Penicillium chrysogenum'

Transcription

1 Penicillin Production by High-Yielding Strains of Penicillium chrysogenum' ERIK G. M. TORNQVIST2 AND WILLIAM H. PETERSON Department of Biochemistry, The development and study of strains of Penicillium chrysogenum that give high yields of penicillin has been a major project at the University of Wisconsin during the past 12 years. Many thousands of cultures have been screened for their penicillin-producing ability in the Botany Department of the University. The methods used for the production and screening of mutants, the genealogy of the outstanding strains and their cultural characteristics have been described recently by Backus and Stauffer (1955). In recent years, interest has centered on cultures that produce no pigment but give high yields of penicillin. Many of the superior cultures have been studied intensively both in shaken flasks and pilot plant fermentations in the Biochemistry Department (Anderson et al., 1953, 1956; Davey and Johnson, 1953; Soltero and Johnson, 1954; and Owen and Johnson, 1955). In the present paper we shall report on factors affecting penicillin production by some of the recent high-yielding strains. These are numbered W50-935, W , W51-20, W51-616, W51-20F3, and W51-20F3-64. MATERIALS AND METHODS The fermentations were run in 500-ml Erlenmeyer flasks containing 100 ml of medium. The flasks were aerated by shaking on a rotary shaker describing a 2-in circle at 250 rpm. The fermentation temperature was 24 to 25 C. As a rule, 3 replicate flasks were used in each experiment. The media had the composition given in table 1. Medium I was used in the first series of fermentations and medium III, a richer medium, was designed on the basis of the data obtained with the first medium. The other media, most of which are more concentrated than III, were' used in a few experiments to show what combinations of corn steep and lactose would be best for the new cultures under our experimental conditions. The ph of the media was adjusted with strong potassium hydroxide to 5.2 to 5.6 before sterilization. Lard oil, containing 3 per cent octadecanol, was used 1 This investigation was supported in part by research grants from Bristol Laboratories, Inc. and Cutter Laboratories. Presented in part before the Fermentation Subdivision of the Agricultural and Food Chemistry Division at the 126th meeting of the American Chemical Society, New York, Fellow of the Sweden-America Foundation. University of Wisconsin, Madison, Wisconsin Received for publication May 17, as an antifoam agent. It was added as described in the individual experiments. Phenylacetic acid (PAA, a solution of the potassium salt containing 5 per cent PAA) was added as a precursor, usually in increments of 0.05 per cent beginning at 24 hours and at intervals of 24 hours thereafter until near the end of the fermentation. From 4 to 6 additions were made giving a total of from 0.2 to 0.3 per cent. In a few experiments, a 5 per cent solution of #3-phenylethylamine (PEA) neutralized to ph 6.0 with H2SO4 was used as precursor. When extra lactose was added to the fermentations, a 15 per cent W/V solution was used. Ammonium acetate was added as a 20 per cent solution. The flasks were inoculated with 4 ml of a mycelium suspension grown in medium containing 6 per cent dextrin and 2 per cent corn steep solids. One hundred ml of the medium in a 500-ml Erlenmeyer flask was inoculated with 5 ml of a spore suspension and the flask was shaken for 40 to 48 hours. The spore inoculum was prepared in the following way: Flat 6-ounce bottles containing about 35 ml of medium (6 per cent honey, 1 per cent Difco Bacto-peptone, 2 per cent agar) were seeded with spores from a soil stock. The bottles were incubated on the flat side at 25 C for 7 to 10 days for growth and sporulation and were stored at 4 C until needed. The spore suspension was made by adding 25 ml of sterile water to a bottle and loosening the spores from the agar surface with an inoculation loop. Analytical methods. Samples of the broth were taken from the fermentations at desired intervals and the ph was measured with a glass electrode. After filtration, certain other analyses were made on the broth. The penicillin titer was determined by a modification of the cylinder-plate method of Schmidt and Moyer TABLE 1. Composition of media used for penicillin production Component* Medium ~ I I II I III IV V VI g per 100 ml Corn steep solids (C. S. S.) Lactose Sodium sulfate or sodium thiosulfate pentahydrate Ratio C. S. S. per lactose * In addition, the medium contained 0.4 per cent CaCO3 unless otherwise stated.

