Sampling, DNA Extraction, and Sequencing

Sludge samples were collected at 23 different time points from MBR. Each time, CO₂ was blown into the MBR to pump out the 50-ml mixed liquor before being collected in a 50-mL falcon tube. The collected sample was centrifuged at 4,000 rpm for 10 min to spin down the cell pellet. The supernatant was discarded, with the pellet aseptically transferred into a sterile 2-ml tube. All samples were stored at -80°C before DNA extraction. The DNA of 23 samples was extracted using the DNeasy Powersoil kit (QIAGEN, Germany) according to the manufacturer’s instructions. The DNA extracts were then subjected to barcoded polymerase chain reaction (PCR) amplification, with the barcodes added to the 5′ end of the PCR primers. The primers 515F (3′-GTGYCAGCMGCCGCGGTAA-5′) and 806R (5′- GGACTACNVGGGTWTCTAAT-3′) were used to amplify the V4 regions of bacterial 16S rRNA genes. PCR reaction solutions (50 μl) were prepared by mixing 25-μl 2 × Premix Taq (Takara Biotechnology, Dalian Co. Ltd., China), 1-μl forward and reverse primers (10 μM), and 3-μl DNA (20 ng/μl) template and nuclease-free water. Thermocycling conditions for PCR were set as follows: 5 min at 94°C for initialization; 30 cycles of 30-s denaturation at 94°C, 30-s annealing at 52°C, and 30-s extension at 72°C; followed by 10-min final elongation at 72°C. PCR products were purified with the TaKaRa MiNiBEST DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Japan) and then quality-checked with gel electrophoresis. Sequencing libraries were generated using NEBNext ^® Ultra™ II DNA Library Prep Kit for Illumina ^® (New England Biolabs, MA, United States) following manufacturer’s recommendations, and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, MA, United States). At last, the library was sequenced on an Illumina Nova6000 platform, and 250-bp paired-end reads were generated (Guangdong Magigene Biotechnology Co., Ltd., Guangzhou, China).

For shotgun metagenomic sequencing, a subset of six DNA samples were selected around six different time points in the four periods of the experiment: day 95 (D95) and day 110 (D110) in the stabilization period (days 51–110); day 180 (D180) in the inhibition period (days 127–188); day 210 (D210) and day 227 (D227) in the adaptation period (days 188–228); and day 264 (D264) from the recovery period (days 228–280). Each of the six samples is a mixture of the sludge from the days mentioned earlier and 3 days before and after. DNA was extracted and assessed before library preparation and sequenced on an Illumina NovaSeq 6000 platform generating 150-bp paired-end reads (Novogene Co., Ltd., Nanjing, China).