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

Biological N fixation (BNF) is traced to the Archean. The nitrogen isotopic fractionation composition (δN) of sedimentary rocks is commonly used to reconstruct the presence of ancient diazotrophic ecosystems. While δN has been validated mostly using organisms grown under present-day conditions; it has not under the pre-Cambrian conditions, when atmospheric O was lower and CO was higher. Here, we explore δN signatures under three atmospheres with (i) elevated CO and no O (Archean), (ii) present-day CO, and O and (iii) future elevated CO, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set from literature for more generalized dependencies of δN and the associated fractionation factor epsilon ( = δN - δN) during BNF in Archaea and Bacteria, including cyanobacteria, and habitats. The ranges between 3.70‰ and -4.96‰ with a mean 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 concentrations, toxin production, and light, although within 1‰. Moreover, correlation showed significant dependency of 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 δN is likely robust when applied to the pre-Cambrian-like atmosphere, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower ) observed in the toxin-producing and 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 into biologically usable NH. During this process, the enzyme nitrogenase preferentially uses light N, resulting in 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 O and CO on this fractionation signal. We find the signal is stable, especially for the primary producers, cyanobacteria, with correlations to CO, light, and toxin-producing status, within a small range. Unexpectedly, we identified higher fractionation signals in toxin-producing and species that offer insight into why some organisms produce these N-rich toxic secondary metabolites.