Jan., 1921] WANN — FIXATION OF FREE NITROGEN 27 
that nitrates have long been regarded as in general the best combined 
nitrogen source for chlorophyll-bearing plants. 
The proof that green plants are capable of fixing free nitrogen is at 
once of real significance, both from a purely scientific and from a practical 
standpoint, even though the plants concerned are members of a low order. 
The question which perhaps at first suggests itself is: Do all green plants 
possess this ability to use the uncombined nitrogen of the air? With the 
exception of the legumes, the evidence is almost entirely negative as regards 
the flowering plants. MoUiard (1916) has recently demonstrated that 
radish plants in pure culture are unable to increase the nitrogen content 
of the culture. The plants were grown in large tubes on nutrient solutions 
containing several different concentrations of ammonium chloride as a 
source of nitrogen, some cultures being supplied with glucose, and others 
being aerated with air charged with carbon dioxide in order to increase 
the photosynthetic activity. In no case did the total nitrogen of the crop 
and residual culture solution exceed that of the seed and original solution, 
even after six weeks' growth. The plants produced, however, were small, 
bearing three or four leaves in addition to the cotyledons, the green weight 
varying from 0.3 to 1.3 gr. Moreover, nitrate nitrogen was not supplied 
as a source of nitrogen in any case, a fact which may be of considerable 
importance in view of the results presented in this paper. It is possible 
that in the case of the higher plants also the proper culture conditions have 
not yet been realized, since in the case of four radish plants grown with the 
roots under aseptic conditions and the tops in free air, gains of 1.32 mg. 
nitrogen per plant were reported by Molliard, but he assumes that this 
nitrogen has come from the ammonia of the air. 
The significance of the legume bacteria has recently been questioned by 
Beijerinck (1918) who argues that, with the exception of lupine and seradella, 
the number and weight of the tubercles is insignificant in comparison with 
the total weight of the plant. The isolated organisms fix nitrogen in pure 
culture only very slightly or not at all, and no fixation could be demonstrated 
with nodules placed in glass jars in which the changes of gases were meas- 
ured. In the case of Robinia the number of tubercles is extremely small 
and fixation in them must be very intense, yet when tested they were 
found to be inactive or nearly so. He concludes that ''the present accepted 
explanation of the behavior of legumes cannot be correct." The situation 
in the higher plants needs to be more carefully and thoroughly investigated. 
Conclusions 
I. Seven species of grass-green algae (Chlorophyceae) exhibited the 
ability to fix nitrogen when grown in pure cultures on mineral nutrient agar 
media containing either ammonium nitrate or calcium nitrate as a souce of 
nitrogen, and glucose. The nitrogen fixed was derived from the free (un- 
combined) nitrogen of the atmosphere. The amounts of fixation ranged 
