Exploring the influence of atmospheric CO₂ and O₂ levels on the utility of nitrogen isotopes as proxy for biological N₂ fixation

Biological N2 fixation (BNF) is traced to the Archean. The nitrogen isotopic fractionation composition (δ¹⁵N) of sedimentary rocks is commonly used to reconstruct the presence of ancient diazotrophic ecosystems. While δ¹⁵N has been validated mostly using organisms grown under present-day conditions; it has not under the pre-Cambrian conditions, when atmospheric pO₂ was lower and pCO₂ was higher. Here, we explore δ¹⁵N signatures under three atmospheres with (i) elevated CO₂ and no O₂ (Archean), (ii) present-day CO₂, and O₂ and (iii) future elevated CO₂, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set from literature for more generalized dependencies of δ¹⁵N and the associated fractionation factor epsilon (ε = δ¹⁵N_biomass – δ₁₅N_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 CO₂ 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 δ¹⁵N 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.