Jan., 1921] 
WANN — FIXATION OF FREE NITROGEN 
23 
increase in nitrogen content of 54 percent. Fixation was secured by these 
four species in both experiments when grown on similar media and under 
similar conditions, so that there seems to be no escape from the definite 
conclusion that these forms do actually use free nitrogen. The amounts 
of fixation secured by these four species on ammonium nitrate with glucose 
in 1919 were considerably lower than found in the 1917-18 experiment on 
a similar medium. As already pointed out, however (page 9), the growth 
produced on this medium in 1919 was very poor, a factor which, it is believed, 
accounts for the smaller fixation. 
This leaves to be considered the case of species no. 11 {Chlorella sp.) 
which is of considerable interest. In series 8A and 9A (the nitrate media 
with glucose) substantial increases in the nitrogen content of the cultures 
occurred, especially on calcium nitrate, where the average of the cultures 
is more than 5.5 mg. above the checks. This same species, however, on 
both nitrate media without a carbon source, .and on the nitrate media with 
mannite, showed distinct losses in nitrogen. These losses ranged from 2 
to 8 mg. and occurred regularly on the media mentioned with this species 
only. This species was used only in the 191 9 experiment, in which acid 
tubes were not inserted between adjacent culture flasks, so that there is no 
means of telling whether the loss occurred as ammonia or as free nitrogen. 
However, that some sort of process resulting in a loss of introgen has taken 
place is apparent, and the results suggest that perhaps both the processes 
of free nitrogen fixation and of denitrification may be going on simul- 
taneously, the former overbalancing the latter when glucose is present. 
As regards the use of free nitrogen by the forms investigated on nitrate 
media to which no carbohydrate energy source was added, the evidence is 
not conclusive. In 191 7- 18 gains of from 0.7 to 1.8 mg. per 100 grams 
medium were recorded for three of the species grown on ammonium nitrate 
(table 4), and one of these species gave an increase of 1.8 mg. on calcium 
nitrate without a carbon source (table 5). In 1919 three out of five species 
showed very slight increases on ammonium nitrate in the absence of a 
carbon source, while on calcium nitrate without glucose or mannite gains 
of from 0.3 to 1.9 mg. per 100 grams medium occurred with three species 
(table 9). As has already been pointed out, however, the growth of all 
species in the absence of glucose is very slow, and, although the cultures 
remain in a healthy condition for long periods of time, the actual amount of 
"crop" produced is slight in comparison with the glucose cultures. That 
some fixation does take place in the absence of glucose seems probable, 
in view of the increases cited above, and the assumption that the amount 
of fixation on one and the same medium is dependent, more or less, on the 
total amount of growth produced does not seem unwarranted. This phase 
of the problem is of considerable importance and is one which should be 
most carefully investigated, as it bears directly on the conditions as they 
exist in nature. It is entirely possible that the conditions realized in the 
