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The recent discovery of comammox (complete ammonia oxidation) Nitrospirahas upended the long-held nitrification paradigm. Although comammox Nitrospirahave been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospirain a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospiraover canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02–0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospirawere identified, which are phylogenetically close to CandidatusNitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR.

Complete ammonia oxidation (i.e., comammox) is a newly discovered microbial process performed by a subset of the Nitrospiragenus, and this unique microbial process has been ubiquitously detected in various wastewater treatment units. However, the operational conditions favoring comammox prevalence remain unclear. In this study, the dominance of comammox Nitrospirain four sponge biofilm reactors fed with low-strength ammonium (NH4+= 23 ± 3 mg N/L) wastewater was proved by coupling 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing. The results showed that comammox Nitrospiradominated in the nitrifying guild over canonical ammonia-oxidizing bacteria (AOB) constantly, despite the significant variation in the residual ammonium concentration (0.01–15 mg N/L) under different sets of operating conditions. This result indicates that sponge biofilms greatly favor retaining comammox Nitrospirain wastewater treatment and highlights an essential role of biomass retention in the comammox prevalence. Moreover, analyses of the assembled metagenomic sequences revealed that the retrieved amoA gene sequences affiliated with comammox Nitrospira(53.9–66.0% read counts of total amoA gene reads) were always higher than those (28.4–43.4%) related to β-proteobacterial AOB taxa. The comammox Nitrospirabacteria detected in the present biofilm systems were close to clade A CandidatusNitrospira nitrosa.