Complete nitrification by a single microorganism

Journal name:
Nature
Volume:
528,
Pages:
555–559
Date published:
DOI:
doi:10.1038/nature16459
Received
Accepted
Published online

Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 18901, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle2. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms3. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

At a glance

Figures

  1. Ammonium oxidation by the enrichment culture.
    Figure 1: Ammonium oxidation by the enrichment culture.

    a, 29N2 (open circles) and 30N2 (filled circles) production from 15NH4+ by the enrichment culture. b, Ammonium (diamonds) oxidation to nitrate (squares) in aerobic batch incubations in the absence (filled symbols) and presence (open symbols) of ATU. Nitrite concentrations were below the detection limit (<5 μM) at all time points. c, Nitrite (triangles) oxidation to nitrate (squares) in aerobic batch incubations. In b and c, total nitrogen balances are indicated (dashed lines). Symbols in all plots represent averages of three individual experiments. Ammonium concentrations were determined in single measurements, other compounds in triplicate. Error bars represent standard deviations of three biological replicates.

  2. In situ detection of Nitrospira and their ammonia-oxidizing capacity.
    Figure 2: In situ detection of Nitrospira and their ammonia-oxidizing capacity.

    a, Co-aggregation of Nitrospira and Brocadia in the enrichment. Cells are stained by FISH with probes for all bacteria (EUB338mix, blue), and specific for Nitrospira (Ntspa712, green, resulting in cyan) and anammox bacteria (Amx820, red, resulting in magenta). b, AMO labelling by FTCP (green). Nitrospira was counterstained by FISH (probes Ntspa662 (blue) and Ntspa476 (red), resulting in white). c, Ammonium-dependent CO2 fixation by Nitrospira shown by FISH-MAR. Silver grain deposition (black) above cell clusters indicates 14CO2 incorporation. Nitrospira was stained by FISH (probes Ntspa476 (red) and Ntspa662 (blue), resulting in magenta). Images in b and c are representative of two individual experiments, with three (b) or two (c) technical replicates each. Scale bars in all panels represent 10 μm.

  3. Schematic illustration of the AMO genomic region in Nitrospira and selected ammonia-oxidizing bacteria.
    Figure 3: Schematic illustration of the AMO genomic region in Nitrospira and selected ammonia-oxidizing bacteria.

    The AMO locus in Nitrospira sp.1 in comparison to sp.2 and the beta- and gammaproteobacterial ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosococcus oceani, respectively. The position of NXR on the AMO-containing Nitrospira contigs is also indicated. Homologous genes are connected by lines. Functions of the encoded proteins are represented by colour, the arrow shows direction of transcription. Numbers specify amino acid identities to Nitrospira sp.1. Parallel double lines designate a break in locus organization. Locus tags for each organism are listed on the right. Genes are drawn to scale. amo, ammonia monooxygenase; bfr; bacterioferritin; ccm, cytochrome c biogenesis; cop, copper transport; cyc, cytochrome c; dct, sodium:dicarboxylate symporter; hao, hydroxylamine dehydrogenase; nxr, nitrite oxidoreductase.

  4. Phylogenetic analysis of the AmoA/PmoA sequence family.
    Figure 4: Phylogenetic analysis of the AmoA/PmoA sequence family.

    Bayesian interference tree (s.d. = 0.01) showing the affiliation of the Nitrospira AmoA. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. Scale bars indicate 10% sequence divergence. a, Radial tree indicating the localization of the novel AmoA/‘unusual’ PmoA sequence group in relation to the main functional groups within the sequence family. Numbers in brackets indicate sequences per group (137 sequences in total). Amo, ammonia monooxygenase; Emo, ethane monooxygenase; Hmo, hydrocarbon/butane monooxygenase; Pmo/Pxm, particulate methane monooxygenase. b, Cladogram detailing the affiliation of the Nitrospira sp.1 (green box) and sp.2 (red box) AmoA sequences within this sequence group. Nitrospira and Crenothrix sequences are depicted in bold. One representative sequence per study is shown for highly similar sequences; numbers in brackets indicate the number of sequences represented.

  5. Ammonium and nitrite conversion by the enrichment culture.
    Extended Data Fig. 1: Ammonium and nitrite conversion by the enrichment culture.

    a, b, Inorganic nitrogen load of the enrichment culture per 24 h cycle (filled symbols) and effluent concentrations (open symbols) for ammonium (a, diamonds) and nitrite (b, triangles). Effluent nitrite concentrations were below the detection limit (<5 μM) at all time points. Data points represent the mean of three technical replicates, error bars the standard deviations of these triplicates. Nitrate concentration in the medium varied between 0.5 and 2.0 mM and total organic carbon (TOC) content between 1.30 and 1.44 ppm, which was due to medium preparation with water obtained directly from the recirculation aquaculture system.

