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

Biological N2fixation (BNF) can be traced to the Archean, over 3 Bya. The nitrogen isotopic fractionation composition (δ15N) of sedimentary rocks is commonly used to reconstruct the presence of diazotrophic ecosystems in the past. While δ15N has been calibrated under modern environmental conditions; it has not under Archean conditions, when atmospheric pO2was lower and pCO2was higher than today. Here we explore δ15N signatures in the laboratory under three simulated atmospheres with (i) elevated CO2and no O2, (ii) present day CO2and O2and (iii) elevated CO2and present day O2, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set with literature data to examine for more generalized dependencies of δ15N 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 CO2concentrations, 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 δ15N 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.