2 278 ERIK G. M. TORNQVIST AND WILLIAM H. PETERSON [VOL. 4 (1944) with Micrococcus pyogenes var. aureus H as the test organism. Residual sugar in the filtered broth was determined (after hydrolysis at 120 C for 30 minutes with 1 N HCl and neutralization) according to the micro method of Shaffer and Somogyi (1933). Soluble nitrogen in the broth was determined by the micro method of Johnson (1941), nitrogen in the washed and dried mycelium by the method of Hiller, Plazin, and van Slyke (1948), and ammonia in the filtered broth by a modification of the method of Umbreit and Bond (1936). One-half ml of the broth was made alkaline with saturated sodium carbonate solution, the ammonia was removed by aeration, collected in standard dilute sulfuric acid, and nesslerized according to Johnson (1941). Residual fat in the broth was extracted by ether from a slightly acidified sample (50 to 100 ml) in a 250-ml separatory funnel. The ether extract was transferred to a weighed 100-ml Erlenmeyer flask, the ether was evaporated, and the fat dried to constant weight at 105 C. Fat in the mycelium was obtained by washing with ether to remove adhering oil, drying, grinding, digesting with boiling methanol, and extracting with ether. The ether was removed from the extract and the residual solids were dried and weighed. The iodine numbers of residual and mycelial fat were determined according to Roseumund and Kuhnhenn (1923). When mycelial dry substance was determined on samples to which no oil had been added, the broth was acidified first to dissolve CaCO3 and other insoluble salts formed during fermentation. The mycelium was filtered off on a Buchner funnel, washed with water, transferred to a weighed Petri dish, and dried to constant weight. Nitrogen in the mycelium was determined by the Kjeldahl method. Preliminary tests on penicillin production by different strains. The most promising new pigmentless strains, W50-935, W , W51-20, and W51-616, were tested simultaneously on medium I with PAA as precursor and lard oil as defoamer. Average yields of 1110 (units) per ml were obtained with W and 1270 u per ml with W The yields of the other cultures were only about half of these values, quite in agreement with the values obtained in the Botany Department. As similar results also have been reported by Soltero and Johnson (1954) in a synthetic medium, only the two higher-yielding strains were given further study. One of the best of the older cultures, W49-133, was included for comparison. Since these cultures had been selected with PEA as precursor, some preliminary tests were made comparing it with PAA. Culture W was used in these tests. PAA proved to be as good a precursor as PEA, or possibly slightly better. It gave the best results when added in small amounts beginning at approximately 24 hours after inoculation and continuing at 24-hour intervals during fermentation. A total of 0.3 per cent of PAA had to be added as compared to 0.15 per cent for PEA. Under such conditions PAA gave 1280 u per ml as compared to 1180 u per ml for PEA. These observations are in agreement with results obtained earlier by Singh and Johnson (1948) with culture Q176. No improvement in penicillin production was obtained when combinations of PEA and PAA were used. PEA proved to be toxic in many cases when added at the beginning of the fermentation. Thus, it almost completely inhibited growth and gave penicillin yields of less than 500 u per ml when 0.25 per cent was added at 0 hour or at 24 hours. An inhibitory level of PEA also could be built up by adding slightly more than 0.05 per cent of PEA at 0 hour and every following 24 hours. The maximum yield dropped to about 900 u per ml. The toxicity was not due to the presence of impurities, as freshly distilled colorless PEA was as toxic as the yellowcolored technical product. By adding PEA before sterilization, the toxic effect was eliminated partly. This indicates that, during sterilization, PEA is either combined with some compound in the medium to form a less toxic substance, or it is changed into a less toxic but still active precursor. After these preliminary tests, a series of runs were made with three of the cultures. The results are summarized in table 2. All of these cultures gave similar penicillin yields. W51-20 seemed, however, to be slightly better than the others. Although promising, W51-20 was not used in further tests because of its somewhat irregular behavior and poor sporulation. While this work was in progress, Backus and Stauffer (1955) isolated several promising substrains of W Two of these, W51-20F3 and W51-20F3-64, did not possess the troublesome qualities mentioned above and were used, therefore, instead of their parent. Importance of amount of inoculum. Results from a great number of experiments indicated that some of the variations in penicillin yield depended upon variations TABLE 2. Penicillin production by strains Chrysogenum (Medium I) of Penicillium No. of Penicillin Time to ph During Lard Culture Ns Maximum Maximum Penicillin Oil RunsMaximum ~Production Added* u/mli hr ml W avg W avg W avg * 0.1 ml oil was added at 24 hr and usually 0.2 ml every 24 hr thereafter.

