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Microbiology. — "Fixation of free atmospheric nitrogen by Azoto- 

 bacter in pure culture. Distribution of this bacterium " By 

 Prof. M. W. Beijkrinck. 



(Communicated in the meeting of May 30, 1908). 



When carbon hydrates are used as source of carbon in Azotobacter 

 cultures, there existed until now some doubt whether the then occur- 

 ring fixation of free nitrogen was originally effected by Azotobacter 

 itself or by other bacteria found in symbiosis with it, because Azoto- 

 bacter in pure culture with carbon hydrates and free nitrogen only, 

 does not show any considerable development. 



For this reason I was formerly of opinion that in such cultures 

 Bacillus radiobacter, a species closely allied to the bacteria of the 

 Papilionaceae, and which is never absent in accumulations of Azoto- 

 bacter, would be the real cause of the nitrogen fixation. l ). 



Continued research, however, rendered this supposition more and 

 more improbable, and the facts which are now to be stated have 

 proved beyond any doubt that the said faculty belongs indeed to 

 Azotobacter itself. 



These facts have regard to the very peculiar relation between 

 Azotobacter and the salts of the organic, acids, more in particular to 

 calcium malate. 



1. Calcium malate as source of carbon. 



When into a wide Erlenmeyer jar a nutrient liquid is introduced 

 of the composition: 100 tap-water, 2 calcium malate, 0.05 K'HPO', 

 with addition of some 10 — 20 cM 3 canal-water, or as much soil 

 for infection, care being taken that the layer of liquid in the jar 

 be not thicker than 2 —5 cM, on cultivation in a thermostat at 

 30° C, usually after 2 or 3 days 2 ) a floating Azotobacter film 

 appears, consisting of strongly motile individuals, and relatively soon 

 obtaining a considerable thickness. Hereby so much calcium carbonate 

 is produced that it forms a closed, floating layer, so to say a cover, 

 on the surface of the liquid. 



If some of this film is inoculated into another jar containing the 

 same medium, corresponding phenomena arc seen when the cull ure 



!) These proceedings of March 1901.Centr.bl. f. Bact. 2te Abt. Bd. 9 pg. 1, 1902. 

 Archives Need. (2) T. 8 p. 190 and 319, 1903. 



2 ) Especially in spring and autumn these experiments succeed. In summer and 

 winter Azotobacter seems sometimes absent in the said quantity of water. 



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