Acetylene Reduction Assay of A. vinelandii

A. vinelandii was grown in 5 mL of each media treatment (Jensen’s, 0.25 g/L glucose, 0.5 g/L glucose, 1 g/L glucose, 5 g/L casamino acids, 10 g/L casamino acids, 20 g/L casamino acids, and 2 g/L glucose 20 g/L casamino acids) for 3 days at 25 °C. Cells were washed, harvested and diluted to 10% before being added to fresh aliquots of the amended media treatments in 30 mL serum bottles. Serum bottles were sealed to be gas-tight and then flushed with argon for 10 min. The serum bottle headspace was injected with 10% vol oxygen and 10% vol acetylene. Nitrogen fixation activity was then measured using the acetylene reduction assay method based on acetylene reduction into ethylene by nitrogenase⁴⁴ . Ethylene concentrations were measured over a 5-day time series using gas chromatography.