Temporal and spatial stability of ammonia-oxidizing archaea and bacteria in aquarium biofilters

Abstract

Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4-5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥ 81-86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium biofilters, and that AOA community composition within a given aquarium is stable over time and across biofilter support material types.

Figure 1. Relative amoA gene abundance of Thaumarchaea and Bacteria in sampled freshwater (F1–F6) and marine (M) aquaria, assuming 1 and 2.5 copies of amoA gene per thaumarchaeal and bacterial cell, respectively.

Figure 2. Denaturing gradient gel electrophoresis of thaumarchaeal 16S rRNA gene amplicons from freshwater (F1–F6) and marine (M) aquarium, with an unweighted pair group method with arithmetic mean (UPGMA) dendrogram representing distances based on Pearson correlations of fingerprint densitometric curves.

Fingerprints have been normalized and aligned. Bands chosen for sequencing are indicated with triangles and numbering on the right side of lanes.

Figure 3. Nonmetric multidimensional scaling (NMDS) ordination of thaumarchaeal 16S rRNA gene DGGE fingerprint.

The two-dimensional stress value for the NMDS was 0.107 based on Bray Curtis distance. Coefficients of determination (R ²) on each axis represent correlations between ordination distances and the corresponding distance matrix.

Figure 4. Maximum likelihood phylogenetic tree of thaumarchaeal 16S rRNA gene sequences and DGGE bands based on 500 bootstrap values.

Only bootstrap values greater than 50% are indicated. The scale bar represents 5% nucleotide divergence.

Figure 5. Spatial AOA distribution in multi-media freshwater biofilters F5 (top panel) and F6 (bottom panel).

Error bars correspond to standard deviations based on triplicate qPCR amplifications. Dashed lines separate sampling days. For sample labels, refer to Table 1.

Figure 6. Denaturing gradient gel electrophoresis of thaumarchaeal 16S rRNA genes from spatially distinct locations within freshwater biofilters F5 and F6 during spatial test.

For detailed characteristics of the samples, refer to Table 1.