214 Journal of Agricultural Research voi. xii. No. 4 



INFLUENCE OF NITRATES ON THE FIXATION OF ATMOSPHERIC NITROGEN BY BACILLUS 



RADICXCOLA 



The ability of B. radicicola to fix atmospheric nitrogen in the absence 

 of the host plant has been studied by numerous investigators. From 

 the results of their work it seems fairly probable that the legume organ- 

 ism can fix nitrogen to a slight extent when growing in a nonsymbiotic 

 state. Beijerinck (j) was one of the earliest to make a study of the 

 possible fixation of atmospheric nitrogen by B. radicicola under these 

 conditions. He found that a small quantity, 0.91 to 1.82 mgm. of nitro- 

 gen was fixed per 100 c. c. of the medium,* Prasmowski (jp, p. 55) and 

 Berthelot (6) concluded as a result of their experiments that when the 

 organism was grown outside the host plant the gain in nitrogen was 

 small. The greatest gain in nitrogen was found by Maze (52) who re- 

 ported an increase of 23.4 mgm. of nitrogen per 100 c. c. of the medium in 

 1 6 days. lyewis and Nicholson {30) found by incubating the cultures for 

 a considerable length of time that a large increase in fixation occurred. 

 Bottomley {8) found that a pure culture of B. radicicola fixed approxi- 

 mately I mgm. of nitrogen in 15 days. Fred (77) made a study of the 

 possible fixation of nitrogen by the legume organism and found that it 

 fixed approximately 1.2 mgm. of nitrogen in 100 c. c. of the medium. 

 He found that on agar films a greater fixation occurred than when the 

 organisms were grown in a liquid medium. 



A few investigators, however, found that no increase»r in nitrogen 

 occurred when B. radicicola was grown outside the host plant. Frank 

 {16) states that in a nitrogen-free medium the legume organisms did not 

 fix enough nitrogen to be accurately measured. Immendorf (25) also 

 found no increase in nitrogen when pure cultures of B. radicicola were 

 grown in soil containing a nitrogen-free solution. 



It will be seen that the majority of investigators, especially the more 

 recent ones, found that a slight amount of atmospheric nitrogen was 

 fixed or assimilated by B. radieicola when grown outside the host plant 

 and on a medium suitable for its development. 



It has already been shown that nitrates cause an increase in the num- 

 ber of B. radicicola when grown in pure culture in sterilized soil. Does 

 such an increase in the number of organisms necessarily mean an in- 

 creased fixation of nitrogen? Three experiments using agar films were 

 carried out in order to determine this point. Erlenmeyer flasks of 

 I -liter capacity containing^ 100 c. c. of mannit agar were used. Before 

 the medium solidified, the nitrates were added in the proportions indi- 

 cated in Table XXVI I. Six flasks for each different quantity of nitrate 

 were prepared, except in one case, as shown in Experiment II. The 

 flasks were plugged with nonabsorbent cotton and sterilized at 10 pounds' 

 pressure for 30 minutes. After cooling, three of each set were inoculated 

 with 5 c. c. of a suspension of B. radicicola in sterile distilled water. 

 The organisms had been growing on mannit agar at 28° C. for six days. 

 The flasks in Experiments I and HI (Table XXVII) were incubated at 



