Bacteriological Studies oe the Fixation of Nitrogen. 39 
Na NOa respectively. After one has seen this experiment there can be 
no questioning the fact, that given a supply of carbon, Na NOa, of and 
by itself, can cause A. chroococcum to produce a chocolate brown to 
black pigment. 
This observation is substantiated by the work of Beijerinck 1 in 
which he has shown that “Pigment formation could also be observed 
in pure cultures if the mannite was replaced by dextrose and nitrate in 
minimum quantities was added.” In the application of these results 
to field conditions, we have a very tenable explanation of the brown 
color of the soil. It has been shown that these soils are abundantly 
stocked with Azotobacter chroococcum, and in the presence of the large 
amount of nitrates which they carry, the inevitable consequence must 
be the production of an intensely brown pigment, which has brought 
the brown spots to our attention. 
In 1904 Heinze 2 expressed the view that possibly the dark color 
of soil was due in a degree to the pigment of A. chroococcum. Lohnis 3 
was not inclined to accept this statement, but Omeliansky and 
Ssewerowa 4 are of the opinion that while it would be a mistake to at¬ 
tribute the dark color of soils to this cause altogether, one has no 
right to deny the possibility of its occurrence. They have shown ex¬ 
perimentally that a brown pigment is produced by Azotobacter in a 
medium containing chalk and hydrolized starch, both of which are 
present in soils as Ca COs and as decomposed plant tissue respectively. 
Therefore, they concluded that, “The part which Azotobacter plays in 
the dark color of the soil is not to be overlooked.” 
The intensely brown pigment which all of our cultures have 
shown in this and the preceding series seems to identify them all as 
varieties of A. chroococcum, and consequently they may be consider¬ 
ed as such in this bulletin. The variation which has been noted 
before is in perfect harmony with the observations of Omeliansky and 
Ssewerowa 5 who state that, “Between the colored and colorless, 
intermediate forms exist in which the pigment formation is more or 
less limited.” 
The Relation of Nitrogen Compounds other than Nitrates to 
THE PRODUCTION OF BROWN PlGMENT. 
Now if nitrates by themselves can bring about pigment production, 
may not the same be equally true of other forms of nitrogen? To 
answer this question, a number of different agars were prepared, each 
containing a different form of nitrogen. The list included nitrogen 
as peptone, asparagin, ammonium chloride, ammonium sulphate and 
1. Cent. f. Bakt. Abt. II, Bd. 7, p. 561. 1901. 
2 . Cent. f. Bakt., Abt. II., Bd. 12 , p. 357; Bd. 16, p. 341. 1906 
3 . Lohnis, Handb. d. Landw. Bakt. p. 712. 
4 . Cent. f. Bakt., Abt. II., Bd. 29 p. p. 649, 650. 1911. 
s . Cent. f. Bakt., Abt. 11 ., Bd. 29, p. 643. 1911. 
