Cellulose nitrate

HPLC autosampler vials were purchased from Waters (LCGC-certified amber glass, 8 × 40 mm), ThermoFisher (polypropylene 8 × 40 mm), and National Scientific (non-silanized and silanized, 1.5 mL vials). 15 mL polypropylene and polystyrene centrifuge tubes were manufactured by Becton Dickinson and Sarstedt, respectively. The 10 × 75 and 12 × 75 mm borosilicate glass tubes were branded as and purchased from VWR. All glass and plastic tubes were new and not previously used. The following 47 mm filters were purchased from Whatman: GF/F, GF/C, 0.2 μm nylon, cellulose acetate, cellulose nitrate, and polyamide; 0.45 μm cellulose acetate; and cellulose nitrate and those from Millipore were 0.22 μm polyethersulfone (PES) and 0.4 μm polycarbonate (Isopore HTTP). HPLC grade water, thiamine hydrochloride, trichloroacetic acid (TCA), sodium hydroxide, and potassium ferricyanide were purchased from VWR or Fisher Scientific. All chemicals were ACS grade. The polypropylene filter flask was manufactured by Nalgene, and the polysulfone filter housing with integrated polypropylene filter support was manufactured by Advantec.

To collect biomass for metagenomic sequencing, we removed larger particles and eukaryotes such as Dunaliella salina, by pre-filtering samples through 3-μm filters (cellulose-nitrate, Millipore). For environmental DNA extraction, we mixed the water samples (120 L) and used 40 L for biomass collection using two strategies. Ten liters of the sample were centrifuged at 4500 rpm (3260×g) for 60 min, and retentate was used for DNA extraction using QIAprep® Miniprep (Qiagen). The second strategy was biomass collection on polycarbonate membrane filters with 0.22-μm pore size (Isopore Membrane Filter, Isopore™ Millipore), and finally DNA extraction from filters using Qiagen DNeasy (Qiagen). The quantity and quality of the extracted DNA were analyzed by a NanoDrop™ One C Microvolume UV–Vis Spectrophotometer and agarose gel electrophoresis.