Acetylene reduction assay (ARA)

The basis for the assay is the fact that nitrogenase, the enzyme complex in diazotrophic microorganisms that reduces nitrogen to ammonia, also reduces acetylene to ethylene. The diazotrophic nature of all the recovered isolates was determined by ARA. Ethylene was quantified by gas chromatography (GC) and the results were expressed in nano moles of C₂H₄ produced h⁻¹ culture⁻¹. One milliliter of pure culture grown in tryptone soy broth (TSB) for 24 h were inoculated onto 10 mL of nitrogen-free semi-liquid medium, with 0.5% mannitol as a carbon source, solidified by 0.3% gellan gum in 20 mL serum bottles and closed with a red rubber septum (SIGMA-ALDRICH, William Freeman and Co., Ltd.) and incubated for 72 h at 28°C. Bottles that showed bacterial growth were assayed for acetylene reduction. Ten percent of the atmosphere in the bottles was replaced with acetylene (C₂H₂), whereas bottles without acetylene were used as the control. After 2 h, at 24°C, 0.25 mL gas samples were taken from each vial and analyzed for amount of ethylene formed using a gas chromatography. These processes were repeated three times from same vial and average were taken for the statistical analysis. A gas chromatograph (Hewlett-Packard 5830A) fitted with a 2–2.1 mm, 80–100 mesh, Poropak R column was used and the oven temperature was adjusted to 70°C. Injection and flame-ionization detector temperatures were adjusted to 150°C. Nitrogen carrier gas flow rate was adjusted to 50 mL min⁻¹.