Microbiology: a Text-book of Microörganisms, General and Applied, Chap III p. 400-416 (1921)
Charles Edward Marshall

Fixation of Atmospheric Nitrogen

In 1901 our knowledge of Azobacteria was enriched by Beyerinck's discovery of a group of large, obligate aerobic bacteria that he designated as Azotobacter. Since that date it has been found that the ability to fix atmospheric nitrogen is possessed also by certain molds and by various species of bacteria. However, this ability is not only extremely variable, but is also very feeble as compared with that of the members of the two groups described by Winogradski and Beyerinck. These two groups may, therefore, be designated as including the nitrogen-fixing bacteria par excellence.

Anaerobic Species.—The species isolated by Winogradski was named by him B. (Clostridium) pasteurianus (Fig. 131). It was found to grow readily under anaerobic conditions in culture solutions containing dextrose and the necessary mineral salts, but no combined nitrogen. The products of growth included protein, butyric and acetic acids, carbon dioxide and hydrogen. In the presence of other bacteria B. (Clostridium) pasteurianus was found to develop also under aerobic conditions. Subsequently studies by Winogradski and other investigators showed that B. (Clostridium) pasteurianus, and varieties of this species are very widely distributed in cultivated soils. More recently Bredeman made a thorough and extended study of anaerobic Azobacteria and demonstrated their almost invariable presence in a large number of soil samples from Europe, Asia and America. In his opinion they correspond more or less closely to B. amylobacter described many years before by van Tieghem.

Fig 131.— B. (Clostridium) pasteurianus, a non-symbiotic nitrogen-fixing organism.
(After Winogradski from Lipman.

Aerobic Species.—A more or less pronounced power to fix atmospheric nitrogen is apparently possessed by a considerable number of aerobic species. Lipman has demonstrated the fixation of small amounts of nitrogen by Ps. pyocyanea and Lohnis secured similar results with Bact. pneumonia, B. lactis viscosus, B. radiobacter and B. prodigiosus. Gottheil has detected fixation by B. ruminatus and B. simplex; Pillai has described a nitrogen-fixing aerobic bacillus, B. malabarensis; Westermann studied a similar organism that he named B. danicus; while Beyerinck and van Delden observed, some years earlier, that, certain strains of B. mesentericus could fix relatively large amounts of nitrogen. Similarly Ps. radicicola has been found to possess a slight, but nevertheless an appreciable power to fix elementary nitrogen in culture solutions or in the soil.

But while nitrogen fixation among aerobic soil bacteria is not as uncommon as was at one time supposed, this function is so feeble and so variable in most instances, as to be of negative, or, at best, of doubtful economic significance. On the other hand, the aerobic, Azotobacter, first described by Beyerinck in 1901, may be regarded not only as possessing a very pronounced ability to fix atmospheric nitrogen, but as playing a role of some moment in maintaining the supply of combined nitrogen in the soil.

To the two species of Azotobacter, A. chroococcum and A. agilis described by Beyerinck and van Delden, Lipman added A. vinelandii (Fig. 132), A. beyerincki and A. woodstownii, and Lohnis and Westermann, A. vitreum. Of these species A. chroococcum and A. beyerincki are most common and are widely distributed in cultivated soils of Europe and America, and probably also of the other continents. They are absent in acid soils deficient in humus, and most common in limestone regions and in irrigated soils rich in mineral salts. Their food requirements are covered by solutions containing potassium phosphate, magnesium sulphate, calcium chloride and ferric sulphate, and some organic nutrient, such as dextrose, saccharose, xylose, mannit, acetate, propionate, butyrate, malate, ethyl alcohol, etc. An alkaline or neutral reaction and the presence of salts of iron are essential for the vigorous development of Azotobader, while humates have been shown by Krzemieniewski to exert a stimulating influence on the growth of these organisms, even though not acting directly as a source of food and energy. As shown by Lipman and others, Azotobacter may gain an increased power of fixing atmospheric nitrogen in the presence of other organisms. It is resistant to drying, notwithstanding the fact that it produces no spores, and has been successfully isolated from soil samples that had been kept in a dry state for several years. For some reason it may be detected in the soil most readily in the fall and winter months.

As to the nitrogen-fixation by fungi, it has been shown elsewhere that the evidence is, if anything, of a negative character. Some algae are able to fix atmospheric nitrogen, especially those that live symbiotically with azotobacter.

Fig. 132. Azotobacter vinelandi, a non-symbiotic nitrogen-fixing organism.
(After Lipman.)

Energy Relations.—In the fixation of nitrogen by bacteria the necessary energy for the process is furnished by the carbohydrates, organic acids, alcohols or other organic nutrients employed in the culture media. Since any given quantity of organic nutrient possesses a definite amount of potential energy the fixation of nitrogen is necessarily limited by the supply of such potential energy. This limitation was already recognized by Winogradski in his experiments with B. (Clostridium) pasteurianus. For every gram of dextrose used up there was produced, on the average, 2 to 3 mg. of combined nitrogen. In the experiments of Bredeman with B. amylobacter, and of Pringsheim with "Clostridium americanum" the amounts fixed were, at times, considerably larger. On the whole, however, it has been proved by a number of investigators that Azotobacter can fix much larger quantities of nitrogen than the anaerobic bacilli. The extended investigations of Lipman showed that A. vinelandii has the ability to fix more nitrogen per unit of organic nutrient consumed than either A. chroococcum or A. beyerincki. Under favorable conditions A. vinelandii may at times fix 15 or even 20 mg. of nitrogen per g. of mannit used up. Krzemieniewski found in experiments with A. chroococcum that additions of humates to the culture solutions increased the nitrogen fixed from a maximum of 2.4 mg. to a maximum of 14.9 mg.

The practical bearing of the foregoing data lies in the fact that the fixation of nitrogen in cultivated soils is limited, among other things, by the energy available, that is, by the quantity of readily decomposable organic residues. An indication as to the extent of these is given by the amount of humus present; nevertheless, this must remain an indication merely, for most of the humus is too inert to serve as a source of energy to Azotobacter. From the data at present available different investigators have estimated the quantity of nitrogen fixed by Azotobacter at 6.8 kg. to 18 kg. (15 to 40 pounds) per acre, per annum. Assuming favorable conditions for fixation, so that 500 g. (1 pound) of nitrogen could be fixed for every 50 kg. (100 pounds) of carbohydrate consumed, it would still take an equivalent of 680 kg. to 1,814 kg. (1,500 to 4,000 pounds) of sugar to produce this quantity of combined nitrogen. It may be noted in this connection that Azotobacter have been demonstrated to live in symbiosis with algae, obtaining thereby the necessary energy for their activities. This may explain, perhaps, the remarkable facts observed by Headden in Colorado, relating to the accumulation of such enormous quantities of nitrate in the soil as to destroy all vegetation. In some instances the nitrates were found to be present to the extent of 90,718 kg. (100 tons), or more (per acre), to a depth of a few inches. If the accumulation of combined nitrogen was due to Azotobacter, as is claimed by Headden, and the bacterial residues oxidized by nitrifying bacteria to nitrates, it is difficult to account for the source of the 1,000 or 2,000 tons of carbohydrates necessarily used up in the process of fixation, unless it could be proved that the energy was furnished by algae.