Exploring the influence of atmospheric CO 2 and O 2 levels on the utility of nitrogen isotopes as proxy for biological N 2 fixation.

Biological N ₂ fixation (BNF) is traced to the Archean. The nitrogen isotopic fractionation composition (δ ¹⁵ N) of sedimentary rocks is commonly used to reconstruct the presence of ancient diazotrophic ecosystems. While δ ¹⁵ N has been validated mostly using organisms grown under present-day conditions; it has not under the pre-Cambrian conditions, when atmospheric p O ₂ was lower and p CO ₂ was higher. Here, we explore δ ¹⁵ N signatures under three atmospheres with (i) elevated CO ₂ and no O ₂ (Archean), (ii) present-day CO ₂ , and O ₂ and (iii) future elevated CO ₂ , in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set from literature for more generalized dependencies of δ ¹⁵ N and the associated fractionation factor epsilon ( ε = δ ¹⁵ N _biomass - δ ¹⁵ N _N2 ) during BNF in Archaea and Bacteria, including cyanobacteria, and habitats. The ε ranges between 3.70‰ and -4.96‰ with a mean ε value of -1.38 ± 0.95‰, for all bacteria, including cyanobacteria, across all tested conditions. The expanded data set revealed correlations of isotopic fractionation of BNF with CO ₂ concentrations, toxin production, and light, although within 1‰. Moreover, correlation showed significant dependency of ε to species type, C/N ratios and toxin production in cyanobacteria, albeit it within a small range (-1.44 ± 0.89‰). We therefore conclude that δ ¹⁵ N is likely robust when applied to the pre-Cambrian-like atmosphere, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower ε ) observed in the toxin-producing Nodularia and Nostoc spp. suggests a heretofore unknown role of toxins in modulating nitrogen isotopic signals that warrants further investigation.IMPORTANCENitrogen is an essential element of life on Earth; however, despite its abundance, it is not biologically accessible. Biological nitrogen fixation is an essential process whereby microbes fix N ₂ into biologically usable NH ₃ . During this process, the enzyme nitrogenase preferentially uses light ¹⁴ N, resulting in ¹⁵ N depleted biomass. This signature can be traced back in time in sediments on Earth, and possibly other planets. In this paper, we explore the influence of p O ₂ and p CO ₂ on this fractionation signal. We find the signal is stable, especially for the primary producers, cyanobacteria, with correlations to CO ₂ , light, and toxin-producing status, within a small range. Unexpectedly, we identified higher fractionation signals in toxin-producing Nodularia and Nostoc species that offer insight into why some organisms produce these N-rich toxic secondary metabolites.
Competing Interests: The authors declare no conflict of interest.