Cellulose nitrate membrane filter

In this study, event-based precipitation samples were collected from June 2014 to May 2016. In total we collected 235 precipitation samples consisting of 201 rainfall events and 34 snowfall events. To reduce evaporation effects on isotopes, samples were transferred from the precipitation collector to sealed glass vials (Qorpak Bottles, Fisher Scientific Co. Germany) immediately after each event. The samples were then stored at 4 °C until isotope analysis. If the precipitation event was finished after midnight, sampling was conducted at the earliest possible time in the morning. Snowfall samples were melted in sealed plastic bags, poured into the vials and then stored. Prior to measurements, samples containing impurities were filtered with 0.45 μm syringe filters (Cellulose Nitrate Membrane Filters, GE Healthcare Co. UK) or centrifuged (Iec Centra CL2 Centrifuge, Thermo Electron Co. USA) depending on the size of the impurities.

Graphene oxide (GO) was purchased from Graphenea. Pyrrole monomer (Py; 97%) was obtained from Merck and distilled prior to use. Nickel nitrate hexahydrate (Ni(NO₃)₂·6H₂O; 98%) and cobalt nitrate hexahydrate (Co(NO₃)₂·6H₂O; 98%) were supplied by Sigma Aldrich. The reducing agent, hydrazine monohydrate (N₂H₄·H₂O, 100%) was supplied by Nacalai Tesque, Inc. Ferric chloride hexahydrate (FeCl₃·6H₂O, 98%) employed as an oxidizing agent was procured from Bendosen. Sodium hydroxide (NaOH; 98.4%) and sodium sulfate (Na₂SO₄; 99%) were obtained from Friendemann Schmidt and Merck, respectively. Indium tin oxide (ITO) coated glass with a sheet resistance of 7 Ω sq⁻¹ acquired from Xin Yan Technology Limited was utilized as a current collector. The cellulose nitrate membrane filters (pore size, 0.45 μm and ∅, 47 mm) and filter papers were received from GE Healthcare Life Science, UK and Whatman, UK, respectively. Deionized water (Millipore Milli-Q, 18.3 MΩ cm @ 25 °C) was used throughout the experimental. All chemicals utilized in this study were of analytical grade and used without any further purification unless otherwise stated.

This protocol was adapted to grow reproducible biofilms under anoxic and normoxic conditions. The method has previously been described (Bjarnsholt et al., 2015 ). The filter biofilms were kept on the same LB plates throughout the experiment. P. aeruginosa was propagated from frozen stock and grown overnight in 20 mL LB medium at 37°C in an orbital shaker at180 rpm. Cultures were adjusted to an optical density (OD₆₀₀ nm) of 0.05 (UV spectrophotometer UV-1800 UV-VIS, Shimadzu corporation, JP) and 10 μL was transferred to the cellulose nitrate membrane filters (25 mm in diameter, GE Healthcare Life Sciences, United Kingdom). Plates were incubated under normoxic and anoxic conditions and kept in plastic bags with wet paper to avoid dehydration. Two filters were sampled per biological replicate (n = 4) on each sampling day (day 1, 3, 7, 15 and 17). Filters were removed, placed in 10 mL tubes containing 5 mL 0.9% NaCl and vortexed thoroughly (1 min) prior to CFU determination. CFU/mL was determined as previously described.