(Gailey et al., 1946) and deterioration of the steep liquor. Penicillin yields and

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THE EFFECT OF ENVIRONMENTAL CONDITIONS ON PENICILLIN FERMENTATIONS WITH PENICILLIUM CHRYSOGENUM X-16121 2 J.

1 THE EFFECT OF ENVIRONMENTAL CONDITIONS ON PENICILLIN FERMENTATIONS WITH PENICILLIUM CHRYSOGENUM X J. J. STEFANIAK, F. B. GAILEY, F. G. JARVIS, AND M. J. JOHNSON Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison, Wisconsin Received for publication April 11, 1946 When penicillin is produced by submerged fermentation in tanks, many factors influence the course of the fermentation and the penicillin yield. Some of these factors are discussed in other reports from this institution. Tank design and the effect of variations in aeration are described by Stefaniak et al. (1946). Gailey et at. (1946) report results obtained with various cultures. Koffler et al. (1945) give data on the metabolic changes occurring in the medium during fermentation. Other studies include the effect of bacteriostatic agents (Knight and Frazier, 1945) and the role of corn steep liquor in penicillin fermentations (Bowden and Peterson, 1946). In the present paper the effects of variation in temperature, pressure, and carbon dioxide tension are reported. Data on the toxicity of various antifoam agents are also given. A comparison of glucose and lactose as carbohydrate sources in tank fermentations is made. The experimental work was performed during a period when numerous variations in penicillin yields were obtained because of the instability of the culture (Gailey et al., 1946) and deterioration of the steep liquor. Penicillin yields and chemical changes in the medium varied considerably during the course of the experiments. EXPERIMENTAL METHODS The culture used was Penicillium chrysogenum X-1612 (Carnegie Institution)- The experimental conditions described by Stefaniak and co-workers (1946) were followed throughout these experiments. The methods for the determination of sugar, ammonia nitrogen, organic nitrogen (soluble nonammonia nitrogen), mycelial nitrogen, and are reported by Gailey et al. (1946). The carbon dioxide content of the exhaust air in penicillin fermentations was determined in the following manner: Three to five cu ft of exhaust air, depending on the carbon dioxide concentration, were bubbled through 20 ml of 1 N sulfuric acid in a 35-by-200-mm pyrex test tube (to remove ammonia), then through 20 ml of 1 N sodium hydroxide solution in a 22-by-760-mm absorption column, and finally through a wet test meter. After the gas sample had been passed through the apparatus the column was washed free of sodium hydroxide solution with 1 This work was done in co-operation with and supported by the Office of Production Research and Development, WPB contract no Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 119

2 120 STEFANIAK, GAILEY, JARVIS, AND JOHNSON [VOL. 52 carbon-dioxide-free water. The resulting solution, after the addition of 10 ml saturated BaCI2 solution, was titrated to the phenolphthalein end point with standard hydrochloric acid. Blanks were determined in the manner described above. In this case a quantity of air from the service line equal to that of the gas smple was used instead of the exhaust air. The difference between the CO2, VOLS. PER MIN. PER PE.NICILLIN UNITS/mt. 10,000 VOLS. CULTURE NH3-N, mg PER L. SUGAR, g. PER L. MYCELIAL-N,mg/lOOrnt C0z AV. 25 to 60 HRS _ 70 - / / / / 600 /t SX "9 40 t -- - = 0 20 \: 200 PE. AT PEN. MAX FIG. 1. 2o FERMENTATION TEMPERATURE, DEG. C. CoMPARIsON OF FERMENTATIONS AT VARious TzMPERATuREs blank titration and the sample titration represented carbon dioxide produced by the culture. EXPERIMENTAL RESULTS Influence of temperature on penicillin fermentaton8. In order to determine the effect of temperature on chemical changes within the medium and on penicillin production, tank fermentations were run at 20 C, 23 C, 26 C, 29 C, and 32 C.

