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Complete nitrification by a single microorganism

Naturevolume 528, pages555–559 (2015)Cite this article

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Abstract

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 twoNitrospira 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 similaramoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novelamoA 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.

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Figure 1: Ammonium oxidation by the enrichment culture.
Figure 2:In situ detection ofNitrospira and their ammonia-oxidizing capacity.
Figure 3: Schematic illustration of the AMO genomic region inNitrospira and selected ammonia-oxidizing bacteria.
Figure 4: Phylogenetic analysis of the AmoA/PmoA sequence family.

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Accession codes

Primary accessions

European Nucleotide Archive

Data deposits

Metagenomic data is available in the European Nucleotide Archive (ENA) under accession numbersCZQA01000001–CZQA01000015 andCZPZ01000001–CZPZ01000036.

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Acknowledgements

We would like to thank K. Stultiens, T. van Alen, J. Frank, P. Klaren, L. Pierson and L. Claessens-Joosten for technical assistance, T. Spanings for biofilter maintenance and C. Herbold for the ANI analysis. We are grateful for the use of the confocal microscope from the Microscopic Imaging Centre (MIC, Radboud UMC, Nijmegen) and would like to thank H. Croes and M. Willemse for technical assistance. The LABGeM team and the National Infrastructure “France Genomique” are acknowledged for support within the MicroScope annotation platform. We are thankful to C. Dupont, A. Santoro and M. Saito for consenting to our use of theNitrospira marina nxrA sequences, which were produced by the US Department of Energy Joint Genome Institute. M.A.H.J.v.K was supported by the Technology Foundation STW (grant 13146), D.R.S. by the BE-Basic Foundation (grant fs7-002), M.A. and P.H.N. by the Danish Council for Independent Research (DFF 4005-00369), M.S.M.J. by the European Research Council (ERC Advanced Grant projects anammox 232937 and Eco_MoM 339880) and the Dutch Ministry of Education, Culture and Science (Gravitation grant SIAM 024002002), B.K. and S.L. by the Netherlands Organization for Scientific Research (NWO VENI grants 863.11.003 and 863.14.019, respectively). The Radboud Excellence Initiative is acknowledged for support to S.L.

Author information

Authors and Affiliations

  1. Department of Microbiology, IWWR, Radboud University, Heyendaalseweg 135, Nijmegen, 6525, AJ, 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. Department of Chemistry and Bioscience, Center for Microbial Communities, Aalborg University, Fredrik Bajers Vej 7H, Aalborg, 9220, Denmark

    Mads Albertsen & Per H. Nielsen

  3. Laboratory for Microbiology, University of Gent, K. L. Ledeganckstraat 35, Gent, 9000, Belgium

    Boran Kartal

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

    Mike S. M. Jetten

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  1. Maartje A. H. J. van Kessel

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  2. Daan R. Speth

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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.

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Extended data figures and tables

Extended Data Figure 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.

Extended Data Figure 2 Metagenome binning.

a,b, Extraction of theNitrospira 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.

Extended Data Figure 3 Phylogenetic analysis of NXR.

Bayesian interference tree (s.d. = 0.0099) showing the affiliation of theNitrospira sp.1 and sp.2nxrA sequences in comparison to other genome-sequencedNitrospira,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 describedNitrospira 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.

Extended Data Figure 4 16S rRNA-based phylogenetic analysis.

Bayesian interference tree (s.d. = 0.0098) showing the affiliation of theNitrospira sp.1 and sp.2 16S rRNA sequences withinNitrospira sublineage II. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. The strongly supported sequence group containing the novelNitrospira spp. catalysing complete nitrification is shaded in grey, the two subgroups containingNitrospira 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 (seeExtended 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 ofNitrospira sublineages I and IV were used as outgroup (n = 24); the outgroup position is indicated by the arrow.

Extended Data Figure 5 Control experiments of AMO-labelling.

a, Cells incubated with the fluorescent dye FTCP (green) were stained by FISH using probes specific forNitrospira (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 thanNitrospira (Ntspa662, red; yellow overlay).c andd, Positive controls: ammonium oxidizing bacteria (c, Nso1225 and Nso190, red) in an aerobic enrichment culture and aNitrosomonas europaea pure culture (d, NEU, red, and EUB338mix, blue) were stained by FTCP (resulting in yellow and white overlays, respectively).e andf, Negative controls: canonicalNitrospira in an aerobic enrichment culture (e, Ntspa662, blue) and aNitrospira 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 andb) or one (c tof) individual experiments, with three technical replicates each. Scale bars in all panels represent 10 μm.

Extended Data Figure 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) inb 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 andb) or triplicate (c andd). Error bars represent standard deviations of three biological replicates.

Extended Data Figure 7 Ammonium and nitrite-dependent CO2 fixation shown by FISH-MAR.

a–d, FISH with probes for all bacteria (EUB338mix, blue), and probes specific forNitrospira (Ntspa662, red; resulting in magenta) and anammox bacteria (Amx820, green; resulting in cyan).a, Ammonia-dependent carbon fixation. OnlyNitrospira 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 activeNitrospira cells on the right side of the same floc.b, Inhibition of ammonia-dependent carbon fixation by ATU.c, Nitrite-dependent carbon fixation. OnlyNitrospira cells incorporated14CO2.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 Table 1 General genomic characteristics ofNitrospira sp.1 and sp.2
Extended Data Table 2 FISH probe specifications
Extended Data Table 3 Metagenome screening forNitrospira-likeamoA sequences

Supplementary information

Supplementary Table 1

Nitrospira sp.1 and sp.2 genes discussed in this study. (XLSX 16 kb)

Supplementary Table 2

Marker HMMs used by CheckM. (XLSX 20 kb)

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van Kessel, M., Speth, D., Albertsen, M.et al. Complete nitrification by a single microorganism.Nature528, 555–559 (2015). https://doi.org/10.1038/nature16459

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Editorial Summary

Time to rethink nitrification

Two groups this week report the enrichment and characterization ofNitrospira species that encode all of the enzymes necessary to catalyse complete nitrification, a phenotype referred to as 'comammox' (for complete ammonia oxidation). Until now, this two-step reaction was thought to involve two organisms in a cross-feeding interaction. Phylogenetic analyses suggest that comammoxNitrospira are present in a number of diverse environments, so these findings have the potential to fundamentally change our view of the nitrogen cycle and open a new frontier in nitrification research.

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