26 CIRCULAR NO, 131, BUREAU OF PLANT INDUSTRY. 



Our studies of the cellulose destroying bacteria have shown that 

 these organisms bring about a rapid destruction of cellulose only 

 when supplied with a considerable quantity of combined nitrogen. 

 It therefore seemed necessary to consider first the effect of combined 

 nitrogen in the culture media on the nitrogen fixing power of Azo- 

 tobacter before undertaking to determine the value of cellulose as an 

 indirect source of energy for nitrogen fixation by these organisms. 



REVIEW OF INVESTIGATIONS ON THE EFFECT OF COMBINED 

 NITROGEN IN THE CULTURE MEDIA ON THE NITROGEN-FIXING 

 PROPERTIES OF MICRO-ORGANISMS. ' 



The synthetic culture media used by different investigators in the 

 study of the nitrogen-fixing properties of micro-organisms, although 

 showing considerable variety in composition, have usually contained 

 little or no combined nitrogen. The absence of nitrogen in these 

 media seems to have resulted from a very general acceptance of the 

 early conclusions of Berthelot and Winogradsky that if an organism 

 is grown in a medium rich in combined nitrogen there is a reduction 

 or total loss of the nitrogen- fixing power exhibited by the organism 

 when grown in nitrogen free media. From a review of the work of 

 these early investigators it would seem that the results secured would 

 hardly warrant a final acceptance of their views without additional 

 proof. 



Studies by Berthelot of nitrogen fixation with pure cultures are 

 limited, and in these studies no special attention was given to the 

 effect of combined nitrogen on the nitrogen-fixing power of the organ- 

 isms. Berthelot believed, however, that a small quantity of com- 

 bined nitrogen was required to give the organisms the mmimum 

 amount of energy necessary to enable them to utilize the free nitrogen 

 of the air. 



Winogradsky soon proved this latter conclusion to be erroneous by 

 demonstrating the nitrogen-fixing power of an impure culture of a 

 butyric ferment in nutrient solutions from which the last trace of 

 nitrogen had been removed. In 13 flasks containing no nitrogen he 

 secured a fixation of from 1.25 to 3 milligrams of nitrogen for each 

 gram of dextrose added. Wlien 2.1 milligrams of combined nitrogen 

 were added to the solution, the fixation for four flasks varied from 

 2.1 to 3.56 milligrams for each gram of dextrose added. With three 

 flasks in which 3, 3.3, and 4 milligrams of combined nitrogen were 

 added the fixation was 2.5, 0.3, and 0.4 milligrams, respectively. It 

 would seem that the addition of 2.1 mifligrams of combined nitrogen 

 had a slight stimulating effect on the nitrogen-fixing organisms, while 

 the addition of larger quantities reduced the quantity of nitrogen 

 fixed. 



1 For bibliography, see p. 34. 

 [Cir. 131] 



