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Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle

Nature volume 549, pages 269272 (14 September 2017) | Download Citation


Nitrification, the oxidation of ammonia (NH3) via nitrite (NO2) to nitrate (NO3), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalysed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira1,2, which alone convert ammonia to nitrate, raised questions about the ecological niches in which comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolate a pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats on the basis of a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, AOA are not necessarily the most competitive ammonia oxidizers present in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analysed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM1 (ref. 3). These results suggest a role for comammox organisms in nitrification under oligotrophic and dynamic conditions.

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European Nucleotide Archive


  1. 1.

    et al. Complete nitrification by Nitrospira bacteria. Nature 528, 504–509 (2015)

  2. 2.

    et al. Complete nitrification by a single microorganism. Nature 528, 555–559 (2015)

  3. 3.

    , , , & Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461, 976–979 (2009)

  4. 4.

    in Nitrification (eds , & ) 347–383 (ASM Press, 2011)

  5. 5.

    in Nitrification (eds , & ) 95–114 (ASM Press, 2011)

  6. 6.

    et al. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437, 543–546 (2005)

  7. 7.

    . et al. Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442, 806–809 (2006)

  8. 8.

    , & Comparison of oxidation kinetics of nitrite-oxidizing bacteria: nitrite availability as a key factor in niche differentiation. Appl. Environ. Microbiol. 81, 745–753 (2015)

  9. 9.

    , , , & Microscale distribution of populations and activities of Nitrosospira and Nitrospira spp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors. Appl. Environ. Microbiol. 65, 3690–3696 (1999)

  10. 10.

    et al. Enrichment and characterization of an autotrophic ammonia-oxidizing archaeon of mesophilic crenarchaeal group I.1a from an agricultural soil. Appl. Environ. Microbiol. 77, 8635–8647 (2011)

  11. 11.

    et al. Cultivation of autotrophic ammonia-oxidizing archaea from marine sediments in coculture with sulfur-oxidizing bacteria. Appl. Environ. Microbiol. 76, 7575–7587 (2010)

  12. 12.

    , & A new perspective on microbes formerly known as nitrite-oxidizing bacteria. Trends Microbiol. 24, 699–712 (2016)

  13. 13.

    et al. Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proc. Natl Acad. Sci. USA 108, 8420–8425 (2011)

  14. 14.

    et al. Cyanate as an energy source for nitrifiers. Nature 524, 105–108 (2015)

  15. 15.

    et al. Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon “Ca. Nitrosotenuis uzonensis” representing a clade globally distributed in thermal habitats. PLoS One 8, e80835 (2013)

  16. 16.

    et al. A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring. Proc. Natl Acad. Sci. USA 105, 2134–2139 (2008)

  17. 17.

    et al. The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations. Environ. Microbiol. 14, 3122–3145 (2012)

  18. 18.

    & Archaeal and bacterial ammonia-oxidisers in soil: the quest for niche specialisation and differentiation. Trends Microbiol. 20, 523–531 (2012)

  19. 19.

    Ammonia oxidation: different niches for bacteria and archaea? ISME J. 4, 1092–1094 (2010)

  20. 20.

    Biochemical basis for whole-cell uptake kinetics: specific affinity, oligotrophic capacity, and the meaning of the Michaelis constant. Appl. Environ. Microbiol. 57, 2033–2038 (1991)

  21. 21.

    et al. Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation. Proc. Natl Acad. Sci. USA 111, 8239–8244 (2014)

  22. 22.

    , & Why is metabolic labour divided in nitrification? Trends Microbiol. 14, 213–219 (2006)

  23. 23.

    , & Freshwater recirculating aquaculture system operations drive biofilter bacterial community shifts around a stable nitrifying consortium of ammonia-oxidizing Archaea and comammox Nitrospira. Front. Microbiol. 8, 101 (2017)

  24. 24.

    et al. Metagenomic analysis of rapid gravity sand filter microbial communities suggests novel physiology of Nitrospira spp. ISME J. 10, 2569–2581 (2016)

  25. 25.

    et al. Metagenomic evidence for the presence of comammox Nitrospira-like bacteria in a drinking water system. MSphere 1, e00054–e00015 (2015)

  26. 26.

    et al. Comammox in drinking water systems. Water Res. 116, 332–341 (2017)

  27. 27.

    et al. AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment. Front. Microbiol. 8, 1508 (2017)

  28. 28.

    & Kinetic characteristics of ammonium-oxidizer communities in a California oak woodland-annual grassland. Soil Biol. Biochem. 28, 1307–1317 (1996)

  29. 29.

    et al. Isolation of ‘Candidatus Nitrosocosmicus franklandus’, a novel ureolytic soil archaeal ammonia oxidiser with tolerance to high ammonia concentration. FEMS Microbiol. Ecol. 92, fiw057 (2016)

  30. 30.

    et al. Growth of nitrite-oxidizing bacteria by aerobic hydrogen oxidation. Science 345, 1052–1054 (2014)

  31. 31.

    , & in Molecular Microbial Ecology (eds & ) 213–239 (Bios-Garland, 2005)

  32. 32.

    , , , & In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67, 5273–5284 (2001)

  33. 33.

    & in The Prokaryotes (eds et al.) 3352–3378 (Springer, 1992)

  34. 34.

    et al. Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon from soil and a member of the archaeal phylum Thaumarchaeota. Int. J. Syst. Evol. Microbiol. 64, 2738–2752 (2014)

  35. 35.

    , & mmgenome: a toolbox for reproducible genome extraction from metagenomes. Preprint at (2016)

  36. 36.

