INDUSTRIAL AND ENGINEERING CHEMISTRY
1202
ACKNOWLEDGMENT
The authors wish t o acknowledge the assistance of H. G. Dawson of the Firestone Research Laboratory for the analytical values of Table VI and to H. H. Miller of the Xylos Rubber Company for various reclaim evaluations and other data. Appreciation is expressed for the encouragement and assistance of F. W. Stavely and R. F. Dunbrook, and for the permission t o publish this manuscript by the managements of the Firestone Tire & Rubber Company and the Xylos Rubber Company.
Vol. 40, No. 7
Ibid., 2,359,122 (1944) ; 2,363,873 (1944) ; 2,372,584 (1945). Lutz, G~ntmi-Ztg.,25, 120-1 (1910). Miller, G. R., in “Chemistry and Technology of Rubber,” bi Davis, C. C. and Blake, J. T., pp. 720-38, New York, Reinhold Publishing Gorp., 1937. Mooney, M., IND. ENG.CHEM.,ANAL.ED.,6, 147 (1934). Neal, A. M., and Schaffer, J. R., Jr., U. 5. Patent 2,333,810 (1943).
Oldham, E. W., Baker, L. M., and Craytor, M. W., IND.ENG. CHEM., ANAL.ED.,8 , 4 1 (1936). Palmer, H. F., and Kilbourne, F. L., Jr., IND.ENG.CHEM.,32, 512 (1940).
Ritter, J. J., and Sharp, E. D., J. Am. Chem. SOL, 59, 2351
LITERATURE CITED
(1937).
Andrews, R. D., Tobolsky, A. V., and Hanson, E. E., J . Applied Phys., 17, 352-61 (1946),
British Ministry of Supply, private communication (June 1943). British Ministry of Supply, private communication (May 1946). Carpenter, A. S., IND. ENG.CHEM.,39, 187 (1947). Dasher, P., U. S. Patents 2,304,548, 2,304,549, 2,304,550, 2,304,551 (1942).
Essex, W. G., U. S. Patent 2,154,894 (1939). Farmer, E. H., in “Advancesin Colloid Science,” Vol. 11,p. 303, New York. Interscience Publishers. 1946. Farmer, E. H,, Trans. Faraday SOC.,42,228-36 (1946). Garvey, B. S., U. S. Patent 2,193,624 (1940). Gillman, H. H., Rubber Aoe (New York), 58, 709-14 (19461, Gumlich, W., U. S. Patent 2,280,484 (1942). Gumlich, W., and Ecker, R., U. S. Patent 2,338,427 (1944). Hughes, A. J., and Amphlett, P. H., Trans. Inst. Rubber Ind., 19, 165 (1944).
I. G. Farbenindustrie, A. G., Kautschuk-Zentrallaboratorium, Leverkusen, “Report on Reclaiming of Buna Vulcanizates with the Aid of Renacit” (Aug. 28, 1945). Ioannue, J. P., U. S. Patent 2,069,151 (1937). Jones, F. A., Owen, E. W. B., Tidmus, J. J., andFraser, E. E., Trans. Inst. Rubber Ind., 19, 190 (1944). Kirby, W. G., and Steinle, L. E., U. 9. Patent 2,279,047 (1942).
Sebrell, L. B., Canadian Patent 289,290 (1929) ; Chem. Zentr.. 103, I, 2392 (1932). Shelton, J. R., and Winn, H., IND. ENG.CHEM.,39, 1133 (1947). Simmons, H. E., War Production Board, Office of the Rubber Director, private communication, Feb. 23 and June 3, 1943. Smith, G. E. P., Jr., “Antioxidant Effects in Natural and Synthetic Rubbers,” Symposium on Degradation and Aging of High Polymers, Polytechnic Institute of Brooklyn, Nov. 30. 1946.
Smith, G. E. P., Jr., Ambelang, J. C., and Gottschalk, G. W., IND. ENG.CHEM.,38, 1166 (1946). Spence, P., and Ferry, J. B., J. Am. Chem. SOC., 59, 1648 (1937). Taylor, H. S., and Tobolsky, A. V., Ibid., 67,2063 (1945). Tobolsky, A. V., Prettyman. I. B.. and Dillon, J. H., J . Applied __ Phys.,-l5, 380 (1944).
U. S. Rubber Co., British Patents 575,545, 575,546, 575,545 (1946).
Waters, W. A., Trans. Faraday Soc., 42,189 (1946), Wolf, G. M.,Deger, T. E., Cramer, H. I., and DeHilster, C. C.. IND.ENG.CHEM.,38, 1157 (1946). Ziegler, K., and Ganicke, K., Ann., 551, 213 (1942). RECEIVED June 10, 1947. Presented at the meeting of the Division of Rubber Chemistry of the AMERICANCHEMICAL SOCIETY, Cleveland, Ohio May 26 t o 28, 1947.
CITRIC ACID Production by Submerged Fermentation with Aspergillus niger PIKG SHU AND MARVIN J. JOHNSON University of Wisconsin,Madison, Wis. Average yields of 72 grams of anhydrous citric acid per 100 grams of added sucrose were obtained by submerged culture of Aspergillus niger in shake flasks on a synthetic medium at an initial sucrose concentration of 140 grams per liter. The fermentation required 9 days. A 70% yield was obtained i n 12 days at a sucrose concentration of 260 grams per liter. Data on the effect of changes i n composition of the medium are presented. The optimal conditions for shake flask fermentations include potassium dihydrogen phosphate above 1 gram per liter, magnesium sulfate heptahydrate above 0.25 gram per liter, iron concentration of 1 mg. per liter, 2.5 grams per liter of nnimonium nitrate, and an initial pH between 2.2 and 4.2.
ONSIDERABLE research effort has been expended on attempts t o develop a submerged fermentation process for citric acid production (16). Amelung (1) reported citric acid production from sucrose by aerating a submerged culture of Aspergillus japonicus. The yield of citric acid was very low. According t o a recent patent of Szucs (fd),citric acid has been successfully produced in submerged cultures of Aspergillus niger. Szucs’ preferred procedure involves transfer of preformed mycelium from a growth medium t o a fermentation medium, and
the use of oxygen or air-oxygen mixture for aeration. Similarly, Karow and Waksman (2, 13) reported the production of citric acid in submerged cuItures of Aspergillus wentii. The maximum citric acid yield is obtained when oxygen is used for aeration, and after the desired growth is obtained the growth medium is replaced by a fermentation medium. Using shake flask technique, Perquin (8)systematically studied the effect of variation of the environmental conditions (both gaseous and liquid phases) on the production of citric acid in submerged cultures of Aspergillus niger. He concluded that the presence of zinc sulfate, potassium chloride, and increased concentration of magnesium sulfate in the liquid phase favored the production of citric acid. On the other hand, the presence of a high concentration of potassium dihydrogen phosphate i n the medium was unfavorable. The use of oxygen or oxygen-air mixture for aeration resulted in a higher citric acid yield. Under all conditions, only a small amount of citric acid was produced. Karow and Waksman ( 2 ) demonstrated the requirement of manganese sulfate for maximum citric acid production by submerged culture of Aspergillus wentii. The substitution of urea for other nitrogenous salts proved satisfactory. They also found that the presence of magnesium sulfate and high concentrations of potassium dihydrogen phosphate in replacement or fermentation medium were unfavorable for citric acid production.
I N D u sT R IA L A N D E N G I N E E R I N G
July 1948
Studies on citric acid production by the surface culture method
(5-6,8, 9) have shown the fermentation to be very sensitive t o changes i n trace metal and phosphate concentrations, nitrogen source, initial pH, and other factors. The optimum conditions vary with the strain of microorganism. No satisfactory conditions for a n efficient one step submerged fermentation for the production of citric acid have been reported. I n the present study, submerged citric acid fermentations were carried out; in these citric acid was produced on the growth medium. Air was used as an oxygen source. The effect of variations in environmental conditions on citric acid yield and on the chemical changes in the medium were investigated; large scale fermentations or fermentations of crude sugar were not attempted. METHODS
The organism used in the experiments was a colony isolated from culture 72-4 ( B ) , which in turn was a colony isolated from Asper illus niger ATCC 1015. The stock culture was carried on soil. h a n s f e r s were made from a soil stock culture through two successive sugar agar slants t o a n agar bottle plate. These plates had an agar surface of approximately 72 sq. cm. and contained 25 ml. of agar medium A (Table I). The inoculated bottle plates were incubated at 30 O C. Spores from 3- to 5-day old bottle plates were suspended in 50 ml. of sterile distilled water, and 1.5 ml. of the suspension were used to inoculate 50 ml. of fermentation medium contained in a cotton-plugged 500-ml. Erlenmeyer flask. These fermentation flasks were incubated at 25" C. on a shaker which moved the flasks in a horizontal circle 1 inch in diameter at a speed of 270 r.p.m. The composition of all fermentation media was that of medium B, Table I, except for components being systematically varied. At intervals of 5, 7, and 9 days, samples were taken. The figures given in the tables are for samples taken at 9 days, except where otherwise stated. Residual reducing sugar was determined by the method of Shaffer and Somogyi (10) and citric acid by the method of Perlman, Lardy, and Johnson ( 7 ) . In the use of this method, it was found t h a t 0.6 N ferrous sulfate solution was much superior to hydrogen peroxide for removal of excess permanganate. Reaction was more rapid, and a slight excess was not detrimental. Titratable acidity was expressed in terms of anhydrous citric acid. Evaporation was corrected by setting up a n uninoculated control flask. All the determined values were calculated on the basis of the sugar concentration of the control flask. D r y weight was determined on the washed mycelia after drying at 110' C. overnight.
TABLE I. COMPOSITION OF MEDIA Constituents Domino morose, g. Bacto agar, g. KHpPOi, g. MgS04.7H10, g. N H I N O ~g., HCI, g.
Medium A Wt.,/Liter' 140 0 20.0 1.0 I
0.25 2.5
..
0.48 3.8
Medium B, Wt. ,/Liter 140.0
cH E M I sT R Y
1203
TABLE11. EFFECTOF SUCROSECONCENTRATION ON CITRIC ACIDPRODUCTION Yield of Citric & - nn -A___ - .-
Available Utilized sugar, sugar,
Sucrose Concn., G./100 MI.
%
% '
Residual Acidity Due Sugar, t o Citric Acid, % of Total G./100 M1.
hlycelial Weight G./100 MI.
OF MAGNESIUM SULFATECONCENTRATION TABLE 111. EFFECT ON CITRICACIDPRODUCTION
Yield of Acid M 80r.7HgO Available sugar, 6oncn,, G./Liter %
Citric on U6ilized Acidity Due sugar, to Citric Acid, % % of Total
Residual Sugar G./100 Ml.
Mycelial Weight G./lOO MI.
that sodium nitrate is a better nitrogen source than ammonium nitrate. This conclusion was not confirmed i n preliminary experiments. The best nitrogen source appeared to be ammonium nitrate which was used in all subsequent experiments. The data of Table IV show that the optimum concentration of ammonium nitrate is in the neighborhood of 2.5 grams per liter. At either side of the optimum concentration, a low acid yield and poor growth were observed. It appeared from preliminary experiments that the phosphate ion might function in some manner other than as a simple nutrient and buffer. It appears t o be related to the acid production rate inasmuch as the most rapid acid production was observed at phosphate concentrations above the optimum value for growth (Table V). As the p H of the medium drops rapidly to approximately 2 a t all phosphate concentrations, the buffering action of phosphate cannot be responsible for the observed effect. It has been well established that there is a n optimum concentration of iron for maximum citric acid production (6). The optimum level as determined in the present study was approximately 1 mg. per liter as shown in Table VI. It is evident from the table that mycelial weight varies directly with iron concentration. At low levels of iron, sugar utilization is poor because of
2:5 0.25 2.5
t o p H 3.8
0.06
TABLEIV. EFFECTO F AMMONIUM NITRATE CONCENTRATION ON CITRICACIDPRODUCTION
0.25
2.2 1.3
