September 19s
INDUSTRIAL AND ENGINEERING CHEMISTRY
grant and also for the interest shown in this work by R. D. Muir, W.L. Gaby, and A. B. Hatch of that company. W. C. Frazier, S. G. Knight, D. Perlman, Dorothy M. Powelson, and P. A. Tetrault made helpful suggestions during the preparation of this manuscript; these were very much appreciated. The authors wish to thank M. P. Backus who furnished the Wisconsin penicillin strains used in thii work; J. H. Noyea of the Schenley Laboratories for his cooperation and gift of crystalline penicillin; A. F. Langlykke of E. R. Squibb and Company for bringing several patents to our attention; and the manufacturers who furnished the surface-active agents and product information. LITERATURE CITED
(1) Behrena, 0.K., in “The Chemistry of Penicillin,” hnceton, N. J., Princeton University Preee, 1948. (2) Benham, R. W., Pro& SOC.Ex&. Biol. Med.,46,176-8 (1941). (3) Cohen, S.,Snyder, J. C., and Mueller, J. H., J . Bact., 41,581-91 (1941). (4) Dubos, R.J., Am. Sciatist, 37,353-70 (1949). (5)Dubos, R. J., J . Ezptl. Med., 85,9-22 (1947). (6)Feeney, R. E., Mueller, J. H., and Miller, P. A., J . Boct., 46, 559-62 (1943). (7)Ibid., pp. 563-71. (8) Fieser, L.,and Fieser, M., “Organic Chemistry,” pp. 390, 398, Boston, Maea., D. C. Heath and Co., 1944. (9) Foster, J. W., and Woodruff, H. B., J . Bat., 47,43-58 (1844).
1823
(10)Gaby, W. L.,and Sawmiller, L. F., unpublished results. (11) Goldschmidt, E.P.,and Koffler, H., unpublished results. (12) Holtman, D. F.,J . B a t . , 49,313-14 (1945). (13) Hutchings, B. L.,and Boggiano, E., J. Biol. Chem., 169,229-30 (1947). (14) Kluener, R. G., J . Bact., 57,101-9 (1949). (15) Koffler, H.,and Cohen, M., Proc. Meet. Soc. Am. Bact., 1, 45 (1948). (16) Koffler, H., and Goldschmidt, M. C., A n . J . Bot., 36,811 (1949). (17)b i n , J., and Lein, P.,J . Bact., 58,595-9(1949). (18) Liggett, R. W., and Koffler, H., Bmt. Revs., 12,297-311 (1948). (19) Phelps, A.S.,U. 8.Patent 2,473,818(1949). (20) Pollock, M.R., Howard, G. A., and Boughton, B. W., Biochem. J., 45,417-22 (1949). (21) Rudert, F. J., U.S. Patent2,374,503(1945). (22) Schmidt, W. H., and Moyer, A. J., J . Bact., 47, 199-209 (1944). (23) Shull, G. M.. Thoma, R. W., and Peterson, W. H., Arch. Biochem., 20, 227-41 (1949). (24) Singh, K., and Johnson, M. J., J . Bact., 56,339-55 (1948). and Koffler. H.. Science. 109.496-6 11949). (25) Starks. 0.. Stefa&k,’J. J., Gailey. F.‘B., Jar&, F. G., and Jo&son, M. J., J . Bact., 52,119-27 (1946). Tanner, F. W., Wickerham, L. J., and Van h e n , J. M.. U.S. Patent 2,445,128(1948). Tomarelli, R. M., Norris, R. F., Gyargy, P., Hassinen, J. B., and Bernhart, F.W., J . Biol. Chem., 181,87948(1949). Williams, V. R., and Fieger, E. A., Ibid., 166,335-43(1946). Williams, W. L., Broquist. H. P.. and Snell. E. E.. Ibid.. 170. . . 619-30 (1947). RECEIVED April 8, 1950.
Penicillin Fermentations in a Waldhof -Type Fermentor W. E. BROW” AND W. H. PETERSON University of Wisconsin, Madison, Wis.