3 1956] PENICILLIN PRODUCTION BY PENICILLIUM CHRYSOGENUiM2 279 '1U500_XO1i 5W51-20F3 / >s W e Inoculum, mg. dry mycelium/100 ml. FIG. 1. Influence of size of inoculum on penicillin production in the amount of inoculum. Figure 1 shows the relation between inoculum size and penicillin yield. A 48-hour inoculum of both W and W51-20F3 was used. The different points on the graphs correspond to 1, 2, 4, and 8 ml of inoculum, containing 15 mg of dry mycelium per ml. Maximum penicillin yields were obtained with the two highest amounts of inoculum. With a smaller inoculum, a slightly higher yield was obtained at 110 hours than at 86 hours. It is evident that the usual amount of inoculum, 4 per cent by volume, was too low for maximum yield. In order to obtain the best results with this amount, the inoculum should contain about 15 mg of dry mycelium per ml. An inoculum of 5 to 6 per cent by volume was indicated, especially for W51-20F3 which appears to require a larger inoculum for maximum yield than does W Therefore, 5 ml of inoculum was used in later experiments. Culture W51-20F3 was a distinctly better penicillin producer at all levels than W A thick inoculum can be obtained by inoculation with a small number of spores and allowing a long growth time, or by inoculation with a large number of spores and allowing a shorter growth time. In shaken flask fermentations, inocula developed in these two ways will be very different because of the varying number of mycelial units. An inoculum from a small number of spores has a tendency to form big pellets in the final fermentation and to give low yields. In pilot plant fermentations this may not be of so great importance as the mycelium is partly broken up by the agitator in the tank. Effect of supplements on penicillin yields with medium I. Since analytical data of previous runs indicated that both ammonia and lactose were exhausted before the end of the fermentation, additions of these compounds and also of oil were made.3 The results from the various additions are given in table 3. Lactose had no appreciable effect but oil increased penicillin production 300 u per ml or about 25 per cent (Run 28). In Run 30 the increase was nearly 45 per cent. Without oil, the penicillin maximum was reached in 111 hours after which the penicillin yield declined to 760 u per ml at 135 hours. With oil, the penicillin content reached 1148 u per ml in 111 hours, rose to 1330 u at 135 hours, and then fell to 1210 u at 159 hours. Oil gave not only faster penicillin production but it also prolonged the period of production. Addition of ammonium acetate at the lower level (100 mg) had no appreciable effect; however, at 250 mg per flask an increase of nearly 20 per cent over the oil figure was obtained. The higher level of ammonium acetate is equivalent to about twice the maximum amount of ammonia nitrogen present in the usual corn steep medium (figures 2 and 3). Perhaps, under more carefully controlled conditions, a mycelium of higher nitrogen content and presumably greater activity could be obtained. Oil utilization and composition of mycelium. When the residual oil was separated from the broth in fermentations with culture W (table 2) about 250 mg of fat per flask was recovered. Therefore, about 75 per cent of the oil was utilized since 1.1 ml or 1.0 g had been added to each flask. The recovered fat was crystalline in nature and had almost the same acid number as the fatty acids from alkali-hydrolyzed oil. This indicated that it was composed almost exclusively of free fatty acids. The iodine number was nearly constant for the three samples, varying from 58.6 to This is lower than the iodine number of the added oil, 65.8, which shows some preferential utilization of the unsaturated part. Oil recovered from other flask experiments gave similar results. As a rule, the best utilization of the oil occurred when it was added in small portions every 24 hours. When 0.5 ml of oil was added at one time, formation of pellets of calcium soap was very marked, and this necessarily affected the utilization of the fatty acids. The effect of oil and lactose additions on the amount of mycelial dry substance formed is given in table 3. The lowest mycelial dry weight was obtained when no additions were made to the medium. When oil or lactose was added, nearly 50 per cent more mycelium was formed. The per cent of nitrogen in the mycelium was 3An increased production of penicillin from the addition of oil has been reported by a number of investigators. We have reviewed the literature on the subject recently (Anderson, Tornqvist and Peterson, 1956) and will not repeat a discussion of it here.

4 280 ERIK G. M. TORNQVIST AND WILLIAM H. PETERSON[ [VOL. 4 TABLE 3. Effect of supplements on penicillin yields and mycelium composition (Culture WV49-133, Medium I) Supplements Maximum Penicillin ph of Penicillin ph ofmycelium Penicillin Dry wt N ~~~~Mycelium N Direct Calculated umi. hr g/loo ml % mig/100 ml mg/100 ml Run 28 None Lactose* Lard oilt Run 30 None Not determined Lard oil Not determined Lard oil and NH4C2H302t (a) Not determined (b) Not determined * Lactose: 0.3 g at 48 hr, and so forth; total 1.2 g/100 ml. t Lard oil: 0.2 ml at 24 hr and every 24 hr thereafter; total, 1.1 ml/100 ml of medium. t NH4C2H302: (a) 25 mg at 48 hr and so forth; total 100 mg/100 ml. (b) 50 mg at 48 hr. and so forth; total 250 mg/100 ml Time, hrs. FIG. 2. Chemical changes in penicillin fermentations (Medium III) S 1000 z highest with added lard oil and lowest when lactose was added. The amount of nitrogen in the mycelium can be obtained in two ways, either from the dry weight of the mycelium and its nitrogen content or by the difference between the total nitrogen and the nitrogen not taken up from the medium. Results from such calculations are given in table 3. The two methods check fairly well except for the fermentation on the basal medium. Values calculated from the nitrogen uptake should be approximately 5 to 10 per cent higher than those obtained from the weight and nitrogen content of the mycelium because of the dilution that resulted from additions of precursor and lactose. Fermentation of a richer medium. The results obtained with medium I indicated that this medium was too dilute to give maximum penicillin production. Therefore, the richer medium III was used for some further studies. The superiority of the more concentrated medium was obvious from the first experiments. Yields from 1400 to 1870 u per ml were obtained with culture. W and from 2000 to 2800 u per ml with culture

5 1956] PENICILLIN PRODUCTION BY PENICILLIUM CHRYSOGENUM 281 Ijg "c, / /.6.5 W51-20F3. The average for the two cultures was about 1550 u per ml and 2400 u per ml, respectively. Although the variation between different runs amounted to as much as 20 per cent, the variation between individual flasks in a particular run seldom amounted to 10 per cent. Chemical changes in typical fermentations with cultures W and W51-20F3 in this medium are shown in figure 2. The more rapid and higher penicillin production of W51-20F3 is especially noticeable. The metabolic rate of this culture was lower than that of W as indicated by its slower utilization of lactose and ammonia. The more even ph plateau of W51-20F3 on the two media seems also to be characteristic of this culture. Of interest also are the changes in the nitrogen content of themycelium. Up to about 30 hours, there was not only a rapid increase in weight of mycelium, but the nitrogen content of the mycelium rose to a maximum of around 10 per cent. It then decreased to about 6 per cent at the end of the fermentation. The decrease in nitrogen content was not the result of a loss of nitrogen from the mycelium as is shown by the constant level of nitrogen present in the filtrate. Other data show that the drop in per cent of nitrogen is balanced by a corresponding increase in weight of mycelium. While it may be that nitrogen compounds are excreted and then reabsorbed to make the new mycelium, the over-all phenomenon is one of translocation of nitrogen within the mycelium. conten 1s penicillin 6.0 Medium / Mediuxn4wVi'5 Mediumn 9 _ Medium V I I I I. I I Time, hrs. FIG. 3. Chemical changes in gradeci media (Culture W51-20F3-64) The data in figure 2 show that the shift in nitrogen content is associated with penicillin formation. Penicillin appears in the medium when the nitrogen content of the mycelium reaches a maximum, and continues to form as the nitrogen content falls, and ceases when the latter approaches a low, constant level. Perhaps at this time the forms of nitrogen in the mycelium are predominantly of an inert chitinous type. A systematic and detailed study of the forms of nitrogen in the mycelium at the two stages should be highly informative. Effect of oil and calcium carbonate additions to medium III. Because of the great stimulation of penicillin production which was obtained by adding oil to medium I, similar experiments were made with medium III. As preliminary tests indicated that oil depressed penicillin production in medium III when only 0.1 per cent CaCO3 was present, fermentations were run with varying concentrations of this compound. Table 4 shows some data from two of these experiments. For the sake of comparison, medium I was also used with culture W The results with that culture will be discussed further. No great differences in penicillin production resulted from variation in CaCO3 level when no oil was added. Real stimulation from the addition of oil was obtained on medium I when 0.4 per cent CaCO3 was added to the medium. With less than 0.2 per cent CaCO3, addition of oil had a depressing effect. As the oil is hydrolyzed rapidly by the mold with formation of free fatty acids and glycerol, oil additions lowered the ph markedly, for example, 6.2 at 51 hours, when no CaCO3

