Dataset: Alkaline phosphatase activity for G. huxleyi CCMP371 grown under low N and low P N:P ratios in laboratory-based culture experiments

For this alkaline phosphatase activity dataset, G. huxleyi CCMP371 was grown under low N and low P conditions by modulating dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorus (DIP) ratios. This experiment began as a single batch culture until a maximum in vivo chlorophyll fluorescence was reached (based on a previous growth experiment; data contributed to BCO-DMO in this same project), which initiated a semi-continuous phase of the experiment. Briefly, CCMP371 (in L1/5-Sargasso seawater base media) was used to inoculate L1/5-artificial seawater base media (ASW) in each of six 2 L Erlenmeyer flasks. The cultures were kept at 18ºC (the optimum in terms of its thermal range; see Krinos et al. 2025) on a 14 h:10 h light-dark cycle with a light intensity of 100 µE m-2 s-1. Growth was monitored daily by in vivo fluorescence and by microscopic cell counts (reported elsewhere). A total of 3 flasks were randomly selected as low N treatment flasks, and the remaining 3 were selected as low P treatment flasks for the semi-continuous phase of the experiment, with daily removal of 0.4 L of culture and replacement with 0.4 L modified L1/5-ASW, with an N:P of 0.5:1 (0.5 µM N and 1 µM P) for low N and an N:P of 125:1 (25 µM N and 0.2 µM P) for low P until steady-state growth, as determined by in vivo fluorescence and microscopic cell counts (reported elsewhere), was maintained. Dilutions were made at the same time each day, roughly 5 h into the light cycle, to avoid diel effects. Daily dilutions continued until the cultures reached a steady state that was maintained for at least 4 days after the daily removal/dilution began. Once the low N and low P triplicate cultures reached steady state, samples were collected for a suite of physicochemical parameters, including alkaline phosphatase activity (reported here).

For APA analysis, samples (5 mL) from each experimental treatment (low N and low P) and replicate were filtered onto 47-mm polycarbonate membranes (0.2 μm) and stored at −20°C until analysis. APA was assayed after Dyhrman and Ruttenberg (2006) on a Biotek Synergy H1 hybrid microplate reader in fluorescence mode (with an excitation wavelength of 360 nm and an emission wavelength of 460 nm) using the fluorogenic phosphatase substrate 6,8-difluoro-4-methylumbelliferyl phosphate. Subsamples (0.2 mL) from each sample were transferred to a black, flat-bottom, untreated 96-well microplate and assayed immediately and then at intervals adjusted to the activity of the sample, such that all readings fell within the linear range of the assay (at least four measurements within 1 h or less). A standard curve from 0 to 600 nM DiFMU in artificial seawater was generated and used to calculate the rate of DiFMUP hydrolysis. Values were normalized per G. huxleyi cell (cell counts reported elsewhere).

Details for the reagents used are as follows:

6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP; Invitrogen/Thermo Scientific D6567); Abs/Em = 358/455

6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU; Invitrogen/Thermo Scientific D-6566); reference standard