  6. Metagenome binning.
    Extended Data Fig. 2: Metagenome binning.

    a, b, Extraction of the Nitrospira sp.1 (a) and sp.2 (b) genome sequences from the metagenome using differential coverage binning. Each circle represents a metagenomic scaffold, with size proportional to scaffold length; the plots contain a total of 47,584 scaffolds. The inlay of each figure shows the secondary binning based on tetranucleotide frequencies, with a total of 331 (a) and 281 (b) scaffolds included. Taxonomic classification is indicated by colour; a total of 3,158 essential marker genes were detected. The extracted bins are enclosed by a dashed line. c, d, Genome contaminations were excluded by generating linkage maps of the final bins of sp.1 (c, 25 scaffolds) and sp.2 (d, 86 scaffolds) using mate-pair sequencing data.

  7. Phylogenetic analysis of NXR.
    Extended Data Fig. 3: Phylogenetic analysis of NXR.

    Bayesian interference tree (s.d. = 0.0099) showing the affiliation of the Nitrospira sp.1 and sp.2 nxrA sequences in comparison to other genome-sequenced Nitrospira, Nitrospina and anammox bacteria. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. NCBI protein accession numbers for all publicly available sequences are indicated, numbers with an asterisk are IMG gene IDs. The described Nitrospira sublineages are indicated by coloured boxes and roman numbers. The scale bar represents 10% sequence divergence. Note the different affiliation of the “Candidatus N. nitrosa” (sp.1) nxrA sequences. The tree contains 25 sequences from 12 species, belonging to 3 different phyla. Sequences from closely related bacterial putative nitrate reductases were used as outgroup (n = 4); the outgroup position is indicated by the arrow.

  8. 16S rRNA-based phylogenetic analysis.
    Extended Data Fig. 4: 16S rRNA-based phylogenetic analysis.

    Bayesian interference tree (s.d. = 0.0098) showing the affiliation of the Nitrospira sp.1 and sp.2 16S rRNA sequences within Nitrospira sublineage II. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. The strongly supported sequence group containing the novel Nitrospira spp. catalysing complete nitrification is shaded in grey, the two subgroups containing Nitrospira sp.1 and sp.2 (in bold) are highlighted by green and red boxes, respectively. N. moscoviensis is depicted in bold for comparison. The curly bracket indicates the target group of the newly designed FISH probe Ntspa476 (see Extended Data Table 2). Scale bar indicates 10% sequence divergence. The tree contains a total of 181 sequences; the size of sequence groups is indicated in brackets. Sequences from members of Nitrospira sublineages I and IV were used as outgroup (n = 24); the outgroup position is indicated by the arrow.

  9. Control experiments of AMO-labelling.
    Extended Data Fig. 5: Control experiments of AMO-labelling.

    a, Cells incubated with the fluorescent dye FTCP (green) were stained by FISH using probes specific for Nitrospira (Ntspa662, red) and all bacteria (EUB338mix, blue). A small cell cluster was stained by FTCP and targeted by both probes (resulting in a white overlay signal), while all other bacteria (in blue) were not or only slightly stained by FTCP. The green signal is due to autofluorescence and unspecific FTCP binding to the floc matrix. b, Anammox cells (Amx820, blue) showed minor staining by FTCP (green), but to a much lesser degree than Nitrospira (Ntspa662, red; yellow overlay). c and d, Positive controls: ammonium oxidizing bacteria (c, Nso1225 and Nso190, red) in an aerobic enrichment culture and a Nitrosomonas europaea pure culture (d, NEU, red, and EUB338mix, blue) were stained by FTCP (resulting in yellow and white overlays, respectively). e and f, Negative controls: canonical Nitrospira in an aerobic enrichment culture (e, Ntspa662, blue) and a Nitrospira moscoviensis pure culture (f, Ntspa662, red, and EUB338mix, blue; magenta overlay) did not show any labelling with FTCP (green). The two bright green structures in (c) and the bright pink signal in (e) are due to autofluorescence. Images are representative of two (a and b) or one (c to f) individual experiments, with three technical replicates each. Scale bars in all panels represent 10 μm.

  10. Batch incubations with nitrite, urea and without substrate.
    Extended Data Fig. 6: Batch incubations with nitrite, urea and without substrate.

    a, b, Nitrite (triangles) oxidation by the enrichment culture to nitrate (squares) in the absence (a) and in the presence (b) of ATU. The ammonia (diamonds) in b presumably stems from biomass decay and is not oxidized owing to ATU inhibition. c, Urea conversion to ammonium (diamonds) and subsequent oxidation to nitrate (squares). d, No-substrate control; minor amounts of ammonium (diamonds) presumably stem from mineralisation of degrading biomass, leading subsequently to nitrate (squares) formation. Symbols in all plots represent averages of three independent incubations; ammonium was determined in single measurements, nitrite and nitrate in duplicate (a and b) or triplicate (c and d). Error bars represent standard deviations of three biological replicates.

