Phosphohydrolysis rates from samples collected in the coastal western North Atlantic on R/V Endeavor cruise EN588 during September 2016 (NCEI Accession 0278879)

Acquisition Description:
Field sampling: Surface seawater (5–35 m) was collected in September 2016 during two sampling campaigns in the coastal western North Atlantic (Diaz et al., 2018; Supplementary Table S1). Three sites were sampled aboard the R/V Endeavor using a Niskin rosette sampler and incubated immediately in order to determine rates of P hydrolysis. Two sites accessible by small boat in Woods Hole Harbor and Buzzard's Bay, MA, were sampled utilizing a peristaltic pump. These samples were transported on ice packs and analyzed for P hydrolysis rates within 5–6 hours of collection. Additional samples were preserved and analyzed for chlorophyll, bacteria and phytoplankton abundance, and soluble reactive P (SRP), as detailed below.

Chlorophyll: In the dark, 250 mL of seawater was filtered onto 25 mm GF/F filters. Samples were stored in the dark at -20C until analyzed according to protocols adapted from Strickland and Parsons (1972). Briefly, samples were extracted in 90% acetone in the dark (4C, 9 hr) and measured using a 10AU fluorometer (Turner). Sample signals were calibrated using a chlorophyll-a standard (Sigma) and were corrected for phaeopigments by accounting for the fluorescence of extracts before and after acidification to 0.003 M HCl.

Abundance of bacteria and phytoplankton: Seawater samples were preserved for flow cytometry with 0.5% glutaraldehyde (final concentration), flash frozen in liquid nitrogen and stored at -80°C until analysis. Bacteria and group-specific phytoplankton counts were conducted on a Guava EasyCyte HT flow cytometer (Millipore). Instrument-specific beads were used to calibrate the cytometer. Samples were analyzed at a low flow rate (0.24 µL s⁻¹) for 3 min. To enumerate bacteria, samples were diluted (1:100) with filtered seawater (0.01 µm). Samples and filtered seawater blanks were stained with SYBR Green I (Invitrogen) according to the manufacturer's instructions and incubated in a 96-well plate in the dark at room temperature for 1 hr. Bacterial cells were counted based on diagnostic forward scatter vs. green fluorescence signals. Major phytoplankton groups were distinguished based on plots of forward scatter vs. orange (phycoerythrin-containing Synechococcus sp.), and forward scatter vs. red (eukaryotes). Size classes of eukaryotic phytoplankton were further distinguished based on forward scatter (pico-, nano- and large eukaryotes).

Soluble reactive P: Seawater samples were collected from Niskin rosette bottles or the peristaltic pump into acid cleaned, high density polyethylene bottles. Samples used for determining in situ SRP concentrations were frozen and stored upright at -20°C until analysis. Field samples and diatom filtrates were both analyzed for SRP using a standard colorimetric method (Hansen and Koroleff, 1999). To determine in situ SRP concentrations in field samples, SRP analysis was conducted using a 4 cm glass spectrophotometry cell on triplicate subsamples, and the detection limit, defined as three times the standard deviation of replicate blank measurements, was 115 nmol L⁻¹ SRP. For incubations to determine P hydrolysis rates (see below), replicate samples were analyzed in clear 96-well plates on a multimode plate reader (Molecular Devices) with a detection limit of 800 nmol L⁻¹ P.

P-hydrolysis of model DOP substrates: Field samples were incubated with the fluorogenic probe 4-methylumbeliferone phosphate (MUF-P) and two inorganic polyphosphate compounds with an average chain length of 3 or 45 P atoms.

Samples were amended with each substrate at a final concentration of 20 M P. This concentration was assumed to be rate-saturating based on preliminary experiments. Hydrolysis of polyphosphates was determined from the production of phosphate using the colorimetric protocol outlined above. Hydrolysis of the fluorogenic probe MUF-P was monitored using a standard fluorescence technique. Briefly, hydrolysis of MUF-P to 4-methylumbellierone (MUF) was measured (excitation: 359 nm, emission: 449 nm) and calibrated with a multi-point standard curve of MUF (10–500 nmol L⁻¹). In both methods, samples were corrected for substrate autohydrolysis by accounting for negative controls, which were filtered (0.2 m) and boiled (99C, 15 min) prior to P amendment in order to eliminate enzyme activity. See Diaz et al. 2018 Frontiers in Marine Science 5: 380 for full methods.