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) can be traced to the Archean, over 3 Bya. The nitrogen isotopic fractionation composition ({delta}15N) of sedimentary rocks is commonly used to reconstruct the presence of diazotrophic ecosystems in the past. While {delta}15N has been calibrated under modern environmental conditions; it has not under Archean conditions, when atmospheric pO2 was lower and pCO2 was higher than today. Here we explore {delta}15N signatures in the laboratory under three simulated atmospheres with (i) elevated CO2 and no O2, (ii) present day CO2 and O2 and (iii) elevated CO2 and 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 {delta}15N during BNF across the Archaea and Bacteria, including cyanobacteria, and habitats. We find a mean {varepsilon}-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 CO2 concentrations, toxin production and light, although within 1 {per thousand}. Moreover, correlation showed significant dependency of the magnitude of {varepsilon} 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 {delta}15N is likely robust when applied to the Archean, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower {varepsilon}) 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 N2 into biologically usable NH3. During this process, the enzyme nitrogenase preferentially uses light 14N, resulting in 15N 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 pO2 and pCO2 on this fractionation signal. We find the signal is stable, especially for the primary producers, cyanobacteria, with correlations to CO2, light and toxin producing status, within a small range. Unexpectedly, we identified higher fractionation signals in toxin producing Nodularia and Nostoc species, that offers insight into why some organisms produce these N-rich toxic secondary metabolites.