Sample Processing, DNA Extraction, and PCR Amplification

The water samples used for the microbiological analyses were sent back to the laboratory in a special ice box on the same day, and the water samples were filtered with a circulating water vacuum pump on a super-clean worktable, and the water samples were filtered through a 0.22-μm filter membrane. To ensure the reliability of the experimental results, three replications were made for each sample. The filtered membrane was preserved at −80°C. To avoid contamination, sterile techniques were used throughout all processes. According to the manufacturer’s protocols, microbial DNA was extracted using the HiPure Soil DNA Kits and HiPure Stool DNA Kits (Magen, China). We amplified the V3-V4 region of the bacterial 16S rRNA genes with the bacterial universal primers 341 F (CCTACGGGNGGCWGCAG) and 806 R (GGACTACHVGGGTATCTAAT). PCR reactions were performed in triplicate, with 50-μl mixtures containing 5 μl of 10 × KOD Buffer, 5 μl of 2.5 mM dNTPs, 1.5 μl of each primer (5 μM), 1 μl of KOD polymerase, and 100 ng of template DNA. The PCR amplification conditions were 95°C for 2 min, followed by 27 cycles at 98°C for 10 s, 62°C for 30 s, and 68°C for 30 s and a final extension at 68°C for 10 min.