    & Short-term assay of soil urease activity using colorimetric determination of ammonium. Biol. Fertil. Soils 6, 68–72 (1988)

  37. 37.

    , , , & Alternative methods for measuring inorganic, organic, and total dissolved nitrogen in soil. Soil Sci. Soc. Am. J. 74, 1018–1027 (2010)

  38. 38.

    Physiological and Chemical Tests for Drinking Water. (Nederlands Normalisatie Instituut, 1966)

  39. 39.

    , & A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 5, 62–71 (2001)

  40. 40.

    Nutrient uptake by microorganisms according to kinetic parameters from theory as related to cytoarchitecture. Microbiol. Mol. Biol. Rev. 62, 636–645 (1998)

  41. 41.

    et al. An acid-tolerant ammonia-oxidizing γ-proteobacterium from soil. ISME J. 11, 1130–1141 (2017)

  42. 42.

    & Competition for ammonium between nitrifying and heterotrophic bacteria in dual energy-limited chemostats. Appl. Environ. Microbiol. 57, 3255–3263 (1991)

  43. 43.

    , & Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts. J. Bacteriol. 120, 556–558 (1974)

  44. 44.

    , & Oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter hamburgensis grown in mixed continuous cultures at different oxygen concentrations. Arch. Microbiol. 161, 156–162 (1994)

  45. 45.

    , , & Influence of starvation on potential ammonia-oxidizing activity and amoA mRNA levels of Nitrosospira briensis. Appl. Environ. Microbiol. 71, 1276–1282 (2005)

  46. 46.

    , , , & The ammonia-oxidizing nitrifying population of the River Elbe estuary. FEMS Microbiol. Ecol. 17, 177–186 (1995)

  47. 47.

    et al. Effects of bacterial community members on the proteome of the ammonia-oxidizing bacterium Nitrosomonas sp strain Is79. Appl. Environ. Microbiol. 82, 4776–4788 (2016)

  48. 48.

    , & Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats. Arch. Microbiol. 157, 436–441 (1992)

  49. 49.

    & Kinetics of nitrite oxidation by Nitrobacter winogradskyi. Biochem. J. 85, 440–447 (1962)

  50. 50.

    , & Kinetics of Nitrobacter agilis at extreme substrate, product and salt concentrations. Appl. Microbiol. Biotechnol. 40, 442–448 (1993)

  51. 51.

    et al. Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation. J. Biosci. Bioeng. 123, 581–589 (2017)

  52. 52.

    , , , & Ammonia-oxidizing bacteria with different sensitivities to (NH4)2SO4 in activated sludges. Water Res. 28, 1523–1532 (1994)

  53. 53.

    Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus. Arch. Microbiol. 147, 126–133 (1987)

  54. 54.

    & A chemical model of seawater including dissolved ammonia and the stoichiometric dissociation constant of ammonia in estuarine water and seawater from −2 to 40°C. Geochim. Cosmochim. Acta 59, 2403–2421 (1995)

  55. 55.

    & Growth and oxidation kinetics of three genera of ammonia oxidizing nitrifiers. FEMS Microbiol. Lett. 7, 213–216 (1980)

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We thank A. Mueller for assistance with the cultivation of AOA strain 5A, M. Palatinszky for assistance with the cultivation of N. uzonensis, J. Vierheilig for help with molecular analyses and the cultivation of N. inopinata, and D. Gruber, N. Cyran, A. Klocker and S. A. Eichorst for assistance with sample preparation for electron microscopy. K.D.K., C.J.S., P.H., S.R. and M.W. were supported by the European Research Council Advanced Grant project NITRICARE 294343 (to M.W.). P.P. and H.D. were supported by the Austrian Science Fund (FWF) project P27319-B21, and A.D. and H.D. were supported by FWF project P25231-B21. K.D.K. and L.Y.S. were supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-03745). M.A. was supported by research grant 15510 from the VILLUM FONDEN.

Author information


  1. Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, Research Network Chemistry meets Microbiology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria

    • K. Dimitri Kits
    • , Christopher J. Sedlacek
    • , Ping Han
    • , Petra Pjevac
    • , Anne Daebeler
    • , Stefano Romano
    • , Holger Daims
    •  & Michael Wagner
  2. Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, Bld 2, 119071 Moscow, Russia

    • Elena V. Lebedeva
    •  & Alexandr Bulaev
  3. Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark

    • Mads Albertsen
  4. Department of Biological Sciences, University of Alberta, CW405 Biological Sciences Building, Edmonton, Alberta T6G 2E9, Canada

    • Lisa Y. Stein


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H.D. and M.W. designed this study and wrote the manuscript with the help of all authors. K.D.K. and C.J.S. performed the kinetic and yield experiments. E.V.L. and A.B. purified N. inopinata. P.H., E.V.L. and A.B. enriched the N. uzonensis-related AOA strain 5A. A.D. and S.R. performed electron microscopy of N. inopinata. P.P. and L.Y.S. helped with data interpretation. M.A. performed purity checks of N. inopinata and AOA strain 5A by Illumina sequencing and bioinformatics analyses.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Holger Daims.

Reviewer Information Nature thanks M. Kuypers, A. Schramm and M. Strous for their contribution to the peer review of this work.

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    Supplementary Information

    This file contains a short formal description of Nitrospira inopinata sp. nov. (as a pure culture of this organism is first described in this manuscript), a Supplementary Discussion of genome-based hypotheses on the niche specialization of Nitrospira inopinata, and Supplementary References.

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