Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A microbial consortium couples anaerobic methane oxidation to denitrification


Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale1,2. Freshwater sediments often receive increased downward fluxes of nitrate from agricultural runoff and upward fluxes of methane generated by anaerobic decomposition3. In theory, prokaryotes should be capable of using nitrate to oxidize methane anaerobically, but such organisms have neither been observed in nature nor isolated in the laboratory4,5,6,7,8. Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate9,10. Here we show that the direct, anaerobic oxidation of methane coupled to denitrification of nitrate is possible. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitrification in the complete absence of oxygen. This consortium consisted of two microorganisms, a bacterium representing a phylum without any cultured species and an archaeon distantly related to marine methanotrophic Archaea. The detection of relatives of these prokaryotes in different freshwater ecosystems worldwide11,12,13,14 indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: AOM is coupled to the denitrification of nitrite by the enrichment culture after 16 months of enrichment.
Figure 2: Phylogeny and fluorescence in situ detection of the archaeal and bacterial members of the consortium mediating AOM coupled to denitrification.


  1. Frankenberg, C., Meirink, J. F., Van Weele, M., Platt, U. & Wagner, T. Assessing methane emissions from global space-borne observations. Science 308, 1010–1014 (2005)

    ADS  CAS  Article  Google Scholar 

  2. Galloway, J. N. et al. Nitrogen cycles: past, present, and future. Biogeochemistry 70, 153–226 (2004)

    CAS  Article  Google Scholar 

  3. Conrad, R. Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO). Microbiol. Rev. 60, 609–640 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Knowles, R. Denitrifiers associated with methanotrophs and their potential impact on the nitrogen cycle. Ecol. Eng. 24, 441–446 (2005)

    Article  Google Scholar 

  5. Shima, S. & Thauer, R. K. Methyl-coenzyme M reductase and the anaerobic oxidation of methane in methanotrophic Archaea. Curr. Opin. Microbiol. 8, 643–648 (2005)

    CAS  Article  Google Scholar 

  6. Strous, M. & Jetten, M. S. M. Anaerobic oxidation of methane and ammonium. Annu. Rev. Microbiol. 58, 99–117 (2004)

    CAS  Article  Google Scholar 

  7. Valentine, D. L. Biogeochemistry and microbial ecology of methane oxidation in anoxic environments: a review. Anton. Leeuw. Int. J. G. 81, 271–282 (2002)

    CAS  Article  Google Scholar 

  8. Mason, I. Methane as a carbon source in biological denitrification. J. Water Pollution Control Fed. 49, 855–857 (1977)

    CAS  Google Scholar 

  9. Orphan, V. J. et al. Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments. Proc. Natl Acad. Sci. USA 99, 7663–7668 (2002)

    ADS  CAS  Article  Google Scholar 

  10. Boetius, A. et al. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623–626 (2000)

    ADS  CAS  Article  Google Scholar 

  11. Kasai, Y., Takahata, Y., Hoaki, T. & Watanabe, K. Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil. Environ. Microbiol. 7, 806–818 (2005)

    CAS  Article  Google Scholar 

  12. Koizumi, Y., Kojima, H. & Fukui, M. Characterization of depth-related microbial community structure in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rDNA and reversely transcribed 16S rRNA fragments. FEMS Microbiol. Ecol. 46, 147–157 (2003)

    CAS  Article  Google Scholar 

  13. Bakermans, C. & Madsen, E. L. Diversity of 16S rDNA and naphthalene dioxygenase genes from coal-tar-waste-contaminated aquifer waters. Microb. Ecol. 44, 95–106 (2002)

    CAS  Article  Google Scholar 

  14. Stein, L. Y., La Duc, M. T., Grundl, T. J. & Nealson, K. H. Bacterial and archaeal populations associated with freshwater ferromanganous micronodules and sediments. Environ. Microbiol. 3, 10–18 (2001)

    CAS  Article  Google Scholar 

  15. Hallam, S. J. et al. Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305, 1457–1462 (2004)

    ADS  CAS  Article  Google Scholar 

  16. Krüger, M. et al. A conspicuous nickel protein in microbial mats that oxidize methane anaerobically. Nature 426, 878–881 (2003)

    ADS  Article  Google Scholar 

  17. Strous, M., Kuenen, J. G., Fuerst, J. A., Wagner, M. & Jetten, M. S. The anammox case—a new experimental manifesto for microbiological eco-physiology. Anton. Leeuw. Int. J. G. 81, 693–702 (2002)

    CAS  Article  Google Scholar 

  18. Nauhaus, K., Treude, T., Boetius, A. & Krüger, M. Environmental regulation of the anaerobic oxidation of methane: a comparison of ANME-I and ANME-II communities. Appl. Environ. Microbiol. 7, 98–106 (2005)

