Comammox Nitrospira among dominant ammonia oxidizers within aquarium biofilter microbial communities

ABSTRACT Nitrification by aquarium biofilters transforms ammonia waste (NH 3 /NH 4 + ) to less toxic nitrate (NO 3 - ) via nitrite (NO 2 - ). Prior to the discovery of complete ammonia-oxidizing (“comammox” or CMX) Nitrospira , previous research revealed that ammonia-oxidizing archaea (AOA) dominated over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Here, we profiled aquarium biofilter microbial communities and quantified the abundance of all three known ammonia oxidizers using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquaria. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples (average CMX amoA genes: 2.2 × 10 3 ± 1.5 × 10 3 copies/ng) and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples (average AOA amoA genes: 1.1 × 10 3 ± 2.7 × 10 3 copies/ng) and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters (average of 3.2 × 10 1 ± 1.1 × 10 2 copies of AOB amoA genes/ng of DNA), except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results demonstrate that comammox Nitrospira plays a previously overlooked, but important role in home aquarium biofilter nitrification. IMPORTANCE Nitrification is a crucial process that converts toxic ammonia waste into less harmful nitrate that occurs in aquarium biofilters. Prior research found that ammonia-oxidizing archaea (AOA) were dominant over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Our study profiled microbial communities of aquarium biofilters and quantified the abundance of all currently known groups of aerobic ammonia oxidizers. The findings reveal that complete ammonia-oxidizing (comammox) Nitrospira were present in all freshwater aquarium biofilter samples in high abundance, challenging our previous understanding of aquarium nitrification. We also highlight niche adaptation of ammonia oxidizers based on salinity. The network analysis of freshwater biofilter microbial communities revealed significant positive correlations among nitrifiers and other community members, suggesting intricate interactions within biofilter communities. Overall, this study expands our understanding of nitrification in aquarium biofilters, emphasizes the role of comammox Nitrospira , and highlights the value of aquaria as microcosms for studying nitrifier ecology.