Nucleopore

Liposomes used in this study contain a 4:4:2:0.5:0.01 molar ratio of 1,2,dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, total ganglioside extract (TGE) and Oregon green DHPE, based on compositions found in previous studies that provide comparative data19 21 (link). The lipids were purchased from Avanti Polar Lipids (Alabster, AL), and Oregon green DHPE was purchased from Molecular Probes, Eugene, OR. To form liposomes, all components were dissolved and mixed in biotechnology grade chloroform (Sigma-Aldrich) in a glass vial. The bulk solvent was first removed by blowing high purity nitrogen and the solution was placed in a desiccator under vacuum for 3 h to ensure complete evaporation of the solvent. GPMV buffer (50 mM Hepes, 150 mM NaCl, 2 mM CaCl₂, pH 7.4) was then added to the vial to re-suspend the dried lipid film to create a 5 mg/mL solution. Liposomes were then extruded ten times through a 100 nm pore size polycarbonate filter (Whatman Nucleopore), and five times through a 50 nm pore size filter.

Samples were collected from luer-lock sampling ports to measure microcystin content, cell concentration/size, and for RNA extraction. Samples (50 mL) for microcystin quantification were collected on 47-mm glass filters (Advantec MFS GF75, 0.3 μm retention) via vacuum filtration, flash-frozen in liquid nitrogen, and stored at −80°C until processing. Samples for RNA (30 mL) were collected on 47-mm diameter polycarbonate filters (Whatman Nucleopore, 1.0 μm pore-size) via vacuum filtration, flash-frozen in liquid nitrogen, and stored at −80°C until extraction.

The LIVE/DEAD® Baclight™ kit (Invitrogen) was used to assess membrane integrity of bacterial cells after exposure to biocides. In brief, an aliquot of 1 mL was filtered through a 0.2-μm Nucleopore® (Whatman) black polycarbonate membrane and stained with 250 μl of SYTO9™ and 50 μl of propidium iodide (PI) in the dark (Barros et al., 2021 (link)). After reacting for 7 min, the stains were filtered and the samples mounted on a slide with immersion oil. Then, samples were observed using a 100x objective oil immersion fluorescence objective of a LEICA DMLB2 microscope incorporated with a mercury lamp HBO/100W/3 and a CCD camera. The optical filters used were a combination of a 480–500 nm excitation filter and a 485 nm emission filter (Chroma 61000-V2 DAPI/FITC/TRITC; Ferreira et al., 2011 (link)). A IM50 software (LEICA) was used to record images. At least 15 images were captured for each sample and the experiments were repeated in three different occasions.

Multilamellar (MLVs) and large unilamellar (LUVs) phospholipid vesicles were prepared as previously described (Martínez-Ruiz et al., 2001 (link); García-Linares et al., 2013 (link); Alm et al., 2015 (link); Rivera-De-Torre et al., 2017 (link)). Briefly, a phospholipid solution in 2:1 (v/v) chloroform/methanol was first dried under a flow of nitrogen and then subjected to vacuum to remove residual solvents. The dry film obtained was used to prepare a lipid dispersion by adding 0.5–2.0 ml of 50 mM sodium phosphate buffer, pH 7.0, briefly vortex mixing, and incubating for 1 h at 37°C. When needed, this suspension of MLVs was further subjected to five cycles of extrusion at 37°C through polycarbonate filters (100-nm pore size) (Nucleopore, Whatman) to obtain a homogeneous population of LUVs. Laser scattering measurements were periodically conducted at the Spectroscopy and Correlation Facility of the Universidad Complutense to confirm LUV size homogeneity.

10 mL of collected water sample were fixed on site with sterile-filtered formaldehyde to the final concentration of 1%, transported to the laboratory and stored at 4 °C in the dark for <48 h. Half ml of the fixed water was filtered onto white 0.2 µm polycarbonate filters (Nucleopore, Whatman), air-dried and mounted on microscopic slides with an anti-fading glycerol mix containing 4′,6-diamidino-2-phenylindole (DAPI) at concentration of 1 µg mL^{−1 57 (link)}. They were stored at −20 °C until processed.
Total bacterial and APB abundance was determined using epifluorescence Zeiss Axio Imager.D2 microscope, as described in Cepáková et al.^{35 (link)}. Minimum 10 microphotographs were taken for every sample under UV/blue emission/excitation channel for DAPI fluorescence (total bacteria), blue/red emission/excitation channel for autofluorescence from Chl a (algae and cyanobacteria), and white light/infrared emission/excitation channel for autofluorescence from BChl a (APB). Minimum 1500 DAPI-stained cells were counted manually in ZEN software. As some part of Chl a autofluorescence is also visible in infrared spectrum, only the cells that did not showed autofluorescence from Chl a were counted as APB bacteria.

Dissolved primary production (¹⁴C-DOCp) rates were determined after a 2–3 h incubation period, from the 72-mL samples sets, using four different types of filters, and after a 9 h incubation, using 0.2-μm PC filters (Whatman, 25 mm ∅). The different types of filters used during the short incubations were borosilicate (25 mm Whatman GF/F, 0.7 μm), silver membrane (SM) (25 mm Steriltech, 0.2 μm), polycarbonate (25 mm Whatman nucleopore, 0.2 μm) and mixed acetate and nitrate cellulose (25 mm Millipore GSWP, 0.22 μm). For each incubation, two 5-mL replicates were taken from each 72-mL bottle and were filtered under low-vacuum pressure. Filtrates were subsequently acidified to a pH of ~2 with 100 μL of 50% HCl and kept overnight in open scintillation vials (20 mL) placed on an orbital shaker. After the removal of inorganic ¹⁴C, 15 mL of scintillation cocktail was added to each filtrate. The radioactivity on each filtrate and filter was determined following the procedure previously detailed in ¹⁴C-POCp measurements section.

Samples of 50 ml were fixed with buffered, sterile‐filtered paraformaldehyde (Penta, Prague, Czechia) to a final concentration of 1%, and 0.5 ml was filtered onto white polycarbonate filters (pore size 0.2 μm, Nucleopore, Whatman, Maidstone, UK). Cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) at concentration of 1 mg L⁻¹ (Coleman, 1980 ). Total and AAP bacterial abundances were determined using an epifluorescence Zeiss Axio Imager.D2 microscope equipped with Collibri LED module illumination system (Carl Zeiss, Jena, Germany). Ten microphotographs were taken for every sample under 325–370 nm excitation and 420–470 nm emission wavelengths for DAPI fluorescence (total bacteria), 450–490 nm excitation and 600–660 nm emission wavelengths for autofluorescence from Chl‐a (algae and cyanobacteria), and combined 325–370 nm, 450–490 nm, 545–565 nm and 615–635 nm excitation and 645–850 emission wavelengths for autofluorescence from BChl‐a (AAP bacteria). As some part of Chl‐a autofluorescence is also visible in the infrared spectrum, only the IR‐positive cells that did not show any autofluorescence from Chl‐a were counted as AAP bacteria (Cottrell et al., 2006 (link)).