Production of Citric Acid by Submerged Fermentation
S.
development
M.MARTIN AND W. R. WATERS
Division of Applied Biology, Nafional Research laboratories, Offawa, Onf., Can.
I
Fermentation Method
N SPITE of the considerable interest in the submerged method
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for the production of citric acid, the literature is somewhat scanty, especially on the fermentation of crude sources of carbohydrate. The bulk of the available information on this subject is contained in the patent literature which is not readily accessible and at times is difficult to interpret. The submerged culture method for the production of citric acid has been reviewed by Perlman ( 7 ) . Recently patents by Woodward et al. ( I S ) , Snell and Schweiger ( l a ) , and Schweiger and Snell (fO)have been assigned to Miles Laboratories, Inc., which is erecting a plant for the production of citric acid based on the submerged process ( 2 ) . Woodward et al. (IS) have described a method of conditioning crude sources of carbohydrate for the production of citric acid based on the principle of ion exchange. Snell and Schweiger (12)described a process in which the cellular morphology of a strain of Aspergillus niger is controlled b y the nutrient balance. I n the latter process nitrogen in the form of ammonium carbonate is added for mold growth and the iron content is maintained below 1 p.p.m. Schweiger and Snell (IO) have described a fermentation process in which decationized raw sugar is fermented in the presence of morpholine in a column-type fermenter. I n this process the fermentation mash is inoculated with spores and aerated vigorously. A yield of 79.9% in 82/3 days is reported, from which a conversion rate of 0.38% conversion per hour can be calculated. Moyer ( 5 ) has recently described a process for the submerged production of citric acid in which the crude source of carbohydrate is treated simply by the addition of methanol. By this method, the addition of 1 t o 3% of methanol to the production medium greatly increased the yield of citric acid and the tolerance t o iron, zinc, and manganese. Another feature of this process is the use of 2% of germinated inoculum. Submerged fermentations were carried out by the shake flask technique and in stainless steel tanks with aeration and agitation. Using commercial glucose as the substrate, Moyer reported a yield of 64.470 on sugar consumed in 8 days in shake flask experiments. This rate is equivalent t o 0.33% conversion per hour. Whether the submerged process with its apparent instability can compete on a commercial basis with the more easily controlled pan fermentation has yet t o be decided. Two of the world's largest producers appear t o be convinced of the virtues of the latter method. Chas. Pfizer and Co. is increasing its output from pan fermentations ( 3 )and Kemball, Bishop and Co. of England is erecting a plant t o employ the surface process at Cornwall, Ontario ( 1 ) . Since there is a large supply of Canadian beet sugar molasses, this substrate rather than cane molasses was chosen for the following study.
INOCULUM. Strain N.R.C. A-1-233 (University of Wisconsin Strain 72-4) of A . niger, used exclusively in this work, was maintained in soil stocks. For use, the culture was transferred serially four times on a synthetic medium similar t o that described by Shu and Johnson (If). Mature spores from the fourth culture (5 t o 7 days old) were used t o inoculate a seed mash. The latter was prepared by diluting sugar beet molasses t o about 12% sugar and adjusting the p H t o 6.0 with hydrochloric acid. After sterilizing, 0.5 t o 0.8 gram of potassium ferrocyanide trihydrate per liter was added t o the hot maph. After cooling, 0.5 gram of dibasic potassium phosphate trihydrate per liter wag added. The evact amount of ferrocyanide added depended on the molasses in use and was determined approximately by trial and error in shake flask experiments. The spores from about a 70-square cm. surface area of agar medium (in a &ounce medicine bottle) suspended in 50 ml. of water were used t o inoculate 500 ml. of seed mash in a 1-liter Erlenmeyer flask. The seed culture was incubated a t 26' C. for 24 hours on a reciprocating shaker with lard oil as the antifoam agent. Usually 3 or 4 seed cultures were set; the best were selected on the basis of macro- and microscopic appearance for use as inoculum.
FERMENTATION. The fermentation mash was prepared in a manner similar t o that used for the seed mash, except t h a t according to t h e molasses sample used the ferrocyanide level was varied from 0.5 t o 1.0 gram per liter and the phosphate level from 0.1 t o 0.5 gram per liter. T o obtain a good precipitate when the mash was treated with ferrocyanide, it was found necessary t o add the latter t o the hot mash or t o a mash which was subsequently heated. It was not necessary, however, t o remove the precipitate and indeed, slightly better results were obtained when the precipitate was left in the mash. The fermentations were carried out in borosilicate glass towertype fermenters (Figure 1)) 6 X 150 cm., each equipped with a medium-porosity sintered-borosilicate glass disk having a n area of about 12 square cm. T o facilitate cleaning, two-piece fermenters with standard joints just above the aeration disks were used. The fermenters were sterilized by intermittent steaming on several consecutive days. Two and one half liters of mash in such a fermenter gave a column about 100 cm. high with a surface area of about 28 square cm. Foaming was a severe problem and no one method appeared capable of coping with it completely. Fairly satisfactory control was obtained by the initial addition of 2 ml. of octadecanol (3% in paraffin oil) per fermenter, adding lard oil as required during the fermentation, and by mechanical foam breakers. Two and one half liters of fermentation mash were added to 2229
v
INDUSTRIAL AND ENGINEERING CHEMISTRY
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each fermenter and oxygenated at a rate of 100 ml. of oxygen per minute for 2 hours. This preliminary oxygenation ensured oxygen saturation, probably served to oxidize any readily oxidizable compounds present, and helped to combat the severe foaming which occurred a t the start of the run. I
MOTOR
H O L E s - - l
SHAFT
SPINNER
'UEE
I
A I R OR OXYGEN
Figure I. lower-Type Fermenter
At the end of this pretreatment period the mash was inoculated with the seed culture using a 2 t o 8'4; inoculum, depending on the quality and density of the seed. Immediately after seeding, aeration of the mash was begun. ltoistened compressed air was passed through the fermenters, beginning a t a rate of about 100 ml. per minute and being increased during the first hour to a final rate of 500 ml. per minute. Aeration \vas continued for 24 hours and considerable growth took place duiing this time. The mash WAR then oxygenated a t a ratr of 500 ml. of oxygen p ~ minute r
Figure 2.
Seed Pellet (300x1 29 houn old
a
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per fermenter until the en3 of tlie fermentation. Perquin (8) showed that using oxygen rathcr than air as the atmosphere increased the yield of citric acid but that oxygen inhibited spore germination and initial stages of growth. Samples were removed for analysis a t frequent intervals t o follow the course of the fermentation. CULTURAL CHARACTERISTICS. The quality of the inoculum is of prime importance. A good seed a t 20 to 24 hours possesses i h e following charact,eristics: Macroscopic characteristics include many small individual colonies about 0.2 t o 0.5 mm. in diameter, a free flowing mash, and some clearing of the mash by trapping of the precipitat.ed matter in the pellets. Microscopic characteristics include pellets well separated one from another with a minimum of fusion, spherical, dense pellets with limited lateral hyphae and with much precipitate enmashed in thc pellet (Figure 2), and individual hyphae, short, grcatly thickened, vacuolated, and granular, with short clublike branches and with particles of precipitate adhering t o the hyphae (Figure 3). These morphological characteristics resemble those described by Snell and Schweiger (12). During the period of about 12 t o 18 hours after inoculation of t'he fermenters, t h e small pellets (about 0.75 t o 1.0 mm. in diameter) begin t o aggregate into loose flocculent masses. By this time the mash begins to clear and appears wine colored by transmitted light, After about 18 hours the aggregates break u p and individual hard, smooth pcllets begin t o form. During this time there is an increase iu foaming and, if the air flow is stopped, the pellets rise t o the top. -4fter about 30 hours serious foaming ceases and the pellets t e d t o settle out if the oxygen supply is shut off. By this time growth has practically ceased. Washed pe1let.s taken from this stage or later are creamy white, smooth, gravelly to t h e touch, and of medium size (about 1.0 t o 2.0 mm. in, diameter) as shown in Figure 4. The size of inoculum used in t h e fermentem should be Buch that fully developed pellets are allowed free movement in the mash. On settling, the pellets should occupy between one fourth and one third of the tot,al volumc.
Analytical Total acid was determined by titration of 1-ml. samples to tlie phenolphthalein end point n i t h 0.1 N sodium hydroxide and is expressed as anhydrous citric acid. Citric acid was determined colorimetrically by the method of' Saffran and Denstcdt, ( 8 ) and is expressed as anhydrous citric acid.
Figure 3.
Seed Pellet (1 200 X) 22 hours old
INDUSTRIAL AND ENGINEERING CHEMISTRY
September 1952 Table 1.
