Winogradsky followed up his work and isolated from soil a large anaerobic spore-forming organism, capable of fixing nitrogen, to which he gave the name Clostridium pasteurianum. In 1901 the investigations of Beyerinck, in Holland, led to the important discovery of a group of large aerobic organisms, which he named Azotobacter. These were found to be very active in fixing free nitrogen. More recently, a number of other nitrogen-fixing bacteria have been described, and the property has been found to exist to a small extent in several previously well-known organisms.
It becomes important to determine which are the groups of bacteria whose nitrogen-fixing powers are of chief importance in the soil.
On account of its energetic fixation of nitrogen in culture media, Azotobacter has attracted the greatest attention of workers. The evidence seems to be consistent with the view that Azotobacter is of importance in the soil. Thus the distribution of Azotobacter would appear to be world-wide. It is found all over Western Europe and the United States. Lipman and Burgess[45] found it in soils collected from Italy and Spain, Smyrna, Cairo, the Fayum, the Deccan in India, Tahiti, Hawaii, Mexico, Guatemala, and Canada. C. M. Hutchinson[29] found it to be distributed throughout India. It was found by Omelianski[55] to be widely distributed in European and Asiatic Russia, and by Groenewege[28] in Java. Ashby[1] at Rothamsted, isolated it from soils from the Transvaal, East Africa, and Egypt. Also, an association has sometimes been found between the ability of a soil to fix nitrogen and the occurrence and vigour of its Azotobacter flora. Thus Lipman and Waynick[46] found that if soil from Kansas were removed to California, its power to produce a growth of Azotobacter, when inoculated into a suitable medium, was lost, and, at the same time, its nitrogen-fixing power was greatly reduced. Moreover, it is known that conditions favourable to the fixation of nitrogen by Azotobacter in cultures on the whole favour nitrogen fixation in soils. The conditions that favour other aerobic nitrogen-fixing bacteria are, however, not sufficiently distinct to make such evidence of great value.
It is usually found that nitrogen fixation is most active in well-aerated soil. Thus Ashby,[1] at Rothamsted, found the nitrogen-fixing power of a soil to decrease rapidly with depth. Similar results were obtained in Utah by Greaves. This suggests, at first sight, that anaerobic nitrogen fixers are unimportant under normal soil conditions. It is, however, quite possible that they may assume an importance when acting in conjunction with aerobic organisms. Thus Omelianski and Salunskov[55] found that beneficial association, or symbiosis, could occur between Azotobacter and Clostridium pasteurianum, the former absorbing oxygen from the surroundings, and thus creating a suitable anaerobic environment for the Clostridium.
The question of symbiosis of nitrogen-fixing bacteria with each other and with other organisms offers an inviting field for research. There is evidence that this factor may have considerable importance. Beijerinck and Van Delden[3] early recognised that Azotobacter in mixed cultures fixed more nitrogen than in pure cultures. Granulobacter, an organism which they found to be commonly associated with Azotobacter in crude cultures, appears to increase its nitrogen-fixing powers (Krzeminiewski).[41] It was also found by Hanzawa[31] that a greater fixation of nitrogen was obtained when two strains of Azotobacter were grown together. A symbiosis between Azotobacter and green algæ has been described, and will be further [discussed] by Dr. Bristol. It is likely that this association may be of importance under suitable conditions on the soil surface where the algæ are exposed to light.
The combination of elemental nitrogen is an endothermic process which requires a very considerable amount of energy for its accomplishment. This fact is well illustrated by the various commercial processes in use for fixation of atmospheric nitrogen. The nitrogen-fixing bacteria obtain this energy from the carbon compounds in the soil. A number of compounds were compared as sources of energy by Löhnis and Pillai,[47] who tested their effect on the amounts of nitrogen fixed by Azotobacter in culture. It was found that mannitol and the simpler sugars give the best results as sources of energy, but that other organic compounds can also be used. Mockeridge[51] has adduced evidence that ethylene glycol, methyl-, ethyl-, and propyl-alcohol, lactic, malic, succinic, and glycocollic acids could also be utilised. Since so large a part of the organic matter added to soil is in the form of celluloses, it is of great importance to ascertain how far these compounds and their decomposition products can be utilised in nitrogen fixation. Stubble, corn-stalks and roots, oak leaves, lupine and lucerne tops, maple leaves, and pine needles may all serve as useful sources of energy to nitrogen-fixing organisms in the soil. Pure cellulose cannot apparently be used as a source of energy, but when acted upon by cellulose decomposing organisms, it becomes available as a source of energy. Hutchinson and Clayton, at Rothamsted, found that a fixation of nitrogen could be brought about by mixed cultures of Azotobacter, and of the cellulose attacking Spirochæta cytophaga, when grown in cultures containing pure cellulose. It is not known how far cellulose decomposition must proceed to produce an effective source of energy, nor what are the substances thus produced that are utilised. This point will not be decided until something more is known of the course of changes in the breaking-down of cellulose in the soil.
The amount of nitrogen fixed per unit of energy material decomposed varies greatly, according to the organism and the conditions. Winogradsky found that his Clostridium assimilated 2-3 mgs. of nitrogen per gram of sugar consumed. Lipman found that Azotobacter fixed 15-20 mgs. of nitrogen per gram of mannite consumed.
Fig. 5.
Caption: Azotobacter. Decrease in efficiency in N fixation with age of culture. (Koch & Seydel.)