CELLULOSE AS A SOUECE OF ENERGY FOE NITEOGEN FIXATION. 27 



The following year Winogradsky continued his studies of the effect 

 of combined nitrogen upon the nitrogen-fixing properties of this 

 butyric ferment associated with two other organisms. In one series 

 of flasks in which the nutrient solutions contained 3 grams of dex- 

 trose and quantities of nitrogen as ammonium sulphate varying from 

 2.1 to 21.2 milligrams the results showed the greatest gain for the 

 flask receiving the smallest quantity of combined nitrogen, and when 

 the combined nitrogen added amounted to 21.2 milligrams there was 

 a loss of 2.2 milligrams. Wmogradsky therefore concluded that for 

 nitrogen fixation the ratio of nitrogen to carbohydrate should not be 

 greater than 6 to 1,000. We should not lose sight of the fact, how- 

 ever, that the results secured by Winogradsky in this experiment may 

 have been determined m a large measure by the impurity of his 

 culture. 



Work by Ashby and by Hoffmann and Hammer has indicated that 

 a large percentage of the nitrogen fixed by impure cultures in solu- 

 tions may be lost through denitrifying processes, especially if the 

 incubation period is prolonged. The latter investigators noted a loss 

 of 38 per cent of the nitrogen fixed in a series of flasks incubated for 

 six weeks. In view of these results it would seem that the gains 

 secured by Winogradsky are of somewhat doubtful value in deter- 

 mining the eft'ect of combined nitrogen on the nitrogen-fixing power 

 of micro-organisms. 



In 1897 Maze reported nitrogen-fixation studies with Bacillus radici- 

 cola in a decoction of white kidney bean to which 2.6 })er cent of 

 saccharose, a little sodium chlorid, and a trace of sodium bicarbonate 

 were added. One hundred cubic centimeters of the solution con- 

 tained 22.4 milligrams of initial nitrogen. However, a total gain of 

 23.4 milligrams of nitrogen was secured, or a gain of 9 milligrams for 

 each gram of sugar added. The nitrogen gain reported in this rich 

 nitrogenous medium is far in excess of gains reported by later investi- 

 gators for B. radicicola in solutions weak in initial nitrogen. 



Saida in 1901 studied the nitrogen-fixing properties of P/^om-aJe toe, 

 Mucor stolonifer, and Aspergillus niger in nitrogen-free and nitrogen- 

 containing solutions. The nitrogen fixation by these organisms was 

 usually stimulated by the addition of small quantities of combined 

 nitrogen. 



Lipman in 1903 studied the nitrogen-fixing properties of Azo- 

 tobacter vinelandii in mannite solutions to which small quantities of 

 nitrogen as potassium nitrate, ammonium chlorid, and peptone were 

 added. The gains secured did not show any appreciable effect from 

 any of the nitrogen compounds employed. However, the quantities 

 of nitrogen added were extremely small, amounting to less than 1 

 milligram to each 100 cubic centimeters of solution. 



[Cir. 131] 



