Jan., 1921] 
WANN — FIXATION OF FREE NITROGEN 
25 
showing increases of from 11 to 18 mg. per 100 cc. culture solution. Remy 
and Rosing (1911) found from 10 to 20 mg. to be the average fixation per 
ICQ cc. culture, or 2 to 15 mg. per gram of mannite consumed. Perhaps the 
most extensive experiments with Azotobacter forms are those of Lipman 
(1903 and 1905), who secured the following gains in nitrogen, each in 100 
cc. of culture solution: A. chroococcum, 4.42 mg. ; A. beyerinckii, 2.37 to 
6.78 mg. ; A. vinlandii, 1.67 to 7.90 mg. in 1903, and 8.36 to 20.99 "^g. in 
1905. The numerous results with mixed cultures and impure soil cultures 
by many investigators show even wider ranges of fixation, and the condi- 
tions of the experiments have been so various that it is difficult to arrive 
at an average, but in many cases it appears to be between 6 and 10 mg. per 
100 grams of culture medium. 
Because of the nature of the experiments reported in this paper it is 
impossible to state in what form the nitrogen is added to the culture. 
Whether or not the process is connected with that of photosynthesis, the 
ratio of nitrogen fixed to sugar respired or to the amounts of nitrate absorbed, 
and the amount fixed per unit dry weight of crop produced are phases of the 
problem which can be merely mentioned at this time. The solution of 
these questions will be simplified by the proper development of liquid 
culture methods, some of which are now in progress. 
Since the results obtained are wholly contrary to the generally accepted 
idea of the relation of green plants to elementary nitrogen, it may be well 
to point out the conditions under which some of the earlier negative results 
with the green algae were obtained. The first nitrogen determinations of 
pure cultures of algae were made by Kossowitsch (1894), who grew a single 
species of Cystococcus (?) on sand moistened with 20 cc. of a mineral 
nutrient solution containing calcium nitrate as a source of nitrogen. The 
cultures, however, grew for only three weeks, because, the author concludes, 
of the lack of nitrates, only 2.5 mg. nitrogen as nitrate having been supplied. 
The addition of more calcium nitrate to the cultures caused them to revive, 
and it was upon this fact that his conclusion was based. However, it is 
not altogether clear that the lack of growth may not have been due to some 
other cause, such as deficiency in calcium, only a "trace" of which was 
present in addition to that added as calcium nitrate. The analyses of the 
cultures and uninoculated checks clearly showed that there had been no 
increase in the nitrogen content of the flasks, either in the presence or in the 
absence of glucose. It is apparent, however, that conditions for a vigorous, 
long-continued growth were not realized in these experiments, hence it is 
not to be expected that appreciable increases in nitrogen should occur. 
As a result of a large number of analyses of pure cultures, Kruger and 
Schneidewind (1900) concluded that the green algae investigated were 
unable to use free nitrogen. They grew a large number of forms on a 
variety of media, using both sand and solution cultures. Two of the media 
contained nutrient solutions free from combined nitrogen, five contained 
