A modified method from a previous study was applied to the analysis. Samples were centrifuged at 4 °C and 5,000 × g for 10 min and DNA was extracted from the bacterial sediment using a DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The quantity of DNA was determined by real-time polymerase chain reaction (PCR) using Qiagen HotStarTaq DNA Polymerase and primers specific for the V4 region of the 16S rRNA gene, along with a probe. The V4 region of the 16S rRNA gene was amplified with specific primers containing adapters and indices for sample identification. Each sample was amplified in duplicate. The PCR reaction contained 18 µL of AccuPrime™ Pfx SuperMix (Invitrogen, Carlsbad, CA, USA), 1.2 µL of each primer (10 µM) and 1.2 µL of extracted DNA. The cycling conditions were as follows: initial denaturation at 95 °C for 5 min, 30 cycles at 95 °C for 15 s, at 55 °C for 30 s and at 68 °C for 1 min, followed by a final step at 68 °C for 5 min. The amplified indexed DNA fragments were purified and brought to the same concentration using the SequalPrep™ Normalization Plate Kit (Applied Biosystems™, Waltham, MA, USA). The amount of balanced PCR products was determined visually by gel electrophoresis. Samples were pooled and the concentration of each pool was measured using the Qubit™ dsDNA HS Assay Kit (Invitrogen). The final pool with approximately 2 nM concentration was sequenced on the Miseq platform (Illumina, San Diego, CA, USA) using the V2 kit of 2 × 250 bp.