Nitrification, the oxidation of ammonia to nitrate via nitrite, is an important process in municipal wastewater treatment plants (WWTPs). Members of the
Nitrospiragenus that contribute to complete ammonia oxidation (comammox) have only recently been discovered and their relevance to engineered water treatment systems is poorly understood. This study investigated distributions of Nitrospira, ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB) in biofilm samples collected from tertiary rotating biological contactors (RBCs) of a municipal WWTP in Guelph, Ontario, Canada. Using quantitative PCR (qPCR), 16S rRNA gene sequencing, and metagenomics, our results demonstrate that Nitrospiraspecies strongly dominate RBC biofilm samples and that comammox Nitrospiraoutnumber all other nitrifiers. Genome bins recovered from assembled metagenomes reveal multiple populations of comammox Nitrospirawith distinct spatial and temporal distributions, including several taxa that are distinct from previously characterized Nitrospiramembers. Diverse functional profiles imply a high level of niche heterogeneity among comammox Nitrospira, in contrast to the sole detected AOA representative that was previously cultivated and characterized from the same RBC biofilm. Our metagenome bins also reveal two cyanase-encoding populations of comammox Nitrospira, suggesting an ability to degrade cyanate, which has only been shown previously for several Nitrospirarepresentatives that are strict nitrite oxidizers. This study demonstrates the importance of RBCs as model systems for continued investigation of environmental factors that control the distributions and activities of AOB, AOA, comammox Nitrospira, and other nitrite oxidizers.