Sampling and analytical procedures:
Bioassay Experiments consisted of incubating, over an incubation period of 48h, surface seawater (5m) with inorganic and organic phosphate compounds (20 µM; final concentration of P) including, polyphosphate (polyp), inorganic phosphate (Pi), nucleotides (ATP, AMP) and phosphonate (Mepn). In each incubation experiment, a control treatment (surface seawater) was considered as well in each bioassay experiment alongside a treatment amended with nitrogen (NH4Cl, NaNO3). These bioassay experiments were conducted at station 1 and stations 3. At each station, inorganic and organic phosphate amendments were performed on seawater with and without nitrogen enrichment.
Alkaline phosphatase activity was determined fluorometrically using 4-methylumbelliferyl phosphate (MUF-P), as representative of phosphomonoesterase activity. Total APA was measured in each experimental bottles in triplicate. Hydrolysis rates were determined by separately incubating 200 µL of seawater with 8 different concentrations (0, 0.1, 0.2, 0.5, 1, 5, 10, 20 µM; final concentrations) of MUF-P in 96-well black microtiter plates. To ensure linearity of the hydrolysis rate, the increase of fluorescence was measured (excitation/emission wavelength: 359/449 nm) at multiple time points over an incubation period of 24 h. Maximum hydrolysis rate (Vmax) was determined, using a nonlinear regression based on the rectangular hyperbolic function following V=Vmax x S/ Km+ S, where S and V are the concentrations of the substrate and the hydrolysis rates, respectively.
Location: Northwestern Atlantic surface waters. Depth: surface-50 m.
Instruments: Reading of fluorescence was performed on a plate reader (SpectraMax® M2, Molecular Devices).
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