Amplicon of UMSR sludge metagenome: day 18
DNA was extracted from each fresh sludge sample using the Bacteria DNA Isolation Kit Components (MOBIO, USA). And then the variable V3 and V4 regions of the bacterial 16S-rRNA gene in the DNA extracts were amplified using the primer pair 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) via PCR. The composition of the PCR products was then detected by pyrosequencing on the Illumina Miseq sequencer platform (Majorbio, Shanghai, China). The obtained raw data was demultiplexed, quality filtered, and analyzed using Quantitative Insights Into Microbial Ecology (QIIME) (version 1.9.1). The optimized sequences were clustered into different operational taxonomy units (OTUs) at a 97% similarity level by Uparse (version 7.0.1090). Taxonomic information of the OTU representative sequences was annotated using the Ribosomal Database Project (RDP) Classifier with a confidence threshold of 70%, referencing the Silva database.