Fermentation of Beet Molasses in Submerged Culturea (61-Hour fermentation)
a
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molasses were fermented simultaneously in duplicate under like conditions it was found that one sample of beet molasses (Chatham 1946) and the sample of blackstrap had unsatisfactory fermentation characteristics under the conditions of test (Table 11). The poor fermentation properties shown by Chatham 1946 beet
All values corrected for evaooratinn
Table 11.
Comparison of Four Molasses Samples by Fermentation"
(Fermentation time, 78 hours) Conversion of AvailTotal Acid as Anhydrous able Su ar to Anh Citric Acid, Citric Acid, drous %trio Ad$-
Sugar Molasses Utilized, Samples % % % % Chatham 79.5 1946 2.4 1.3 9.9 Chatham 1947 93.0 8.6 70.7 10.9 Chatham 93.0 1949 8.6 66.0 9.4 Brit. West Indies black1.1 89.0 strap 1.1 9.0 Each value is the average of two fermentations and is not corrected for evaporation losses (less than 10%). b Inoculum grown in mash prepared from Chatham 1949 molasses with 0.8 gram K4Fe(CN)s.3HzO and 0.5 gram KzHPOr.3HzO/liter: fermentation mashes prepared from various molasses samples were treated b y the addition of 0.5 gram KaFe(CN)s.3HzO; 0.13 gram KeHP04.3HaO per liter and 8% inoculum was used in each case.
Sugar was determined after a 15minute hydrolysis in 1.5 hydrochloric acid by the method of Sumner as described by Kolmer and Boerner (4). Sugar conversion values are based on the conversion of available sugar t o anhydrous citric acid and were calculated by the formula
% ' conversion
=
Figure 4.
molasses may have been due t o original characteristics of the molasses or t o changes resulting from storage. Figure 6 shows the course of the 4 fermentations when the duplicate values are averaged. When Chatham 1949 beet molasses was used in both the seed
Table 111.
Experimental and Results
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.
Effect of Varying Size of InoculumG (Fermentation time, 119 hours)
grams citric acid X 100 grams available sugar
Evaporation was calculated on the basis of total change in volume less volume of samples. I n many cases it was impossible t o calculate the loss due t o evaporation because of changes in mash volume as a result of foaming over.
Pellets from Fermenter (20x1
60 hours from seeding fermenter
Size of Inoculumb, % 3.OC 4.5d 6.0C
Total Acid as Citric Acid,
%
7.4
9.0 10.2
Anhydrous Citric Acid,
%
6.0 7.0 7.8
Conversion of Available Sugar t o Anhydrous Sugar Citric Acid, Utilized,
%
%
48.5 54.5 61.0
86.8 93.5 94.0
a Values not corrected for evaporation losses (less than 10%). b T h e seed and fermentation mashes contained 0.8 and 0.5 pram K4F* (CN)s.3HzO and 0.5 and 0.13 gram K2HP04.3HzO per liter, respectively. C Average of two fermentations. d One fermentation.
When five simultaneous, and as nearlv as eossible identical, fermentations were carried out on Chatham 1947 beet molasses the data shown in Table I were obTable IV. Fermentation of Chatham 1950 Molassesn tained. The values given in the table are from Conversion of the 61-hour samples, although some of the inAvailable Sugar to Anhydrous Sugar AnhydrouR Total Acid as dividual fermentations reached their maximum Citric Acid, Utilized, Citric Acid, Citric Acid, % % yields at 68 hours. I n no case was the difNo. Time % % 68 97.1 1 70 9.3 8.9 ference due to sampling time greater than 0.5% 72 96.2 2 70 9.7 9.3 total acid or 0.2% citric acid. The course of the 63 (agprox.) 91.7 (aPProx.) ab 65 8.5 8 . 2 (approx.) 4 Lost because of fermentation when sugar, total acid, and citric .... frothing ... 6a 88.0 acid values for the five fermentations are averaged 5 881/z 8.3 7:s 57 90.2 6 88'/2 7.5 7.4 and plotted against time is shown in Figure 5. a All values corrected for evaporation. inoculum was grown 24 hours in mash prepared The average conversion rate of 0.93% confrom Chatham 1950 molasses and contaihing 0.5 gram K2HP04.3H20 per liter and varying amoupts of KrFe!CN)e.3HzO (1 and 2, 3 and 4, and 5 and 6 contained 0.3, 0.5, and 0.7 gram version per hour illustrates the rapidity of this per liter, respectively). The amount of inoculum used varied in accordance with the micro- and macroscopic appearance of the seed. The fermentation mash was prepared from fermentation. Chatham 1950 molasses and contained 0.5 gram KzHPOa.3HzO and 0.5 gram KiE"e(CNbWhen three samples of beet molasses, from 3Hz0 per liter. b This fermentation frothed over to some extent and evaporation could not be deterthe same refinery but from different years, and mined. The average evaporation was used in the calculations. one sample of British West Indies blackstrap
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8
6
I-
z W
0
a
24
2
0 20
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40
60
80
TIME, H O U R S
Figure 5.
Fermentation of Beet Molasses in Submerged Culture Average lot live fermenbtionr
and fermentation mashes, increasing the inoculum from 3 to 670 resulted in increased yields of citric acid (Table 111). However, as low as 2% inoculum was the best for other samples of molasses. I n the foregoing runs a considerable difference exists bet,ween total acid and citric acid. Quantitative partition chromatography has shown this t o be due to oxalic and gluconic acids. The ratio of citric t o total acid appears t o be a function of the molasses used, for when a sample of Chatham 1950 molasses was fermented under various conditions the ratio was nearly 1.0 (Table IV). Similar ratios have been obtained in other runs with this molasses under similar and other conditions. The rapidity of the fermentation is again vrdl illustrated, with conversion rates of 1.02 and 0.9770 per hour for the two fermentations with low fertrocyanide in the seed.
Discussion Through application of the fermentation method described, good yields of citric acid from sugar beet molasses were obtained in a n unusually short time for the citric acid fermentation. In addition, the use of a small inoculum ( 2 to l0TG) of germinated spores and young pellets decreased the time required in the fermentation vessel and made possible the use of a small propagation vessel run in conjunction with a larger fermenter. The successful use of a small inoculum also suggests the use of several propagation stages for building up a large volume of seed. These considerations, together with the use of a crude molasses mash treated only by the addition of potassium ferrocyanide and supplemented with phosphate,
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indicate commercial possibilities in this process. Expensive treatments such as ion exchange, clarifications, or filtration are unnecessary. On the other hand, the use of large quantities of pure oxygen, although satisfactory for laboratory scale experiments, would be a disadvantage commercially. Since, however, only a small percentage of the oxygen supplied is utilized by the mold, it becomes feasible t o recycle the effluent oxygen after removal of carbon dioxide. The individual principles utilized in the process are not new t o the citric acid fermentation but rather a combination of the “prior art” has resulted in a very rapid fermentation. In spite of the high oxygen demand, a relatively small aniount is utilized in the fermentation. A few experiments indicate that less than 2y0 of the available oxygen is used. That rapid aeration is essential has, however, been demonstrated. When, for example, aeration was discontinued for as short an interval as 30 minutes during the first 24 hours, acid production was generallq late in starting or even eliminated. When oxygenation was interrupted there was usually little or no acid production throughout the subsequent fermentations. B similar effect was noted when the mycelium packed on the aeration disk. Ferrocyanide and phosphate levels depended on the molasses sample and may be determined approximately by shake flask experiments. The two appeared t o be interrelated, phosphate trnding to reduce the toxicity of the ferrocyanide. Since sugar beet molasses is high in nitrogen, supplements of this element were not necessary. About 10% of the total nitrogen is utilized during the fermentation and the remainder probably represents nitrogen in a form less readily available t o the organism. I n the production of citric acid by the submerged culture method there are many factors difficult t o control. However, rigid experimental control usually ensures reasonable duplication from run to run. Major differences in yield within a given run are usually caused by uneven seeding, failure t o pool spore BUSpensions or seed cultures where two or more of each are used, variation in rates of aeration or oxygenation, or cracked aeration disks. Although the fermenters were run open little difficulty waR experienced with contamination. The shake flask data mentioned in (6) suggests that beet niolasses from sources other than Chatham can be fermented; however, it has not been actually demonstrated that they can be fermented by the special technique outlined in this paper.
LO-:*;;;rU
I947 x
x-
CHATHAM
1949
W
P 0 4
a
I-
A-h-CHATHAM
1946
A
A
60
Figure
BO TIME, H O U R S
IO0
6. comparison of Molasses by Fermentation Yields Average of two lermentationr
!a
September 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
It is hoped that within the next year it will be possible t o undertake pilot plant trials of this process. Meanwhile work on the fermentation method and also on some of the more fundamental aspects of the problem oi citric acid formation are being continued in the laboratory. Acknowledgment
The authors wish t o acknowledge the technical assistance of Joseph Langevin and James Slobodian. literature Cited (1) Can. Chem. Process I d s . , 35, 266 (1951). (2) Chem. Eng. News, 28, 3982 (Nov. 13, 1950). (3) Chem. Inds., 68, 20 (1951).
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(4) Kolmer, J. A,, and Boerner, E., “Approved Laboratory Technic,” New York,Appleton-Century Co., 1945. (5) Moyer, A. J., Proc. SOC. Am. Bact., 51st Gen. Meeting, 1951. (6) National Research Council, Can., Div. Applied Biology, Quart. Rept., 3, No. 2 (1951). (7) Perlman, D., Econ. Botany, 3, 369 (1949). (8) Perquin, L. H. C., thesis, Delft, 1938. (9) Saffran, M., and Denstedt, 0. F., J . Bhl. Chhem., 175, 849 (1948). (IO) Schweiger, L. B., and Snell, R. L. (to Miles Laboratories, Inc.), U. S. Patent 2,476,159 (July 12, 1949). (11) Shu, P., and Johnson, M. J., IND.ENG.CHEM.,40, 1202 (1948). (12) Snell, R. L,and Schweiger, L. B. (to Miles Laboratories, Inc.), U.8. Patent 2,492,667 (Dee. 27, 1949). (13) Woodward, J. C,, Snell, R. L., and Nickols, R. S., Ibid., 2,492,673 (Dec. 27, 1949). RQCEIVEDfor review November 19, 1951. ACCEPTED April 24, 1952. Issued as Paper 139 on Usee of Plant Products and as N.R.C. 2774.
fngFnyring
Precision liquid Feeder
Ppocess development I
B. W. JONES, S. A. JONES, AND M. E. NEUWORTH Pittsburgh Consolidation Coal Co , Library, Pa.
F
E E D I N G of high melting organic compounds and viscous with a properly dimensioned cylinder and grooved piston, the 0 tarry fractions at low nonpulsating reproducible rates was an ring is squeezed, resulting in a hydraulic seal. 0 ring seals can essential requirement for quantitative investigation of specific be ueed for pressures up t o 3000 pounds per square inch. Howorganic reactions in this laboratory. In addition, continuous ever, operating pressures approaching this limit would require subfeeding of these materials was required in amounts up t o 500 stituting a steel cylinder. Steam jacketing of the cylinder permits grams against variable pressures and with a maximum error of the feeding of compounds which melt below 100’ C. Modi1%. A variety of liquid feeders has been fying the method of heating or substitutdescribed recently by Lundsted et al. (1) ing a different heating fluid would permit and Michaeli (9). Several are also availhandling - matorids having a much higher able commercially. However, none of the rqelting point. Feed rates from 0.8 to 13 PLUNGER a designs described would meet all these rem1. per minute can be obtained with the ’O-RING quirements. present design. The design of a feeder based on a motorDetails of the feeder are shown in Figdriven syringe was undertaken. A syringe ure 1. Feeding is effected by pushing the feeder is particularly well suited for the plunger through the cylinder a t a condelivery of a completely nonpulsating flow stant rate. The cylinder i? large enough to hold the entire charge t o be fed. of liquid independent of back pressure It is fabricated from precision-bore glass variations. Fabrication of a conventubing with an inside diameter tolertional syringe of the desired capacity from ance of *0.0002 inch. This tubing can be obtained from Fischer & Porter Co., either metal or glass was considered imHatboro, Pa. The connection between practical. Tolerances between the plunger the cylinder and a metal reaction sysCLUTCH and the syringe barrel cannot be made tem is made by means of a glass t o close enough t o assure a liquid seal where metal ball and socket joint or glass t o metal pipe connection. The plunger any appreciable back pressure is encounhas 8 groove in its periphery which tered. Development of a successful design carries an 0 ring gasket, assuring a liqwas accomplished by utilizing an “0” uid t,ight seal with the cylinder walls. It is equipped with a small drain cock ring-sealed metal piston in a precisionwhich permits drainage of the cylinder bore glass cylinder. An 0 ring is a round without disassembling the unit. This is cross section, perfectly circular rubber ring convenient for cleaning the feeder, particularlv when the cvlinder contains cormolded to extremelv close tolerances. It is rosive *chemicals. -b rings are availgenerally fitted into a rectangular, maFigure 1. Schematic Diagram able in a sufficient variety of matechined groove. When used in conjunction of Feeder rials t o handle most liquids. The authors
