Penicillin from Chemically Defined Media PETER HOSLER AND MARVIN J. JOHNSON Universify o f Wisconsin, Madison, W i s .
T
HE study of factors affecting the production of penicillin
Chemically defined media, referred to as synthetic media, have been useful in the study of nutritional requirements and the effect of various adjuvants. These media involve the use of glucose, lactose, a nitrogen source, a t least one organic acid, and essential minerals. In a quantitative study of mineral requirements made by Jarvis (6), the elements phosphorus, sulfur, magnesium, potassium, iron, zinc, and copper were found essential. However, even with the addition of precursor, yields did not equal those obtained with corn steep liquor media. Soltero ( l a ) obtained hiah (commrable to ours) on svnthetic media in - vields " , shaken flasks, using continuous feeding.
by submerged fermentation .has received much attention, but practically all the work has been done with a modified corn steep liquor-lactose medium, first described by Moyer and Coghill (7). Brown and Peterson ( 2 ) reported penicillin fermentations in 30-liter laboratory fermentors, using the culture Penicillium chrysogenum Q176, and a steep liquor-lactose medium. They found optimal conditions included aeration a t 0.64 liter of air per liter of medium per minute, agitation a t 580 r.p.m., and a pH range from 6.8 to 7.4. Under best conditions they obtained 1800 units of penicillin per ml.
AIR FILTERS
AIR EXHAUST INOCULATING
SAMPLE LIkE REFERENCE CELL
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GLASS ELECTRODE
SPRECURSOR SOLUTION U G a
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-
L
RUBBER -TUBING
BAFFLE
AGITATOR
AIR SPARGER
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METERING PUMP
; Figure 1.
Thirty-Liter Fermentor Assembly
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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In the present study, high penicillin yields were obtained on synthetic media, with the w e of continuously fed glucose to maintain the desired nutrient level and automatic p H control t o eliminate the need for chemical buffers. Since there are no natural precursors in the chemically defined media, as there are in corn steep liquor media, better control can be ha.d over the type of precursor utilized, especially in the synthesis of special types of penicillin. For this reason, Thorn and Johnson ( I S ) used chemically defined media for the synthesis of various aliphatic penicdlins in shake flask fermentations.
Table 1. -~
3Iediutn
Glucose b (NFIdsS04 KHzPOI NalSOl IClgS01.7H20 FeSOc7I110 MnSOc4HsO ZnSOr.7HzO CuSOn CaCk CaSOa CaCOjb
Fermentation Equipment Featured Automatic Feeds, Defoaming, and pH Controls
0
/
100
150
TIME, ROURS
Figure 9 .
7.;
...
0 7 0.18 0.05 0 05 0,008 0 08 2.0
...
10.0 0.4 7.5 1 0 0 7 0.18 0.05 0.05 0.008 0.0.; 2.0
Medium C 40.0 16.0 3.0 0.50
0.25 0.10 0 03 0>02 0.004
...,
1 i :0
The culture Penicillium chry8ogenzm Q178 was used in all the experiments. Fifteen-liter fernienations were run, with aeration at the rate of-one volume of air per volume of medium per minute and agitation a t 400 r.p.m. The temperature was 25" C . The first experiments were made with an excess of aninioriiuni sulfate in the medium and with pH cont,rol by sodium hydroside addition. Medium -1,Table I, was used for these esperiments. Later experiments involved the use of gaseous ammonia for p H control and nitrogen source. These fermentations were carried out on medium B, Table I. Ammonia N added during the fermentation averaged 2.0 grams per liter. PotasRium phenylacetate was added a t the rate of 0.0042% per hour. This \vas mixed with glucose solution. and the mist'ure mas fed on an hourly schedule. Antifoain oil consisted of lard oil containing 6% Alkaterge C (Commercial Solvents Corp., Terre Haute, Ind.). Approximately 400 ml. of oil was used during the eourse of the fermentation. The complete fermentor
MYCELIAL NITROGEN
50
10.0 10.0
Grams per Lite1 Medium Ba
Penicillium chrysogenurn 4176 Was Used in All Runs
/
'1
Aa
actunted only nhen the p H dropped belon the contioi point, and only the amount of ammonia required br the mold as supplied. In some of the first fermhtations, an ewess of ammonium sulfate n a s supplied in the medium, and the pH n a s controllcti by the automatic addition of sodium hydroxide solution, but the equipment n as essentially the same Air was sterilized by filtration through cotton-packed filter3 in series \Tith the spargers. The main sparger was a ling OF stainless steel tubing, Fhich had siu 'ls2-inchholes. The animoniaair sparger was an open tube.
d
"t4
Composition of Media
Additional glucose was slowly added t o t h e fermentation during the penirillin producing phase. a t rates specified in the text. b Ghicose a n d CaCOa were each sterilized separately in distilled w a t e r and added a t time of inoculation.
Thirty-liter fermentors of the type described by Rirett et ul. ( 8 ) were modified to provide automatic sugar and precursor addition. automatir defoaming, and automatic p H control. Figuie 1 is a diagrammatic draviing of the apparatus. The 12 x 18 inch stainless steel fermentor body is shown transparent, for clarity. The fermentor body was flanged a t the top and was bolted to the square fermentor head. The sugar and precursor solution was contained in a 4-liter glass bottle and as pumped to the fermentor through 1/4 X I / , & inch lubber tubing, by a rotary-type metering pump, driven by a small electiic motor. The pump operated 4 minutes per hour, to deliver about 25 ml. of solution. The electronically controlled antifoam system is described completely by Anderson et al. ( 1 ) . When foani contacts the foam electrode, an electronic relay activates the motor of the metering valve. The antifoam oil tank is under such a n air pressure that oil is added to the fermentor a t a rate of about 1.0 nil. per minute.
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Vol. 45, No. 4
Chemical Changes in a Lactose Fermentation (Medium A, Table I) with 2.5% Lactose pH controlled with 2 N sodium hydroxide
pH was controlled by the automatic addition of gaseous ammonia. The glass electrode n-as obtained from the National Technical Laboratories, Pasadena, Calif. It contained 1.0 M potassium chloride buffered a t p H 6 with 1.0 JI phosphate. The reference cell was made of borosilicate glass and was filled n i t h 1.0 M potassium chloride. Both electrodes contained a silver-silver chloride half-cell. Both electrodes were set in I/*inch steel pipe with rubber mountings; the steel pipe was screwed into the fermentor head. The electrodes supplied the input for a Beekman Model R p H indicator. This in turn operated through an electronic relay system to control a solenoid valve on a cylinder of ammonia. I n series with the p H control mechanism was an electronic timer, supplying an electrical pulse 0.7 second long. B:,attaching a counter to the solenoid valve, a record could be kept of the amount of ammonia added. The ammonia entered a n air stream and passed into the fermentor through the ammonia-air sparger. Thus the ammonia addition process was
I
Figure 3.
Chemical Changes in a Continuous Feed Glucose Fermentation (Medium A, Table I)
Glucose feed rate, 0.041 7%/hour;
p H controlled by addition of sodium hydroxide
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INDUSTRIAL AND ENGINEERING CHEMISTRY
assembly, including the electrodes, was autoclaved for 90 minutes a t 15 pounds steam pressure, transferred t o the cooling bath, maintained under positive air pressure and inoculated when cold. Glucose for initial growth was sterilized in a separate container, and added to the fermentor at the time of inoculation. Unless otherwise specified] the fermentations were held at p H 5.5 for the first 18 hours for mycelial growth, then the p H control point was raised to the penicillin producing range, usually 7.4. Continuous feeding of glucose and potassium phenylacetate was also initiated at this time. The vegetative inoculum was grown as follows:
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runs and the continuous feed glucose runs. I n an attempt to accelerate penicillin production early in the fermentation, runs without a separate growth phase were set up. Thew fermeiitations were on medium B, except that the initial p H was adjusted t o 7.0, there was no sugar present initially in the fermentation,
A soil stock of spores was used to inoculate 6-ounce bottles containing 20 ml. of the sporulation medium described by Gailey et al. ( 4 ) . Spore suspensions from two such bottles were used t o inoculate eight 500-ml. Erlenmeyer flasks, each containing 100 ml. of inoculum medium (medium C, Table I). After 44 hours of incubation on a rotary shaker (2-inch circle, 250 r.p.m.) t h e 800 ml. of inoculum was added to the fermentor through the inoculating port. Samples were removed a t least every 12 hours and stored for nnalysis as described by Gailey et al. ( 4 ) . Penicillin was assayed by the Oxford cup method with Micrococcu.La pyogenes var. aureus as the organism and penicillin G as the standard (8, 9). The p H of the samples was determined immediately after being taken by means of a glass electrode p H meter. Residual sugar was determined by the method of Shaffer and Somogyi (IO). Total nitrogen was determined on an aliquot of unfiltered broth a t the end of the fermentation. A 50-ml. portion was digested with concentrated sulfuric acid until colorless, then a suitable aliquot was taken for the Nesslerization and colorimetric measurement described by Johnson (6). Soluble Kjeldahl nitrogen was a 6 o determined by the method of Johnson (6). Total nitrogen a t any time could be interpolated from the initial and final values, together with the information from the ammonia addition counter, described previously. Mycelial nitrogen was calculated by subtracting the soluble nitrogen from the total nitrogen a t any given time.
150
TIME, HOURS
Figure
4. Chemical Changes in a Continuous Feed Glucose Fermentation (Medium B, Table I)
Glucose feed rate, 0.042% per hour,
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MYCELIAL NITROGEN
pH controlled by addition of ammonia
'1: /
Glucose Replaced lactose in Fermentation Figure 2 shows the chemical changes in a fermentation containing lactose. Medium A was used, with the exception that lxcstose was substituted for continuously added glucose. The nialimum yield, 660 units of penicillin per ml., is typical of fermentations that use lactose in synthetic media. The rate of Iactose utilization was approximately 0.03% per hour, which may be compared to the glucose feed rates used in later experiments. A typical continuous feed glucose fermentation, in which an excess of ammonium sulfate was used, is represented in Figure 3. Medium -4 was used with total nitrogen at 2.12 grams per liter. After the initial sugar was consumed in the first 18 hours, the sugar level was practically zero, varying from 0.0301,to less than 0.01% during the 1-hour feeding cycle. The peak penicillin yield was 1560 units per ml. a t 139 hours; the average rate of penicillin production is thus 11.2 units per ml. per hour. This rate of penicillin production is used as a figure of merit, so that runs of different lengths may be compared. Gaseous Ammonia Furnished Nitrogen and Controlled pH Figure 4 typifies the fermentations in which gaseous ammonia was used t o control the p H and supply nitrogen. Medium B was used, with a total of 2.10 grams of nitrogen per liter supplied by the end of the fermentation. The peak yield was 1740 units per ml. a t 138 hours, t o give an average penicillin production rate of 12.7 units per ml. per hour. Apparently there is little difference between the runs in which ammonium sulfate was present in excess and the runs in which ammonia was fed t o the fermentation. There is a marked difference, however, between the lactose
SED
I
0
50
I
TIME, HOURS
100
150
Figure 5. Chemical Changes in a Continuous Feed Glucose Fermentation without Separate Growth Phase ( M e d i u m B, Table I) Glucose feod rete, 0.0294% per hour
and the continuous feed additions of glucose and precursor weie started a t the beginning of the fermentation. I n this manner it was hoped that any lag in penicillin production due t o adaptation to the continuous feeding eystem might be avoided. Figure 5 shows the chemical changes for a typical fermentation of this type. The p H was constant in the range 7.0 to 7.5, the sugar level was less than 0.0370, and soluble nitrogen was approximately constant a t 0.5 gram of nitrogen per liter. Total nitrogen was 2.41 grams per liter a t the end of the fermentation. The peak penicillin yield was 1774 units per ml. at 114 hours, t o give a n average production rate of 15.5 units per ml. per hour. Coni pared t o the other types of fermentations described, the imposition of penicillin producing conditions during growth was beneficial to yield. Optimum pH for Penicillin Production, 7.4
In previously reported studies of optimum p H for penicillin production, the rate of sugar utilization has been a dependent,
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INDUSTRIAL AND ENGINEERING CHEMISTRY
0.07% per hour gave good rates of penicillin production. Below a feed rate of 0.03% per hour, there is insufficient nutrient t o support the mycelium, and autolysis occurs early in the fermentation. Above a feed rate of 0.07% per hour, the mold growth is very thick, and either air or nutrient becomes limiting. Between these values, there is a balance between the tendency t o grow and the tendency to autolyze, which results in good penicillin production. The optimum feed rate for fermentations without a separate growth phase, such as the one shown in Figure 5, was similarly determined, with the results shown in Figure 8. Data are included
x
aY
go
a
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1
I
7.0
8.0
7. 5
Vol. 45, No 4
PH
Figure 6.
Optimum p H for Penicillin Production (Medium A, Table I) Glucose feed rate 0.035% per hour
rather than an independent variable-tbat is, the rate of lactose hydrolysis may be a function of the pH, so that changing the p H would simultaneously change the effective sugar feed rate. Several runs were made on medium A, with glucose feed rates of approximately 0.035% per hour, under conditions of differing pH plateaus during penicillin production. The results are expressed in Figure 6. Penicillin production rate was chosen to 0.’025 GLUCOSE FEED RATE,
Figure 8.
