalaldehyde (OPA) fluorescence method. Samples were placed in the sample trays and only opened immediately before the sample probe took the sample. The baseline water used for ammonium determination was freshly prepared Milli-Q (MQ) water. The next parameters to be determined were dissolved nitrate and silicate. The methods used were Cd reduction and diazo dye for nitrate & nitrite (hereafter referred to as Nox), and molybdate blue in the presence of oxalic acid for Silicate. The baseline water used for these determinations was also MQ water. Finally, a 3rd run was carried out using an ultra-low level phosphate (DIP) method involving using a 100 cm long flow cell (LWCC) and molybdate blue determination derived from Murphy and Riley (1962). The blank used for phosphate determination was surface seawater, whose DIP value was determined in a previous experiment in which we determined a refractive index correction by removing the ascorbic acid reagent. It was not possible to use MQ as the baseline, as has been used elsewhere because the MQ water in our lab (derived from desalinated water) contains ~20 nM DIP.

Picophytoplankton abundance using flow-cytometry
Water samples (1.5 ml) were collected from each sampling depth, put in cryo-vials (Nunc), and fixed with 7.5 µl 25% glutaraldehyde (Sigma). Vials were incubated in the dark for 10 min, flash-frozen in liquid nitrogen, and stored in a -80 degree C freezer. Before analysis, samples were thawed in the dark at room temperature. Analysis was performed using a BD Canto II flow-cytometer with 2 μm diameter fluorescent beads (Polysciences, Warminster, PA, USA) as a size and fluorescence standard. Three types of phytoplankton cells were identified based on their natural auto-fluorescence: Prochlorococcus, Synechococcus, and picoeukaryotes. Cells were differentiated based on cell chlorophyll (Ex482nm/Em676nm, PerCP channel) and phycoerythrin fluorescence (Ex564nm/Em574nm), and by the size of the cell (forward scatter). Data were processed using FlowJo software. Flow rates were determined several times during each running session by weighing tubes with double-distilled water, and counts of the standard beads were used to verify a consistent flow rate.

Additional data related to this study: Amplicon sequencing of picoplankton ITS (Internal Transcribed Sequence) are available as NCBI BioProject PRJNA802375. NanoSIMS analyses of flow-corted Prochlorococcus cells are presented in a manuscript by Wu et al (currently under review, doi: 10.1101/2022.01.14.476346).