Dataset: Phosphorus uptake - bulk
Deployment: AE0810

Sample collection: 
The data presented in this study were collected on 7 cruises throughout the Western North Atlantic Ocean (cruise X0606, X0705, X0804, BVAL 39, BVAL 46, AE1206, and AE1319). Data for kinetics experiments were collected from throughout the western North Atlantic, from roughly 55oN in the Labrador Sea to ~21oN, just north of Puerto Rico. All sample depths were <200m. All samples for Pi uptake rates and kinetics experiments were collected in acid-cleaned Niskin bottles attached to a CTD rosette and kept in subdued lighting until experiments were initiated (< 1 h).  Samples for whole community ambient uptake rates were collected from ~4 depths in the upper 60 m, while samples for taxon-specific ambient uptake rates were collected from 5 m, 40 m, and the deep chlorophyll maximum (DCM; ranging from 80 to 120 m) (27). Trichodesmium colonies were collected from the near surface (roughly within the top 20 m) by vertically hauling a handheld 100 µm net.  Single colonies were transferred a second time into fresh 0.2 µm-filtered water to reduce contamination of closely associated organisms, and subsequently separated by morphotype (either ‘puff’ with radial trichomes or ‘raft’ with parallel trichomes); only data for ‘rafts’ are presented here.

Dissolved inorganic and organic, and particulate nutrients:
Samples for NO3-/NO2-, NO2- and PO4-3 are gravity filtered through 0.8 µm Nucleopore polycarbonate filters using acid cleaned in-line polycarbonate filter holders, then frozen (-20oC) in HDPE bottles until analysis (Dore et al., 1996).  Tests of frozen versus refrigerated samples have indicated no significant difference between storage methods (Dore et al., 1996).  Nutrient samples prior to ~2003 were analyzed on a modified Technicon Autoanalyzer and samples post ~2003 were analyzed on an Alpkem Flow Solution IV; both instrumental setups have comparable sensitivity and method detection limits validated by 6-month sample overlap on both instruments. During every sample run, commercially available certified standards, OSIL and Wako Chemical, are analyzed to maintain data quality, as well as ‘standard water’ from 3000 m which serves as an internal standard.

Soluble reactive phosphate (SRP) concentrations in the euphotic zone of the Sargasso Sea are below analytical detection limits (~20 nmol kg-1) of standard nutrient autoanalyzer configurations.  To resolve the low concentrations of SRP in the surface waters at BATS the Magnesium Induced Co-precipitation method, referred to as MAGIC-SRP measurements (Karl and Tien, 1992; Rimmelin and Moutin, 2005), was used starting in late 2004.  Several modifications to the method were made and detailed in Lomas et al. (2010a).  Sample accuracy was checked on each run with a certified OSIL nutrient standard.  The method detection limit following this protocol is ~1 nmol kg-1 with a precision of + 5% at 5 nmol kg-1.

Particulate organic carbon (POC) and nitrogen (PON) samples are filtered on precombusted (450oC, 4h) Whatman GF/F filters and frozen until analysis Steinberg et al., 2001a.  Samples are analyzed on a Control Equipment 240-XA or 440-XA elemental analyzer standardized to acetanilide.  Particulate phosphorus samples (PPhos) are analyzed using a ash-hydrolysis method (Lomas et al., 2010a).  Oxidation efficiency and standard recovery is tested with each sample run using an ATP standard solution and a certified phosphate standard (OSIL Phosphate Nutrient Standard Solution).  Method precision is ~9% at 2.5 nmol kg-1 (the lowest concentrations typically observed well below the euphotic zone), and ~1% at 15 nmol kg-1 (typical euphotic zone concentrations).  The method detection limit, defined as three times the standard deviation of the lowest standard (2.5 nmol kg-1) is ~0.5 nmol kg-1.    

33Phosphate incubations:
The approach for ambient whole community and population-specific uptake rate measurements were previously published (Casey et al, 2009). Briefly, duplicate aliquots of 10 ml seawater were amended with 0.15 µCi (~80 pmol L-1) additions of H333PO4 (3000 Ci mol-1; PerkinElmer, USA), and incubated for 30 - 60 min in subdued lighting (~100 µmol photons m-2 s-1) at ~23oC. This temperature was within ~3oC of the coolest/warmest in situ temperature from which the samples were collected. The duration of each incubation varied depending on turnover time of the added isotope, such that efforts were made to keep uptake to <25% of the tracer added.  Duplicate killed control incubations were conducted for each station. Killed controls were amended with paraformaldehyde (0.5% final concentration) for 30 min prior to the addition of isotopic tracer and incubation. Whole community incubations were terminated by filtration onto 0.2 µm polycarbonate filters that were subsequently placed in glass scintillation vials.  Population-specific ambient uptake incubations were terminated by the addition of paraformaldehyde (0.5% final concentration), and stored at 4oC until sorting (<12 h) as described in the next section. 

Whole community and population-specific kinetics experiments were conducted by adding 0.15 µCi (~80 pM) of H333PO4 to ~10 replicate 10 ml seawater samples that were further amended by increasing additions of ‘cold’ KH2PO4 up to 100 nM.  Samples were incubated as above, but the incubations were terminated by the addition of KH2PO4 to a final concentration of 100 µM (28). Whole community samples were filtered onto 0.2 µm polycarbonate filters, and rinsed with an oxalate wash (29). Surface bound phosphate in population-specific samples was accounted for by subtracting 33P counts for sorted populations to which 100 µM phosphate had been added prior to addition of the isotopic tracer. It is assumed that addition of such a high level of phosphate would result in negligible uptake of radioactive phosphate and thus any signal was attributed to surface absorption; this correction was always <2-3%. Population-specific kinetics experiments for samples collected in the deep chlorophyll maximum were first gravity concentrated and resuspended in phosphate-free Sargasso Sea surface water prior to incubation as described. Population-specific samples were stored at 4oC in the dark until sorting (<3 h) as described in the next section. Kinetics experiments for Trichodesmium spp. were conducted in the same manner as above for whole community samples but with picked and rinsed colonies and increasing additions of ‘cold’ KH2PO4 up to 1000 nM. 

Flow cytometry analysis and cell sorting:
Samples were sorted on an InFlux cell sorter (BD, Seattle, WA) at an average flow rate of ~40 µL min-1.  Samples were sorted for Prochlorococcus, Synechococcus, and an operationally defined eukaryotic algae size fraction (eukaryotes >2 µm). A 100 mW blue (488 nm) excitation laser was used. After exclusion of laser noise gated on pulse width and forward scatter, autotrophic cells were discriminated by chlorophyll fluorescence (>650 nm), PE (585/30 nm), and granularity (side scatter). Sheath fluid was made fresh daily from distilled deionized water (Millipore, Billerica, MA) and molecular grade NaCl (Mallinckrodt Baker, Phillipsburg, NJ), pre-filtered through a 0.2 µm capsule filter (Pall, East Hills, NY), and a STERIVEX sterile 0.22 µm inline filter (Millipore, Billerica, MA). Mean coincident abort rates were < 1% and mean recovery from secondary sorts (n = 25) was 97.5 ± 1.1% (data not shown).  Spigot™ (BD Seattle, WA) and FCS Express V3™ (DeNovo Software, Seattle, WA) were used for data acquisition and post-acquisition analysis, respectively. Sorted cells from each sample were gently filtered onto 0.2 µm Nucleopore polycarbonate filters, rinsed with copious amounts of 0.2 µm filtered seawater, an oxalate wash(29), and placed in a 7 ml scintillation vial for liquid scintillation counting.