\‘”j 5
B
X
a
I
l5
X
0
0.’05
0.I
s
PER CENT PER AOUR
Optimum Feed Rate for Penicillin Production
Fermentations without separate growth phase; medium B, Table I, p H plateau 7.3 Culture P. chrysogenum Q 1 7 6 Culture P. chryrogenum W49-133; conditions identical to those for Q 1 7 6
3”8B1Q:
E a i
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5-
x
,
E O0 1 Figure 7.
I
0.025 0.05 0.075 GLUCOSE FEED RATE, PER CENT PER AOUR
Optimum Feed Rate for Penicillin Production X
A
0
p H plateau Medium A Table I. Medium A: Table I; Medium 8, Table 11
7.4 1.0% initial sugar 2.0% initial sugar 1.0% initial sugar
express yield. These data indicate that p H 7.4 is optimum under the conditions specified, which verifies the findings of other workers. Brown and Peterson ( 2 ) found pH 7.2 to be optimum for penicillin production in 30-liter fermentors TTith steep liquorlactose medium. Jarvis and Johnson ( 5 ) found that pH 7.4 gave the maximum yields on synthetic medium in shaken flask experiments, while Singh and Johnson (11) reported an optimum p H range during fermentation of 7.6 to 7.9 when phenylacetic acid was added to synthetic medium in shaken flasks. A p H plateau of 7.4was used in subsequent experiments concerning the glucose feed rates. Sugar Feed Rates of 0.03 to 0.07 Gave Highest Production Figure 7 shows the results of several runs made with 1.0% initial sugar, with a p H plateau of 7.4, under differing rates of glucose feed. Data were taken from runs on medium 4, with pH controlled by additions of sodium hydroxide, and from runs on medium B, with p H controlled by additions of ammonia. Two runs were made with 2y0 initial sugar, in medium A, but since the yields were no higher than previously obtained, no further investigation was made. Sugar feed rates from 0.03 t o
from runs made with the pigmentless culture P . chrysogenum W 49-133; the fermentation conditions were identical to those used for strain Q176. The optimum feed rate in this case, 0.03% per hour, is more sharply defined than in the previous case. At the relatively high feed rate of 0.04% per hour, the mycelium became quite thick early in the fermentation, so that a t about 40 hours it began to autolyze-that is, the mycelial growth was exponential with time, but the sugar feed rate was linear. Therefore, it was possible for the mycelial population to exceed the capabilities of the constant nutrient feed, and thus the mycelium starved. I n spite of the low optimum feed rate, yields with t,his method were better than yields from other methods. Literature Cited (1) Anderson, R. F., Whitmore, L. M., Jr., Brown, 1%E., ‘. Peterson,
W. H., Churchill, E. W.,Roegner, F. R., Campbell, T. H., Backus, M. P., and Stauffer, J. F., IND. ENG.CHEM.,45, 768
(1953). (2) Brown, W. E., and Peterson, W.H., Ibid., 42, 1769 (1950). and Woodruff, H. B., J . Bact., 47, 43 (1944). (3) Foster, J. W., (4) Gailey, F. B., Stefaniak, J. J., Olson, B. H., and Johnson, M. J., IND. ENG.CHEM.,52, 129 (1946). (5) Jarvis, F. G., and Johnson, M. J., J . Am. Chem. Soc., 69, 3010 (1947). ( 6 ) Johnson, M. J., J . Bid. Chem., 137, 575 (1941). (7) M,oyer, A. J., and Coghill, R. D., J . Bact., 51, 79 (1946). (8) Rivett, R. W., Johnson, hf. J., and Peterson, TV. H., IND. ENG. CHEM.,
42, 188 (1950).
(9) Schmidt, W. H., and Moyer, A. J., J . Bact., 47, 199 (1944). (10) Shaffer, P. A., and Somogyi, M., J . Biol. Chem., 100, 695 (1933). (11) Singh, K., and Johnson, kl. J., J . Bact., 56, 339 (1948). (12) Soltero, F. V.,and Johnson, M. J., Applied Microbid., 1, 52 (1953). (13) Thorn, J. A,, and Johnson, M. J., J . Am. Chem. Soc., 72, 2062 (1950). RECEIVEDfor review October 3, 1952. ACCEPTEDJ a n u a r y 16, 1953. Published with t h e approval of the director of the Wisconsin Agricultural Experiment Station. Supported in p a r t by grants by Bristol Laboratories, In?., and Chas. Pfiaer a n d Co., Inc.
