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Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr


Nitrogen is an essential nutrient for all organisms that must have been available since the origin of life. Abiotic processes including hydrothermal reduction1, photochemical reactions2, or lightning discharge3 could have converted atmospheric N2 into assimilable NH4+, HCN, or NOx species, collectively termed fixed nitrogen. But these sources may have been small on the early Earth, severely limiting the size of the primordial biosphere4. The evolution of the nitrogen-fixing enzyme nitrogenase, which reduces atmospheric N2 to organic NH4+, thus represented a major breakthrough in the radiation of life, but its timing is uncertain5,6. Here we present nitrogen isotope ratios with a mean of 0.0 ± 1.2‰ from marine and fluvial sedimentary rocks of prehnite–pumpellyite to greenschist metamorphic grade between 3.2 and 2.75 billion years ago. These data cannot readily be explained by abiotic processes and therefore suggest biological nitrogen fixation, most probably using molybdenum-based nitrogenase as opposed to other variants that impart significant negative fractionations7. Our data place a minimum age constraint of 3.2 billion years on the origin of biological nitrogen fixation and suggest that molybdenum was bioavailable in the mid-Archaean ocean long before the Great Oxidation Event.

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Figure 1: Sedimentary δ15N through time.
Figure 2: Whole-rock δ15N versus TOC.
Figure 3: TOC and δ15N versus Mo and V enrichment factors.


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This work was funded by the Virtual Planetary Laboratory at the University of Washington, a Geological Society of America graduate student research grant to E.E.S., and by an Agouron Institute grant to R.B. We thank N. J. Beukes for access to the Witwatersrand samples, and the University of Washington Isolab for technical support.

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Authors and Affiliations



R.B. collected samples from the Soanesville Group; B.M.G. collected samples from the Witwatersrand Supergroup; R.B. and E.E.S. collected samples from the Fortescue Group and Nullagine Group; E.E.S. and M.C.K. performed the analyses; E.E.S. drafted the manuscript with advice and contributions from R.B.; B.M.G. and M.C.K. commented on the draft.

Corresponding author

Correspondence to Eva E. Stüeken.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Organic carbon (a) and bulk nitrogen (b) isotopes as a function of total abundances.

Correlation coefficients refer to data points and trend lines of the same colour. See Supplementary Information 3 for discussion. Each sample was analysed in two or three replicates.

Extended Data Figure 2 Crust-normalized elemental abundances.

a, Normalized relative to average upper continental crust; b, normalized relative to average upper Archaean crust (Methods). Some data points in b are missing because concentrations for those elements in average upper Archaean crust were not provided. In both parts, elements are sorted by relative abundance in upper continental crust.

Extended Data Figure 3 Reproducibility of the UW-McRae in-house standard over a range of sample sizes.

Filled diamonds, analysed with regular CN method; grey circles, analysed with a soda-lime column to scrub CO2 from the gas stream; horizontal lines, total average (solid) ± 1 s.d. (dashed).

Extended Data Table 1 Comparison of kerogen and whole-rock data for samples with sufficient material from the Soanesville Group and the Witwatersrand Supergroup

Supplementary information

Supplementary Information

This file contains Supplementary Information 1-3, Supplementary Tables 1-4 and additional references. (PDF 832 kb)

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Stüeken, E., Buick, R., Guy, B. et al. Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr. Nature 520, 666–669 (2015).

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