Measurements of Nitrogenase Activity of Gluconacetobacter diazotrophicus Colonies on Solid Medium

The nitrogenase activity of Gluconacetobacter diazotrophicus colonies on solid LGI-P medium was examined by H2 evolution and acetylene reduction assays under 2 kPa and 20 kPa 02 partial pressures (p02).Colonies incubated at 20 kPa of p02 produced large quantities of extracellular polysaccharides and formed thick pellicles, while colonies at 2 kPa of p02 formed very thin pellicles.H2 evolution rate was reduced for both cultures in the presence of acetylene, but not abolished even at 30% acetylene.Because of this incomplete saturation of nitrogenase by acetylene, the acetylene reduction assay underestimated at least 13 to 17% of total nitrogenase activity of G. diazotrophicus colonies.The percentage of remaining H2 produced in acetylene reduction assay showed no significant difference between colonies incubated at 2 kPa and 20 kPa of p02, suggesting that the incomplete saturation of nitrogenase by acetylene was not caused by the pellicle as diffusion barrier.Our study shows that to measure total nitrogenase activity (TNA) of G. diazotrophicus by acetylene reduction assay, the H2 evolution rate should be monitored.