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Published January 1, 2019 | Supplemental Material
Journal Article Open

Effect of iron limitation on the isotopic composition of cellular and released fixed nitrogen in Azotobacter vinelandii

Abstract

Most biological nitrogen transformations have characteristic kinetic isotope effects used to track these processes in modern and past environments. The isotopic fractionation associated with nitrogen fixation, the only biological source of fixed nitrogen (N), provides a particularly important constraint for studies of nitrogen cycling. Nitrogen fixation using the 'canonical' Mo-nitrogenase produces biomass with a δ^(15)N value of ca. −1‰ (vs. atmospheric N_2). If the 'alternative' V- and Fe-only nitrogenases are used, biomass δ^(15)N can be between −6‰ and −7‰. These biomass values are assumed to be relatively invariant and to reflect the cellular level expressed isotope effect of nitrogen fixation. However, field and laboratory studies report wide ranges of diazotrophic biomass δ^(15)N (from −3.6‰ to +0.5‰ for Mo-based nitrogen fixation). This variation could be partly explained by the release of dissolved organic N (DON) that is isotopically distinct from biomass. The model nitrogen fixer Azotobacter vinelandii secretes siderophores, small molecules that aid in Fe uptake and can comprise >30% of fixed nitrogen. To test whether siderophores (and other released N) can decouple biomass δ^(15)N from the isotope effect of nitrogen fixation we measured the isotopic composition of biomass and released N in Fe-limited A. vinelandii cultures fixing nitrogen with Mo- and V-nitrogenases. We report that biomass δ^(15)N was elevated under Fe limitation with a maximum value of +1.2‰ for Mo-based nitrogen fixation. Regardless of the nitrogenase isozyme used, released nitrogen δ^(15)N was also 2–3‰ lower than biomass δ^(15)N. Siderophore nitrogen was found to have a slightly higher δ^(15)N than the rest of the DON pool but was still produced in large enough concentrations to account for increases in biomass δ15N. The low δ^(15)N of siderophores (relative to biomass) is consistent with what is known from compound specific isotope studies of the amino acids used in siderophore biosynthesis, and indicates that other amino-acid derived siderophores should also have a low δ^(15)N. The implications for studies of nitrogen fixation are discussed.

Additional Information

© 2018 Elsevier Ltd. Received 11 April 2018, Accepted 22 September 2018, Available online 1 October 2018. We thank M.A. Weigand for her help and guidance with the denitrifier method. We are also grateful to X.T. Wang, E.R. Kast, V. Luu, J.A. Lueders-Dumont, S.E. Fawcett and A.R. Babbin for helpful discussions and assistance with methods. This work was supported by the Princeton Environmental Institute, the National Science Foundation (grant numbers EAR-1631814 and OCE 1657639) and NASA 80NSSC17K0667.

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August 22, 2023
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