6 282 ERIK G. MI. TORNQVIST AND WILLIAM H. PETERSON [V'OL. 4 TABLE 4. Effect of oil and calcium carbonate on penicillin production Max Penicillin Hr to Max ph Range Medium Yield, u/mi Yield p ag Culture MediuN CaCO3 Nio oil oil* oil o Oil No oil Oil* W I III W51-20F3 III * 0.2 ml lard oil was added at 24 hr and every 24 hr thereafter. was added. On medium III, no stimulation by oil was observed even when 0.4 per cent CaCO3 was included in the medium. The depressing effect of oil on the penicillin production, however, was even greater with only 0.1 per cent CaCO3 in this medium than it was with medium I. The results with culture W51-20F3 on medium III were quite similar to those for W Penicillin production did not vary much with different amounts of CaCO3 when no oil was used, but the tendency toward lower yields in the oil-low CaCO3 fennentations was marked still more than for culture W The penicillin yields obtained when oil and adequate CaCO3 were used were in no cases definitely higher than when no oil was added. The lower ph values that resulted from oil additions are of particular interest and are given in detail in figure 4. The ph dropped markedly with decreasing concentrations of CaCO3 and reached a minimum around 65 hours. The ph drop was small and of about the same magnitude with 0.4 and 0.6 per cent CaCO3 present in the medium. This would be expected as 0.4 per cent CaCO3 is about the amount needed for neutralization of the acids liberated from the oil, and additional CaCO3 would be of no benefit. With no CaCO3 the ph never reached the level for good penicillin production, that is, ph 7 to 7.5. The same unfavorable but less marked ph picture was noted with 0.1 per cent CaCO3 in the medium. The variation in penicillin production was as striking as that for ph. The more even the ph values the better was the yield, a result that emphasized the long-recognized importance of proper ph control. Further data on strength and balance of media. The improvement in yields obtained with medium III suggested that even this medium might be too weak for maximum penicillin production by the pigmentless Time, brs. FIG. 4. Effect of calcium carbonate on ph and penicillin production in medium containing oil (Culture W51-20F3, Medium III, Table 4). cultures. Therefore, some experiments were made in which all six media listed in table 1 were used. Figure 3 shows the results from one of these experiments, in which culture W51-20F3-64 was used. This culture is one of a hundred single spore cultures selected from W51-20F3, and gave about the same yields as the parent culture in screening tests. As was the case with the other cultures, the penicillin yields increased with increasing concentration of nutrients up to medium V where the yield was 1980 u per ml. Medium VI which was slightly richer than medium V gave somewhat lower yields, probably because some other condition, such as air supply, had become the limiting factor. With better aeration as in pilot plant fermentors, it should be possible to use such a heavy medium with formation of more nitrogen-rich mycelium and higher penicillin yields. An interesting result from these fermentations is observed when data within each pair of media are compared. In each pair, the highest yields were obtained from the medium with the lowest corn steep-lactose ratio. From this, it appears that, in addition to a medium of optimal concentration, a proper balance must exist between corn steep solids and lactose in order to obtain maximum yields. The best value for this ratio seems to be about 0.6. The nitrogen content of the mycelium,4 in general, increased with increasing concentration of corn steep solids. In general, the mycelium with the highest nitro- 4The determination of mycelial nitrogen on samples from shaken flasks was somewhat difficult. During the early stages in the fermentation, the amount of mycelium was small. During later stages it was easy to obtain the actively growing mycelium in the flask mixed with old mycelium which had been sticking to the wall of the flask. Great care was taken to avoid this, but it was not possible to avoid such mixing in every case. This might explain some unexpected irregularities in the curve for mycelial nitrogen.

