Tff system

Water samples at 3.0, 5.0, and 5.5 m depth were collected from Lake Shunet (54° 25’N, 90° 13’E) on July 21, 2010. The collection procedure was described in our previous research¹⁴ . 20 L of water were pumped from each depth into four sterile 5-L polypropylene bottles. Water was filtered through 10-μm plankton net to remove large particles. The water was stored for 3 h in the bottles, and part of the water was then transferred into sterile 2.0-ml screw tubes (SSIbio^®) and stored at −80 °C until DNA extraction. The rest of water was concentrated using tangential flow filtration (TFF) system (Millipore) with 0.22-μm polycarbonate membrane filters. The bacteria in the retentate were then retained on cellulose acetate membranes (0.2 μm pore size; ADVANTEC, Tokyo, Japan) and stored at −80 °C until DNA extraction. The physicochemical properties of samples were mentioned in a previous study¹⁴ . The pH was 8.1, 7.6, and 6.7; water temperature was 15.5, 9.5, and 7.5 °C; and salinity was 26, 40, and 71 g/L in the 3.0-, 5.0-, and 5.5-m samples, respectively.

GVH was passed through ultrafiltration (UF) membranes (molecular weight cut-offs of 1 kDa, 5 kDa, and 10 kDa) using a laboratory-scale tangential flow filtration (TFF) system (Millipore, Burlington, MA, USA). GVH was subjected to molecular weight fractionation to obtain peptides with molecular weights <1 kDa (1 kDa or smaller), 1–5 kDa (between 1 and 5 kDa), 5–10 kDa (between 5 and 10 kDa), and >10 kDa (10 kDa and larger). All recovered fractions were lyophilized and stored at −80 °C until use.