Exploring the influence of atmospheric CO₂and O₂levels on the utility of nitrogen isotopes as proxy for biological N₂fixation

Biological N 2 fixation (BNF) can be traced to the Archean, over 3 Bya. The nitrogen isotopic fractionation composition (δ 15 N) of sedimentary rocks is commonly used to reconstruct the presence of diazotrophic ecosystems in the past. While δ 15 N has been calibrated under modern environmental conditions; it has not under Archean conditions, when atmospheric pO 2 was lower and pCO 2 was higher than today. Here we explore δ 15 N signatures in the laboratory under three simulated atmospheres with (i) elevated CO 2 and no O 2 , (ii) present day CO 2 and O 2 and (iii) elevated CO 2 and present day O 2 , in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set with literature data to examine for more generalized dependencies of δ 15 N during BNF across the Archaea and Bacteria, including cyanobacteria, and habitats. We find a mean ϵ-value of -1.38 ± 0.95, for all bacteria, including cyanobacteria, across all tested conditions. The expanded data set reveal correlations of isotopic fractionation of BNF with CO 2 concentrations, toxin production and light, although within 1 ‰. Moreover, correlation showed significant dependency of the magnitude of ϵ to species type, C/N ratios and toxin production in heterocytous 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 Archean, 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.