Apr 21 ,1933 
Protein Synthesis by Azotobacter 
265 
The reaction of this solution was readjusted to a Ph of 7.0 to 7.4, 
filtered if necessary. The required quantities were placed in flasks and 
sterilized in the autoclav at 20 pounds pressure for 30 minutes. 
methods of analysis 
Total nitrogen determinations and sugar analysis of the Azotobacter 
cultures were made at frequent intervals. 
In all cases the total nitrogen was determined by the usual standard 
methods. Unless otherwise noted, 50 cc.portions of the medium in dupli¬ 
cate were used. The figures referred to in the tables denote the average 
of th^ duplicate analyses. The net gain in nitrogen is recorded in all 
cases, unless otherwise stated, as milligrams of nitrogen in each 100 cc. 
of medium. In other words, the figures refer to the quantity of nitrogen 
fixed per gram of dextrose, since each 100 cc. of medium contains this 
quantity qf sugar. 
Sugar determinations were made according to the method proposed 
by Shaffer (9). The copper resulting from the reduced Fehlings was 
determined by colorimeter readings. Duplicate readings were made 
each time, and the average of these was recorded. As a general rule, 
50 cc. of the medium were used. This was diluted to 100 cc., and 20 cc. 
of this filtrate were used for reduction. The figures given, unless 
otherwise stated, refer to grams of dextrose per 100 cc. of medium. 
COMPOSITION OF THE AZOTOBACTER CELL 
The composition of the Azotobacter cell has been determined by 
several investigators. Gerlach and Vogel (j) report a protein content of 
Bo per cent. 
Lipman (2) reports the nitrogenous composition of the Azotobacter 
membrane as 10.45 per cent total nitrogen, 6.39 per cent nonbasic 
nitrogen, 2.76 per cent basic nitrogen, and 0.98 per cent ammonia nitro¬ 
gen. He observed that lead acetate precipitated practically all of this 
nitrogen in young or old cultures, while phototungstic or tannic acid 
would not precipitate nearly as much. He believed that in young cul¬ 
tures this nitrogen substance is in a soluble form and not precipitated by 
phosphotungistic acid, but as the culture grows older this soluble nitro- 
genous material is converted into an insoluble and more complex protein. 
Stoklasa ( 4 ) cultivated Azotobacter in liquid cultures. The growth 
was collected on a filter and. washed. The nitrogen content of the washed 
cells was 11.3 per cent. 
Hoffmann and Hammer (6) studied the composition of Azotobacter 
and report analyses much lower in protein. The organism was grown 
on Ashby agar plates. The growth was scraped off, washed, and dried. 
The maximum protein content recorded was 17.75 per cent. They 
suggest that the wide difference in their results, as compared with those 
of other investigators, was due to the jellylike material which in 
liquid cultures is filtered out of solution. In their method this material 
is included in the analysis. This increases the total residue, and since 
this substance is thought to be carbohydrate, the total percentage of 
nitrogen is decreased. The phosphorus content (P2O5) varied from 2.51 
per cent to 2.97 per cent. Increasing with the age of the culture. Stok¬ 
lasa (4) reports the PjOg content to be 4.93 per cent. 
Omeliansky (7) grew Azotobacter on dextrose mineral salt agar for 
six days at a temperature of 30® C. An analysis of this growth is 
