The purpose of the present investigation was to study the effect of some chemical, physical and biological factors on growth, efficiency of nitrogen fixation and nitrogenase activity of Azotobacter chroococcum.
From biochemical studies with cell-free preparations of various nitrogenfixing microorganisms reported in the literature, it can be concluded that three major requirements have to be met before N 2
fixation can proceed viz. the presence of ATP, a powerful reductant, and the appropriate enzyme system (nitrogenase). In intact (living) cells there must also be carbon skeletons for accepting the fixed nitrogen, to allow the process to continue for any length of time. Otherwise, fixed nitrogen accumulates within the cell and will impair the continuation of nitrogen fixation by repressing the synthesis of nitrogenase.
To investigate whether the degree of oxidation and the molecular size (chain length) of organic carbon sources affect the efficiency of nitrogen fixation (defined as the amount of nitrogen fixed per g of carbon compound consumed), some Azotobacter
strains, especially those of A. chroococcum,
were cultivated in modified Burk's liquid medium supplied with a number of hexoses, hexitols, fatty acids (as the sodium or calcium salts) and primary alcohols (chapter 3). Since the assimilability of the hexoses tested and that of the corresponding hexitols was different, the hexose-hexitol comparison could not be used for achieving the purpose of this study (Table 3.1).
Ethyl alcohol gave higher efficiency values of nitrogen fixation than acetate. Propanol and butanol were less efficient than propionate and butyrate, probably due to the inhibitory effect of these alcohols on the growth of the azotobacters at the concentrations used (Tables 3.2 and 3.3).
Increased efficiency of N 2
fixation occurred with increased length of the carbon chain of fatty acids up till four carbon atoms (Table 3.5). The efficiency of the nitrogen fixation of Azotobacter
cultures supplied with butyrate was found to be considerably higher than that of cultures with glucose. This higher efficiency might be due to the greater reducing capacity of the former compound and/or the production of more ATP per weight unit of butyrate. Since azotobacters have very high Q O2
-values, it is more likely that these organisms suffer from insufficient reducing capacity and carbon skeletons than from insufficient energy for growth. Therefore, the greater reducing capacity per weight unit of butyrate was obviously the cause of the higher efficiency of N 2
fixation with this compound as compared to that with glucose.
The results of the experiments reported in chapter 4 show that increased O 2
supply of A.chroococcum
up to a certain level favoured the nitrogenase activity of the cells (Figures 4.1 B and 4.2). This was presumably due to the improved supply of ATP and NADPH 2
resulting from an increased catabolism of carbon compounds including respiration.
Upon further increase of the O 2
supply of the cells, nitrogenase activity decreased, presumably as a result of competition between respiration and nitrogenase activity for reductants. If azotobacters were exposed to excess oxygen for a short time, and then were returned to the optimum pO 2
, the cells immediately resumed their optimum nitrogenase activity (YATES, 1970). If this exposure to excess oxygen was continued for a prolonged period, return to the optimum pO 2
gave only a poor recovery of the nitrogenase activity (Fig. 4.3). This low recovery was presumably due to the inactivation of the oxygen-sensitive component of the nitrogenase.
The optimum level of O 2
supply for nitrogenase activity was dependent on the cell density of the Azotobacter
cultures. Those with high cell densities fixed N 2
(and grew) at an O 2
supply which inhibited N 2
fixation (and growth) of cultures with low cell densities, apparently due to a more efficient removal of oxygen (achieved by cell respiration) in the former culture.
In agreement with results of earlier investigations reported in the literature (cf. DALTON and POSTGATE, 1969), in the present study it was found that lowering the pO 2
increased the cell yield of A. chroococcum
grown with N 2
as the source of nitrogen. Such an effect was not observed when urea had been supplied as the nitrogen source (Table 4.1). These results suggest that the decreased cell yield of A.chroococcum,
grown at high pO 2
with N 2
as the nitrogen source was due to the increased utilization of carbon compounds for respiratory protection of nitrogenase.
Although it is generally believed that nitrogen fixation in Azotobacter cul
tures is confined to proliferating cells, there are some indications reported in the literature concerning the ability of non-growing azotobacters to fix N 2
(see 5. 1). These reports, however, are not supported by critical quantitative studies on cell growth.
In chapter 5, various methods have been described to stop the growth of azotobacters. These methods include: application of chloramphenicol, depriving the bacteria of K +
or Ca 2+
, and incubating the cells under air containing 10%C 2
Although the elimination of cell growth by the addition of chloramphenicol was found to be accompanied with the suppression of N 2
fixation (Table 5.3), the latter process was mostly less severely depressed than cell proliferation (Tables 5.1 and 5.2).
To explain the reduced N 2
fixation by non-growing chloramphenicol-treated azotobacters, it should be considered that although the synthesis of nitrogenase was prevented, the activity of the enzyme in the non-growing cells could have been retained for a prolonged period. This would lead to the accumulation of soluble nitrogenous compounds including NH4+
, which are interfering with the nitrogenase activity of living cells (7.3.1).
