Gf f glass microfiber filter

Subsamples of 2 mL from each exponentially growing culture were fixed in acidic Lugol’s iodine solution and the cell concentration was then estimated using Sedgwick–Rafter chambers. Other subsamples of 40 mL of the same culture were filtered through GF/F glass microfiber filters 25-mm in diameter (Whatman, Maidstone, England) using a vacuum pump; the residual medium volume was estimated using glass graduated cylinders. Each cell-containing filter was placed in a 1.5 mL Eppendorf tube to which 750 µL of 0.05 M acetic acid was then added. The sample was then sonicated (1 min, 50 Watts), centrifuged at 17,968× g for 10 min, and the supernatant was transferred to a new 1.5-mL Eppendorf tube and processed as described above. The supernatants were combined (final volume 1500 µL) and kept at −20 °C until toxin analysis.

To assess the impact of the treatments on the elemental stoichiometry of phytoplankton communities, samples of 200 mL were filtered at the onset and the end of the incubation period on pre-combusted and pre-washed GF/F glass microfiber filters (Whatman, 25 mm, pore size 0.7 μm). All filters were stored individually in Eppendorf tubes at −20°C. For POC and PON analyses, filters were dried for at least 48 h at 60°C in a drying chamber before being wrapped in tin foil and analyzed with an elemental analyzer (Elementar vario MICRO cube). Particulate P was measured by spectrometric determination (Thermo Scientific Multiskan Spectrum) of orthophosphate (Grasshoff et al., 1999 (link)). Dissolved inorganic nitrogen (DIN), phosphorus (DIP) and silicate (DSi) were measured at the beginning of the experiment and after the 72-h incubation period to evaluate nutrient changes in our treatments. A volume of 50 mL was filtered through nylon membrane filters (Merck, 47 mm, pore size 0.2 μm) and stored at −20°C. The samples were measured with an autoanalyzer (Alliance Instruments GmbH) after a modified method after Grasshoff (Seal/Alliance methods; (Grasshoff et al., 1999 (link)).

Total phosphorus (TP) was measured using the molybdenum-blue method, after potassium persulfate digestion. DOC was measured on a TOC1010 total carbon analyzer (OI Analytical) using a high-temperature sodium persulfate oxidation method. For Chla determinations, we filtered one liter of water on 47mm-diameter GF-F glass microfiber filters (Whatman), extracted the filter in hot ethanol, stored it overnight at 4°C, then determined the absorbance of the extract at 665nm and 750nm, using a UV/Vis UltroSpec 2100 spectrophotometer (Biochrom Ltd.). We have used this estimate for all subsequent analyses, but note that there was good agreement between the spectrophotometric and the HPLC-based chlorophyll estimate described in the previous section.

Water was filtered through 0.7 µm GF/F glass microfiber filters (Whatman, pre-combusted 400°C, 4 h), acidified with HCl (25%, analysis grade, Carl Roth) to pH 2 and stored at 4°C in the dark. Analysis of DOC concentrations was performed via high-temperature catalytic oxidation method^{45 (link)} using a Shimadzu (Japan) TOC-VCPH/CPN Total Organic Carbon Analyzer equipped with ASI-V auto sampler. We controlled the accuracy of the measurement for every run by analyzing deep-sea reference samples provided by the University of Miami (Dennis Hansell). The error of DOC was below 2.8 µmol C L^-1.