Evaluation of argon-induced hydrogen production as a method to measure nitrogen fixation by cyanobacteria

The production of dihydrogen (H₂ ) is an enigmatic yet obligate component of biological dinitrogen (N₂ ) fixation. This study investigates the effect on H₂ production by N₂ fixing cyanobacteria when they are exposed to either air or a gas mixture consisting of argon, oxygen, and carbon dioxide (Ar:O₂ :CO₂ ). In the absence of N₂ , nitrogenase diverts the flow of electrons to the production of H₂ , which becomes a measure of Total Nitrogenase Activity (TNA). This method of argon-induced hydrogen production (AIHP) is much less commonly used to infer rates of N₂ fixation than the acetylene reduction (AR) assay. We provide here a full evaluation of the AIHP method and demonstrate its ability to achieve high-resolution measurements of TNA in a gas exchange flow-through system. Complete diel profiles of H₂ production were obtained for N₂ fixing cyanobacteria despite the absence of N₂ that broadly reproduced the temporal patterns observed by the AR assay. Comparison of H₂ production under air versus Ar:O₂ :CO₂ revealed the efficiency of electron usage during N₂ fixation and place these findings in the broader context of cell metabolism. Ultimately, AIHP is demonstrated to be a viable alternative to the AR assay with several additional merits that provide an insight into cell physiology and promise for successful field application.