Apr. 3 x, 1933 
Protein Synthesis by Azotobacter 
267 
SOURCES OF ENERGY 
There is a wide variety of substances available as energy for Azoto¬ 
bacter which differ greatly in their value as sources of energy. Not only 
is there a difference in the value of the sources of energy for nitrogen 
fixation, but the eflBLciency of the same material for azofication varies, 
according to different investigators. This is illustrated in the work of 
Ivdhnis and Pillai (j), Hoffmann and Hammer (6), and Mockenridge (10). 
There appears to be, however, a rather uniform opinion that a mannite 
solution will furnish energy for the fixation of the largest quantity of 
nitrogen. As a result, mannite is the carbohydrate employed almost 
universally in azofication experiments. 
As a preliminary study, ^e comparative value of various substances 
as sources of energy for Azotobacter was determined. One per cent of 
the test material was substituted for mannite in Ashby’s medium con¬ 
taining calcium carbonate. Flasks of each medium were inoculated with 
a pure culture of Azotobacter and aerated at 30® C. for six days. The 
average quantity of nitrogen fixed per gram of test substance for the two 
cultures studied were as follows: 
Mgm. 
Potassium acetate 
Dextrose. 
Saccharose. 
Mannite. 
Molasses. 
Lactose. 
8 . o 
7.8 
7*7 
7.2 
3-0 
2.4 
Dextrose has been preferred in this laboratory as a source of energy for 
studying the nitrogen-fixing ability of different cultures and their rate 
of fermentation. Quantitative determination can be easily made and, 
as shown by these experiments with aerated cultures, it is an efficient 
source of energy for azofication. 
While no special endeavor has been made to study the nitrogen-fixing 
ability of different Azotobacter cultures with the object of classifying 
them on such a basis, over 20 strains have been studied. The nitrogen 
fixation for these cultures grown in dextrose medium has varied from 
7.20 mgm. to 18.72 mgm. per gram of dextrose. Many of the same 
cultures have been repeatedly used for the past two years, and the varia¬ 
tions in the quantity of nitrogen fixed at different times by the same 
cultures is as much as the difference between various cultures. This 
variation, or at least a large portion, may be attributed to the different 
intensities of aeration to which the cultures were submitted. The aim 
was to aerate all cultures alike, but such is impossible unless the aeration 
is mechanically controlled. This suggests that the optimum quantity of 
air necessary for the maximum growth of Azotobacter should be deter¬ 
mined. 
YIELD OF AZOTOBACTER GROWTH 
Cultures of Azotobacter cultivated by vigorous aeration in a liquid 
medium will exhibit a vigorous growth within two to four days. The sugar 
is rapidly consumed, and a corresponding increase in the nitrogen con¬ 
tent of the liquid is noted. If the culture produces pigment, coloration 
of the medium is observed. In some cultures this coloration is black; in 
others brown. In those cultures which fail to produce pigment the solu¬ 
tion becomes thick and milky in appearance. Pure cultures maintain 
an alkaline reaction in the absence of calcium carbonate in the medium 
throughout growth and emit a rather pleasant odor. 
