GC–MS analysis of hydrocarbon products

A. vinelandii strain expressing the V-nitrogenase was grown as described above until the cell density started to plateau, when the flask was capped airtight, followed by addition of ¹³CO at a concentration of 15% to the gas phase of this culture. The culture was grown for another 8 h before 250 μl headspace sample was taken and analysed by GC–MS using a Thermo Scientific Trace 1300 GC system coupled to a Thermo ISQ QD (Thermo Electron North America LLC, Madison, WI)^4,5. Specifically, a 250 μl gas sample was injected into a split/splitless injector operated at 120 °C in in split mode, with a split ratio of 5. A 1 mm ID liner was used to optimize the sensitivity of gas separation, which was achieved on an HP-PLOT-Q capillary column (0.320 mm ID × 30 m length, Agilent Technologies, Santa Clara, CA) that was held at 40 °C for 2 min, heated to 180 °C at a rate of 10 °C min⁻¹ and held at 180 °C for 1 min. The carrier gas, helium, was passed through the column at a rate of 1.1 ml min⁻¹. The mass spectrometer was operated in electron impact ionization mode and the identities of C₂H₄, C₂H₆ and C₃H₈ were confirmed by comparing their masses and retention times with those of the Scott standard alkane and alkene gas mixture.