STUDIES ON AZOTOBACTEE CHROOCOCCUM BEIJ. 363 



causes a loss of this element under special conditions that are 

 not as yet completely understood. This loss may later be 

 replenished by this same organism in the process of '4ater life." 

 Apparently this second phase, phase of replenishment of the 

 lost nitrogen, is directly connected with the second phase of the 

 carbon metaboUsm studied in the preceding pages, since it 

 appears to take place after the reducing sugar has either dis- 

 appeared from solution or has been transformed into a non- 

 reducing substance. That this assumption is justified is shown 

 by the fact that the nitrates have a special importance in the 

 carbon metabolism as is also shown by the data in the chapters 

 on the carbon relations. 



Nitrates and filtration of media 



-^rom the work of Allen (1919) we gather that filtration of a 

 culture solution, under the conditions designed to remove the 

 phosphates quantitatively, makes it unsuited for the develop- 

 ment of Azotobacter chroococcum. Although the addition of 

 calcium carbonate to the filtered solutions acted as a slightly 

 beneficial agent in bringing about nitrogen fixation, it was not 

 until phosphate was added in the form of tricalcium phosphate 

 that any appreciable nitrogen fixation took place. Unfor- 

 tunately the criterion used by Allen in drawing his conclusions^ 

 is subject to objection in the light of the above results so that in 

 the following study the sugar consumption and production of 

 bacterial substance was used as a measure of growth rather 

 than the fixation of atmospheric nitrogen. The phosphates 

 were replaced by nitrates, since some traces of phosphorus in 

 the soluble form were undoubtedly present in the solution. 



Experiment 69. A solution of the following composition was 

 prepared and filtered, after a short standing at room tempera- 

 ture, over a coarse filter paper. 



Deep well water 500.00 cc. 



K2HPO4 0.25 gram 



NaCl 0.25 gram 



FeS04 + 7H2O 0.02 gram 



Glucose 5 . 00 grams 



• Fixation of atmospheric nitrogen. 