Although the addition of chloramphenicol to cell-free extracts did not inhibit the activity of nitrogenase (Fig. 5.4), a ready decline of this activity was observed when the antibiotic was added to living cells. This was the case both under air (where N 2
fixation was possible; Table 5.4) and under air containing 10 % C 2
(where N 2
fixation was prevented; Figs. 5.1 and 5.2). These results suggest that the depressing effect of chloramphenicol on the N 2
fixation by living, nongrowing azotobacters was due to a competition between the antibiotic and nitrogenase for reductants. The presence of accumulated NH4+
may aggravate this effect (7.3.1)
cultures of potassium ions, reduces and finally stops the growth of the cells by preventing protein synthesis. Although nitrogen fixation follows this tendency, similar to that of chloramphenicol-treated cells it is less severely affected by K deficiency than growth (Table 5.5). This indicates that the effect of K deficiency on nitrogenase activity is indirect viz. by effecting the accumulation of soluble nitrogenous compounds, including ammonia, which depresses nitrogenase activity (see 7.3. 1). Decreased metabolic activity of the cells, resulting in shortage of reductants and energy, may also be involved in the reduced nitrogenase activity of K-deficient cells.
In contrast to Azotobacter
cells deprived of K +
, which continue to fix small amounts of N 2
, even when cell growth is almost completely arrested, Ca-deficient cells under similar conditions cease to fix N 2
(Table 5.5). The cause of the suppression of N 2
fixation in Ca-deficient cells in unknown, but is most probably due to the decreased metabolic activity of such cells.
That non-growing cells of azotobacters are able to preserve a high nitrogenase activity for a prolonged period was clearly shown by incubating cultures of nitrogen-fixing cells under air containing 10 % acetylene. Under such conditions, owing to the high affinity of nitrogenase for C 2
, the fixation of N 2
is completely suppressed, resulting in the existence of non-growing cells which do not fix N 2
and as a consequence do not accumulate ammonia in their pool. This enables the nitrogenase to function for a prolonged period (Fig. 5.5).
In the present study, it was shown for the first time that living cells of azotobacters are able to produce H 2
when being incubated under air containing 10 % acetylene. Although cell-free extracts of azotobacters are able to evolve H 2
when incubated under argon with C 2
or N 2
, H 2
evolution has never been observed to take place in living cells incubated under air (Tables 5.6 and 5.7). These differences presumably depend on the fact that part of the reductants and the ATP which in growing N 2
-fixing cultures are used for the assimilation of fixed nitrogen (ammonia) are available for H 2
production when the cells are incubated under air containing 10 % acetylene.Azotobacter
cells are known to excrete small amounts of nitrogenous compounds into the medium (cf. RUBENCHIK, 1960). In an experiment with 5 different strains of Azotobacter,
7-13 % of the total nitrogen fixed was excreted by the cells (Table 6.1). Chapter 6 describes two efforts to remove the excreted nitrogenous compounds and to study the effect of this removal on the nitrogenase activity of cultures of A. chroococcum.
These efforts include: (1) harvesting, washing, and resuspending growing cells in a fresh medium containing the same amount of sugar as present in the discarded supernatant, and (2) growing A. chroococcum
in the presence of a Rhodotorula sp.
The first method gave a slight, but consistent increase in nitrogenase activity (Fig. 6.1). Azotobacter
cells grown with yeast cells achieved a higher efficiency of nitrogen fixation than pure cultures only when subjected to excess oxygen (Table 6.2). This beneficial effect was apparently due to the consumption by the yeast cells of excess oxygen, thus creating better conditions for nitrogen fixation, rather than to the removal of excreted ammonia as was suggested when starting these experiments. Similar findings have been reported by KALININSKAYA (1967) in an association of a nitrogen-fixing Mycobacterium
sp with a yeast.
The effect of combined nitrogen on the synthesis and the functioning of nitrogenase in A. chroococcum is
described in chapter 7. Addition of various forms of combined nitrogen to growing N 2
-fixing cultures showed (Figs. 7.1 and 7.2) that (a) ammonium ions and nitrate caused a considerable decline of the nitrogenase activity, (b) Casamino acids and amides slightly reduced this activity, and (c) amino acids had no effect on the nitrogenase activity presumably due to the poor uptake of these compounds by the cells. In general it is assumed that the reduction of the nitrogenase activity of living Azotobacter
cells by a supplemented nitrogenous compound depends on the readiness of conversion of this compound to ammonium ions. In the case of nitrate the possibility of a competition between the reduction of this compound and the nitrogenase activity for reductants can not be excluded (Figs. 7.7 and 7.8).
The decline in nitrogenase activity of Azotobacter
cells upon the addition of NH4+
was assumed to be due to the combined effect of (1) competition between nitrogenase activity and ammonia assimilation for reductants and/or ATP, and (2) repression of nitrogenase synthesis (Fig. (7.3.1). If the amount of NH4+
added to the culture was large enough, eventually degradation of the enzyme also took place (Table 7.1). From the absence of an effect upon the addition of NH4+
to cell-free extracts (Fig. 7.4) it was concluded that feedback inhibition did not participate in the decline of nitrogenase activity in vivo
caused by the addition of NH4+
to living cells.
To decide which part of the depression by NH4+
was due to 'competition' and which part to repression of the nitrogenase synthesis, a comparison was made between the nitrogenase activity of living cells, measured at various periods, after the addition of NH4+
, and that of cell-free extracts made from the same cells, at the same time (Table 7.1). As the loss of the nitrogenase activity in living cells started immediately and proceeded at a much faster rate than that of cell-free preparations of the same culture, it is concluded that competition for reductants and/or ATP between nitiogenase activity and NH4+
assimilation was responsible for the immediate decline of nitrogenase activity of Azotobacter
cells supplied with NH4+
. The repression of nitrogenase synthesis upon the addition of NH4+
was not immediate and proceeded at a relatively slow rate.
Although the immediate decline of the nitrogenase activity in living cells supplied with NH4+
might be due to uncoupling of the oxidative phosphorylation, the concentrations of NH4+
used in this investigation were much lower than the values reported in the literature to be responsible for that effect.