But where there is an excess of carbohydrate or other non-nitrogenous source of energy available in the soil, the case is different. Here the organisms have a supply of energy which enables them to multiply rapidly until the organic nitrogen is insufficient for their needs. Hence they turn to the ammonia and nitrate present in the soil, and build up their proteins from this source. Doryland[17] has shown that many common soil ammonifiers assimilate ammonia and nitrate when supplied with carbohydrate. There may thus be a temporary loss of nitrate from soil when sugar, starch, straw, or such materials are added to it. (4) _Fixation of Free Nitrogen._ The bacteria that we have so far considered take up their nitrogen directly from compounds containing this element. There remain, however, a comparatively small but very important group of bacteria possessing the power of causing elemental nitrogen to combine, and of building it up into their proteins. This fixation of nitrogen by micro-organisms is a vital step in the economy of nature. Losses of nitrogen from the land are continually occurring through the washing-out of nitrates by rain, and through the evolution of gaseous nitrogen during the processes of decay. To maintain the supply of combined nitrogen which is essential to living organisms, there must therefore be a compensating process by which the supply of nitrogen compounds in the soil is kept up. It was discovered in the middle of the nineteenth century that if soil were kept moist and exposed to the air, there was an increase in the amount of nitrogen compounds present. Berthelot, in 1893, studied the nitrogen relationships of soil, and recognised that this fixation of nitrogen in soil was the work of micro-organisms. 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.