Aquarium nitrification revisited: Thaumarchaeota are the dominant ammonia oxidizers in freshwater aquarium biofilters

Abstract

Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in many terrestrial and aquatic environments. Although nitrification is the primary function of aquarium biofilters, very few studies have investigated the microorganisms responsible for this process in aquaria. This study used quantitative real-time PCR (qPCR) to quantify the ammonia monooxygenase (amoA) and 16S rRNA genes of Bacteria and Thaumarchaeota in freshwater aquarium biofilters, in addition to assessing the diversity of AOA amoA genes by denaturing gradient gel electrophoresis (DGGE) and clone libraries. AOA were numerically dominant in 23 of 27 freshwater biofilters, and in 12 of these biofilters AOA contributed all detectable amoA genes. Eight saltwater aquaria and two commercial aquarium nitrifier supplements were included for comparison. Both thaumarchaeal and bacterial amoA genes were detected in all saltwater samples, with AOA genes outnumbering AOB genes in five of eight biofilters. Bacterial amoA genes were abundant in both supplements, but thaumarchaeal amoA and 16S rRNA genes could not be detected. For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonium concentration. DGGE of AOA amoA genes revealed variable diversity across samples, with nonmetric multidimensional scaling (NMDS) indicating separation of freshwater and saltwater fingerprints. Composite clone libraries of AOA amoA genes revealed distinct freshwater and saltwater clusters, as well as mixed clusters containing both freshwater and saltwater amoA gene sequences. These results reveal insight into commonplace residential biofilters and suggest that aquarium biofilters may represent valuable biofilm microcosms for future studies of AOA ecology.

Figure 1. Relative gene abundances of Bacteria and Thaumarchaetoa in aquaria.

Relative 16S rRNA (A) and amoA (B) gene abundances for bacteria and thaumarchaea in saltwater (SW1–SW8) and freshwater (FW1–F27) aquaria and in aquarium supplements (SP1, SP2). These data were calculated from duplicate qPCR amplifications.

Figure 2. Freshwater aquaria ammonium concentrations and relative thaumarchaeal amoA gene abundances.

AOA amoA gene copies are expressed as a percentage of the total amoA gene copies (per ng DNA). The coefficient of determination (R²) for the linear regression is 0.7249. The associated Pearson correlation coefficient (r) is −0.8518, with an associated p-value of <0.001. See Table S1 for all sample data.

Figure 3. Denaturing gradient gel electrophoresis of thaumarchaeal amoA gene amplicons.

Saltwater (A) and freshwater (B) fingerprints have been normalized and aligned. Bands chosen for sequencing are indicated with triangles: white triangles correspond to bands appearing in Figure 4. Black triangles represent failed sequencing reactions. Clustering of freshwater and saltwater fingerprints (C) is based on nonmetric multidimensional scaling (NMDS) using Pearson correlations of background-subtracted densitometric curves.

Figure 4. NMDS ordination of translated thaumarchaeal amoA gene sequences.

Sequences were obtained from clone libraries and DGGE bands derived from freshwater and saltwater aquarium filter samples. The inset panel provides a summary of the number of freshwater and saltwater clone library sequences contained within each cluster. Sequences obtained from DGGE bands correspond to the white triangles in Figure 3, and are labelled with sample and band numbers. Selected Genbank sequences from uncultivated clones and reference strains are included for comparison. The sampling environment and Genbank accession numbers for all reference sequences are included within parentheses.