Bacteria and the Nitrogen Cycle.—The second great group of compounds left in the soil by plants and added thereto by manure are the nitrogen compounds such as proteins and their derivatives. Soil organisms themselves provide a further source of such compounds. The decomposition of organic nitrogen com pounds can be brought about by a very large number of bac teria, actinomycetes and fungi and the great complexity of the soil microflora has rendered it impossible to isolate and classify more than a small fraction of these, or to follow the stages through by which proteins are decomposed. During the process, the nitro gen is released as ammonia. The production of ammonia is, how ever, a by-product in the economy of the bacteria, which are deriving energy by decomposing the organic compounds. They can indeed be prevented from attacking the proteins, if a more readily available non-nitrogenous energy source is offered them. Thus the production of ammonia in soil can be depressed by the addition of sugar or straw.
The ammonia that is produced in soil does not usually accumu late there, but is oxidised successively to nitrite and to nitrate. An interesting organism Nitrosomonas is able to oxidise ammonia to nitrite. Pure culture study has shown that this organism derives its carbon by synthesis from a process which requires a considerable expenditure of energy. It is unable to utilise organic compounds as sources of carbon. The energy necessary for as similation of is obtained by oxidising ammonia to nitrite. The nitrifying organisms are normally very sensitive to acidity and will not work efficiently unless the nitrous acid that they produce is neutralised by an available base which in soil is usually calcium carbonate. Since however, nitrification will take place in certain acid forest soils, there are probably strains of the organ ism unusually tolerant of acid conditions. A second ammonia oxidising organism named Nitrosococcus has been described. It has been suggested that this is a stage in the life-history of Nitrosomonas.
The nitrites produced from ammonia do not accumulate in the soil but are rapidly oxidised to nitrates. An organism named Nitrobacter, capable of bringing this about, has been studied in pure culture. It resembles Nitrosomonas in deriving its carbon from and obtains the necessary energy for this by oxidising nitrites. The activity of both Nitrosomonas and Nitrobacter, when grown in artificial media is hindered by the presence of soluble organic compounds. It is probable that in the soil they are less sensitive, since the formation of nitrate occurs rapidly in such media as richly manured soils and in the purification of sewage.
The nitrates produced in the soil are the principal source of nitrogen available to crop plants. They can also supply nitrogen to the majority of soil micro-organisms. When the soil contains an excess of available carbohydrate materials the energy supplied by these enables an immense multiplication of micro-organisms to take place and these obtain their nitrogen by assimilating ni trates and ammonium compounds, thus depleting the soil of nitrogen that would otherwise have been available to the crop plants. This is the principal reason why the ploughing in of straw and similar substances, in which the ratio of carbon to nitrogen is high, has a depressing action on the crop. The nitrogen locked up in the cells of micro-organisms is, of course, not lost to the soil, but is released by their death and decomposition, so that the loss of nitrates is only temporary. Indeed at times when the soil is uncropped, this assimilation of nitrates may be beneficial since it lessens the loss of nitrogen through the leaching action of rain. When the soil has become exhausted of its available nitrogen compounds, any further supply of carbohydrates can only be utilised by those micro-organisms that possess the power of taking up the free nitrogen in the soil atmosphere. A number of bacteria
are now known that possess the power of assimilating or "fixing" elemental nitrogen. The first of these to be described was an anaerobic spore-forming rod, Clostridium pasteurianum. It has been shown that this organism increases rapidly in water-logged soil to which sugar is added, so that it may be of importance in wet soils. Recently, moreover, it has been found that Clostridium and various aerobic bacteria can grow in mixed culture in the presence of air, the aerobic bacteria presumably removing oxygen and creating an anaerobic environment for Clostridium. When sugar or mannitol is added to well aerated soil, however, aerobic nitrogen-fixing bacteria become predominant. Amongst these the most abundant is Azotobacter. The widespread distribution of Azotobacter and the fact that it is the chief form that appears when soluble carbohydrates are added to soil makes it probable that this organism is chiefly responsible for nitrogen fixation. It is very sensitive to acidity and does not develop in soils whose reaction is on the acid side of pH 6-o. It also requires a con siderable supply of phosphates, a deficiency of which limits its development in certain soils. Various other aerobic nitrogen fixing bacteria have been described but are probably of less im portance in the soil than Azotobactei-. The physiology of both Azotobacter and Clostridium pasteurianum have been much studied under laboratory conditions. The process by which nitro gen is fixed is not yet understood but it is probable that the nitrogen is combined with hydrogen and not with oxygen. It is claimed on thermodynamical grounds that this combination re quires no expenditure of energy. In culture solutions, however, about ioo grams of dextrose are utilised by Azotobacter for every gram of nitrogen fixed, though the figure is very variable. No doubt the large amount of dextrose consumed also supplies the energy needed to build up protein from the simpler com pounds that are first produced. Clostridium pasteurianum, with an anaerobic metabolism, consumes about 400 grams of dextrose per gram of nitrogen fixed. The difference between Azotobacter and Clostridium in this respect is due to the degree to which the carbohydrate is decomposed. Azotobacter breaks down the sugar almost completely to and water, whereas with Clostridium, the decomposition is only partial, fatty acids, alcohol and H2 being produced as well as This partial decomposition pro duces far less energy per gram of sugar utilised than is obtained by Azotobacter. Indeed, the amount of nitrogen fixed per unit of energy made available, is higher in the case of the Clostridium. The efficiency of nitrogen fixation by Azotobacter is claimed to be higher in the soil than when it is grown in solution. More over, under natural conditions, it is probable that nitrogen fixing bacteria are assisted by the presence of other organisms which remove the by-products of their metabolism. Impure cultures have been found to fix more nitrogen than pure cultures, while the presence of protozoa has been found to stimulate fixation. The association of nitrogen fixing bacteria with green plants pro vides a special case of symbiosis. In this case the green plant, in addition to removing by-products, also supplies the bacteria with carbohydrate produced by photosynthesis. The association of nitrogen-fixing bacteria with algae is a simple case. Of greater importance, however, is their association with higher plants, espe cially legumes. The organism forming nodules on the roots of legumes was first isolated in 1888 by Bejerinck who named it Bacillus radicicola, and since then has formed the subject of a great deal of research. The organism passes a portion of its life in the soil where it can exist for years without the presence of its host plant. It exists in several cell forms which appear to con stitute a definite life-cycle.