202 Nitrogen Fixation— Nonsymbiotic 



Kostytschev and Winogradsky demonstrated that ammonia is pro- 

 duced in cultures of Azotobocter. They conckided that this am- 

 monia is the first stage in the fixation of nitrogen b)- bacteria. Burk 

 found that ammonia was present in the older cultures and concluded, 

 therefore, that it is rather a secondary decomposition product. The 

 inability to find ammonia in \oung cultures of the organism speaks 

 against its being the first step in the fixation process. 



Blom suggested the following reactions as explanations of the 

 mechanism of the fixation of nitrogen, through the h\drox\lamine 

 stage. This was produced by cultures of Az. ogilis, iron serving as a 

 catalyst: 



1. N^N (atmospheric) :;=i N^X (solution). 



2. 2(Cat. Fe++) + N^N ^ (Cat. Fe++)2-N=^N. 



3. (Cat. Fe++)2-N=N + iR.O ^ 



(Cat. Fe++)2 • HOXH— HXOH. 



4. (Cat. Fe++)2- HOXH— HXOH + ^2H+ ^ 



2(Cat. Fe+++) + '^HOXH.. 



5. (Cat. Fe+++) + H ^ (Cat. Fe++) + H+. 



The hydrox) lamine, once produced, will iuteract with oxalacetic 

 acid to give rise to oximes. which are changed to aspartic acid, as 

 shown later. 



According to Winogradsky, both anaerobic and aerobic bacteria 

 produce ammonia out of the nitrogen gas and nascent hydrogen with 

 which they come in contact. In the case of the anaerobes, the 

 hydrogen is formed during the butxric acid fermentation. In the 

 case of Azofobacter, an enzyme, azoh)'drase, concerned in the am- 

 monia synthesis is believed to be produced. A part of the am- 

 monia is immobilized by the growing cells, and a part is excreted 

 into the medium. The molecular nitrogen acts as a h\drogen ac- 

 ceptor, the action of the enzyme continuing even after the death of 

 the cells. 



Wieland also considered that the action of the hydrogen acceptors 

 formed in the cells of nitrogen-fixing bacteria does not depend upon 

 oxygen for hydration, but rather upon the molecular nitrogen with 

 which the hydrogen forms ammonia, perhaps through the hydrazine 

 stage in a manner similar to the Haber synthesis. 



Virtanen found aspartic acid in young cultures of Azotobacter 

 before ammonia could be detected; this led him to conclude that 