A laboratory Waldhof-type fermentor, holding 30 liters, suitable for use in penicillin fermentation is described. When operated in the conventional manneri.e., without antifoam-low fermentor capacity (9.5 liters) and limited production of penicillin (600 units per ml.) resulted. When antifoam agents-e.g., Alkaterge C-were used, the volume of medium in the fermentor could be increased to 17 liters and the yield of penicillin was raised to over 2000 units per ml. These broths assayed approximately 97% penicillin G. In the standard type of fermentor, utilization of lactose is often irregular and leads to unsuitable pH condition-Le., above 8.0. In Waldhof fermentations rapid and uniform utilization of sugar and lower and more constant pH values (ca. 7.0) were obtainable. These results are probably related to better aeration of medium with Waldhof fermentor.
A
MAJOR problem in deep tank fermentations, such as those of yeast and penicillin, is to provide sufficientair for growth and functioning of the submerged cells and a t the same time to cope with the foam that inevitably accompanies aeration and agitation. Many antifoam materials and typea of fermentors have been developed to eliminate or minimhe the foam problem. Perhaps the most succeasful fermentor for this purpose is that known as the Waldhof, which was developed at Waldhof-Mannheim, Germany, for use in the production of yeast (9). Thia fermentor is so constructed that the agitator-aerator forces the liquid a t the bottom of the tank outward and upward and simultaneously aerates it. The liquid, together with foam generated by aeration and agitation, returns to the bottom of the fermentor 8 Praent
address. Merok & Co. Ltd., Vallefield, Que., Canada.
through a central cylinder or draft tube which prevents the accumulation of nonbreaking foam on the top of the liquid. As the foam passes through the draft tube and out through the agitator it is subjected to a beating action that destroys more or 1- of it. Saeman (8)has described an open laboratory Wddhof fermentor which has been successfully applied to the continuous production of fodder yeast from wood sugar (6). Two features of t h u fermentor, efficiency of aeration and a p parent freedom from need for antifoam materials, made it desirable to determine its applicability to penicillin fermentation. A modified Waldhof-type fermenter was constructed which differed somewhat from the preceding designs in the method of aeration and agitation, but more particularly in being a closed container that can be operated under aseptic conditions. FERMENTATION EQUIPMENT
A drawing of the fermentor ia given in Figure 1. Because it is essentially a modification of the standard 3O.liter fermentor (8,7), only the internal parts peculiar t o the Waldhof type are detailed. All parts within the fermentor are made of stainless steel. Draft tube A, 6 inches in diameter and 10 inches in length, is suspended from the top of the fermentor by four supports, B. The lower edge of the draft tube has a 1-inch bevel. Agitator plate C, 6 inches in diameter, is mounted on the lower end of shaft D immediately below the draft tube. On the upper side of the agitator plate are four curved blades, E, 0.5 X 4 inches, which extend 1.25 inches beyond the edge of the plate. Sparger ring F, 7.25 inches in diameter, has six ‘/winch holes. The shaft is rotated clockwise, so that medium is withdrawn from the bottom of the draft tube. The medium, which is mixed thoroughly with air by the action of the draft tube, is forced upward and ultimately spills
INDUSTRIAL AND ENGINEERING CHEMISTRY
1824
over thc top of the draft tube, through which it is drawn down to the &,ator, thus completing the cycle. A small baffle plate, G, 2.5 X 3 inches, is mounted on one of the supports of the draft tube. The lower edge of the baffle plate and the upper edge of the draft tube are on the same level. This bafFle prevents coning of the liquid up the walls of the container by spilling the foam and liquid into the draft tube. Once good mycelial growth is obtained, no coning of the liquid occurs. The electrode, H , used for foam detection, is placed so that the tip is 1 inch above the upper rim of the draft tube. When foam comes in contact with this electrode, an automatic device ( 2 )adds antifoam to the fermentor.