    CAS  Google Scholar 

  19. Koga, Y., Morii, H., Akagawa-Matsushita, M. & Ohga, M. Correlation of polar lipid composition with 16S rRNA phylogeny in methanogens. Further analysis of lipid component parts. Biosci. Biotechnol. Biochem. 62, 230–236 (1998)

    CAS  Article  Google Scholar 

  20. Blumenberg, M., Seifert, R., Reitner, J., Pape, T. & Michaelis, W. Membrane lipid patterns typify distinct anaerobic methanotrophic consortia. Proc. Natl Acad. Sci. USA 101, 11111–11116 (2004)

    ADS  CAS  Article  Google Scholar 

  21. Blumenberg, M., Seifert, R., Nauhaus, K., Pape, T. & Michaelis, W. In vitro study of lipid biosynthesis in an anaerobically methane-oxidizing microbial mat. Appl. Environ. Microbiol. 71, 4345–4351 (2005)

    CAS  Article  Google Scholar 

  22. Knittel, K., Lösekann, T., Boetius, A., Kort, R. & Amann, R. I. Diversity and distribution of methanotrophic archaea at cold seeps. Appl. Environ. Microbiol. 71, 467–479 (2005)

    CAS  Article  Google Scholar 

  23. Islas-Lima, S., Thalasso, F. & Gomez-Hernandez, J. Evidence of anoxic methane oxidation coupled to denitrification. Water Res. 38, 13–16 (2004)

    CAS  Article  Google Scholar 

  24. Waki, M., Tanaka, Y., Osada, T. & Suzuki, K. Effects of nitrite and ammonium on methane-dependent denitrification. Appl. Microbiol. Biotechnol. 59, 338–343 (2002)

    CAS  Article  Google Scholar 

  25. Eisentraeger, A., Klag, P., Vansbotter, B., Heymann, E. & Dott, W. Denitrification of groundwater with methane as sole hydrogen donor. Water Res. 35, 2261–2267 (2001)

    CAS  Article  Google Scholar 

  26. Costa, C. et al. Denitrification with methane as electron donor in oxygen-limited bioreactors. Appl. Microbiol. Biotechnol. 53, 754–762 (2000)

    CAS  Article  Google Scholar 

  27. Raghoebarsing, A. A. et al. Methanotrophic symbionts provide carbon for photosynthesis in peat bogs. Nature 436, 1153–1156 (2005)

    ADS  CAS  Article  Google Scholar 

  28. Hinrichs, K. U., Hayes, J. M., Sylva, S. P., Brewer, P. G. & DeLong, E. F. Methane-consuming archaeabacteria in marine sediments. Nature 398, 802–805 (1999)

    ADS  CAS  Article  Google Scholar 

Download references


We thank B. Kartal, J. van de Vossenberg and M. Schmid for discussions, and J. G. Kuenen and M. Wagner for critical reading of the manuscript. We thank J. Eigensteyn and W. Geerts for technical support. M.S. and K.F.E. are supported by a VIDI grant from the Dutch Science Foundation (NWO). Help from G. Boedeltje in choosing the sampling location is also gratefully acknowledged. Author Contributions M.S., A.A.R., A.J.P.S. and K.T.P-S. performed the sampling; A.A.R. the enrichment, A.A.R., A.P. and K.F.E. the batch experiments and labelling; W.I.C.R., S.S. and J.S.S.D. the biomarker analysis; A.A.R. and K.T.P-S. the FISH analysis; A.A.R. the molecular analysis; and A.A.R. and H.J.M.O.C. the phylogeny and probe design. The research was conceived by M.S. and M.S.M.J., and pilot experiments were performed by K.T.P-S. A.A.R., A.P., K.F.E., S.S., J.S.S.D., H.J.M.O.C., M.S.M.J. and M.S. contributed to interpreting the data and writing the paper.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Marc Strous.

Ethics declarations

Competing interests

The 16S rRNA gene sequences have been deposited in GenBank under accession numbers DQ369741 (archaeal sequence) and DQ369742 (bacterial sequence). Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Data

This file contains the figure legends for Supplementary Figs 1 and 2, Supplementary References, and Supplementary Methods. (DOC 30 kb)

Supplementary Figure 1

This file contains Supplementary Fig. 1. (PDF 127 kb)

Supplementary Figure 2a

This file contains Supplementary Fig. 2a. (PDF 22 kb)

Supplementary Figure 2b

This file contains Supplementary Fig. 2b. (PDF 20 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Raghoebarsing, A., Pol, A., van de Pas-Schoonen, K. et al. A microbial consortium couples anaerobic methane oxidation to denitrification. Nature 440, 918–921 (2006).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing