CARBON AND NITROGEN CYCLES IN THE SOIL 197 



accounting for nearly half the sugar. A little alcohol was 

 found, but practically no non-W)latile acid. There was a dis- 

 tinct relationship between the amount of nitrogen assimilation 

 and the sugar decomposed, each milligram of nitrogen fixed 

 requiring the oxidation of about 5Q0 milligrams of sugar. 



Three organisms were present, a Clostridium and two 

 bacteria, and they obstinately refused to be separated by the 

 method of successive cultures. Not until recourse was had to 

 anaerobic conditions were the two bacteria suppressed and the 

 Clostridium obtained pure. The bacteria having been isolated, 

 it appeared that the Clostridium alone possessed the power of 

 fixing nitrogen, but a fresh difficulty now arose because in 

 pure cultures the organism would work only under anaerobic 

 conditions. Only when the protective bacteria were simul- 

 taneously present did fixation go on in presence of air. The 

 organism was called Clostridium pasteurianum : ^ it formed 

 rods i'2 fj, thick and i '5 to 2 ytt long and also spores (312). 



In order to simplify the bacterial flora Winogradsky had 

 heated his soil to 75°, thereby killing non-spore formers, but 

 later on Beijerinck (14 and 15), working with unheated soil 

 discovered three other nitrogen-fixing organisms ; Azotobacter 

 chroococcum (so called because, as it ages, it turns brown and 

 finally almost black), Granulobacter and Radiobacter.^ Of 

 these azotobacter is the most active ; it forms large cocci, or 

 rods, 4 to 6 yu, in thickness.^ It differs in two important respects 

 from Clostridium : (i) it is aerobic ; (2) it produces practically 

 no butyric acid. Its effects can be studied by inoculating O'l 

 to 0-2 grm. of soil into 100 c.c. of tap water containing 2 per 

 cent, mannitol, -02 per cent. K2HPO4, and sufficient CaCOg, 

 and keeping for some weeks at 27" to 30° C. in a thin, well- 

 aerated layer* in an Erlenmeyer flask. Azotobacter fixed 



^ For further investigation of Clostridium see Omelianski, Rome Bull., 1917, 

 8, iigo. 



2 Since shown by Stoklasa (2756) to possess only slight nitrogen-fixing 

 power. 



* Lohnis and Smith describe cyclical changes through which azotobacter is 

 considered to pass {yourn. Ag. Res., 1916, 6, 675). 



* Later on Beijerinck used calcium malate in place of sugar, and showed also 

 how to make plate cultures of the organisms (16). 



