330 The Physiology of Plants BOOK in 



cerned formed a number of masses of zoogloea. He kept 

 the acid neutralized and the fermentation went on so long 

 as any sugar remained in the fluid. When it was finished 

 fungal and algal intruders were able to grow in the culture 

 fluid, proving that combined nitrogen was then present, 

 and consequently that it had been formed in the process 

 of the fermentation. Chemical analysis also showed this 

 to be the case. The zoogloea on . examination proved to 

 contain two filamentous bacteria and a species of Clos- 

 tridium. These were successfully isolated, with, however, 

 very great difficulty, the Clostridium proving far from 

 easy to cultivate. So isolated, their respective activities 

 were determined. 



Winogradsky ascertained that the presence of the three 

 organisms is necessary for fixation unless oxygen is excluded 

 from the culture. The actual fixation is carried out by 

 the Clostridium, which is strictly anaerobic. The part 

 played by the other two organisms is the consumption of 

 the free oxygen of the fluid. For this purpose they can be 

 replaced under appropriate conditions by other organisms. 

 Only after the action of these oxygen-consuming microbes 

 can the Clostridium begin to develop. Clostridium has 

 since been carefully examined by its discoverer and its 

 peculiar powers and properties are known. He did not 

 learn, however, anything as to the form of the combination 

 into which the atmospheric nitrogen is made to enter. 



Winogradsky claimed to have determined that Clostri- 

 dium and forms related to it are the only microbes with 

 the capacity for fixing nitrogen. This is in opposition to 

 Berthelot, in whose opinion the power is far more widely 

 spread. Berthelot's views were shared by Beijerinck, who 

 claimed it for a bacterium he called Azotobacter, and for 

 certain Cyanophyceae, including Nostoc and Anabcena. He 

 based upon his observations a special class of physiological 

 organism, to which he gave the name ' oligonitrophilous '. 



