cale Production of Azotobacter SYLVAN B. LEE1 AND R. H. BURRIS University of Wisconsin, Madison, Wis.
gated in connection with the development of a technique for growing azotobacter on a large scale.
The production of azotobacter by a largescale technique is described. The final fermentation is carried out in a 200-gallon pilot-plant yeast fermenter. Yields were obtained of 10-11 pounds of cell paste (85 per cent moisture) in 300 liters of medium in 32 hours. The efficiency of the conversion of sugar to cells w a s 15 per cent. The total nitrogen fixed was 32 mg. per 100 ml., equal to about 96 grams in the fermenter. Assays of the cells for several members of the vitamin B complex show that azotobacter cells grown in a nitrogen-free sucrosemineral salts medium have a vitamin content equal or superior t o that found in yeast. When grown in a molasses medium, the cells are higher in biotin, thiamine, and pantothenic acid than when grown in a sucrose medium, and are slightly lower in nicotinic acid and riboflavin. A number of cell-free enzymes have been prepared from cells grown by this method. The results are discussed as to possibilities of improving the yields when growing the organisms on a large scale. Several uses are suggested.
Fermenta tion Methods Azotobacter vinelandit' is grown in Burk's ( 1 ) mineral salts solution plus 2.0 per cent sucrose, sodium molybdate (0.1 p. p. m. molybdenum), and ferrous sulfate (3 p. p. m. iron). In certain experiments high-test Cuban molasses replace sucrose as the carbohydrate source. The temperature for growth is 30" C., the pH 7.2. First-generation cultures are grown in twelve 6-ounce bottles containing 15 ml. of medium per bottle. After 24 hours 12 Roux bottles, each containing 100 ml. of medium, are inoculated with these cultures. After a second incubation period of 24 hours, each of six 10-liter bottles containing 6 liters of medium are inoculated with two Roux bottle cultures. As shown in Figure 1, the bottles are closed with sterile units consisting of rubber stoppers fitted with necessary glass and rubber tubing connections. A porous stone diffuser connected to the inlet glass tubing is suspended in the medium. The medium is aerated rapidly through the porous stone diffusers with air sterilized by passage through a large cotton filter. Smaller cotton filters are attached to each bottle. After 24 hours the final culture is inoculated with the bottle (third-generation) cultures. The apparatus for growing the final culture (Figure 2) is a 200-gallon copper fermenter used for the pilot-scale production of yeast. Its fittings are steam and cold water lines, a line furnishing air filtered through cotton, and a supplementary air line from a motor-driven blowx. This air, which is not filtered, is used only when large volumes of air are
T
HE availability of an abundant supply of azotobacter would greatly facilitate many researches. Recent contributions in the field of biological nitrogen fixation have supplied evidence for the similarity of the nitrogenfixing reaction in the symbiotic system (nitrogen fixation through association of root nodule bacteria and leguminous plants) and the free-living nonsymbiotic system of the organisms of the family Azotobacteriaceae. The nonsymbiotic system of azotobacter offers certain advantages for studies on the mechanism of the nitrogen-fixing reaction, especially those which deal with the enzyme systems concerned directly or indirectly in the fixation reaction. A study of the cell-free enzymes of azotobacter may give valuable leads to the mechanism of the reaction. Large quantities of cells are a prerequisite for such studies. Various microorganisms have proved to be excellent sources of vitamins and a variety of other biological products. Since azotobacter possesses the highest respiration rate of any known organism, it might be expected to contain a high level of respiratory enzymes and vitamins; this point was investi1
Present address, General 3Iills, Inc
, Uinneapolis,
FIGURE 1. TEN-LITERBOTTLES FOR SECONDGENERATION CULTURES
Minn
354
March, 1943
INDUSTRIAL AND ENGINEERING CHEMISTRY
needed and danger from contamination is at a minimum. The steam for heating or water for cooling is supplied through an interior coil; the air line is fitted inside the fermentation tank with large porous stone diffusers. The medium (250-300 liters) is placed in the fermenter and sterilized for 2 hours a t 10 pounds steam pressure. The medium is cooled by passing cold water through the inner cop er coil and is then inoculated with 36 liters of the 24-hour thirggeneration culture. The medium in the fermenter is aerated rapidly for 30-34 hours, during which time periodic samples are removed from a jet at the bottom of the fermenter for microscopic examination and for determinations of total sugar, total nitrogen, soluble nitrogen, pH, dry weight of cells, and Qo, (cu. mm. oxygen uptake per mg. dry weight of cells per hour). Qo, (N) values (cu. mm. oxygen uptake per mg. cell nitrogen per hour) are calculated from the Qo, values and the percentage nitrogen in the cell.
355
during this run which was stopped after 33.5 hours. After 19.5 hours, 6.25 mg. of nitrogen had been fixed per 100 ml. of medium; the final fixation was 12.2 mg. per 100 ml. Subsequent experiments indicate that the fixation in this experiment may have been limited by diminished carbohydrate supply (2 per cent sucrose furnished). The total yield of cell paste was 1675 grams with a moisture content of 85.2 per cent. This is equivalent to 248 grams of dry cells. Microscopic examinations showed no contamination during the period of growth. EXPERIMENT 2. Sucrose was again used as the carbohydrate source, and aeration was by cotton-filtered air from the 30pound pressure line. Periodic analyses were made for total
~~
TABLEI. c
~ i Mg.~ Nitrogen/100 ~ , Ml. D~~ wt., Total N Sol. N mg./100 ml. Hours 0 1.19 10.8 8 3.78 0:36 33.5 14 9.99 0.79 77.7 20 26 32 a
14.60 17.15 19.13
1.08 1.17 1.23
SUMMARY O F ANALYTICAL
Experiment 2 Sugar Oxygen Uptake Qoz Qcz (N) mg./100 h. Cu. mm./hr./ml. 1306 ... 1132 550 1'6'40 16,200 1700 2185 18,500
..
p35]
118.4 147.6 156.0
.
2205 1970 1675
1570a 1404 1271
1865 1330 1070
16,300 12,300 9,400
RESULTS 7
pH 7.2 7.3 7.0
Total N , mg./100 ml. 2.19 4.35 12.00
7.0 6.8 6.6
24.28 29.32 33.40
Experiment 3 D r y wt., Sugar mg./100 ml. mg./lOO'ml. 50.9 1950 67.3 1750 124.2 1388 223.1 273.1 314.8
698 396 144
pH 7.2 7.22 7.05 7.04 7.07 7.72
Sucrose added t o fermentation.
Nitrogen determinations are made by the semimicro-Kjeldahl method of Umbreit and Bond (9). Soluble nitrogen is determined in the same manner on an aliquot of the supernatant obtained in making the dry weight determination. Total dry weights are obtained by centrifuging 100-ml. qliquots in celluloid cups at 4500 r. p. m. for 15 minutes; the cells are resuspended in distilled water and centrifuged for 10 minutes at 3500 r. p. m. in tared glass centrifuge cups. The cells are then dried at 95" C. and weighed. Total sugars are run by the method of Stiles, Peterson, and Fred (7). QO values are measured by making the proper dilution of the cells, plkcing them in the Warburg microrespirometer, and observing the rate of ox gen uptake for 30 minutes at 32' C. pH determinations are ma& with the Beckman pH meter. At the end of the fermentation the cells are concentrated in a power-driven separator of the ty e used in the manufacture of yeast. Two passages of the final Erment through the separator reduce the final volume from 300 to approximately 50 liters, or a sixfold concentration of the bacterial cells. The cell cream is then concentrated to a paste in a Sharples supercentrifuge at a speed of 35,000 r. p. m. A yeast press was not available, but it would perhaps be equally suitable for recovering the cells.
Fermentation Efficiency EXPERIMENT 1. This preliminary experiment was made to test the methods and become familiar with the operation of the equipment. Cotton-filtered air from a 30-pound pressure line was used throughout. No extensive analyses were made
TABLE11. EFFICIENCY OF CELL PRODUCTION AND h FIXATION Mg. Per 100 Time, Hours
N fixed
D r y wt.
Sugar used
0-8 8-14 14-20 20-26 26-32
2.56 6.24 4.63 2.53 1.98
Experiment 2 22.7 174 34.3 298 40.7 298 29.0 166 8.8 133
0-8 8-14 14-20 20-26 26-32
2.16 7.65 12.28 5.04 4.08
Experiment 3 16.14 200 362 56.9 98,Q 690 50.0 302 41.7 252
N Fixed, Mg./g. Sugar
T
Sugar Converted to D r y Cells,
~
~
FIGURE 2.
~
~
FOR
~
~
~
FERMENTER FINALCULTURES
200-GALLON
COPPER
%
14.7 20.9 15.5 15.3 14.9
13.1 11.5 13.7 17.5 6.6
10.8 21.2 17.8 16.7 16.2
8.2 15.7 14.33 16.55 16.19
nitrogen, soluble nitrogen in the supernatant after cells were removed, dry weight of cells, total sugar, QOZ,and pH. MicroscoDic examinations were also made at each harvest. The analytical results of experiment 2 are given in Table I and a graph of the data in Figure 3. The efficiency of production during various periods is summarized in Table 11. The total yield of cell paste (Figure 4) was 2350 grams. The cell paste contained 79.6 per cent moisture, corresponding t o a yield of 480 grams of dry cells. The dried cells contained
INDUSTRIAL AND ENGINEERING CHEMISTRY
356
11.98 per cent nitrogen; thus 57.5 grams of nitrogen were fixed. Figure 3 shows that the logarithmic growth phase was attained quickly (as would be expected with the large inoculum) and continued through the first 14 hours. Table I1 indicates that the greatest efficiency of production was attained in the log phase during the 8-14 hour period. During this time 20.9 mg. nitrogen were fixed per gram of sucrose utilized, as compared t o 14.7 mg. fixed in the previous 8-hour period (the first 8 hours of the run) and 15.5 mg. during the subsequent 6-hour period. However, the dry weight of cells obtained per unit of sucrose utilized was somewhat lower during this period. I n other experiments the efficiency in converting sugar to cells was also higher during the log phase. DRY WEIGHT
-
A I
0 NITROGEN FIXED 0 DRY WEIGHT n~ A SUGAR CONSUMED
V."
the 14-hour point and continued throughout. The analytical data (Table 11) show clearly the effect of additional aeration during the 14-20 hour period. The comparable results of experiments 2 and 3 during this period are as follows: Total Increase during 14-20 Hr. Period over Preceding 14 Hr., Expt. 2 Expt. 3 Total nitrogen fixed 46.0 102.0 Total cell yield 52.8 79.50 Total sugar utilized 60.2 119.5 Smaller percentage increases in cell yield in Expt. 3 can be explained by t h e high 0-hour dry weight due to t h e higher solids content of t h e molasses medium.
v0
The effect of additional aeration is further emphasized by comparing the two experiments during the 8-14 hour period when the rates of aeration were identical: Total Increase during 8-14 Hr. Period over Preceding 8 Hr., % Expt. 2 Expt. 3 162.0 176.0 132.0 85.0 171.0 181.2
CONSUMED
FIXED
I
Total nitrogen fixed Total cell yield Total sugar utilized 3.0-
1.5
-
2.5-
2.0-
2 FIGCRE 3. GRAPHOF DATAFOR EXPERIMENT
The over-all efficiency (0-32 hours) was 16.8 mg. nitrogen fixed per gram of sucrose utilized, an excellent result for this reaction. Cellular material represented 13.5 per cent of the sugar utilized. The rate of respiration was highest during the log phase when the &os was 2185'and the Qo, (N) was 18,500. EXPERIMENT 3. Since molasses furnishes carbon more economically than commercial sucrose, high-test Cuban molasses was tried as the source of carbohydrate. The culture was aerated with cotton-filtered air from the 30-pound laboratory air pressure line during the first 14 hours, and then aerated more rapidly with unfiltered air from the motor-driven blower during the remainder of the fermentation. Samples were removed periodically for analysis. The results are given in Tables I and I1 and Figure 5. The growth curve is typical. The lag phase continued about 6-8 hours, followed by the log phase which lasted through the 20-hour period; a t that time the phase of negative acceleration was reached. I n experiment 2 the log phase lasted only until the 14-16 hour period and had reached the phase of negative acceleration a t 20 hours. The difference can be explained largely by the fact that in experiment 3 a greater rate of aeration (use of motor-driven blower) was started a t
Vol. 35, No. 3
The latter table shows that, during a period in which the rate of aeration was identical in both experiments, the rate of nitrogen fixation, increase in cell yields, and sugar utilization were nearly the same. It is impossible to say definitely what the effect of still greater rates of aeration would be; owing to the highly aerobic nature of azotobacter, one would expect further increasing rates of fixation with increasing rates of aeration although the degree of increase per unit increase in aeration would become less. This has proved to be true in the production of commercial yeast in grain and molasses media. I n experiment 3, 31.2 mg. nitrogen were fixed per 100 ml. of medium in 32 hours which is excellent for large-scale procedures. This represents a total for the 300 liters of 93.6 grams nitrogen fixed. The net cell yield was 263.9 mg. per 100 ml. or a total of 792 grams of dried cells, corresponding
YIELD,2350 GRAMS,OF CELLPASTE (100-ML. FIGCRE4. TOTAL GRADUATE SHOWN FOR COMPARISON)
IN CELLSOF Azotobacter tinelandii TABLE111. VITAMINSOF B COMPLEX
Biotin Cells Hydro- Unhydrolyzed lyzed
Riboflavin Nicotinic acid Medium Medium b
(cells Cells* removed)
Sucrose Molassesa sascarbohydrate carbohydratesource source Brewer's yeast, &v. a
350 304 50
. ...
1 .. 0,
(cells Cells removed) 590 480 550
Values for cells expressed in micrograms per gram d r y cells.
. .. ...
3.0
b Values for medium (cells removed) expressed in micrograms per ml.
24 . 52 63 2.0
0.039 0.44 0.8
Medium (cells removed) 0 .0098 .. .
....
~
Thiamine Medium (cells Cells removed)
3963 . 0 40
...
0.0
...
Pantothenic Acid Cells Medium Hydro- Unhydro(cells lyzed lyzed removed) 152 59 0.77 184 70 150 50
.. *.
March, 1943
INDUSTRIAL AND ENGINEERING CHEMISTRY
t o 5270 grams (11.6 pounds) of cell paste with a moisture content of 85 per cent. Table I1 shows that the greatest efficiency in nitrogen k a tion was again evident during the log growth phase (8-14 hour period) when 21.2 mg. nitrogen were fixed per gram sucrose used. As regards cell yield from sugar utilized, the greatest efficiency was unexpectedly attained during the latter part of the fermentation, This may be explained in part by the more rapid rate of aeration during this part of the fermentation. The differences in efficiency are small, however, and may not be significant. The over-all efficiency (0-32 hours) was 17.3 mg. nitrogen fixed per gram of sugar used; 14.6 per cent of the sugar utilized was converted to cells.
Bacteria in Preparation of Cell-Free Enzymes Many of the cells obtained in the foregoing experiments have been used in the preparation of cell-free enzymes. The techniques for obtaining the cell-free preparations and the results were reported by Lee, Burris, and Wilson (8). Oxalacetic acid and a-ketoglutaric acid decarboxylases, hydrogenase, cytochrome oxidase, and dehydrogenases for succinic, malic, and lactic acids have been demonstrated in cellfree preparations of azotobacter.
Vitamins i n Azotobacter Current interest in microorganisms as sources of vitamins led to an examination of azotobacter for various members of the B complex. The results are given in Table 111. Assays have been run on the cells grown on a medium containing pure sucrose (experiment 2) and molasses (experiment 3) as the carbon sources. Average values for the amount of these vitamins in brewer’s yeast are included for comparative purposes, Microbiological assay methods were used for biotin (4), pantothenic acid (8), riboflavin (6),and nicotinic acid (6). The thiochrome method was used in the thiamine determinations (W). Results of experiment 2 show that Azotobacter uinelundii, growing in a nitrogen-free mineral salts medium, synthesizes various members of the B complex to such an extent that its cells are a t least equal t o brewer’s yeast in all vitamins for which assays were made and are superior in their contents of biotin and riboflavin. When grown in molasses medium (experiment 3) azotobacter cells again exceed brewer’s yeast in contents of riboflavin and biotin and, in addition, are higher in thiamine. The organism when grown in molasses contains more biotin, thiamine, and pantothenic acid than when grown in sucrose medium. On the other hand, molasses-grown cells appeared to be slightly lower in riboflavin and nicotinic acid.
