234 METABOLISM 



capable of fulfilling this protective role in the same way. The organism 

 must first operate on the culture medium by absorbing the oxygen before 

 Clostridium can begin to combine the nitrogen ; the protecting organism must 

 also obtain combined nitrogen in the first instance from the nutritive solution ; 

 later on Clostridium provides for it in this respect. It would thus appear that 

 organisms which make very small demands on nitrogenous compounds will 

 be the most suitable forms to accompany Clostridium. This condition is 

 fulfilled by the two Bacteria present in the 20ogloeae because such traces of 

 nitrogen as are unavoidably present as impurities in the reagents employed 

 are sufficient for the purpose, although Winogradsky showed that a minute 

 addition at first of ammonia or nitric acid induced much more rapidly 

 both fermentation and nitrogen combination. 



Working on his previous experiences, Winogradsky has since then been able 

 to isolate Clostridium pasteurianum by a second method much more rapidly and 

 effectively. He added a trace of garden soil to a non-nitrogenous nutritive solu- 

 tion, and allowed a stream of nitrogen gas to pass through the fluid. A drop 

 of this solution was after a certain time transferred to a fresh nutritive solution, 

 identical in character, and this process was repeated several times. The 

 final culture was heated to 80° C. after Clostridium had formed its spores, 

 so that all admixtures of foreign organisms were killed. The result was a pure 

 culture of the spores of Clostridium. 



How nitrogen assimilation is carried out in this case is, however, quite 

 unknown. We know neither the primary nor the final products of assimila- 

 tion ; we do not know whether ammonia is formed and made further use of 

 or whether a complicated nitrogenous substance, e. g. some kind of proteid, 

 arises at once. One of Winogradsky's researches on this subject shows 

 that the nitrogen occurs chiefly in an insoluble organic condition, and 

 only in small quantities as soluble compounds in the nutritive solutions. The 

 latter, which possibly becomes free only when the Clostridium cells die, serves 

 as a nutrient for other organisms, especially for the two concomitant Bacteria. 



Clostridium pasteurianum is an anaerobe. It acts fermentatively on cane 

 sugar, dextrose, laevulose, and certain other carbohydrates ; but it is unahle 

 to make use of starch, cellulose, lactose, and higher alcohols. According to 

 Winogradsky (1902), the products of fermentation are, on the one hand, 

 butyric and acetic acids (about 45 per cent, of the sugar), and carbon-dioxide 

 and water on the other (about 55 per cent, of the sugar). The fermentation 

 acts as a source of energy, especially for the purpose of combining atmospheric 

 nitrogen, and so we cannot wonder that Winogradsky succeeded in establish- 

 ing quite definite numerical relations between the amount of sugar used up, 

 and the gain in nitrogen (viz. i g. of sugar fermented for every 2-5-3 mg. of 

 nitrogen combined), but he was unable to say whether the sugar was employed 

 only as material for fermentation, or whether it was also nutritive. There 

 is no evidence at present available, however, to show whether Clostridium 

 withdraws not merely nitrogen but also carbon from the air, or whether it 

 assimilates carbon-dioxide like nitro- and sulphur-bacteria. 



Berthelot had imagined that the capacity for fixing free nitrogen was 

 possessed by many micro-organisms, Winogradsky finds it limited to Clos- 

 tridium and forms related to it ; at least these forms are the only ones which 

 are able to commence and carry on vegetative growth without combined 

 nitrogen. Following on Winogradsky's researches, it has often been stated 

 that other organisms also are able to make use of atmospheric nitrogen. Thus 

 Beijerinck (1901) observed a bacterium {Azotobader) of unusually large 

 size, which forms no spores, and which on that account could not be isolated 

 by Winogradsky's method ; it develops on organic nutrients in presence 

 of air, especially on mannite, propionic acid, &c., without any combined 