3 1946] ENVIRONMENTAL FACTORS IN PENICILLIN FERMENTATIONS 121 These experiments were performed during a period when considerable difficulty was encountered with culture variation and when penicillin yields of only 250 to 350 units per ml were being obtained. The composition of the medium used was as follows: lactose 3 per cent, steep liquor solids 4 per cent, and calcium carbonate 1 per cent. Figure 1 summarizes the data for these experiments. The values given represent 2 fermentations at 20 C, 3 fermentations at 26 C, and 4 fermentations at each of the other temperatures. When the temperatures of penicillin fermentations were raised, the rate of sugar utilization and the rate coz PENICILLIN NH3-N MYCELIAL-N SUGAR ORGANIC-N a, EtH z~~~~~~oo MYC.-N NH-N HOURS FIG. 2. PENICILLIN FERMENTATIONS AT 2-LB PRESSURE PER SQ INCH (Values are averages from 4 fermentations.) CO2, vols. per minute per 1,000 vols. culture; NHs-N, mg per 10 ml; sugar, grams per 100 ml; penicillin, units per ml; mycelial-n, mg per 100 ml; organic-n, mg per 100 ml. of carbon dioxide production increased. It is apparent that the culture utilized the available nutrients more rapidly at the higher temperatures. The ammonia nitrogen maximum, which usually occurs at 18 to 24 hours, was not increased appreciably until the incubation temperature was raised to 32 C. The high ammonia maximum at this temperature was correlated with the fact (not shown in figure 1) that, although rapid oxidation of steep liquor nitrogen compounds occurred, growth during the initial period was slow; therefore less ammonia was assimilated. When the fermentations were conducted at temperatures above 26 C, autolysis occurred before the fermentations were terminated because of the exhaustion of the available nutrients. The and the ammonia content of the

4 122 STEFANIAK, GAILEY, JARVIS, AND JOHNSON [V>OL. 52 medium rose very rapidly when autolysis occurred. Penicillin yields were not appreciably affected at temperatures below 32 C. The maximum yield at this temperature was considerably below the maximum at any other temperature. With the exception of the 32 C fermentations, the penicillin yields at 50 hours were similar. The amount of penicillin produced after 50 hours in fermentations conducted at 29 C and 32 C was very small. Effect of pressure on the fermentation. A series of experiments in which the internal pressure of the tanks was maintained at 2,20, and 40 lb per sq inch gage co? PENICILLIN NH3 -N MYCELIAL-N SUGAR ORGANIC-N c02 8 /., PEN MYC.-.N 200 I ~~~ORG -N 2 SUG HOURS FIG. 3. PENICILLIN FERMNTATiONS AT 20-LB PRESSURE PER SQ INCH (Values are averages from 3 fermentations.) CO2, vols. per minute per 1,000 vols. culture; NHs-N, mg per 10 ml; sugar, grams per 100 ml; penicillin, units per ml; mycelial-n, mg per 100 ml; organic-n, mg per 100 ml. pressure were performed. The data are summarized in figures 2, 3, and 4. The medium used in these experiments contained 4 per cent lactose, 4 per cent steep liquor solids, and 1 per cent calcium carbonate, except in one 20-lb run in which 3 per cent lactose was used. The rate of metabolism (rate of carbon dioxide production) was higher at the 20- and 40-lb pressures; nevertheless the sugar utilization was about the same in all cases. The plateau was lowered at the higher pressures. The observed change with pressure approximates that calculated from the increase in partial pressure of carbon dioxide at the higher tank pressures. The ammonia maximum was depressed with increasing pressure. At 40-lb pressure the amount of ammonia nitrogen present after

5 1946] ENVIRONMENTAL FACTORS IN PENICILLIN FERMENTATIONS hours was very low and may have limited penicillin formation. Somewhat heavier growth occurred at the higher pressures. The highest penicillin yields were obtained at 20-lb pressure, although a yield in excess of 400 units per ml has been obtained at 2-lb pressure. The penicillin yields at 40-lb pressure were consistently low. In summary, it may be concluded that pressure has but little effect on penicillin production, except that at high pressures the slow rise decreases the COz PENICILLIN NH53 -N MYCELIAL-N SUGAR ORGANIC-N 10, CO? // W ' HOURS FIG. 4. PENICILLIN FERMENTATIONS AT 40-LB PRESSURE PER SQ INCH (Values are averages from 4 fermentations.) C02, vols. per minute per 1,000 vols. culture; NH3-N, mg per 10 ml; sugar, grams per 100 ml; penicillin, units per ml; mycelial-n, mg per 100 ml; organic-n, mg per 100 ml. rate of penicillin formation during the early stages of the fermentation, andithe low ammonia levels present at the later stages may limit penicillin formation. Effect of carbon dioxide tension on the fermentation. In industrial penicillin fermentations in large tanks the carbon dioxide content of the medium and the exhaust air are usually higher than in our tanks. This occurs because at low aeration rates the efficiency of air utilization is higher (Stefaniak et al., 1946, figure 10), and because the greater depth of medium results in better air utilization. High carbon dioxide tensions also occurred in the medium in our high-pressure fermentations. In order to determine whether high carbon dioxide levels had any effect on the fermentations other than a lowering of, fermentations were set up in which the air used for aeration contained 2 per cent

