Fixation of free atmospheric nitrogen by Azoto- 



bacter in pure culture. 



Distribution of this bacterium. 



Proceedings of the Section of Sciences, Kon. Akademie van Wetenschappen, Amster- 

 dam, Vol. XI, 1908, p. 67 74. - Verscheen onder den titel Binding van vrije atmo- 

 spherische stikstof door Azotobacter in reincultuur. Verspreiding dezer Bacterie in 

 Verslagen Kon. Akademie van Wetenschappen, Wis-en Natuurk. Afd., Amsterdam, 



Deel XV, 1908, biz. 4653. 



When carbon hydrates are used as source of carbon in Azotobacter cultures, 

 there existed until now some doubt whether the then occurring fixation of 

 free nitrogen was originally effected by Azotobacter itself or by other bacteria found 

 in symbiosis with it, because Azotobacter 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 radio- 

 bacter, a species closely allied to the bacteria of the Papilionaceae, and which is 

 never absent in accumulations of Azotobacter, would be the real cause of the nitrogen 

 fixation 1 ). 



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. 



i. 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 4 , with addition of some 

 10 20 cM 3 canal-water, or as much soil for infection, care being taken that the 

 iayer of liquid in the jar be not thicker than 2 5 cM, on cultivation in a thermostat 

 at 30 C., usally 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. 



l ) These proceedings of March 1901. Centrbl. f. Bact. 2te Abt. Bd. 9 pg. i, 1902. 

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



especially in spring and autumn these experiments succeed. In summer and 

 zotobacter seems sometimes absent in the said quantity of water. 



