A. vinelandii has been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered around A. vinelandii 's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targeted in vitro measurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts the in vivo activity of each nitrogenase isoenzyme are lacking. This article aims to provide in vivo nitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation.