Nitrogen by Azotobader and the Growth of the Organism . 88 1 
Table IV. 
Flask 
No. 
Culture Solution. 
Time 
taken . 
Nitrogen 
Content. 
Gain in 
Nitrogen. 
Average 
Gain in 
Nitrogen. 
Relative 
Gain on 
1 grm . 
1. 
IOO C.C. ] 
[ % solution— sterile 
0-69 mg. 
2. 
„ with 
3 - 
Azotobader 
>9 *3 
10 days 
10 „ 
9*21 mg. 
8.93 mg. 
8.52 mg. 
8-24 mg. 
| 8*38 tag. 
8 38 mg. 
5 * 
50 c.c. I 
% solution — sterile 
0.71 mg. 
6. 
„ with 
7* 
Azotobader 
8 „ 
8 „ 
5*27 mg. 
5.12 mg. 
4.56 mg. ; 
4-4 1 mg. ' 
| 4-48 mg. 
4-48 x 2 — 
8*98 mg. 
9. 
25 C.C. I 
% solution — -sterile 
0.71 mg. 
10. 
, , with 
Azotobader 
7 » 
1*84 mg. 
1.T3 mg. 
| i-i 3 mg. 
1. 13 x 4 - 
11. 
n v 
7 
1*84 mg. 
1. 13 mg. 
4.52 mg.. 
The flasks used were all of the same size, namely Erlenmeyer flasks of 
300 c.c. capacity ; hence the depth of the liquid, and consequently the 
aeration, varied with the quantity of solution supplied. It follows that the 
organisms in the 50 c.c. cultures obtained a better aeration than those 
in the 100 c.c., and this fact probably accounts for the slightly greater 
nitrogen-fixation, relatively, in the former than in the latter. Directly 
opposed to this is the comparatively small increase in the 25 c.c. cultures, 
though the organisms in this case possessed the greatest advantage as 
regards aeration. It is a well-known fact, however, that when supplied with 
soluble nitrogen, Azotobader does not fix until this available nitrogen has 
been consumed, so the very small fixation in the 25 c.c. cultures is probably 
explained by the assumption that the Bacteria first of all utilize the small 
amount of nitrogen present in the medium, as shown by the control, using 
at the same time a portion of the mannite as a source of energy. When 
the process of nitrogen-fixation began, a relatively much smaller amount of 
mannite would be available as a source of energy in these cultures than in 
the 50 and 100 c.c. ones, and hence a smaller gain in nitrogen would be 
expected. All the materials used were supposed to be chemically pure, but 
every control so far examined shows the presence of nitrogen. The growths 
were apparently perfectly healthy, but it is fairly evident that the organisms 
used throughout the series were more or less weakened by continued 
growth under artificial conditions, since the fixation per gramme of carbo- 
hydrate consumed amounted to only between 8 and 9 mg., while in the 
earlier part of the work the organisms originally obtained from the same 
source gave fixations amounting to 14 mg. However, Hoffmann and 
Hammer obtained good fixations in 25 c.c. of 1 per cent, solution, so that, 
when multiplied by four, their results gave fixations of 14*40 mg. on 1 grm. 
carbohydrate used- — results which are not comparable with any of those 
obtained in 25 c.c. cultures in the course of the present work. 
3 m 
