Freshwater Recirculating Aquaculture System Operations Drive Biofilter Bacterial Community Shifts around a Stable Nitrifying Consortium of Ammonia-Oxidizing Archaea and Comammox Nitrospira

Recirculating aquaculture systems (RAS) are unique engineered ecosystems that minimize environmental perturbation by reducing nutrient pollution discharge. RAS typically employ a biofilter to control ammonia levels produced as a byproduct of fish protein catabolism. (ammonia-oxidizing), , and (nitrite-oxidizing) species are thought to be the primary nitrifiers present in RAS biofilters. We explored this assertion by characterizing the biofilter bacterial and archaeal community of a commercial scale freshwater RAS that has been in operation for >15 years. We found the biofilter community harbored a diverse array of bacterial taxa (>1000 genus-level taxon assignments) dominated by (~12%) and (~9%). The bacterial community exhibited significant composition shifts with changes in biofilter depth and in conjunction with operational changes across a fish rearing cycle. also were abundant, and were comprised solely of a low diversity assemblage of (>95%), thought to be ammonia-oxidizing archaea (AOA) from the presence of AOA ammonia monooxygenase genes. were present at all depths and time points. However, their abundance was >3 orders of magnitude less than AOA and exhibited significant depth-time variability not observed for AOA. Phylogenetic analysis of the nitrite oxidoreductase beta subunit ( ) gene indicated two distinct populations were present, while were not detected. Subsequent identification of ammonia monooxygenase alpha subunit genes in conjunction with the phylogenetic placement and quantification of the genotypes suggests complete ammonia-oxidizing (comammox) and nitrite-oxidizing populations co-exist with relatively equivalent and stable abundances in this system. It appears RAS biofilters harbor complex microbial communities whose composition can be affected directly by typical system operations while supporting multiple ammonia oxidation lifestyles within the nitrifying consortium.