Dataset: Phytoplankton phosphonate utilization

Axenic cultures of the pico-prasinophyte Micromonas commoda (CCMP 2709), the coccolithophore Emiliania huxleyi (CCMP 2090), and the diatom Phaeodactylum tricornutum (CCMP 2561) were obtained from the National Center for Marine Algae and Microbiota (Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine). The cultures remained axenic throughout the experiments as determined by SYTO-staining and flow cytometric counting on a BD FACSJazz cell sorter; all cultures were free of bacteria during these experiments. Phytoplankton were grown in artificial sea water amended with L1 media with (P. tricornutum) or without (M. commoda and E. huxleyi) silica. The P source was added separately to achieve the desired growth conditions; Pi-replete media contained 36 µM PO₄³⁻, the Pi-deficient condition received 0.1 µM PO₄³⁻, and the phosphonate treatments received either 36 µM MPN or 2-AEPN. The Pi-deficient treatment (0.1 µM) represents a control for the low level of contaminating Pi measured in the phosphonate media; thus, an increase in growth in the MPN and 2-AEPN conditions above that measured in the Pi-deficient condition is due to phosphonate utilization. The potential for abiotic breakdown of phosphonate to Pi was investigated in media-only tubes exposed to the experimental temperature and light conditions for 10 days. Pi levels did not change throughout the experimental period (MPN average Pi = 0.11 µM ± 0.02; 2-AEPN average Pi = 0.10 µM ± 0.02), strongly supporting the notion of active enzymatic breakdown of phosphonates for growth. Natural P concentrations are much lower than those used in this study; the replete nutrient concentrations were used to support high cell yields necessary for analytical measurements. Cultures were acclimated to the four growth conditions described above as they had been maintained in each P treatment for a minimum of two transfers (20 days). Cultures were grown at 20°C in a 14h light/10h dark cycle at ~100 µE m⁻² s⁻¹ with a starting concentration of ~1x10⁴ cells mL⁻¹ in 25 mL culture volumes. Phytoplankton growth was monitored by fluorescence measurements using a Turner TD-700 fluorometer and cell counts analyzed by flow cytometry. Specific growth rates (μ) were calculated from the linear regression of the natural log of cell counts during the exponential growth phase of cultures. Quadruplicate cultures were setup for each treatment; three replicates were harvested in the late exponential phase of growth for physiological measurements, while the fourth was used to monitor cell abundances later in the growth cycle.

Physiological measurements:
Cell samples (5 mL culture volume) for particulate P were collected onto precombusted 25 mm Whatman glass fiber filters, rinsed with 0.17 M Na₂SO₄, and stored frozen at -20°C until analysis. Determinations were made as previously described (Lomas et al. 2010). Briefly, filters were rinsed with 0.017 M MgSO₄, dried at 90°C, and combusted at 500°C for 2 h. Upon cooling, 0.2 M HCl was added and hydrolyzed at 80°C for 30 min. After cooling, mixed reagent was added, the samples were centrifuged, and absorbance was read at 885 nm using a Genesys 10 spectrophotometer.

Alkaline phosphatase content (APA) measurements were made by quantifying the hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate using a Molecular Devices FilterMax F5 microplate reader. Abiotic substrate hydrolysis was accounted for in killed controls that were boiled and cooled prior to substrate addition, as well as in media-only controls. The fluorescent reference standard, 6,8-difluoro-4-methylcoumarin was used to calculate the rate of hydrolysis, which was then normalized to cell abundance to determine APA per cell.