Nitrification rates in Arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea

Abstract

The functioning of Arctic soil ecosystems is crucially important for global climate, and basic knowledge regarding their biogeochemical processes is lacking. Nitrogen (N) is the major limiting nutrient in these environments, and its availability is strongly dependent on nitrification. However, microbial communities driving this process remain largely uncharacterized in Arctic soils, namely those catalyzing the rate-limiting step of ammonia (NH3) oxidation. Eleven Arctic soils were analyzed through a polyphasic approach, integrating determination of gross nitrification rates, qualitative and quantitative marker gene analyses of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and enrichment of AOA in laboratory cultures. AOA were the only NH3 oxidizers detected in five out of 11 soils and outnumbered AOB in four of the remaining six soils. The AOA identified showed great phylogenetic diversity and a multifactorial association with the soil properties, reflecting an overall distribution associated with tundra type and with several physico-chemical parameters combined. Remarkably, the different gross nitrification rates between soils were associated with five distinct AOA clades, representing the great majority of known AOA diversity in soils, which suggests differences in their nitrifying potential. This was supported by selective enrichment of two of these clades in cultures with different NH3 oxidation rates. In addition, the enrichments provided the first direct evidence for NH3 oxidation by an AOA from an uncharacterized Thaumarchaeota-AOA lineage. Our results indicate that AOA are functionally heterogeneous and that the selection of distinct AOA populations by the environment can be a determinant for nitrification activity and N availability in soils.

Figure 1

Relationship between gross nitrification and NH₃ oxidizer populations. In situ and potential gross nitrification rates (without or with amendment with NH₄Cl to a final concentration of 1.7–2.5 mℳ, respectively), abundance of AOA and AOB (amoA copies g⁻¹dw soil) and proportion of archaeal amoA sequences associated with the dominant AOA clade in the clone library from each soil. The hierarchical clustering dendrogram represents relationships between AOA populations and is based on weighted UniFrac pairwise distances (with normalized branch weights). All nodes of the dendrogram have Jackknife support values of 100% (after 1000 permutations). The scale represents the distance between populations in UniFrac units. Arch, archaeal; Bac, bacterial; ND, not detected. *Below estimated quantification limit in the highest amount of template tested but detected with the end point PCR assay.

Figure 2

Maximum-likelihood phylogenetic tree of archaeal amoA. Phylogeny is based on 51 sequences (590-bp long) and includes a representative of each phylotype (⩾93% nucleotide identity), closest BLAST hits with GenBank and major reference sequences. Tree was calculated based on the GTR+I+G model of DNA evolution (see Supplementary Information for details). Percentages of bootstrap replicates (1000 replicates) supporting the nodes are represented by filled circles: black, ⩾90% gray, ⩾80% and white, ⩾70%. Phylotypes from this study, sequences from cultured organisms and metagenomic clones are indicated in bold. Reference sequences are named as ‘environmental source (accession number)'. Different symbols represent different samples and are color-coded according to soil ecosystem. Numbers following the symbols represent the number of sequences obtained from the sample associated with the respective phylotype. Names on the right-hand side of the tree represent the main AOA clades defined in this study.

Figure 3

Relationship between AOA distribution and soil properties. CCA biplot of AOA phylotype relative abundances and selection of physico-chemical parameters (model 1). Symbols represent the 11 AOA phylotypes and are color-coded according to major origin of sequences. Symbols with two colors correspond to phylotypes dominating two distinct soil types. Symbols with more than one phylotype name represent overlapping phylotypes. The percentages of phylotype distribution variance explained by the two principal canonical axes are represented close to the axes. Conditional variables are represented by the black arrows.

Figure 4

Growth and activity of AOA in enrichment cultures. NO₂⁻ production (solid lines), NH₄⁺ consumption (dashed lines) and thaumarchaeal 16S rRNA gene copies at days 26 and 40 (bars) in nine enrichment cultures from moss tundra (Lon-mt2) and frost boil (Knu-fb) soils incubated at 4, 20 or 28 °C. Figures depict three example cultures out of nine cultures incubated at each temperature, exhibiting identical NH₃ oxidation activity. AOB were not detected in any of the cultures. Filled circles and bars: black, Knu-fb cultures; gray and white, two different Lon-mt2 cultures, respectively.