DINITROGEN FIXATION BY AZOTOBACTER CHROOCOCCUM AND ITS ISOCITRATE DEHYDROGENASE ACTIVITY: A QUICK METHOD FOR SCREENING NITROGEN FIXING STRAINS

Abstract

Nitrogenase activity in the rhizosphere of sugarcane (Saccharum officinarum Linn.), and its associated soil was measured by reduction of acetylene. Zero order kinetics and first order kinetics of acetylene reduction (C₂H₂→C₂H₄) were used to describe the activity of N₂ fixers from these soils. Arranged in descending order of nitrogen-fixing (acetylene-reducing) potential, the sites examined were highland (arable), mesic meadow, peat polygon trough, transition zone between mesic meadow and peat polygon trough, lowland (flooded with water) and virgin flooded land. On most of the land, sugarcane has been under cultivation for centuries. In total, 9 acetylene reducers were isolated, the remaining being oligonitrophiles. In addition to using the saturation kinetic values to describe the activity of dinitrogen fixers, first order kinetics of several potential energy substrates were also compared. Carbohydrate concentrations (glucose, maltose, arabinose, mannose and raffinose) between 25 and 100μg/ml were used since the Michaelis-Menten plot indicates that between this range first order kinetics were measured. The results indicated that various carbohydrate additions had different effects on C₂H₂ reduction.
Because of the relatively high temperature requirements for Azotobacter chroococcum development, tropical areas appear more favorable for a higher incidence of this bacterium. Optimal temperatures for acetylene reduction were between 25°-30°. Acetylene reduction was pH-dependent, the optimum occurring between pH 6.4-7.5. Nitrogenase activity was almost completely inhibited by air and anaerobic conditions, and was optimum at around pO₂ 0.06atm.
The reduction of 2, 3, 5-triphenyl tretrazolium chloride to triphenyl formazan has been used to estimate the dehydrogenase activity of Azotobacter strains in vivo. A significant correlation has been obtained between isocitrate dehydrogenase reactions and the oxygen uptake of a number of isolates; the production of the formazan was used as an index of endogenous respiration. A similar relationship was also obtained between dehydrogenase reactions and bacterial numbers. A relationship between acetylene reduced per gram of organic carbon utilized and the dehydrogenase reactions has been recorded. This investigation also proposes an index for quick screening of nitrogen fixation in Azotobacter.