Synechococcus Growth– Axenic Synechococcus WH8102 (open ocean strain) and WH5701 (coastal strain) were obtained from the National Center for Marine Algae and Microbiota (NCMA, Bigelow Laboratories, East Boothbay, Maine). Both strains were grown in batch culture using SN media (Waterbury et al. 1986) made with aged, filtered (0.2 µm), and autoclaved (120°C, 30 minutes) seawater from station ALOHA (A Long-term Oligotrophic Habitat Assessment). At the late-exponential phase, cultures were transferred in triplicate to one of two SN media: (1) +Pi (45 µmol L^-1 KH₂PO₄, following Waterbury et al. (1986)) and (2) -Pi (no KH₂PO₄ added; Pi below detection limit). All cultures were incubated at 25°C on a 12h:12h light cycle at 130 µmol m^-1 s^-1 in sterile culture flasks with a vent cap (0.22 µm hydrophobic membrane).

DOP Hydrolysis– Representative phosphoester (P-ester) and polyphosphate (PolyP) hydrolysis rates were determined as in Diaz et al. (2018). The nucleotide AMP was used as a model P-ester, 3-PolyP as a model PolyP, and ATP as a representative containing both P-ester and P-anhydride bonds. Two separate experiments were conducted with both Synechococcus strains to determine DOP hydrolysis in the presence (whole cell) and absence (cell-free filtrate) of cells (Diaz et al. 2018, 2019). The +Pi and -Pi cultures were subsampled approximately every three days over ~20 days to obtain whole cell and cell-free DOP hydrolysis results along each phase of the cellular growth curve. To prepare cell-free filtrates, the +Pi and -Pi culture aliquots were aseptically filtered (0.2 µm) at each subsampling day. To determine DOP hydrolysis rates, the production of Pi was measured over time. Aliquots (200 µL) of +Pi and -Pi treatments were amended with a single DOP substrate (3-PolyP, ATP or AMP; 20 µmol L^-1 P, final concentration) in triplicate wells of a non-treated standard 96-well transparent microplate and incubated over 6-hours. An unamended control treatment to track Pi concentrations in the cultures over time were prepared in triplicate and monitored in parallel. Pi (or soluble reactive phosphorus) was measured using a standard colorimetric protocol (Hansen and Koroleff 1999) on a multimode plate reader (SpectraMax® M2, Molecular Devices). Absorbance read at 880 nm was calibrated using a standard curve of monopotassium phosphate (0, 0.5, 1, 2, 5, 10, 20, 40, 50, 75 µmol L^-1; KH₂PO₄). The average detection limit of Pi using this method, defined as three times the standard deviation of the triplicate blank measurements, was 0.125 ± 0.005 µmol L^-1. The calibration curve was prepared with 0.2 µm filtered ALOHA seawater with a Pi background concentration below the detection limit.

Organism identifiers (Life Science Identifier, LSID):
Synechococcus, urn:lsid:marinespecies.org:taxname:160572