  11. Ammonium and nitrite-dependent CO2 fixation shown by FISH-MAR.
    Extended Data Fig. 7: Ammonium and nitrite-dependent CO2 fixation shown by FISH-MAR.

    ad, FISH with probes for all bacteria (EUB338mix, blue), and probes specific for Nitrospira (Ntspa662, red; resulting in magenta) and anammox bacteria (Amx820, green; resulting in cyan). a, Ammonia-dependent carbon fixation. Only Nitrospira cells were active, as indicated by silver grain deposition. Note the inactive anammox cells on the left side of the smaller floc, co-localizing with highly active Nitrospira cells on the right side of the same floc. b, Inhibition of ammonia-dependent carbon fixation by ATU. c, Nitrite-dependent carbon fixation. Only Nitrospira cells incorporated 14CO2. d, No-substrate control. Images are representative of two individual experiments, with two technical replicates each. Scale bars in all panels represent 10 μm.

Tables

  1. General genomic characteristics of Nitrospira sp.1 and sp.2
    Extended Data Table 1: General genomic characteristics of Nitrospira sp.1 and sp.2
  2. FISH probe specifications
    Extended Data Table 2: FISH probe specifications
  3. Metagenome screening for Nitrospira-like amoA sequences
    Extended Data Table 3: Metagenome screening for Nitrospira-like amoA sequences

Accession codes

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Author information

Affiliations

  1. Department of Microbiology, IWWR, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands

    • Maartje A. H. J. van Kessel,
    • Daan R. Speth,
    • Huub J. M. Op den Camp,
    • Boran Kartal,
    • Mike S. M. Jetten &
    • Sebastian Lücker
  2. Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark

    • Mads Albertsen &
    • Per H. Nielsen
  3. Laboratory for Microbiology, University of Gent, K. L. Ledeganckstraat 35, 9000 Gent, Belgium

    • Boran Kartal
  4. Department of Biotechnology, TU Delft, Julianalaan 67, 2628 BC Delft, the Netherlands

    • Mike S. M. Jetten

Contributions

M.A.H.J.v.K and S.L. executed experiments and analysed data. D.R.S. and M.A. contributed to metagenomic data analyses. M.A. and P.H.N. performed sequencing, assembly and binning. M.A.H.J.v.K., H.J.M.O.d.C., B.K., M.S.M.J. and S.L. planned research. M.A.H.J.v.K., B.K. and S.L. wrote the paper. All authors discussed results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Metagenomic data is available in the European Nucleotide Archive (ENA) under accession numbers CZQA01000001CZQA01000015 and CZPZ01000001CZPZ01000036.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Ammonium and nitrite conversion by the enrichment culture. (60 KB)

    a, b, Inorganic nitrogen load of the enrichment culture per 24 h cycle (filled symbols) and effluent concentrations (open symbols) for ammonium (a, diamonds) and nitrite (b, triangles). Effluent nitrite concentrations were below the detection limit (<5 μM) at all time points. Data points represent the mean of three technical replicates, error bars the standard deviations of these triplicates. Nitrate concentration in the medium varied between 0.5 and 2.0 mM and total organic carbon (TOC) content between 1.30 and 1.44 ppm, which was due to medium preparation with water obtained directly from the recirculation aquaculture system.

  2. Extended Data Figure 2: Metagenome binning. (225 KB)

    a, b, Extraction of the Nitrospira sp.1 (a) and sp.2 (b) genome sequences from the metagenome using differential coverage binning. Each circle represents a metagenomic scaffold, with size proportional to scaffold length; the plots contain a total of 47,584 scaffolds. The inlay of each figure shows the secondary binning based on tetranucleotide frequencies, with a total of 331 (a) and 281 (b) scaffolds included. Taxonomic classification is indicated by colour; a total of 3,158 essential marker genes were detected. The extracted bins are enclosed by a dashed line. c, d, Genome contaminations were excluded by generating linkage maps of the final bins of sp.1 (c, 25 scaffolds) and sp.2 (d, 86 scaffolds) using mate-pair sequencing data.

  3. Extended Data Figure 3: Phylogenetic analysis of NXR. (447 KB)

    Bayesian interference tree (s.d. = 0.0099) showing the affiliation of the Nitrospira sp.1 and sp.2 nxrA sequences in comparison to other genome-sequenced Nitrospira, Nitrospina and anammox bacteria. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. NCBI protein accession numbers for all publicly available sequences are indicated, numbers with an asterisk are IMG gene IDs. The described Nitrospira sublineages are indicated by coloured boxes and roman numbers. The scale bar represents 10% sequence divergence. Note the different affiliation of the “Candidatus N. nitrosa” (sp.1) nxrA sequences. The tree contains 25 sequences from 12 species, belonging to 3 different phyla. Sequences from closely related bacterial putative nitrate reductases were used as outgroup (n = 4); the outgroup position is indicated by the arrow.

