, Volume 127, Issue 2–3, pp 189–198 | Cite as

Alternative nitrogenase activity in the environment and nitrogen cycle implications

  • Xinning ZhangEmail author
  • Darcy L. McRose
  • Romain Darnajoux
  • J. P. Bellenger
  • François M. M. Morel
  • Anne M. L. Kraepiel
Biogeochemistry Letters


Biological nitrogen fixation, the main natural input of fixed nitrogen into the biosphere, is catalyzed by Mo-, V-, or Fe-only nitrogenase metalloenzymes. Although “alternative” V- and Fe-only nitrogenase genes are found in many environments, the contribution of these isoenzymes to N2 fixation is unknown. Here we present a new method (ISARA, isotopic acetylene reduction assay) that distinguishes canonical Mo and alternative nitrogenase activities based on in vivo 13C fractionation of acetylene reduction to ethylene (13εMo = 13.1–14.7 ‰, 13εV = 7.5–8.8 ‰, 13εFe = 5.8–6.5 ‰). ISARA analyses indicate significant contributions of alternative nitrogen fixation in boreal cyanolichens and salt marshes (~10–40 % acetylene reduction, ~20–55 % N2 fixed). These results affect the quantitative interpretation of natural abundance 15N data or traditional acetylene reduction assays. They also invite a reexamination of the conditions under which the different nitrogenase isozymes are active and suggest significant interactions between the cycles of nitrogen and trace metals.


Nitrogen fixation Alternative nitrogenase Trace metals Nitrogen cycle Stable isotopes 



We gratefully acknowledge the Princeton Environmental Institute (Grand Challenge Program), Andlinger Center for Energy and the Environment (Princeton U.), NSF Geobiology (GG-1024553), and the Canadian Research Chair in Terrestrial Biogeochemistry (CRC-950-219383) for funding; M. Saito, J. Waterbury, and I. Valiela (WHOI); Socièté des Espaces de Plein Air (Québec); T. Thiel (U. Missouri); S. Oleynik (Princeton U.); N. Van Oostende (Princeton U.) for their aid in field investigations, the provision of bacterial cultures, or advice on isotope analyses. The authors declare no conflict of interest.

Author contributions

A.M.L.K. and X.Z. designed the experiments; X.Z., D.L.M., A.M.L.K, R.D. performed the experiments, X.Z., D.L.M., R.D. analyzed the data; J-P.B. contributed materials; X.Z, D.L.M., F.M.M. and A.M.L.K co-wrote the paper.

Supplementary material

10533_2016_188_MOESM1_ESM.pdf (840 kb)
Supplementary material 1 (PDF 840 kb)


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Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Xinning Zhang
    • 1
    Email author
  • Darcy L. McRose
    • 1
  • Romain Darnajoux
    • 3
  • J. P. Bellenger
    • 3
  • François M. M. Morel
    • 1
  • Anne M. L. Kraepiel
    • 1
    • 2
  1. 1.Department of GeosciencesPrinceton UniversityPrincetonUSA
  2. 2.Department of ChemistryPrinceton UniversityPrincetonUSA
  3. 3.Department of ChemistrySherbrooke UniversityQuébecCanada

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