A Major Error in the Acetylene Reduction Assay: Decreases in Nodular Nitrogenase Activity Under Assay Conditions

Observations on both attached nodulated roots and detached nodules have revealed that nitrogenase activity in many legume species declined rapidly in the presence of acetylene, with a concurrent reduction in respiration. The reduction began within a few minutes of exposure to acetylene and continued for 30–60 min before a new steady-state was attained. A similar decline in H<inf>2</inf> evolution and respiration was observed when N<inf>2</inf> was replaced with argon or helium. This suggests that the decrease is linked to the cessation of ammonia production. Measurements of 15N<inf>2</inf> uptake demonstrated that it is the pre-decline rather than final rate of ethylene production which represents the real rate of nitrogenase activity. The implications of these findings for the interpretation of acetylene reduction and hydrogen evolution data are considered.