Phylogenetic Analysis of Attached Microbial Communities in Aerobic and Anoxic Media for the Removal of Wastewater Nitrogen

The removal of nitrogen compounds in wastewater has been successfully developed with various activated sludge-based processes. Microorganisms immobilized in media would enhance biological efficiency by the increase in biomass concentration; however, the microbial community composition in media has not been revealed. Attached microbial communities on immobilization media were analyzed after the operation of the wastewater treatment process, comparing aerobic and anoxic reactors. A modified Ludzack–Ettinger (MLE) process was operated with immobilized media with polyvinyl alcohol and polyethylene glycol. The mixed liquor suspended solid (MLSS) concentration in an aerobic reactor was maintained at 50,000 mg/L and 40,000 mg/L in an anoxic reactor by the media. A maximum of 99% of ammonium nitrogen from the influent was calculated to be oxidized; however, the organic nitrogen produced from microbial growth reduced the overall oxidation rate. The denitrification rate increased with the addition of glucose to adjust the carbon-to-nitrogen (C/N) ratio. Based on the total nitrogen concentration, the nitrogen removal efficiency was calculated to be 48.2% following the adjustment of the C/N ratio. A phylogenetic analysis of the microbial community in immobilized media using next-generation sequencing (NGS) revealed the dominance of nitrifying and denitrifying microorganisms in the aerobic and anoxic reactors, respectively. Sequences amplified using V3–V4 region primers of the 16S rRNA gene yielded 531,188 base pairs (bp) and 396,844 bp reads from the aerobic and anoxic reactors, respectively. Operational taxonomic units (OTUs) were identified at both the phylum and genus levels, with a total of 594 from the aerobic reactor and 375 from the anoxic reactor. Proteobacteria was the dominant phylum in both the aerobic and anoxic reactors, comprising 39.7% of the aerobic reactor and 65.9% of the anoxic reactor. The dominant genera in the aerobic reactor were Nitrospira and Povalibacter. Forty-five percent of the total number of OTUs consisted of known nitrification-related genera in the aerobic reactor. In contrast, the dominant genera in the anoxic reactor were Desulfomicrobium, Desulfobulbus, and Methyloversatilis. A total of 63% of the genera associated with denitrification, including Dechloromonas and Flavobacterium, were found in the anoxic reactor. The population of microorganisms in each reactor was compared in terms of diversity by the QIIME 2 algorithm. The Chao1 index values of α-diversity were 606.05 for the aerobic reactor and 415.53 for the anoxic reactor, indicating greater population diversity in the aerobic reactor compared to the anoxic one. The widespread distribution of nitrification activities among various groups has led to diverse population characteristics in the aerobic environment, particularly within the attached community. The microbiological community present in immobilized aerobic and anoxic media will contribute to future microbial studies on wastewater treatment processes.