FIXATION OF NITROGEN 55 



Ng-fixation. After exposing cultures of CI. pasteurianum in 

 the log phase of growth to Ng^^, the concentration of N^^ 

 in the NH3 isolated from the medium was greater than that 

 of the amide nitrogen of the cell protein which was in turn 

 greater than the average level of the isotope in the pro- 

 teins as a whole. This indicated that the excreted NH3 

 was a product of the fixation processes and did not arise 

 by deamination of amino-acids [51]. The excretion of NH3 

 only occurs when the organisms are grown in certain media, 

 and it is apparently an expression of a deficiency, probably 

 of suitable organic acceptors, since supplementing the 

 medium with biotin, ^-aminobenzoic acid and a-keto- 

 glutarate completely stopped the excretion of NH3 and yet 

 had little effect on the rate of N2-flxation [43]. 



Role of hydroxylamine in N2-fixation 



Because leguminous plants apparently excreted only 

 aspartic acid and substances related to it [38, 5], Virtanen 

 originally concluded, especially in view of the presence of 

 the oximino-compound, that hydroxylamine (NHgOH) was 

 the key product of the fixation mechanism. He proposed 

 that NH2OH condensed with oxaloacetate, a substance 

 known to be present in nodulated roots, thus forming 

 oximinosuccinic acid which was then reduced to aspartic 

 acid. Many other workers were unable to repeat these experi- 

 ments, and following a successful visit to Virtanen's labora- 

 tory, Wilson concluded that excretion was observed only 

 when the rate of photosynthesis was not sufficient to supply 

 enough materials for the utilization of all the products of 

 the fixation mechanism [41]. The later discovery [39] that 

 glutamic acid was present amongst the excreted substances 

 implied that many of the results of the leguminous plant 

 experiments could also be interpreted in favour of the 

 importance of NH3 in the fixation mechanism. Whilst it 

 seems most probable that nitrogen enters into organic com- 

 bination in the form of NH3 and not NHgOH, the possi- 

 bility that NHgOH is a precursor of the NH3 , and perhaps, 

 in certain circumstances, reacts directly with an organic 

 acceptor, has not yet been excluded [cf. 37]. 

 5 