7 1956] PENICILLIN PRODUCTION BY PENICILLIUM CHRYSOGENUM 283 gen content was also the best penicillin producer. The nitrogen content reached a maximum around 45 to 50 hours and then fell steadily for about 50 hours, after which it remained relatively unchanged. The fall in nitrogen and a steady or slowly rising ph are the outstanding features of the penicillin phase. It is probable that maximal penicillin yields will be obtained if and when these two factors can be controlled. The culture is, of course, the prime factor, but maintenance of a vigorous physiologic state is also of great importance. SUMMARY Some new pigmentless strains of Penicillium chrysogenum have been tested for penicillin-producing ability on corn steep-lactose media in shaken flasks. The use of a suitable precursor as well as the importance of using a proper inoculum was investigated. Yields up to 1150 u per ml were obtained on a standard medium [2 per cent corn steep solids (C. S. S.) and 2.5 per cent lactose] with the best culture. Addition of oil, preferably in small portions every 24 hours during fermentation, stimulated penicillin production, giving yields of about 1800 u per ml. More mycelium and higher yields (up to 2800 u per ml) were obtained in a richer medium (2.5 per cent C. S. S., 4 per cent lactose). Oil did not stimulate penicillin production in this medium, indicating that the oil acted primarily as an extra source of energy. The effect of oil in both media depended upon the amount of calcium carbonate present. If less than 0.4 per cent calcium carbonate was present, oil depressed the yield because of the low ph that followed the rapid hydrolysis of the oil by the mold lipase. Experiments in graded media indicated that a C. S. S. concentration of about 3 per cent and a ratio between C. S. S. and lactose of 0.6 gave optimal results. Penicillin production was associated in all cases with a decrease in the nitrogen content of the mycelium from approximately 9 per cent to approximately 6 per cent. This decrease was not the result of a net excretion of nitrogen, which indicates that more mycelium of lower nitrogen content was formed. A mycelium of high nitrogen content and a suitable ph are two factors favorable to good penicillin production. REFERENCES ANDERSON, R. F., WHITMORE, L. AM., JR., BROWN, W. E., PETERSON, W. H., CHURCHILL, W. W., ROEGNER, R. F., CAMPBELL, T. H., BACKUS, M. P., AND STAUFFER, J. F Penicillin production by pigment-free molds. Ind. Eng. Chem., 45, ANDERSON, R. F., TORNQVIST, E. G. M., AND PETERSON, W. H Penicillin production: Effect of oil on pilot plant fermentations. Agri. and Food Chem., 4, BACKUS, M. P., AND STAUFFER, J. F The production and selection of a family of strains in Penicillium chrysogenum. Mycologia, 47, DAVEY, V. F., AND JOHNSON, M. J Penicillin production in corn steep media with continuous carbohydrate addition. Appl. Microbiol., 1, HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D A study of conditions for Kjeldahl determination of nitrogen in proteins. J. Biol. Chem., 176, JOHNSON, M. J Isolation and properties of a pure yeast polypeptidase. J. Biol. Chem., 137, OWEN, S. P., AND JOHNSON, M. J The effect of temperature changes on the production of penicillin by Penicillium chrysogenu?n W Appl. Microbiol., 3, ROSENMUND, K. W., AND KUHNHENN, W A new method for the determination of the iodine number in fats and oils by the use of pyridine sulfate dibromide. Z. Nahr. Genussm., 46, SCHMIDT, W. H., AND MOYER, A. J Penicillin. I. Methods of assay. J. Bacteriol., 47, SHAFFER, P. A., AND SOMOGYI, Ml Copper-iodometric reagents for suigar determination. J. Biol. Chem., 100, SINGH, K., AND JOHNSON, MI. J Evaluation of precursors for penicillin G. J. Bacteriol., 56, SOLTERO, F. V., AND JOHNSON, M. J Continuous addition of glucose for evaluation of penicillin-producing cultures. Appl. MIicrobiol., 2, UMBREIT, W. W., AND BOND, V. S Analyses of plant tissue: Application of a semi-micro Kjeldahl method. Ind. Eng. Chem. Anal. Ed., 8,