FIXATION OF ATMOSPHERIC NITROGEN 559 



ing into consideration the combined nitrogen brought down by the rain- 

 fall and the nitrates washed out by the water. These results were 

 confirmed in a series of other experiments by Berthelot as well as by a 

 number of other writers. 2 Although Berthelot did not succeed in isolat- 

 ing any organisms, he found that the bacteria require combined carbon 

 and hydrogen and enough combined nitrogen to promote their initial 

 growth, and that, when the amount of combined nitrogen available is 

 increased, the bacteria prefer to use this combined nitrogen rather than 

 to fix atmospheric nitrogen. Berthelot demonstrated conclusively that 

 in bare soil, free from vegetation, bacteria exist which are capable of 

 fixing atmospheric nitrogen; these organisms, he found, act best at tem- 

 peratures of 10° to 40°C, in the presence of sufficient oxygen and with 

 an optimum amount of moisture (from 2-3 to 12-15 per cent) . 



This work led to the important contributions of Winogradsky and 

 Beijerinck, as pointed out above. Winogradsky 3 carefully freed the 

 nutrient solutions from all traces of nitrogen and used only mineral 

 salts with 4 per cent glucose as a source of energy. He obtained a gain 

 of 24.68 mgm. and 28.87 mgm. of nitrogen per liter of medium, after 15 

 and 20 days' incubation. A slight addition of ammonium salt or nitrate 

 stimulated the growth and butyric acid fermentation of the organism, 

 but not nitrogen fixation. When more than 0.6 gram ammonia or ni- 

 trate nitrogen were added per 100 grams of sugar, nitrogen-fixation 

 ceased entirely; in other words, 6 parts of combined nitrogen for 1000 

 parts of sugar is just sufficient to prevent any fixation of gaseous nitro- 

 gen. In the absence of combined nitrogen, 2.4 to 3 mgm. of nitrogen 

 were fixed by the organism for every gram of glucose supplied. As a 

 result of this evidence it was concluded that growth and nitrogen fixation 

 are two distinctly separate phenomena; the nitrogen fixing bacteria 

 grow as other organisms do in the presence of available energy and 

 combined nitrogen, but, in the absence of combined nitrogen and in the 

 presence of available energy, they are able to obtain their nitrogen from 

 the atmosphere. 



At first Azotobacter was considered to be the most active nitrogen- 

 fixing organism, since it fixed as much as 15 to 20 mgm. of nitrogen per 

 gram of sugar, while the CI. pastorianum fixed only 2 to 3 mgm. of nitro- 



2 For detailed review of earlier literature consult Voorhees and Lipman, 1907 

 (p. 491); Koch, A. Lafar's Handb. techn. Mykol., 3: 1. 1907; Lohnis, 1910 

 (p. xiii), Omeliansky, W. L. Monogr. 5 Russian Acad. Sci., Petrograd. 1923. 



3 Winogradsky, S. 1893 (p. 107). 



