Biological Nitrogen Fixation 117 



conventional reactions, such as reductive assimilation and 

 transamination. But direct evidence for NH3 eluded us 

 until just a few years ago when we began the tracer studies 

 on Clostridium pasteurianum. Traditionally, this organism 

 is a sluggish fixer and was believed to be both inefficient 

 and slow. But a study of its physiology and the employment 

 of modern methods of culture (for example, shake flasks) 

 demonstrated that fixation by this organism was almost as 

 good as by Azotohacter (10). 



When both the rate and the extent of fixation by the 

 Clostridia had been thus increased, the organism excreted 

 large quantities of ammonia into the medium — it seemed 

 that a medium good enough for a sluggish fixer w^as defi- 

 cient for this new intensive mode of life. The organism 

 apparently was fixing nitrogen more rapidly than it could 

 be assimilated and was discarding the excess into the 

 medium (10). This was direct evidence for ammonia as the 

 end product of fixation, provided that the excreted am- 

 monia could be demonstrated to be "juvenile" nitrogen — 

 recently fixed, and not merely breakdown products of the 

 cells. Before the time of isotopic tracing, this would have 

 been a difficult assignment, but now it was readily tested 

 by employing labeled N2^^. Since the excreted ammonia-N 

 was considerably higher in its level of tracer than any other 

 compound found, and was followed by substances such as 

 amides, known to be storage compounds for ammonia, it 

 was concluded that this provided the long-sought-for isola- 

 tion of the key intermediate in biological nitrogen fixation 



(21). 



Our satisfaction with this result was somewhat allayed by 

 the fact that we could not repeat it with Azotobacter. This 

 organism would not excrete nitrogenous compounds in 

 spite of all our efforts; the growth of the cell apparently 

 always keeps ahead of its rate of fixation. Somewhat in 

 desperation, the isotopic dilution method was tried — a 

 method in which we did not have much hope, since it re- 



