The functional dominance and metabolic diversity of comammox Nitrospira in recirculating aquaculture systems

•Comammox bacteria and/or AOB numerically dominated ammonia oxidizer in RAS.•Comammox became functionally dominant across active AOMs revealed by DNA-SIP.•Nitrospira nitrificans and clade A.2 members acted as the active comammox species.•Comammox MAGs harbor distinct capability of stress resistance and metabolic diversity. As a newly discovered group of ammonia-oxidizing microorganisms, complete ammonia oxidizing (comammox) Nitrospira has been widely found in various oligotrophic ecosystems. However, their activity and ecological niche is still unclear in recirculating aquaculture systems (RAS). This study aimed to compare the abundance and activity of comammox Nitrospira, ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), and elucidate metabolic versatility of comammox Nitrospira in RAS. Quantitative PCR (qPCR) results showed that either comammox Nitrospira or AOB numerically predominated, while comammox Nitrospira and AOA shared similar low ammonia niches. Specifically, DNA-based stable isotope probing in conjunction with high-throughput 16S rRNA gene amplicon sequencing revealed that comammox Nitrospira accounted for 79.1 %, 97.5 %, 91.9 % and 97.6 % in the active ammonia-oxidizing community in four selected typical samples representing high abundance of comammox, AOA, and AOB, respectively. Phylogenetic analysis of heavy fraction DNA further identified novel comammox species from Nitrospira nitrificans cluster and clade A.2 acting as active species in different freshwater aquariums. Moreover, metagenome-assembled genome analysis revealed them as novel species with stress resistance and metabolic diversity compared with known comammox Nitrospira. This study underscores the dominant role of comammox Nitrospira as active ammonia-oxidizers in RAS and presents two novel comammox MAGs with metabolic flexibility, enriching our understanding of the nitrification process in oligotrophic artificial ecosystems. [Display omitted]