222 BACTERIOLOGICAL CHEMISTRY 



can then be found in the medium after the organisms have 

 been removed by centrifugalisation ; nitrites or nitrates 

 were not detected. These experiments support Winograd- 

 sky's theory that the nitrogen is fixed by reduction to 

 ammonia, which is then converted to amino-compounds ; 

 further evidence is the fact that the fixation is inhibited 

 by the presence of ammonia and of nitrate, and that 

 nitrate is reduced to ammonia, as was shown by Stoklasa. 



The fixation of nitrogen seems to be intimately bound 

 up with the growth of the organism since the ratio of 

 nitrogen fixed to glucose fermented (supplying the energy 

 required) is highest in the early stages when growth is 

 rapid, but falls off with the age of the culture until finally 

 no more nitrogen is fixed, though glucose continues to be 

 fermented ; nitrogen is no longer fixed at this stage 

 because there is no further growth, and therefore no 

 synthetic requirements, but glucose continues to be 

 fermented by the enzymes which have already been 

 produced, and liberated by autolysis of the old cells. 



The optimum pH for nitrogen fixation by A. agile is 

 between 7-6 and 7-8. Calcium, which can be replaced 

 by strontium which is rather less effective, is necessary 

 for nitrogen fixation by Azotobacter, at a concentration 

 of 2x 10~* molar. Molybdenum at a concentration of 

 10~^ molar, vanadium at 2 x 10~^ molar and iron at 

 10"^ molar stimulate growth and nitrogen fixation of 

 Azotobacter. 



CI. butyricum, after continued cultivation on labora- 

 tory media, tends to lose its nitrogen fixing power. How- 

 ever, the power can be restored by cultivation in sterile 

 soil, a procedure somewhat analogous to animal passage 

 in the restoration of the virulence of pathogens. Azoto- 

 bacter shows no tendency to such loss of activity. 



Besides the free-living bacteria which fix nitrogen there 

 is a group which live in symbiosis with the leguminous 

 plants. The bacteria grow in the nodules on the roots of 

 the plants which supply the carbon and energy require- 



