BACTERIA FIXING ATMOSPHERIC NITROGEN 111 



in morphology to the other Clostridia, but was capable of fixing larger 

 quantities of nitrogen, perhaps due to its aerobic nature. Bredemann, 

 after examining a large number of Clostridia, came to the conclusion 

 that they are all capable of developing more or less even in the presence 

 of air and fix nitrogen. The presence of 30 mgm. of oxygen per 1 liter 

 of air will still allow spore germination. 



The optimum temperature for the development of the organism is 

 28° to 30°C. The spores are not destroyed at 75° even at the end of 15 

 hours; at 100°C the spores are destroyed in five minutes. 



CI. pastorianum utilizes glucose, maltose, lactose, levulose, sucrose, 

 galactose, maltose, raffinose, dextrin, inulin, glycerol, mannite and 

 lactates. Winogradsky found that the nitrogen source greatly in- 

 fluences the nature of the carbon sources that can be utilized. The 

 greater the concentration of sugar the lower is its economic utilization, 

 3.2 mgm. nitrogen being fixed per gram of glucose in 0.5 per cent solu- 

 tion, 2 mgm., in 2 per cent solution and 1.2 mgm. in 4 per cent solution. 

 In the presence of combined nitrogen, nitrogen fixation decreases and 

 comes to a standstill, when the solution contains more than six parts of 

 combined nitrogen in one thousand parts of solution (Winogradsky). 

 Omeliansky, however, observed some fixation even with a concentration 

 of 16 parts of combined nitrogen. 



The optimum reaction for the growth of CI. pastorianum is pH 6.9 

 to 7.3, but it still develops well at pH 5.7. It can withstand a greater 

 acidity than proteolytic anaerobes like Bac. putrificus, from which it can 

 be thus freed. 37 The addition of CaC0 3 to the glucose medium has a 

 favorable effect, in neutralizing the acids formed ; MgC0 3 is less favora- 

 ble. CI. pastorianum can thus withstand a greater acidity than Azoto- 

 bacter, whose limit is pH 6.0. In acid soils (more acid than pH 6.0) 

 Azotobacter is inactive while the butyric acid bacteria may still be 

 abundant. 



When freshly isolated, the Clostridium fixes more nitrogen than when 

 cultivated for a long time in artificial media. The culture can be 

 invigorated by growing it in Winogradsky's liquid medium, to which 

 enough ammonium sulfate is added so as to offer the organism less 

 nitrogen than is needed for the complete decomposition of the sugar. 

 By transferring from this culture, when gas formation ceases, normal 

 growth and nitrogen fixation is obtained. Bredemann 38 invigorated the 

 culture by passing it through soil. 



"Dorner, 1924 (p. 165). 



38 Bredemann, G. Die Regeneration des Stickstoffbindungsvermogens der 

 Bakterien. Centrbl. Bakt. II, 23: 41-47, 385-568. 1909. 



