Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. I. Free cell cultures

As part of an investigation into the use of biological nitrogen fixation for fertilizer ammonia production, continuous culture studies of respiration and nitrogen fixation in the aerobic bacteria Azotobacter vinelandii under oxygen-limited conditions were conducted. Respiration and growth rates followed Monod forms with respect to dissolved oxygen concentration. However, specific nitrogen fixation rate and nitrogenase activity exhibited maximum values at dissolved oxygen concentrations of ca. 0.02 mM (10% of air saturation). These results suggest careful control of oxygen in the environment is necessary to optimize fixed nitrogen production by this organism.     

Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii.

The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced. The higher the amplitude, the lower the MMW(max). As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system.