6 124 STEFANIAK, GAILEY, JARVIS, AND JOHNSON carbon dioxide. This resulted in a carbon dioxide content of from 2.5 per cent to 3.0 per cent in the exhaust air. The penicillin yield, the rate of carbon dioxide production, and the chemical changes in these fermentations (except for a somewhat lower level) were similar to those of a typical fermentation (figure 3). It is apparent that increased carbon dioxide tension in the medium does not affect penicillin fermentations. CO2 NH3-N SUGAR I ~1z -I I I \ PENICILLIN MYCELIAL-N ORGANIC-N \CO. MYCI-N 4 < PE / % 'ORG-N NHY-N- 'SUGAR HOURS FIG. 5. A PENICILLIN FERMENTATION WITH GLUCOSE AS THE CARBOHYDRATE SOUaCii IN THE MEDIUM* CO2, lr. per minute per 1,000 vols. culture; NHs-N, mg per 10 ml sugar, grams per 100 ml; penicillin, units per ml; mycelial-n, mg per 100 ml; organic-n, mg per 100 ml. 100 [VOL. 52 Glucose as a carbohydrate source. Several fermentations were performed to determine whether good penicillin yields could be obtained when glucose is used in the fermentation medium as a carbohydrate source. Figure 5 summarzes the data for one of these experiments. The rate of carbon dioxide production was high during the initial 26 hours of the fermentation and then decreased very * The constituents of the medium were as follows: glucose 40 grams, steep liquor solids 40 grams, calcium carbonate 10 grams, and water to 1 liter.

1946] ENVIRONMENTAL FACTORS IN PENICILLIN FERMENTATIONS 125 rapidly. The glucose and the available ammonia nitrogen were completely utilized in 30 hours.

7 1946] ENVIRONMENTAL FACTORS IN PENICILLIN FERMENTATIONS 125 rapidly. The glucose and the available ammonia nitrogen were completely utilized in 30 hours. After the disappearance of available carbohydrate and ammonia nitrogen, the nutrients furnished by the steep liquor solids permitted continued growth. When this source of nutrient material could not meet the demands of the culture, autolysis occurred. During autolysis the ammonia nitrogen content and the of the medium rose and the mycelium nitrogen decreased. Penicillin formation stopped when autolysis began. Ammonia assimilation during glucose utilization took place at a more rapid rate than ammonia formation from steep liquor nitrogen compounds. Since the ammonia concentration decreased rather than increased, the rise was delayed. The increase in after glucose exhaustion may be attributed to lactate oxidation (Koffler et al., 1945). It may be concluded that the superiority of lactose over glucose as a carbohydrate source lies in its slow availability to the culture. In lactose fermentations, ammonia formation from steep liquor nitrogen compounds and lactate utilization bring about an early rise. Autolysis is delayed because of the long period required for complete lactose oxidation. On the other hand, in glucose fermentations the value during glucose oxidation is too low for rapid penicillin formation, and the early occurrence of autolysis limits penicrnin production after exhaustion of the glucose. Toxicity of antifoam agents. A number of shake-flask experiments were conducted to test various antifoam agents for toxicity. Table 1 summarizes the data for two toxicity experiments conducted in shake flasks. As may be seen, small amounts of any antifoam agent increased the yield over the controls, except in the case of vegifat Y. This has been found generally true of antifoam agents in shake-flask experiments and is apparently the result of the increased oxygen supply when no foam is present in the flask. When penicillin yields are considered, lard, lard oil, and 3 per cent octadecanol in lard oil were not toxic at any of the concentrations used. However, the levels were progressively lowered with increasing concentrations of these antifoam agents. It will be noticed that the decrease in level follows the degree of toxicity very closely. The foam-breaking capacity of the antifoam agents tested varied considerably. Nopco defoamer and vegifat Y were the most effective foambreaking agents and were also the most toxic. Three per cent octadecanol in soybean oil was toxic in shake-flask fermentations, nevertheless this same material was not toxic when used as an antifoam agent for tank fermentations. The antifoam agent regularily used to control foam in our tank fermentations was 3 per cent octadecanol in lard oil. Before the installation of a good method for antifoam agent addition, several fermentations were completed in which 4 liters of this material were added during the first 5 to 10 hours of the fermentation. The rise and the carbon dioxide maximum were delayed. In these fermentations, 7.0 was generally reached at 35 to 40 hours. Of all the antifoam agents tested 3 per cent octadecanol in lard oil appeared to be the most useful. However, no systematic study of antifoam agents has been attempted with pilot plant equipment.

126 STEFANAK, GAILEY, JARVIS, AND JOHNSON [VOL. 52 TABLE 1 Toxicity of antifoam agents in shake-flask fermentations CONC. 4 DAYS 5 DAYS 6 DAYS 7 DAYs ANTr- _M X. ANTOAM USED FOAM PZN. DAY mz Pen.

8 126 STEFANAK, GAILEY, JARVIS, AND JOHNSON [VOL. 52 TABLE 1 Toxicity of antifoam agents in shake-flask fermentations CONC. 4 DAYS 5 DAYS 6 DAYS 7 DAYs ANTr- _M X. ANTOAM USED FOAM PZN. DAY mz Pen. p Pen. Pen. Pen. u/ml CENT p u/mi U/mi u/mi u/mi None Lard oil Soybean oil % Octadecanol in soybean oil None % Octadecanol in lard oil Vegifat Y Lard Nopco defoamer Corn oil Five-hundred-ml Erlenmeyer flasks containing 100 ml of medium were incubated in a reciprocating shaker (4-inch stroke, 90 cycles per minute at 23 C). Each flask was inoculated with 5 ml of vegetative inoculum grown on 6 per cent dextrin +2 per cent steep liquor solids medium. The medium used for the toxicity tests was as follows: Lactose grams Steep liquor solids grams CaCO, grams NaNO3... KH2PO... MgSO4.7H,O... The double lines separate experi- Distilled water to 1 liter Each number represents an average value for 3 flasks. ments performed 1 week apart. 1.5 grams 0.25 grams 0.12 grams

19461 ENVIRONMENTAL FACTORS IN PENICILLIN FERMIENTATIONS 127 ACKNOWLEDGMENT The work reported here is part of a large co-operative project on penicillin that has been done at the University of

9 19461 ENVIRONMENTAL FACTORS IN PENICILLIN FERMIENTATIONS 127 ACKNOWLEDGMENT The work reported here is part of a large co-operative project on penicillin that has been done at the University of Wisconsin under government contract. Additional funds were furnished by the Heyden Chemical Corporation and Lederle Laboratories, Inc. The authors are indebted to Dr. W. H. Peterson for counsel in the planning and execution of the work. Credit is due Miargaret Larson for the penicillin assays. SUNMMARY Penicillin yields were not appreciably affected by incubation temperatures ranging from 20 C to 29 C, but at 32 C, yields were definitely lower. Metabolic processes were more rapid at higher temperatures. Fermentations were usually conducted at a tank pressure of 20 lb per sq inch. Reducing this pressure to 2 lb per sq inch did not affect the fermentation, but at a tank pressure of 40 lb per sq inch, penicillin yields were reduced. Increased carbon dioxide tension did not affect penicillin fermentations. The metabolic changes in fermentations in which glucose was the carbohydrate source were compared with those on standard lactose medium. Toxicity tests of antifoam agents in shake-flask experiments have been performed. Of the agents found to be nontoxic, 3 per cent octadecanol dissolved in lard oil has been found to be the best agent for controlling foam in tanks. REFERENCES BOWDEN, J. P., AND PETERSON, W. H The role of corn steep liquor in production of penicillin. Arch. Biochem. In press. GAILEY, F. B., STEFANIAK, J. J., OLSON, B. H., AND JOHNSON, M. J A comparison of penicillin-producing strains of Penicillium notatum-chrysogenum. J. Bact., 52, KNIGHT, S. G., AND FRAZIER, W. C The control of contaminants in penicillin fermentations by antiseptic chemicals. J. Bact., 50, KOFFLER, H., EMERSON, R. L., PERLMAN, D., AND BURRIS, R. H Chemical changes in submerged penicillin fermentations. J. Bact., 50, STEFANIAK, J. J., GAILEY, F. B., BROWN, C. S., AND JOHNSON, M. J Pilot plant equipment for submerged penicillin production. Ind. Eng. Chem., Ind. Ed. In press.

Respiration of Penicillium chrysogenum in Penicillin Fer rnent at ions

336 ROLINSON, G. N. (1952). J. gen. Microbiol. 6, 336-343. Respiration of Penicillium chrysogenum in Penicillin Fer rnent at ions BY G. N. ROLINSON Research Department, Bacteriology Division, Boots Pure