Comparative Study of Penicillin Production with Vegetativeand Spore Inoculum of Penicillium chrysogenum1

B. K. BHUYAN,2 B. N. GANGULI, AND D. GHOSH

Biochemistry Division, Antibiotics Research Centre, Hindustan Antibiotics, Ltd., Pimpri (near Poona), India

Received for publication May 20, 1960

In commercial penicillin production the fermentationmedium is usually seeded with a mycelial (vegetative)inoculum of Penicillium chrysogenum prepared by thefollowing transfers: spores -+ germinator tank -- seedtank. However, depending on the fermentation con-ditions, the operation in the germinator tank can besuccessfully eliminated. Goldschmidt and Koffler (1950)used spore-inoculated fermentations in their study ofthe effect of surface active agents on penicillin yields.Rolinson and Lumb (1953) reported that the effect ofhigh aeration on lactose utilization appeared to bepeculiar to fermentations inoculated with spores. Craw-ford et al. (1952) obtained identical yields of cephalo-sporin P with both spore and vegetative inoculum.Backus and Stauffer (1955) used spores inoculateddirectly into the fermentation medium to test theirmutant strains of P. chrysogenum. However, comparisonof penicillin yield and the fermentation cycle (timetaken to reach the maximal yield) between fermenta-tions inoculated directly with spores and with vegeta-tive inoculum has not been reported for the high yield-ing strains of P. chrysogenum. During the early days ofpenicillin fermentation with very low yielding strains,Foster, Woodruff, and McDaniels (1946) observed thatin spore-inoculated fermentation in aerated and agi-tated laboratory fermentor, maximal titre (50 u perml) was reached at 120 hr as compared to 72 hr withvegetative inoculum. The method of direct inoculationof spores, if successful under industrial fermentationconditions, would eliminate one of the major inter-mediate stages in penicillin fermentation with a con-sequent economy in operations. To be consideredequally efficient, different fermentation conditionsshould yield the same amount of penicillin in the samefermentation cycle. This paper reports a comparativestudy of penicillin production in shake flask and agitatedand aerated fermentors with spores and vegetativeinoculum.

MATERIALS AND METHODS

Fermentation TechniquesA commercial strain of P. chrysogenum HA-9 (selec-

tion from a Russian strain) was used in these studies.I Part of the work was presented at the First Scientific Ses-

sion of the Association of Microbiologists of India, Calcutta,November, 1959.

2 Present address: The Upjohn Co., Kalamazoo, Michigan.

Preparation of spores. Spores obtained by sporula-tion on sterilized barley grains were suspended in waterand used for inoculation. Thirumalachar and Gopal-krishnan (1958) reported that on agar slants the my-celium continued to produce new crops of spores forlong periods so that the spores taken from an agarslant were of different ages. In contrast, when sporu-lated on barley grains the vegetative hyphae collapsedafter 8 to 10 days due to the drying of the grains andthe mass of spores was left as a powdery coating. Thusspores of nearly the same age were obtained by thismethod. The method of sporulation on barley grainswas as follows. The spores were obtained from an agarslant as a suspension in 20 to 25 ml of honey-peptonesolution. The suspension was mixed with 100 g of pre-viously sterilized barley grains and incubated at 25 C.Sporulation was complete at the end of 8 days andapproximately 40 billion spores were obtained as awater suspension per flask. Spores were inoculatedeither directly into the fermentation medium or intothe seed medium for preparation of vegetative inocu-lum.

Preparation of seed. The seed medium contained(per L) cornsteep liquor, 31.6 g; sucrose, 18.6 g; NaNO3,2.8 g; MgSO4c7H20, 0.068 g; KH2PO4, 0.31 g; andCaCO3, 3.2 g. The medium was adjusted to pH 5.5before sterilization. No pH adjustment was neededafter sterilization. A spore suspension (usually contain-ing 10 to 20 million spores) was inoculated into theseed medium. The seed flasks were incubated on arotary shaker (250 rpm describing a circle of 2-in. di-ameter) for 48 hr at 25 C. Ten milliliters of thevegetative growth were used to inoculate 100 ml of thefermentation medium.

