X-axis: Days.
Y-axis: Milligrams of Nitrogen fixed per gram of dextrose consumed.
It is found, however, that in liquid culture, the ratio of nitrogen fixed to carbohydrates oxidised varies according to the age of the culture, falling off rapidly as the age increases[42] ([Fig. 5]). This decreasing efficiency in cultures may be due to the accumulation of metabolic products such as would not occur under soil conditions. Indeed, the efficiency of Azotobacter in a sand culture has been found by Krainskii[39] to be considerably greater than in solution. It is thus probable that in soil the nitrogen-fixing organisms are less wasteful of energy material than under the usual laboratory conditions. It is to be hoped that future research will indicate what are the conditions that produce the greatest economy of energy material in nitrogen fixation.
The fixation of nitrogen in soil is depressed by the presence of considerable amounts of nitrates. This is, in all probability, due to the fact that nitrogen-fixing organisms are able to utilise compounds of nitrogen where these are available. The energy needed to build up amino-acids and proteins from nitrate or ammonia is, of course, far less than that required to build up these substances from elemental nitrogen. It is, therefore, not surprising that where nitrate is available, Azotobacter will use it in preference to fixing atmospheric nitrogen.[5]
TABLE III.—ASSIMILATION OF NITRATES.
By Azotobacter in Pure Culture—(Bonazzi).
| Nitrate and Nitrite Present. | Organic Nitrogen and Ammonia Present. | Total Fixed or Lost. | |
|---|---|---|---|
| mgs. | mgs. | mgs. | |
| Culture with nitrate— | |||
| At beginning | 8·55 | 0·76 | — |
| After growth | 0·2 | 8·71 | - 0·4 |
| Culture without nitrate— | |||
| At beginning | — | 0·76 | — |
| After growth | — | 4·50 | + 3.74 |
| (Growth period—24 days at 25° C.) | |||
The chemical process by which nitrogen is fixed is quite unknown, although a number of speculative suggestions have been made. The appearance of considerable amounts of amino acids in young cultures of Azotobacter suggests that these may be a step in the process, but at present the data are too inconclusive to form a basis for theorising.
Azotobacter is very rich in phosphorus, an analysis of the surface growth in Azotobacter cultures, made by Stoklasa, giving about 60 per cent. of phosphoric acid in the ash. In cultures it has been found that a considerable amount of phosphate is needed to produce full development. As would be expected, therefore, nitrogen fixation in soil is often greatly stimulated by the addition of phosphates. Christensen has, indeed, found soils where lack of phosphate was the limiting factor for Azotobacter growth.
Azotobacter is very intolerant of an acid medium, and is very dependent on the presence of an available base. In cultures this is usually provided in the form of calcium or magnesium carbonate. Gainey[21] found that Azotobacter occurred in soils having an acidity not greater than PH 6·0, and Christensen,[7], [9] in Denmark, has found a close association between the occurrence of Azotobacter in soils and the presence of an adequate supply of calcium carbonate. So close was this association that he devised a technique based on this fact for detecting a deficiency of lime in a soil sample.
In addition to the groups already discussed, there is a remarkable and important group of nitrogen-fixing bacteria that inhabit and can carry on their functions within the root tissues of higher plants. It has been known at least from classical times that certain leguminous plants would, under suitable conditions, render the soil more productive. On the roots of leguminosæ small tubercles are commonly found. These were noted and figured by Malpighi in the seventeenth century, and for a long time were regarded as root-galls. As was described in [Chapter I.], the true nature of these tubercles was finally elucidated by Hellriegel and Wilfarth in 1886. As the result of a series of pot experiments, they made the very brilliant deduction that the ability to fix nitrogen, possessed by the legumes, was due to bacteria associated with them in the tubercles.