Nylon membrane filter

Extracts of fern leaves were obtained by solvent extraction, as described by Grzeszczuk et al. [48 (link)]. The solvent was a mixture of methanol (Merck, Darmstadt, Germany) with distilled H₂O (7:3, v/v). Dried leaves were ground in a laboratory mill, then 0.5 g samples were transferred into 50 mL Falcon tubes (Bionovo, Legnica, Poland) and mixed with 40 mL of the mixture. The solutions were then extracted for 30 min in an ultrasonic cleaner (Elmasonic S30H, Elma Schmidbauer GmbH, Singen, Germany) and centrifuged for 5 min at 5000 rpm (Centrifuge 5418 Eppendorf, Warsaw, Poland). The extracts were filtered through 0.22 µm nylon membrane filters (Merck, Darmstadt, Germany). Three repetitions of each extract were prepared and stored in a freezer at −20 °C.

The amount of encapsulated CLO per mg of dry LS was determined by disgregating 50 mg of LS in 5 mL of ethanol under stirring (300 r.p.m.) at 60 °C for 2 h.
The samples were filtered (nylon membrane filters, 0.2 μm pore size, Merck Millipore, Milan, Italy) and analyzed by high-performance liquid chromatography (HPLC) for CLO content, as previously reported [19 (link)]. HPLC determinations were performed using a two-plungers alternative pump (Jasco, Tokyo, Japan), an UV-detector operating at 210 nm, and a 7125 Rheodyne injection valve with a 50 μL loop. The samples were loaded on a stainless steel C-18 reverse-phase column (15 × 0.46 cm) packed with 5 μm particles (Hypersil BDS, Alltech, Fresno, CA, USA).
The elution was performed with a mobile phase containing methanol/water 80:20 v/v at a flow rate of 0.8 mL/min. The retention time of CLO was 6.8 min.
CLO encapsulation efficiency (EE) was calculated as follows [11 (link)]:
All data were the mean of four determinations on different batches of the same type of LS.

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)).