Low diversity and microdiversity of comammox bacteria in wastewater systems suggests wastewater-specific adaptation within the Ca. Nitrospira nitrosa cluster

Abstract

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%) across systems was significantly higher than that of the Nitrosomonas-like ammonia oxidizers (73%) and the Nitrospira-like nitrite oxidizer MAGs (75), 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.

Co-occurrence and cooperation between comammox and anammox bacteria in a full-scale attached growth municipal wastewater treatment process

“…Our previous work demonstrated that comammox bacteria are most prevalent in nitrogen removal systems treating wastewater with an attached growth phase or long solids retention times, and they often co-occur with strict AOB and Nitrospira-NOB 8 . Further, a large portion of comammox bacteria detected in wastewater systems, including those in our past studies 8,9 , belong to clade A1 comammox bacteria and are affiliated with Ca. Nitrospira nitrosa-like populations [10][11][12] .…”

“…MAGs associated with Brocadia (n=2) and Nitrospira (n=3) were obtained from biomass attached to IFAS media even though anammox bacteria were not found in our past study 8,9 . Nitrospira and Brocadia MAGs represented 6.53  0.34 % and 6.25  1.33% of total reads in the sample.…”

“…In order to calculate the relative abundances of the nitrifying bacteria, MAGs generated from this and the past study 9 were grouped and dereplicated using drep v2.5.4 46 at 95% ANI with completeness and contamination thresholds set to 50% and 10%, respectively. Only MAGs with genome coverage (proportion of the genome covered by at least one read) higher than 50% in each sample were used to calculate relative abundances.…”

“…The implications of operating at much lower DO, such as those suggested in other studies 6,18 (less than 1 mg/L), may limit comammox bacterial ammonia oxidation such that they are unable to produce nitrite for anammox bacteria. MAGs from this IFAS system 9 . Two amoA gene sequences in the metagenomic assembly were aligned using BLAST with previously assembled MAGs obtained from the same IFAS system.…”

“…In this study, we report the co-occurrence and cooperation of comammox and anammox bacteria for nitrogen removal in a full-scale integrated fixed film activated sludge (IFAS) system. Our previous studies at the Hampton Roads Sanitation District (HRSD) James River Treatment Plant (JRTP) in Virginia, USA found high abundance of comammox bacteria in the attached growth phase with their concentration routinely exceeding those of canonical AOB 8,9 . Follow-up experiments to determine the nitrification kinetics of comammox bacteria indicated the potential for the presence of anammox bacteria including nitrogen loss consistent with anammox stoichiometry.…”