Low Diversity and Microdiversity of Comammox Bacteria in Wastewater Systems Suggest Specific Adaptations Within the Ca. Nitrospira Nitrosa Cluster

Studies have found Ca . Nitrospira nitrosa-like bacteria to be the principal or sole comammox bacteria in nitrogen removal systems for wastewater treatment. In contrast, multiple populations of strict ammonia and nitrite oxidizers co-exist in similar systems. This apparent lack of diversity is surprising and could impact the feasibility of leveraging comammox bacteria for nitrogen removal. We used full-length 16S rRNA gene sequencing and genome-resolved metagenomics to compare population-level (i.e., species) diversity of comammox bacteria with that of strict nitrifiers in full-scale wastewater treatment systems and assess whether these observations were consistent or diverged at the strain-level. Full-length 16S rRNA gene sequencing indicated that while Nitrosomonas -like bacteria exhibited higher population-level diversity, the effective microdiversity of most Nitrospira -like bacteria were comparatively higher except for one Nitrospira Lineage II population. Comammox bacterial metagenome assembled genomes (MAGs) were associated with Ca . Nitrospira nitrosa. The average amino acid identity between comammox bacterial MAGs (93% ± 3) across systems was significantly higher than that of the Nitrosomonas -like ammonia oxidizers (73%±8) and the Nitrospira -like nitrite oxidizer MAGs (75%±13), suggesting that the same comammox population was detected in all systems. Comammox bacteria and some ammonia oxidizers MAGs were significantly less microdiverse than most ammonia and nitrite oxidizers. Interestingly, strain-resolved analysis also indicates that different nitrogen removal systems harbor different comammox bacterial strains within the Ca . Nitrospira nitrosa cluster. These results suggest that comammox bacteria associated with Ca . Nitrospira nitrosa have low species- and strain-level diversity in nitrogen removal systems and may thus harbor specific adaptations to the wastewater ecosystem. ### Competing Interest Statement The authors have declared no competing interest.