2.3. Samping and Analytical Methods

The operating performance of the reactors was evaluated by analyzing influent and effluent samples on a daily basis. Samples were subsequently filtered through a 0.22 μm membrane filter. Influent and effluent samples were collected daily for both MBR and MBfR to analyze the concentration of NH4+-N, NO3−-N, and TN according to a standard method provided by the American Public Health Association (APHA). NO2−-N concentrations were determined using a colorimetric assay based on sulfanilamide and N-(1-naphthyl) ethylenediamine dihydrochloride. The absorbance was measured at 540 nm in a spectrophotometer (UV6100, METASH, Shanghai, China). The pH and DO were monitored in situ via a PLC system that was connected with pH and DO probes.

The biomass samples were sent out for microbial structure analysis at Novogene Co., Ltd. (Beijing, China). The relative abundance of partial nitrification bacteria, denitrification bacteria, and hydrogen autotrophic denitrifying bacteria were determined by high-throughput sequencing analysis. The V4-V5 hypervariable region of the 16s RNA gene was amplified by polymerase chain reaction (PCR). The amplified PCR used the bacterial primers 515F(5′-GTGCCAGCMGCCGCGGTAA-3′) and 907R(5′-CCGTCAATTCCTTTGAGTTT-3′). The operational taxonomic units (OTUs) of the representative sequences were annotated with the Ribosomal Database Project (RDP) classifier method and the Greengene database [21] for species annotation analysis (the threshold value was set to 0.8–1). The sequence number of each sample was normalized and the trimmed sequences were grouped into OTUs using 97% identity thresholds. [22]. Kingdom, phylum, class, order, family, genus, and species were used to analyze the community composition of each sample. Taxonomic classification at the genus level was performed using the RDP algorithm to classify the representative sequences from each OTU.