Response of Microbial Community Structures and Functions of Nitrosifying Consortia to Biorefractory Humic Substances

Biorefractory humic substances (HS) that are ubiquitously present in nitrogen-rich wastewater streams, such as landfill leachate and livestock waste after anaerobic digestion, can potentially impact nitritation-anammox processes. In this study, multiple sequencing methods (e.g., 16S rRNA sequencing, clone library analysis, and metagenome sequencing) were employed to reveal the response of nitrosifying microbiota to HS at various concentrations (0-50 mg/L). Long-term reactor operation revealed that the nitrite yield was overall stable during all of the experimental days; however, fluctuations were observed as a result of sudden HS loads. The characterization by 16S rRNA sequencing indicated a decreased abundance of the phylum Proteobacteria (from 90.8% in HS0 to 52.1% in HS50) and an increased abundance of the phylum Bacteroidetes (from 4.7% in HS0 to 35.3% in HS50) upon exposure to HS. Both 16S rRNA sequencing and metagenome sequencing revealed that the family Nitrosomonadaceae, which was dominated by the genus Nitrosomonas, dramatically decreased, i.e., from ca. 70% in HS0 to ca. 40% in HS50. Both amoA-based clone libraries and metagenome sequencing suggested a substantial shift of AOB species, e.g., the emergence and enrichment of Nitrosomonas mobiliz upon exposure to HS. Interestingly, the amoABC gene was initially inhibited by 5 mg/L HS and then recovered at a higher level of HS (50 mg/L). In comparison, levels of hao gene were reduced with increasing HS (0, 5, and 50 mg/L). In addition, the abundance of genes assigned to membrane transport decreased after the addition of HS, and this reduction was likely associated with the electron shuttle roles of HS. Overall, the findings of this study provide insights into the response of core species and key genes to HS in nitritation systems.