CARBON AND NITROGEN CYCLES IN THE SOIL 91 



n-butyric and acetic acids in the proportion of three or four molecules 

 of the former to one molecule of the latter, the two acids together 

 accounting for nearly half the sugar. A little alcohol was found, but 

 practically no non-volatile acid. There was a distinct relationship 

 between the amount of nitrogen assimilation and the sugar decomposed, 

 each milligram of nitrogen fixed requiring the oxidation of about 500 

 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 only work under anaerobic con- 

 ditions. Only when the protective bacteria were simultaneously present 

 did fixation go on in presence of air. The organism was called Clostri- 

 dium Pasteurianum : it formed rods i "2 /* thick and 1-5 to 2 fi long, 

 and also spores (314). 



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 Radio- 

 bacter.^ Of these azotobacter is the most active ; it forms large cocci, or 

 rods, 4 to 6 /i in thickness, and does not produce spores. It differs in 

 two important respects from Clostridium : (i) it is aerobic; (2) it pro- 

 duces practically no butyric acid. Its effects can be studied by inoculat- 

 ing O'l to 0'2 grams of soil into 100 cc. of tap water containing 2 

 per cent, mannitol, 02 per cent. K2HPO4, and sufficient CaCOj and 

 keeping for some weeks at 27° to 30° C. in a thin, well-aerated layer 2 

 in an Erlenmeyer flask. Azotobacter fixed more nitrogen than Clostri- 

 dium per gram of sugar decomposed ; Gerlach and Vogel's (104) 

 results are : — 



' Since shown by StoUasa (276) to possess only slight nitrogen-fixing power. 

 " Later on Beijerinck used calcium malate in place of sugar, and showed also how to 

 make plate cultures of the organisms (16). 



' The sugar was not all used up in this experiment. 



7* 



