Occurrence and temporal dynamics of denitrifying protist endosymbionts in the wastewater microbiome

Effective wastewater treatment is of critical importance for preserving public health and protecting natural environments. Key processes in wastewater treatment, such as denitrification, are performed by a diverse community of prokaryotic and eukaryotic microbes. However, the diversity of the microbiome and the potential role of the different microbial taxa in some wastewater treatment plant setups is not fully understood. We aimed to investigate the presence and diversity of denitrifying bacteria of the candidate family Azoamicaceae that form obligate symbioses with protists in wastewater treatment plants. Our analyses showed that denitrifying endosymbionts belonging to the Ca. Azoamicus genus are present in 20-50% of wastewater treatment plants worldwide. Time-resolved amplicon data from four Danish wastewater treatment plants showed high temporal fluctuations in the abundance and composition of the denitrifying endosymbiont community. Fifteen high-quality metagenome-assembled genomes of denitrifying endosymbionts, four of which were circular, were recovered. Genome annotation showed that a newly described, globally widespread species, Ca. Azoamicus parvus, lacked a nitrous oxide reductase, suggesting that its denitrification pathway is incomplete. This observation further expands the diversity of metabolic potentials found in denitrifying endosymbionts and indicates that the contribution of microbial eukaryote holobionts to the wastewater ecosystem dynamics of nitrogen removal and greenhouse gas production may have been underestimated and needs further study.