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was :tttributed to the very efficient aeratioii of the medium, the standard fermentors were operated under modified conditions that were believed to be optimal-viz., 16 liters of medium, aeration at 0.73 liter per liter per minute, and agitation a t 450 r.p.m. To increase the dispersion of air throughout the medium, two of the four agitator blades were set vertically, while the other two blades were set at an angle of 30" to the horizontal. Under these conditions agitation of the medium was so violent that agitation rates higher than 450 r.p.m. could not be used. Samples for analyses were removed periodically through the sampling tube by air pressure. The methods of storage ( 3 ) and analysis ( 2 ) are described elsewhere. Two methods of operation of the Waldhof fermentor were investigated. I n the first procedure, no antifoam was added during the fermentation, as is the practice in operating the Waldhof type of fermentor in yeast production (8, 9). It was found necessary, however, to add 5 ml. of the antifoam mixture to the medium before sterilization to prevent the formation of the initial light foam over which the fermentor had no control. Because low penicillin yields were obtained when the fermentor was operated in this manner, a second procedure was tried. This method involved the addition of antifoam to the medium whenever it was needed to prevent the formation of excess foam. Twenty milliliters of antifoam were added to each fermentor before sterilization. On the average 150 to 200 ml. of antifoam were necessary to control foam production in 120 hours' fermentation time. In both types of fermentation the decrease in volume of medium by evaporation was less than 5%, providing the air was humidified before it entered the fermentor. EXPERIMENTAL DATA
-I-I - h I
12"
d Tap View of Agitator Plate
&!--
OPERATIONWITH No ADDEDANTIFOAM.Because no attempt was made to control foam production during the course of the fermentation, emulsification of the medium began 15 to 20 hours after inoculation, and within the next 5 to 10 hours reached an equilibrated state. One serious drawback to the ferment,or was immediately observed. Only 9.5 liters of medium and inoculum could be used in the fermentor under such conditions. This volume was critical, because a greater volume foamed out whereas a smaller volume was insufficient for adequate circulation.
6"
Figure 1. Laboratory Waldhof-Type Fermentor A.
Draft tuhe
B . Support C. Agitator plate
D. Shaft
E. F. G. If.
Agitator blades Sparger Baffleplate Foam-detecting electrode
FERMENTATION METHODS
Thc mold culture, Penicillium chyysogenumQ-176 ( I ) , was used in 311 the experiments. The mediums used are given in connection with each table or figure. Unless other conditions are indicated, the fermentations were agitated a t 540 r.p.m. and aerated a t 0.46 liter of air per liter of medium per minute (l./l./min.). Phenylacetic acid was added as the sodium salt at the rate of 0.05% every 12 hours starting at the 12th hour of fermentation (2, IO). The fermentors were autoclaved at 15 pounds' steam pressure for 90 minutes and then cooled immediately, under air pressure, in a water bath. Five per cent vegetative inoculum was grown and added to each fermentor (2'). Lard oil containing 3% Alkaterge C (Commercial Solvents Corporation, Terre Haute, Ind.) was used as the antifoam agent. The incubation temperature was 26' to 28' C. The standard glass fermentor (2)was used for comparison with the Waldhof type. Because the success of the Waldhof fermentor
0
20
40
60 80 TIME, HOURS
100
Figure 2. Progressive Chemical Changes in Fermentation without Addition of Antifoam Cerelose 1 %, lactose 2.596, corn steep solids 3 %, sodium sulfate 0.1 %, total phenylacetic acid 0.35%. Fermentation volume 9.5 liters. Aeration 0.62 L/l./min., agitation 540 r.p.rn.
Figure 2 shows the chemical changes of one of the better fermentations obtained witshout the addition of antifoam. Approximately 600 units of penicillin were obtained in 72 hours. The low initial pH, 4.5, was necessary in order to ensure pH suited to penicillin production in the later stages of the fermentation. For
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INDUSTRIAL AND ENGINEERING CHEMISTRY
this reaaon no calcium carbonate could be used in the medium. Particularly significant was the complete absence of the period of lactose adaptation that is frequently encountered in the usual method of fermentation ( 8 , 4, 6). Despite attempts to increase yields by lengthening the time of fermentation, penicillin production could not be extended beyond 72 hours. Invariably, autolysis of the mycelium set in at this time, even though nutrients were not lacking and the pH of the medium was satisfactory for penicillin production. Possibly this can be explained on the basis that the mold received an inadequate supply of air in the later stages of fermentation, because of the stable emulsion that was formed.
