16s rDNA of activated sludge
First, the DNA of the sample were extracted using a tool-kit (E.Z.N.A. Soil 165 DNA Kit, Omega Bio-tek, USA) according to the guidance. Second, the DNA extract on a 1% agarose gel was analyzed in a UV-Vis spectrophotometer (NanoDrop 2000 Thermo Scientific, USA) to ensure the DNA concentration and purity. Third, the samples were sent to a commercial company (Majorbio Company, China) for gene sequencing. Briefly, hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified in a PCR (GeneAmp 9700 PCR thermocycler, ABI Company, USA) with the primer pairs 338F (5'-CCTACGGGAGGCAGCAG-3') and 806R (5'-ATTACCGCGGCTGCTGG-3'). The amplification conditions of PCR were as follows: initial denaturation at 95  for 3 min, followed by 35 cycles of denaturing at 95  for 30 s, annealing at 60  for 30 s and extension at 72  for 40 s, and single extension at 72  for 10 min. After extracting PCR products from 2% agarose gel, the DNA were additionally purified using a tool-kit (AxyPrep DNA Gel Extraction Kit, Axygen Biosciences, USA) and quantified in a next generated sequencer (Quantus Fluorometer, Promega, USA).