112 Perspecflves in Microbiology 



except combined nitrogen; the legume, inoculated with 

 the proper bacteria, was grown in the outer glazed pot; the 

 nonlegume, in the inner unglazed one. 



Lipman noted that, at times, the nonlegume grew as 

 though it were being supplied with nitrogen from some 

 source and assumed it to be the legume. "Noted" is used 

 advisedly in this connection, because many of his observa- 

 tions were merely that; but, as these observations were 

 confirmed by analyses, Lipman proposed that nitrogenous 

 compounds were excreted from the nodules of the legume 

 to its companion. This explanation was too bizarre for the 

 time and was immediately challenged and then forgotten. 



In the late twenties, A. I. Virtanen in Finland, using a 

 similar type of crude apparatus, made the same kind of 

 observation and, unaware of Lipman's earlier experi- 

 ments, described once again this curious phenomenon in 

 mixed cropping. By now, however, improvements in plant 

 physiological techniques had been made, so he quickly 

 abandoned experiments with the crude clay pot and con- 

 centrated on investigations using the definitely more so- 

 phisticated type of apparatus illustrated in Figure 2. The 

 details of this technique have been described in Virtanen's 

 stimulating account of his experiments (13). Here we need 

 observe only that his apparatus provided him with a 

 method for studying the excretion under "sterile" con- 

 ditions, that is, in absence of microorganisms other than 

 the root nodule bacteria. 



This improvement in technique enabled him to recover 

 sufficient excreted nitrogen to characterize it even by the 

 relatively insensitive methods available at that time. Be- 

 cause these excreted products appeared to be almost ex- 

 clusively aspartic acid and its decarboxylation product, 

 P-alanine, Virtanen proposed that the mechanism of nitro- 

 gen fixation proceeded by way of hydroxylamine, which 

 then entered the amino acid pool via aspartic acid (13). It 

 is unnecessary to note that we challenged this interpreta- 



