The fixed nitrogen sensitivity of biological nitrogen fixation in salt marshes sediments from the northeastern United States

Biological nitrogen fixation, the main input of fixed N into ecosystems, converts inert N₂ gas into bioavailable ammonium in an energetically costly reaction catalyzed by the prokaryotic metalloenzyme nitrogenase.  The high ATP and reductant requirements of N₂ fixation explain why this process is highly regulated in diazotrophs, with the presence of ammonium inhibiting nitrogenase expression and activity. Yet, several reports of N₂ fixation in ammonium- and nitrate-rich (10 to 300 µM) benthic environments challenge our understanding of a key environmental sensitivity of N₂ fixation. Field studies point to heterotrophic sulfate reducers as the likely diazotrophs in these benthic settings, but the fixed N sensitivity of sulfate-reducing diazotrophs is not well understood due to a dearth of culture studies. Additionally, assays of N₂ fixation in incubations rarely involve parallel measurements of dissolved inorganic nitrogen, possibly leading to experimental bias in favor of detecting activity under ammonium-replete initial conditions.

To help reconcile the environmental results, we investigate the ammonium sensitivity of N₂ fixation using the acetylene reduction assay and ¹⁵N₂ tracer methods in i) the model sulfate-reducing diazotroph, Desulfovibrio vulgaris str. Hildenborough (DvH), ii) four enrichment cultures from salt marsh sediments of New Jersey, and iii) slurry incubations of sediments collected from three northeastern salt marshes. In all instances, we found that ammonium strongly inhibits biological nitrogen fixation, with nitrogenase activity only detectable when ammonium concentration is below a threshold of 10 µM (slurry incubation) or 2 µM (pure cultures, enrichments). Amendment of ammonium quickly inhibits nitrogen fixation and nitrogenase activity only resumes  once ammonium is depleted to the threshold level. Ammonium additions to actively fixing samples show complete inhibition of N₂ fixation within several hours post-addition. 

Our measurements of the ammonium sensitivity of benthic N₂ fixation are consistent with the traditional understanding of nitrogen fixer metabolism and with early findings of Postgate et al. (1984) demonstrating that N₂ fixation by the sulfate reducer Desulfovibrio gigas is inhibited by ammonium levels that exceed 10 µM. These results help clarify a long-standing paradox in benthic nitrogen cycling. We suggest that prior observations of N₂ fixation at elevated ammonium levels could reflect methodological artifacts due to very fast depletion of ammonium during activity assays, legacy N₂ fixation activity associated with incomplete inhibition by ammonium, or spatial heterogeneity. Further work to standardize fixed N sensitivity assays could help with cross-study comparisons and with clarifying inconsistencies in our understanding of how environmental fixed nitrogen levels control nitrogen fixation.