0.2 μm polycarbonate filters

A total of 20 surface water samples were collected from Lake Bosten between 2010 and 2011. These samples were obtained from five different sampling sites, as illustrated in Figure 1. The collection of water samples followed a seasonal schedule, with undisturbed duplicate water columns being collected in August 2010 (summer), October 2010 (fall), January 2011 (winter), and May 2011 (spring). To preserve the integrity of the samples, they were transferred to sterile containers immediately after collection. Subsequently, subsamples of 500–1,000 mL from each water column were filtered through 0.2 μm polycarbonate filters (Millipore) in the laboratory, using a vacuum pump. The filtered samples were carefully stored at −80°C until DNA extraction for 16S rRNA gene analysis. The remaining water samples were transported to the laboratory for chemical analysis.

Sampling for the bacterial community was performed at monthly intervals. Since the selected reservoirs were shallow, samples were collected from approximately 50 cm below the surface by using a five-liter organic glass water sampler, which was cleaned by rinsing with water from the specific location and sealed after overflow of sampling water to avoid any air bubbles. Reservoirs were sampled within a 2–3 h duration (9:00–12:00 A.M.) on the same day. Concurrent with bacterioplankton sampling, the water temperature and transparency were determined in situ. Samples were stored in 2-L pre-sterilized polypropylene bottles, transported to the laboratory and stored in the dark at 4 °C until analysis. In the lab, 1000–2000 mL water samples were filtered by 0.2-μm polycarbonate filters (diameter 50 mm, Millipore, Billerica, MA, USA) and the polycarbonate filters were then stored at −20 °C until DNA extraction. Environmental variables were measured for water samples, including total phosphorus, total nitrogen, Chlorophyll a, Dissolved Organic Carbon (DOC), Permanganate Index, and the modified trophic state index (TSI_M), using standard methods stated in the literature [22 (link)].

Arabian Sea depth profiles are described in (Pitcher et al., 2011 (link)). Large-volumes of seawater (200–1700 L) were filtered through 142-mm diameter 0.2-μm polycarbonate filters (Millipore, Billerica, MA). Filters were cut into fragments prior to extraction. Cells were lysed by bead-beating with 1.5 g of sterile 0.1 mm zirconium beads (Biospec, Bartlesville, OK) in a extraction buffer containing 10 mM Tris-HCl pH 8, 25 mM Na₂ EDTA pH 8, 1% (v/v) sodium dodecyl sulfate (SDS), 100 mM NaCl, and molecular biology grade water. Samples were incubated at 70°C for 30 min and then extracted with phenol-chloroform (Sambrook et al., 1989 ). After extraction, DNA was precipitated using ice-cold ethanol, dried, and re-dissolved in 100 μl of 10 mM Tris-HCl, pH 8. Total nucleic acid concentrations were quantified spectrophotometrically (Nanodrop, Thermo Scientific, Wilmington, DE, USA) and checked by agarose gel electrophoresis for quality. Extracts were kept frozen at −80°C.