TIME, HOURS
Figure 3. Progressive Chemical Changes in Fermentation with Addition of Antifoam Cerelose l%,lactose 2.5%, corn stee solids 4 % calcium carbonate 0.5%, sodium sulfate 0.1% total phenylacetic acid 0.35%. Lard oil containing 6 % Alkaterge C, 200 ml. Fermentation volume 17 liters. Aeration 0.36 l./l./min., agitation 540 r.p.m.
OPERATIONWITH ADD~TIONOF ANTIFOAM. The first sdvantage accruing from operation of the fermentor with added antifoam was the possibility of using a greater volume of medium. Approximately 17 liters were required to fill the fermentor to a point a t which.proper circulation of the medium was obtained. Because the electrode which initiated the addition of antifoam was only 1 inch above the upper lip of the draft tube, very little foam was allowed to accumulate. Consequently emulsification of the medium was impossible. The following fermentations were, therefore, run with 16 liters of medium plus added inoculum. EFFECTOF AERATIONRAT&. Two rates of aeration, 0.09 and 0.45 liter per liter per minute, were compared. The medium wed in this experiment contained 1.0% cerelose, 4.5% lactose, 4.0% corn steep solids, 0.1% sodium sulfate, 0.2% calcium carbonate, and 0.3% phenylacetic acid. No significant differences were noted between fermentations conducted under either condition. At the lower rate of aeration 952 units of penicillin per ml. were produced in 80 hours. The mycelium present at 50 hours, measured as grams per liter of mycelial nitrogen, was 1.63 grams per liter. Corresponding values obtained at the higher level of aeration were 940 units of penicillin per ml. and 1.51 grams per liter of mycelial nitrogen. EFFECT OF COMPOSITION OF MEDIUM. The effect of the composition of the medium is summarized in Table I. Maximum yields of 1400 to 1500 units of penicillin per ml. were obtained when 4.0% corn steep solids and 4.5% lactose were present in the medium. Only 929 units per ml. were obtained when the corn steep solids content was 2.0%. With 2.5% lactose in the medium 1075 units per ml. were produced in 95 hours. Supplementation of an initial content of 2.5% lactose with an additional 2.0% lactose at 60 hours resulted in a faster fermentation than did an initial concentration of 4.5% lactose with no subsequent additions. No difference in penicillin yields was noted with the two treatments. The effect of different concentrations of calcium carbonate on the fermentation is also shown in Table I. With 0.1% calcium carbonate in the medium the initial pH of the fermentation wm
1825
4.7 and the average pH after 30 hours was only 6.3. Because of this low pH only 1071 units of penicillin per ml. were obtained. When the calcium carbonate content was increased to 0.3 and 0.5%the initial pH values were 5.7 and 5.9, and the corresponding average pH figures at 30 hours were 6.9 and 7.0. At the two higher levels of calcium carbonate, approximately 1500 units of penicillin per ml. were obtained in 95 hours. It has, however, been observed repeatedly that the pH plateau of the fermentation cannot be reliably regulated by the initial pH of the medium. Undoubtedly one of the main variables concerned is the effect of differences in the amounts and times of addition of antifoam. For this reason mediums were subsequently prepared with 0.2 to 0.3% calcium carbonate. After the customary p H rise the fermentations were adjusted to approximately pH 7.0 by the addition of either 2 N sulfuric acid or 2 N sodium hydroxide. EFFECT OB ANTIFOAM.The effect of three antifoam materials on penicillin production was studied with medium containing 1.0% cerelose, 4.5% lactose, 4.0%corn steep solids, 0.3% calcium carbonate, 0.1% sodium sulfate, and 0.5% phenylacetic acid. Triplicate fermentations averaged 2020 units of penicillin per ml. in 125 hours when lard oil containing 6% Alkaterge C was used as the antifoam agent. This yield of penicillin i s compared to approximately 1650 units per ml. in 125 hours, when either lard oil alone or lard oil containing 2% octadecanol was used. Results of duplicate fermentations were averaged for both of these fermentations. The stimulation of penicillin production was not noted when lard oil containing 3% Alkaterge C was the antifoam agent. The average quantity of each antifoam used in 125 hours was 415 ml. of lard oil, 280 ml. of lard oil containing 2% octadecanol, and 245 ml. of lard oil containing 6% Alkaterge C. CHEMICAL CHANOES IN MEDIUMS.The chemical changes in the medium of a typical fermentation are shown in Figure 3. Because the sugar content of the medium was only 1.0% cerelose and 2.5% lactose, the fermentation time was too short for maximum yields of penicillin. Nevertheless, over 1500 units of penicillin per ml. were obtained in 95 hours. Characteristic of fermentations run in the Waldhof fermentor is the rapid utilization of sugar. No slackening in the rate of sugar utilization was noticeable when the cerelose was gme and lactose was the only carbohydrate source remaining in the medium. At 60 hours the sugar in the medium was approaching a rather low concentration. Meanwhile the pH of the medium started to rise and the rate of penicillin formation decreased. By 95 hours the pH of the medium was above 8.0 and penicillin production ceased. Accompanying the autolysis of mycelium was an accumulation of ammonia nitrogen in the medium. Figure 4 shows the chemical changes of a similar fermentation in which lactose was not the limiting factor. In addition to the initial concentration of 1.0% cerelose and 2.5% lactose in the
OB COMPOSITION OF MEDIUM ON TABLE I. EFFECT FERMENTATION^
Component Varied Corn steep solids Lactose Calcium carbonateC
Component,
% 2.0 4.0
2.5 4.5c 4.5 0.1
0.3
Averawe pHC
Maximum Penicillin, Unita/Ml.
Time to Max Penicillin, Hours
6.8 7.0
929 1465
79 95
7.0 6.9 7.1
1075 1449 1508
95 108 123
6.3
1071 1481
110 95
6.9
7.0 1450 95 Basic medium contained cerelose l.O%, lactose 4.5%, corn stoep 4.0%* 0.5
0
sodium sulfate 0.1?&, and calrium Carbonate 0.5%. Phenylacetic acid WRI added as sodium salt a t rate of 0.05% every 12 hours starting at 12th hour. All fermentations adjusted to pH 7.0 a t 30 hours, except those in which calcium carbonate i _ x varied ~ ~ . b Average p€I of fermentation from 30 hours on. In other feriuentaC 2.5% lactose added initially and 2.0% a t 60 hours. tions total lactose added initially.
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1826
TIME, HOURS
Figure 4. Progressive Chemical Changes in Fermentation with Additions of Antifoam and Lactose Cerelose 1%, lactose 7.75% corn steep solids 4 % calcium carbonate 0.570, sodium sulfate 0.17, total phenylacetic acid 0.6%. Lard oil containing 6 % Alkatergc C, 370 ml. Fermentation volume 17 liters. Aeration 0.36 l./l./min., agitation 540 r.p.m.
medium, two additions of 2.0% lactose were made a t 56 and 100 hours, and one addition of 1.2570lactose at 132 hours. Inasmuch as the lactose content of the medium was no longer limiting, over 2100 units of penicillin per ml. were produced in 100 hours. Then, despite the presence of available carbohydrate, a favorable pH, and heavy mycelial growth, the accumulation of penicillin ceased abruptly. I t is doubtful that the organism is incapable of producing more penicillin. It seems probable that penicillin production certsed either because of some deficiency in the medium or the accumulation of an inhibitory metabolic product in the later stages of the fermentation. It may be significant that ammonia nitrogen, which was present in only small amounts during the period of active penicillin production, began to accumulate in the medium a t approximately the same time the maximum penicillin titer was reached.
