DENITRIFICA TION A ND NITROGEN FIX A TION 233 



very thorough elucidation of the details of the part played by certain Bacteria 

 in this process, Berthelot (1892) having previously shown that nitrogen 

 combination must be due to the activity of Bacteria. 



WiNOGRADSKY (1895) made use of the experience he had gained in culti- 

 vating sulphur- and nitro-bacteria, and began his studies on the organism 

 which combined nitrogen by preparing a nutritive solution, which, in addition 

 to dextrose, contained the usual salts, save that no nitrogen compounds were 

 present. He hoped by this ' selective ' mode of culture to provide the organism 

 he was in search of with all the conditions it required, and so isolate it, by 

 shutting out ordinary soil -bacteria which were unable to grow save in the 

 presence of combined nitrogen. His expectations were not disappointed. 

 After the culture fluid had been placed at the bottom of a glass jar and inocu- 

 lated with a small quantity of arable soil, a vigorous formation of butyric 

 acid soon began to take place, and irregularly spherical masses of zoogloeae made 

 their appearance. When the acid was neutralized, the fermentation continued 

 without intermission until the whole of the sugar was used up. Apart from the 

 products of fermentation, the fluid had undergone essential alterations, as was 

 shown by the fact that when fermentation had finished. Fungi appeared on the 

 zoogloeae, and that after these had destroyed the butyric acid. Algae introduced 

 themselves. None of these 

 organisms could exist in the /Q 



original fluid, seeing that it ^ ^ 



was destitute of combined «,f '^ f^/^«iv 

 nitrogen ; their appearance ^J '^* ^'^% ^ 



after fermentation demon- y 



strated the fact that com- / 

 bined nitrogen was present / 



afterwards, and chemical ana- Fig. 4.. Closh-idium pasteuyianum. /, Vegetative rods. 



Ivsis confirmed this. -' Sporo^enous spindle-shaped rods, y, Burst spindles with spores. 



^ -.,. . , .' ,. 4, Germinating; spores. (After WlNOGR.'iuSKY.) From FISCHER 



Microscopical mvestiga- (Vories. a. Bakt. 2nd ed.). 

 tion of the zoogloeae disclosed 



the presence of two filamentous Bacteria, and a species of Clostridium (i. e. a bac- 

 terium which swells into a spindle form when spore formation takes place). Isola- 

 tion and cultivation of the filamentous Bacteria was easily effected, and it was 

 found that they were ordinary saprophytic forms which required extremely little 

 nitrogen, but were quite unable to cause it to enter into combination ; more- 

 over, they were found not to be the cause of butyric acid fermentation. Interest 

 thus became concentrated on the third form, Clostridium pasteurianum, which, 

 morphologically, must be ranked along with the butyric acid Bacteria pre- 

 viously referred to, capable like them of inducing butyric acid fermentation, 

 but which differs greatly from them in its behaviour to nitrogen. The isolation 

 of Clostridium presents many and great difficulties, and is only successfully 

 accomplished if it be sown on carrots in vacuo. If a pure culture on this 

 medium be once more placed in the original non-nitrogenous nutritive solution, 

 fermentation and nitrogen combination do not take place. Both processes 

 commenced at once when Winogradsky added the two bacterial forms present 

 with Clostridium in the zoogloeae, or when he excluded oxygen entirely. The 

 significance of the three Bacteria was thus explained. When alone, Clo- 

 stridium pasteurianum is able to combine nitrogen ; it is strongly anaerobic 

 and thus, in a pure culture, is capable of growth only when oxygen is 

 quite excluded. In nature, however, it is able to live in the aerated upper 

 regions of arable soil if the two other Bacteria associated with it protect 

 it from the action of oxygen. These two Bacteria fulfil no specific function 

 in themselves ; they may be replaced by other appropriate organisms which 

 consume oxygen, e. g, Hyphomycetes. Not every organism, however, is 



