Dataset: AR16_Water_Column

All data collected from a modified procedure as described in Van Mooy et al (2015).

Sampling - Samples were taken at depths corresponding to 30% PAR (10-36 m). At Station 6, samples were taken at depths 5 m, 25 m, 60 m, 90 m, 110 m, 150 m, 205 m, 250 m, and 500 m. Subsamples for incubations were dispensed from the Niskin bottle directly into triplicate acid-cleaned 50 mL polycarbonate bottles and processed as described below.

 

Phosphate uptake rates - The incubation bottles were carried to a laboratory van that was designated solely for work with radioactive isotopes.  Each incubation bottle was spiked with approximately 10 µCi of 33P-phosphoric acid.  The final concentration of 33P-phosphate in the incubations was less than 10 pmol L-1, which was likely two orders of magnitude smaller than ambient phosphate concentrations. The bottles were capped and mixed by gently inverting.  To account for any abiotic adsorption of the radioactive tracer, additional triplicate 50 mL subsamples were spiked with 10% paraformaldehyde prior to the addition of the 33P-phosphoric acid. These “kill controls” were used for blank subtractions in uptake and reduction rate calculations. All bottles were placed in a flow-through on-deck incubator that was maintained at surface seawater temperatures by continually flushing it with the surface seawater from the ship’s pumping system.  Temperature in the incubators was occasionally monitored with a waterproof temperature logger (Onset).  The incubators used blue transparent film to achieve a light intensity to mimick 30% PAR. For the depth profile at Station 6, the incubators used a combination of neutral density screening and blue transparent film to achieve a light intensity to mimick PAR throughout the water column while samples with less than 1% PAR were placed in black plastic bags for complete darkness. Incubations proceeded for an average of 3.32 h (0.95 to 10.5 h) then 5 mL of sample was vacuum filtered (approximately 200 mbar) onto 25 mm diameter 0.2 µm pore size polycarbonate membranes (Millipore). The membranes were quickly rinsed three times with freshly filtered (0.2 µm pore size polycarbonate membrane) surface seawater.  The membranes were then immediately placed in a liquid scintillation vial containing 10 mL of UltimaGold liquid (Perkin Elmer) scintillation cocktail, which was then shaken vigorously.  After resting for a few hours, the 33P-radioacitivity in the vials was determined using a liquid scintillation counter (Perkin Elmer).  A steady-state phosphate turnover rate was calculated by dividing the total 33P radioactivity retained on the membranes by the total 33P radioactivity added to the incubations and the incubation time.

 

Phosphate reduction to intracellular P(III) compounds – The remaining 45 mL of sample was vacuum filtered as described above. Next, the membranes were immersed in 1.0 mL of ultra-high purity (UHP) deionized water (18 MΩ*cm) in a cryovial (Fisher).  The vials were immediately capped and immersed in liquid nitrogen for approximately 10 min, before they were immersed in boiling-hot water for 10 min, and then vigorously shaken.  This freeze-thaw cycle was repeated two additional times, after which generally little discernable cellular debris was visible.  Next, 100 µL aliquots of the samples were injected onto an IC system (Dionex) which pumped an eluent gradient of 23 mmol L-1 to 90 mmol L-1 sodium hydroxide through an IonPac AS18 (Dionex) column at a rate of 1.0 mL min-1.  An ion suppressor using UHP water as a regenerant removed sodium hydroxide from the eluent. Three fractions were collected in 40 second intervals at retention times where pure standards of (1) methyl-phosphonate, 2-hydroxethyl-phosphonate, and (3) phosphite elute (fraction 2 did not have a known phosphonate compound at time of analysis) and the 33P-radioactivity determined as described above. The 33P-radioactivity of the three fractions was summed, corrected for dilution, and then divided by the 33P-radioactivity from the parallel 33P-phosphate uptake subsamples to determine the fraction (%) of 33P uptake that was incorporated into P (III) compounds.  Environmental triplicates were averaged, and the standard deviation was propagated as analytical error.  All samples were processed at sea in May 2017 except samples from Station 9, which were flash-frozen in liquid nitrogen, transported to the laboratory in a cryogenic dry shipper, and stored in liquid nitrogen until their analysis in June 2017.  

 

MAGIC Soluble reactive phosphorus (SRP) concentrations.  SRP (i.e. phosphate) was determined in seawater samples (done in triplicate) and incubations using MAGnesium Induced Coprecipitation (MAGIC) as described by Karl and Tien (1992).

 

Total particulate phosphorus (TTP) concentrations. TPP was determined in seawater samples using a wet chemical oxidation method using potassium persulfate as described in Suzumura (2008). Briefly, 1 to 2 liters of seawater were filtered onto 47 mm 0.2 µm pore size polyvinylidene fluoride membranes (Millipore) and frozen (-80°C) until analysis. One fourth of these filters were cut with clean stainless-steel scissors and placed in 8 mL glass vials for oxidation. 2 mL of 5% (0.19 M) persulfate was added to each vial and the samples were then autoclaved for 30 minutes at 120°C. To remove any residual material, the samples were filtered through 0.45 µm syringe filters (Millipore Millex-HV). The persulfate was shown to inhibit color development when greater than 2%, therefore, the samples were diluted to 0.5% (0.019 M). As with the SRP samples, the TPP samples were analyzed via the molybdenum blue method using a spectrophotometer (Thermo).