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tained in a fermentation for over 70 hours. The neutral pH of the broth was undoubtedly the major factor responsible. VARIATIONAMONG HIGH-YIELDING FERMENTATIONS, The results of seven fermentations in three different experiments under optimal conditions are shown in Table 11. The yields of penicillin at 120 hours varied from 1975 to 2195 units per ml. Two broths with activities of 2020 and 1900 units of penicillin per ml. a t the time of w a y contained 98.9 and 96.7% penicillin G, respectively. No significant difference in yields was noted whether the glass or stainless steel fermentors were used. The 4.5% lactose in the medium was sufficient to give a fermentation time of at least 130 hours. This rate of lactose consumption was made possible by reducing the incubation temperature to 25' C. AI1 the sugar in the medium was added before sterilization. The fermentations were usually adjusted to pH 7.0 with either acid or alkali, after the characteristic pH rise. Subsequent control of the pH was not necessary. The steady utilization of lactose apparently assured a constant pH. Sufficient lactose was present to prevent autolysis until maximum penicillin production was reached. The Waldhof fermentation was also compared with that in the standard fermentor. These fermentors contained 16 liters of medium of the same composition as that used in the Waldhof fermentor, with the exception that 3.0% lactose was used. Two such fermentations yielded approximately 1900 units of penicillin per ml. in 120hours. The yields were slightly lower than those obtained in the Waldhof fermentor, but the difference may not be significant. The fermentors were modified as indicated to obtain more effective dispersion of the air. ACKNOWLEDGMENT
The authors wish to acknowledge their indebtedness to Margaret Larson for the penicillin assays and to Sanford Anderson for construction of the equipment. The research was aided by grants from Commercial Solvents Corporation and Heyden Chemical Corporation. LITERATURE CITED
TABLE11. VARIABILITY AMONG HIGH-YIELDING FERMENTATIONS Penicillin, Units /M1. Antifoam Fermentor Type 90 hours 120 hours Added, M1. 1770 160 Glass Waldhof" 1980 1730 335 Glass' Waldhof' 1380 2000 150 Glass' Waldhof' 2195 1530 160 Glass' Waldhof" 2175 1560 80 Glass' Waldhof" 2130 1550 90 Steel ' Waldhof" 1975 120 1400 Steel: Waldhofa 1960 110 1390 Glass, standardb 1870 150 1560 Glass, standard b 5 Medium in Waldhof fermentors. Cerelose l.O%, lactose 4.59 corn steep solids 4.0%, calcium carbonate 0.25%, sodium sulfate O.l$,' total phenytacetic acid, 0.45%. Initial PH 5.5: average p H from 30 hours on Expt.
7.0.
b Medium in standard fermentors, same as above except 3.0% lactose (instead of 4.5%) used.
(1)Backus, M.P., Stauffer, J. F., and Johnson, M. J., J.Am. Chem. Soc., 68, 152 (1946). (2)Brown, W.E.,and Peterson, W. H., IND.ENQ.CHEM., 42, 1769 (1950).
(3)Gailey,-F. B.,Stefaniak, J. J., Olson, B. H., and Johnson, M. J., J. Bact., 52, 129 (1946). (4) Gordon, J. J., et al., J. Gen. Miwobiot., 1, 187 (1947). (5)Harris, E. E.,Hannan, M. L., and Marquardt, R. R., I N r . ENG. CREM.,40, 2068 (1948). (6)Jarvis, F. G., and Johnson, M. J., J. Am. Chem. SOC..69, 3010 (1947). (7) Rivett, R.W., Johnson, M. J., and Peterson, W. H., IND.ENG. CHEM.,42, 188 (1950). (8) Saeman, J. F., Anal. Chem., 19, 913 (1947). (9)Saeman, J. F., Locke, E. G., and Dickerman, G. K., Office of Technical Services, U.S. Dept. Commerce, FIAT Final Rept. 499 (1945). (10) Singh, K.,and Johnson, M. J., J. Bact., 56, 339 (1948). RECEIVED July 18,1949. Published with the approval of the director of the Wisconsin Agricultural Experiment Station.
The chemical changes indicated in Figure 4 demonstrate some interesting points regarding the penicillin fermentation. It has been emphasized that fermentations conducted in the Waldhof fermentor are characterized by the rapid adaptation of the mold to lactose in the medium. This characteristic ensures constancy in the pH of the fermentation. As long as lactose was used at a rapid rate in the fermentation no serious change in pII was encountered. Consequently pH control, such as has been found desirable in the standard type of fermentor (W), was not necessary. The only pH adjustment required in these high-yielding fernientations was the adjustment of the pH plateau to a level at or near 7.0 after the normal pH rise. The experiment further demonstrated that the period of high penicillin production may be main-
