FIXATION OF ATMOSPHERIC NITROGEN. 269 



CHEMICAL AND BIOLOGICAL RELATIONS. The second and third 

 quarters of the nineteenth century saw the birth of a number of theories 

 dealing with this problem. It was suggested that nitrogen compounds 

 may be formed in the soil by the oxidation of nitrogen to nitric acid. Com- 

 pounds of iron, manganese and lime were supposed in some way to make 

 such oxidation changes possible. It was likewise suggested that nascent 

 hydrogen may be generated in the decomposition of organic matter in the 

 soil, and reacting with elementary nitrogen, may give rise to ammonia. 

 The various hypotheses were not supported by experimental proof; 

 moreover, the situation was complicated by the knowledge, based on 

 empirical observations, that crops of the legume family seemed to be 

 more or less independent of the supply of combined nitrogen in the soil. 

 Indeed, clovers and other legumes had, apparently, the ability to increase 

 the content of combined nitrogen in the soil as was indicated by the ex- 

 periments of Boussingault and of Lawes and Gilbert. Finally, the 

 mystery was solved by the investigations of Berthelot and of Hellriegel 

 and Wilfarth who furnished the proof that elementary nitrogen may be 

 utilized by plants when certain biological relations are met. These 

 relations involve the presence and activities of microorganisms that by 

 themselves, or in conjunction with higher plants, make available to grow- 

 ing vegetation the great store of atmospheric nitrogen. 



NON-SYMBIOTIC FIXATION OF NITROGEN. 



HISTORICAL. Non-symbiotic nitrogen fixation, or Azofication, has 

 already been defined as the production of nitrogen compounds out of 

 atmospheric nitrogen by bacteria independently of higher plants. The 

 part played by bacteria in this process was not recognized until 1885, 

 when Berthelot published some of his data on the accumulation of com- 

 bined nitrogen in uncropped soils. His results seemed to explain a 

 number of scattered observations, made since the middle of the century, 

 on the apparent increase of the nitrogen content of cultivated soils. 



While Berthelot's experiments proved that the nitrogen gains occurred 

 only in unsterilized soils and were, therefore, due to microorganisms, 

 it remained for Winogradski to demonstrate, in 1893, that the formation 

 of nitrogen compounds by certain types of bacteria may be accomplished 

 in culture media nearly or quite devoid of combined nitrogen. Soon 

 after that he succeeded in isolating his organisms in pure culture, and 



