250 AZOF 1C AT ION 



tion of the process was that the nascent hydrogen evolved during 

 the fermentation process reacted with the free nitrogen of the air. 

 Others considered that the active agents were compounds of iron, 

 manganese, and lime existing in the soil and in some way acting 

 as catalytic agents. 



Berthelot's discovery interested Winogradsky who commenced 

 work which eventually bridged the chasm. He employed, as a 

 medium, a nutritive solution free from combined nitrogen, but con- 

 taining mineral salts and dextrose. Fifteen separate species of soil 

 bacteria were isolated, but only one a long sporebearing bacillus 

 which developed normally in the absence of combined nitrogen and 

 seemed to produce butyric fermentation fixed nitrogen to any 

 appreciable degree. Quantitative tests showed that the maximum 

 fixation was attained where no combined nitrogen was purposel 

 added, and that on the addition of such, fixation of nitrogen was 

 diminished. For example, several determinations gave the following 

 results: 



N as NH 3 in dextrose solution . . 2.1 4.2 6.4 8.5 21.2 

 Nfixed 7.0 5.0 5.5 3.6 2.2 



The presence of combined nitrogen tends to decrease fixation. 

 He concluded that in order for any gain to be made, the ratio of the 

 combined nitrogen to the sugar should not exceed 6:1000. Because 

 of the characteristic formation of clostridia in his cultures, Wino- 

 gradsky named the organism Clostridium pasteurianum. The con- 

 clusion which the author reached, however, was that the power of 

 fixing nitrogen is not general among microorganisms, but confined 

 to a few special forms. 



Following Winogradsky, Caron made some interesting discoveries. 

 He found that soils under leafy crops contain greater numbers of 

 bacteria than those under grasses. He also observed that the bac- 

 terial flora of soils in the spring are different from *those in the fall 

 both quantitatively and qualitatively. He used in vegetation 

 experiments pure cultures of the bacteria most frequently 

 encountered in natural soils. Some soils were inoculated with 

 bouillon culture, whereas others received only sterile bouillon. 

 The crop yields were usually in favor of the inoculated plots, but 

 showed variations from season to season. Exceptionally good results 

 were obtained with a sporebearing bacillus which he termed Bacillm 

 ellenbachensis. 



Caron's work led to the commercial exploitation of his cultures, 

 one of which, "alinit," was the subject of much study and contro- 

 versy. This culture was found to contain, according to Severin, two 

 closely-related bacilli which he chose to designate as B. ellenbachensis 

 A and B. These had the power to fix nitrogen to some extent. Tests 