Advantages of the Method Although azotobacter is a rapidly growing, highly aerobic organism, about 15 per cent of the total sugar consumed in these experiments was recovered in dry weight of cells. This is a good yield when one considers that this organism has a very high rate of respiration and would be expected to convert most of an available carbon source to carbon dioxide and water. One must also consider that, as the organism was grown in a nitrogen-free medium, its nitrogen supply was obtained through the fixation of atmospheric nitrogen. Energy for this fixation process is furnished by the carbon source in the medium which would further reduce the efficiency of the organism in converting a carbon source to cellular material. Yeast grown under optimum conditions in grain wort medium with rapid aeration commonly give yields of 30 per cent dry yeast from the sugar utilized. This is superior to that obtained with azotobacter. However, the excellent results with azotobacter thus far indicate that studies to determine the
357 SUGAR CONSUMED
-
i
25-
-
-
2.3.
2.1
0 NITROGEN FIXED DRY WEIGHT
0.9
A SUGAR CONSUMED 0.6
‘9
HOURS
2,0
0.3
--
2.6
1.9-
-2.2 2A
1.7-
-1.5-
FROM EXPERIMENT 3 FIGURE 5. GRAPHOF DATA
optimum conditions of aeration, sugar concentration, salt mixture, age of inoculum, conditions for growing inoculum, and other factors may aid not only in increasing the efficiency of the process but also in cutting down the time of fermentation and increasing the yield of cells per unit volume of medium. The organism grows rapidly in an inexpensive mineral salt-sugar medium. Molasses is perhaps the cheapest source of sugar for the medium during peacetime, and it is an excellent carbohydrate source for the growth and fixation of nitrogen by azotobacter in this process. Further studies may show that the mineral salts contributed to the medium by the molasses may allow reduction in the quantities of salts which must be added. Several points favor the growth of azotobacter on a large scale: (1) The organism grows rapidly in an inexpensive nitrogen-free medium with acceptable yields. The use of such a medium would greatly reduce troubles from contamination. (2) Azotobacter can synthesize vitamins to such an extent that the cells are equal or superior to yeast in vitamin content. Thus it has potential use as a food supplement comparable to the present day use of yeast. (3) The interesting biochemical nature of the organism-namely, high rate of respiration-makes it a potential source for study, isolation, and manufacture of new biochemical compounds.
Acknowledgment The copper fermenter was kindly loaned to the University of Wisconsin by the Red Star Yeast Company of Milwaukee. The authors are indebted to various members of the Departments of Agricultural Bacteriology and of Biochemistry of the university for the vitamin assays.
Literature Cited (1) Burk, Dean, and Lineweaver, Hans, J . Bact., 19, 389 (1930). (2) Hennessy, D. J., and Cereoedo, L. R., J . Am. Chem. Soc., 61, 179 (1939). (3) Lee, S. B., Burris, R. E., and Wilson, P. W., Proc. SOC.Exptl. Biol. Med., 50, 96 (1942).
(4)Shull, G. M., Hutohintcs, B. L., and Peterson, W. H.. J . Biol. Chem., 142, 913 (1942). ( 5 ) Snell, E. E., and Strong, F. M., IND. ENO.CHEM.,ANAL.ED., 11, 346 (1939). (6) Snell, E. E., and Wright, L. D., J . Biol.Chem., 139, 675 (1941). (7) Stiles, H. R., Peterson, W. H., and Fred, E. B., J . Bact., 12, 427 (19261. (8) Strong, F.M., Feeney, R. E., and Earle, Ann, IND. ENG.CHEM., ANAL.ED.,13, 566 (1941). (9) Umbreit, W. W., and Bond, V. S., Ibid., 8, 276 (1936). THISreaeerch was supported in part by grants from the Rockefeller Foundation and from the Wisconsin Alumni Research Foundation.
