Efforts toward optimization of aerobic biohydrogen reveal details of secondary regulation of biological nitrogen fixation by nitrogenous compounds in Azotobacter vinelandii

Biological nitrogen fixation (BNF) through the enzyme nitrogenase is performed by a unique class of organisms known as diazotrophs. One interesting facet of BNF is that it produces molecular hydrogen (H₂) as a requisite by-product. In the absence of N₂ substrate, or under conditions that limit access of N₂ to the enzyme through modifications of amino acids near the active site, nitrogenase activity can be redirected toward a role as a dedicated hydrogenase. In free-living diazotrophs, nitrogenases are tightly regulated to minimize BNF to meet only the growth requirements of the cell, and are often accompanied by uptake hydrogenases that oxidize the H₂ by-product to recover the electrons from this product. The wild-type strain of Azotobacter vinelandii performs all of the tasks described above to minimize losses of H₂ while also growing as an obligate aerobe. Individual alterations to A. vinelandii have been demonstrated that disrupt key aspects of the N₂ reduction cycle, thereby diverting resources and energy toward the production of H₂. In this work, we have combined three approaches to override the primary regulation of BNF and redirect metabolism to drive biological H₂ production by nitrogenase in A. vinelandii. The resulting H₂-producing strain was further utilized as a surrogate to study secondary, post-transcriptional regulation of BNF by several key nitrogen-containing metabolites. The improvement in yields of H₂ that were achieved through various combinations of these three approaches was compared and is presented along with the insights into inhibition of BNF by several nitrogen compounds that are common in various waste streams. The findings indicate that both ammonium and nitrite hinder BNF through this secondary inhibition, but urea and nitrate do not. These results provide essential details to inform future biosynthetic approaches to yield nitrogen products that do not inadvertently inhibit BNF.