HISTORICAL 249 



We know that soil gains in nitrogen are often due to microorganisms, 

 either living free in the soil or in company with the higher plants. 

 The production of nitrogen compounds out of atmospheric nitrogen 

 by bacteria independent of higher plants is designated non-symbiotic 

 nitrogen-fixation, or azofication. When fixation is accomplished by 

 bacteria living in connection with and receiving benefit from higher 

 plants, it is called symbiotic nitrogen-fixation. 



As early as 1SS3 Berthelot undertook the study of soils as 

 regards their relationship to free and combined nitrogen, and as a 

 result of these studies he was the first definitely to recognize that 

 gains which occur in bare unsterilized soils are due to microscopic 

 organisms. He found that when 50 kgm. of arable soil were exposed 

 to air and to rain in a vessel for seven months, after allowing for the 

 small amount of combined nitrogen brought down by the rain, there 

 was a gain in nitrogen of more than 25 per cent. In another experi- 

 ment in which the soil was first washed free from nitrates, there was 

 a gain of 46 per cent. Many other experiments showed gains from 

 10 to 15 per cent. Berthelot was not content with the bare knowl- 

 edge that nitrogen is fixed in the soil by living organisms, but con- 

 tinued his work with the idea of isolating some of these organisms. 

 With the aid of Guignard, he made soil inoculation into sterile 

 bouillon and from this prepared gelatin plates. Cultures were taken 

 from the colonies growing on the plates and bacteria were tested 

 for their nitrogen-fixing power. His results were conclusive that 

 there exist within the soil chlorophyll-free bacteria capable of fixing 

 atmospheric nitrogen. His work had shown that these organisms 

 act best at summer temperatures, between 50 and 104 F., in the 

 presence of a good supply of oxygen, a proportion of water in the 

 soil not exceeding 12 to 15 per cent, and not falling below 2 to 3 

 per cent. 



They require carbon, hydrogen and enough combined nitrogen 

 to promote initial growth. The nitrogen, gained by the soil was 

 proteinaceous in nature, being insoluble in water. Although some 

 of his soils had gained large quantities of nitrogen, he considered 

 that the fixation of atmospheric nitrogen by microorganisms has 

 its limits, since the organisms isolated drew from the atmosphere 

 only so long as the amount fixed in the medium was not great. Heat- 

 ing the soil to 230 F. immediately stopped the process. 



Prior to this a number of chemists, notably Konig and Kiesow, 

 Armsby, Birner, Kellner, Deherain and Avery had found that when 

 organic matter in one form or another undergoes fermentation there 

 is frequently an increase of nitrogen in the fermenting substance. 

 Armsby states it thus: "We must conclude that decaying organic 

 substances in the presence of caustic alkali are able to fix free 

 nitrogen without the gain being manifest as nitric acid or ammonia, 

 and probably without the formation of these bodies." His explana- 



