194 Nitrogen Fixation— Nonsymbiotic 



tained 1,120,000 Clostridiwn and 98,700 Azotobacter cells per gram 

 of soil. 



It is essential to keep in mind that, out of a hundred living cells of 

 CI. pasteurianwn found in a culture, only very few are able to de- 

 velop into colonies or give positive growth on artificial culture media. 

 For this reason, the abundance in the soil of anaerobic bacteria 

 capable of fixing atmospheric nitrogen must be considered very ex- 

 tensive. Further, CI pasteurianwn is found in soils that are much 

 too acid for the favorable development of Azotobacter, in the growth 

 of which an acid reaction becomes a limiting factor. This tends to 

 add further weight to the claim of the potentially greater importance 

 of the anaerobic than the aerobic bacteria as nonsymbiotic nitrogen- 

 fixing organisms in the soil. 



When freshly isolated from the soil, the Clostridia fix more nitro- 

 gen than after they have been cultivated for a long time in artificial 

 media. Cultures kept in collections for a long time can be invig- 

 orated by growing them in liquid media to which enough ammonium 

 sulfate is added to offer the organism less nitrogen than is needed 

 for the complete decomposition of the sugar. By transferring such 

 cultures, when gas formation ceases, to fresh media, normal growth 

 and nitrogen fixation are obtained. 



Nitrogen fixation was shown by Bredemann to be a common prop- 

 erty of the butyric acid bacilli. Some of these organisms are strict 

 anaerobes, whereas others are less sensitive to oxygen. Although 

 more tolerant of acidity than Azotobacter, they have a definite opti- 

 mum at approximately neutral reaction. In pure cultures, 2-3 mg 

 nitrogen is fixed per gram of sugar decomposed, although some 

 strains may fix as much as 5 to more than 6 mg. The mechanism of 

 fixation is explained as a direct reduction of elementary nitrogen to 

 ammonia by nascent hydrogen. Bacillus asterosporus is a faculta- 

 tive anaerobe. It was found capable of fixing small amounts of 

 nitrogen, 1-3 mg per gram of sugar. 



Aerobic Bacteria 



When a simple medium containing tap water, 0.02 per cent 

 K2HPO4, and 2 per cent glucose is inoculated with soil and incu- 

 bated, anaerobic and certain other bacteria are obtained. When 

 the glucose is replaced by mannitol or by propionate of potassium 

 or sodium, aerobic bacteria predominate. Beijerinck first isolated 

 one of these organisms, which he described as Azotobacter chroococ- 

 cum. It is found in soils and manures. On repeated transfer to fresh 