  4. Extended Data Figure 4: 16S rRNA-based phylogenetic analysis. (526 KB)

    Bayesian interference tree (s.d. = 0.0098) showing the affiliation of the Nitrospira sp.1 and sp.2 16S rRNA sequences within Nitrospira sublineage II. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. The strongly supported sequence group containing the novel Nitrospira spp. catalysing complete nitrification is shaded in grey, the two subgroups containing Nitrospira sp.1 and sp.2 (in bold) are highlighted by green and red boxes, respectively. N. moscoviensis is depicted in bold for comparison. The curly bracket indicates the target group of the newly designed FISH probe Ntspa476 (see Extended Data Table 2). Scale bar indicates 10% sequence divergence. The tree contains a total of 181 sequences; the size of sequence groups is indicated in brackets. Sequences from members of Nitrospira sublineages I and IV were used as outgroup (n = 24); the outgroup position is indicated by the arrow.

  5. Extended Data Figure 5: Control experiments of AMO-labelling. (1,114 KB)

    a, Cells incubated with the fluorescent dye FTCP (green) were stained by FISH using probes specific for Nitrospira (Ntspa662, red) and all bacteria (EUB338mix, blue). A small cell cluster was stained by FTCP and targeted by both probes (resulting in a white overlay signal), while all other bacteria (in blue) were not or only slightly stained by FTCP. The green signal is due to autofluorescence and unspecific FTCP binding to the floc matrix. b, Anammox cells (Amx820, blue) showed minor staining by FTCP (green), but to a much lesser degree than Nitrospira (Ntspa662, red; yellow overlay). c and d, Positive controls: ammonium oxidizing bacteria (c, Nso1225 and Nso190, red) in an aerobic enrichment culture and a Nitrosomonas europaea pure culture (d, NEU, red, and EUB338mix, blue) were stained by FTCP (resulting in yellow and white overlays, respectively). e and f, Negative controls: canonical Nitrospira in an aerobic enrichment culture (e, Ntspa662, blue) and a Nitrospira moscoviensis pure culture (f, Ntspa662, red, and EUB338mix, blue; magenta overlay) did not show any labelling with FTCP (green). The two bright green structures in (c) and the bright pink signal in (e) are due to autofluorescence. Images are representative of two (a and b) or one (c to f) individual experiments, with three technical replicates each. Scale bars in all panels represent 10 μm.

  6. Extended Data Figure 6: Batch incubations with nitrite, urea and without substrate. (86 KB)

    a, b, Nitrite (triangles) oxidation by the enrichment culture to nitrate (squares) in the absence (a) and in the presence (b) of ATU. The ammonia (diamonds) in b presumably stems from biomass decay and is not oxidized owing to ATU inhibition. c, Urea conversion to ammonium (diamonds) and subsequent oxidation to nitrate (squares). d, No-substrate control; minor amounts of ammonium (diamonds) presumably stem from mineralisation of degrading biomass, leading subsequently to nitrate (squares) formation. Symbols in all plots represent averages of three independent incubations; ammonium was determined in single measurements, nitrite and nitrate in duplicate (a and b) or triplicate (c and d). Error bars represent standard deviations of three biological replicates.

  7. Extended Data Figure 7: Ammonium and nitrite-dependent CO2 fixation shown by FISH-MAR. (665 KB)

    ad, FISH with probes for all bacteria (EUB338mix, blue), and probes specific for Nitrospira (Ntspa662, red; resulting in magenta) and anammox bacteria (Amx820, green; resulting in cyan). a, Ammonia-dependent carbon fixation. Only Nitrospira cells were active, as indicated by silver grain deposition. Note the inactive anammox cells on the left side of the smaller floc, co-localizing with highly active Nitrospira cells on the right side of the same floc. b, Inhibition of ammonia-dependent carbon fixation by ATU. c, Nitrite-dependent carbon fixation. Only Nitrospira cells incorporated 14CO2. d, No-substrate control. Images are representative of two individual experiments, with two technical replicates each. Scale bars in all panels represent 10 μm.

Extended Data Tables

  1. Extended Data Table 1: General genomic characteristics of Nitrospira sp.1 and sp.2 (79 KB)
  2. Extended Data Table 2: FISH probe specifications (191 KB)
  3. Extended Data Table 3: Metagenome screening for Nitrospira-like amoA sequences (430 KB)

Supplementary information

Excel files

  1. Supplementary Table 1 (16 KB)

    Nitrospira sp.1 and sp.2 genes discussed in this study.

  2. Supplementary Table 2 (20 KB)

    Marker HMMs used by CheckM.

Additional data