Complete ammonia-oxidizing Nitrospira and ammonia-oxidizing archaea as biological supplements for freshwater aquaria

Abstract

Biological filtration in newly established aquaria depends on the establishment of nitrifying microorganisms that prevent accumulation of toxic ammonia and nitrite. Many aquarists use commercial nitrifying supplements to accelerate biofilter establishment and reduce ammonia and nitrite accumulation. However, the microbial composition and in situ efficacy of these products remain poorly characterized. We evaluated the ability of enrichment cultures containing comammox Nitrospira (CX) and the ammonia-oxidizing archaeon Ca. Nitrosotenuis aquarius (AQ) to serve as aquarium nitrifying supplements, tested alongside a conventional supplement (CS) containing ammonia-oxidizing bacteria. Inoculation of new aquaria containing zebrafish ( Danio rerio ) with nitrifying supplements resulted in distinct nitrifier profiles within biofilters of each treatment group corresponding with significant reductions in peak ammonia and nitrite concentrations, which were most evident when applied at the maximum tested supplement dose. Treating aquaria with CX, AQ, or CS maintained ammonia and nitrite at low concentrations, and CX effectively prevented ammonia and nitrite accumulation through all stages of aquarium operation and biofilter establishment. After removing all fish from the aquaria, ammonia “spike-in” experiments demonstrated biofilters treated with CX and AQ conferred long-term protection against nitrite accumulation following acute ammonia loading events. Whereas 16S rRNA gene sequences associated with CS were rarely detected within biofilter profiles, those associated with CX and AQ established early and persisted temporally. Overall, results demonstrate how nitrifiers might be differentially adapted to home aquarium conditions and the potential of AOA and comammox Nitrospira to improve biofilter establishment and maintaining safe water quality in newly established freshwater aquaria. Importance: Establishing nitrifying microbial populations in freshwater aquarium biofilters is required for maintaining water quality and preventing ammonia and nitrite toxicity. Although new aquaria may be seeded by commercial nitrifying supplements, the microbial composition and in situ efficacy of these cultures remain poorly characterized. This study tests the ability of aquarium-derived enrichment cultures of comammox Nitrospira and the ammonia-oxidizing archaeon Candidatus Nitrosotenuis aquarius for use as nitrifying supplements. Results demonstrate that both cultures augment nitrification within newly established freshwater aquaria, especially at increased dosages. Compared to an existing supplement containing ammonia-oxidizing bacteria, the comammox-containing enrichment maintained ammonia and nitrite oxidation activity within filters to prevent ammonia and nitrite accumulation. These results connect microbial community establishment with a practical bioaugmentation application and demonstrate the suitability of supplement microorganisms to establish and persist within biofilter biofilm and reveal that inoculum size is an important consideration for validating nitrifying cultures for supplement application.