influent microbes
Bacterial genomic DNA of influent and biofilm was extracted using the PowerSoil DNA extraction kit (MO BIO Laboratories, Inc., Carlsbad, CA) according to the manufacturer’s protocol. The V1–V3 region of bacterial 16S rRNA genes was amplified using a broadly conserved primer set (8F: 5’-AGAGTTTGATCCTGGCTCAG-3’; 533R: 5’-TTACCGCGGCTGCTGGCAC-3’). The PCR mixture (total volume of 100 μL) contained the following components: 5 U of Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), 1×Pfu reaction buffer, 0.2 mM of dNTPs (TaKaRa, Dalin, China), 0.1 mM of each barcoded primer and 20 ng of genomic DNA template. Thermal cycling consisted of initial denaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and an extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min. The amplicons were gel purified using the TaKaRa Agarose Gel DNA Purification Kit (TaKaRa, China) and quantified with NanoDrop. The purified 16S rRNA gene amplicons (200 ng) were pooled and subjected to pyrosequencing using the ROCHE 454 FLX Titanium platform (Roche, Basel, Switzerland) at the National Human Genome Centre of China at Shanghai, China (CHGC).