Fermentation conditions. All fermentations werecarried out in duplicate on the rotary shaker at 25 Cin 500- ml Erlenmeyer flasks containing 100 ml ofmedium. The control fermentation medium contained(per L), lactose, 40 g; cornsteep liquor, 10 g; peanutmeal, 30 g; CaCO3, 6 g; Na2SO4, 1.2 g; MgSO4 7H20,0.068 g; phenylacetic acid, 1 g; and groundnut oil,1 ml. The pH of the medium was adjusted to 7.0 beforesterilization. Usually the pH decreased on steriliza-tion and was readjusted to 6.8. In spore inoculatedfermentations the medium was inoculated with approx-imately 10 million spores unless otherwise indicated.

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Analytical Procedures

Penicillin in the centrifuged broth was estimated by amodified iodometric method of Alicino (1946). ThepH was determined with a glass electrode. Sugars weredetermined by the method of Shaffer and Somoygi(1933). Lactose was estimated after hydrolysis with2 N HCl in the autoclave at 120 C for 30 min. Ammonianitrogen was determined by the method of Gaileyet al. (1946). It was not possible to determine the my-celial nitrogen since the medium contained insolublenitrogenous materials. Mycelial dry weight was de-termined by filtering the broth in a Buchner funnel,washing once with 0.1 N HC1 and twice with water,and finally drying overnight at 90 C. Rate of oxygenuptake and the respiratory quotient were determinedmanometrically as described by Vinze and Ghosh(1959).

RESULTS AND DISCUSSION

Preliminary shake flask experiments clearly indicatedthat penicillin yields were practically the same whenthe fermentation medium was inoculated either withvegetative seed or directly with spores. Data from atypical experiment are presented in table 1. Detailedstudies were, therefore, undertaken to find the optimalconditions for fermentations with spore inoculum.

Effect of initial pH. The effect of the initial pH of themedium on penicillin yield with spore inoculum isshown in table 2. The maximal penicillin yield wasobtained when the initial pH of the medium was ad-

TABLE 1Penicillin yield with vegetative and spore inoculum

Inoculum* Maximal Penicillin Yields

u/ml hr.

Vegetative ......................... 2,660 144Spore ........................... 2,795 144

* Ten milliliters of vegetative growth or approximately 10million spores were used to seed 100 ml of fermentation me-dium.

TABLE 2Effect of initial pH of medium on penicillin yield in spore-

inoculated fermentations

Initial pH Maximal Penicillin Yield pH Plateau

u/mi hr

5.5 2,020 144 7.066.0 2,300 144 7.026.5 2,360 144 7.327.0 2,700 144 7.417.5 2,310 144 7.60

Controlt 2,510 144 7.22

* Average pH values during 72 to 120 hr.t Control medium inoculated with "vegetative" seed.

justed to 7.0. It was found that the average pH valuesof the media from 72 to 120 hr were in a region suit-able for penicillin production in all cases.

Effect of pH on growth. The effect of pH on growthof the mold with spore inoculum was studied in a me-

dium containing (per L): cornsteep liquor, 50 g, andsucrose, 40 g. This soluble medium was used to avoiderrors in determination of mycelial dry weights. Themedium was adjusted to different initial pH valuesand re-adjusted at these levels manually at 10- to 12-hrintervals. Mycelial dry weights were determined after48-hr of growth. The optimal pH for the growth of themold was again found to be near 7.0 (figure 1). Chemi-cal analysis at the end of 48 hr showed that growthwas not limited by the exhaustion of sugar or nitrogen.As the highest penicillin yield was also obtained when

the initial pH of the medium was 7.0, in all subsequentfermentations the medium was initially adjusted topH 6.8 to 7.0 as a standard practice.

Optimal spore concentration. Optimal spore concen-

go-6go.6

0

.1E40

pH

Figure 1. Effect of pH on the growth of Penicillium chrysoge-num HA-9. The medium contained (per L): cornsteep liquor, 50g; and sucrose, 40 g. The media were maintained at their re-

spective pH levels by manual adjustment of pH.

TABLE 3Effect of spore concentration on penicillin yield

Spore~ Dry WeightInoculum Concn in Seed Maximal Penicillin

Medium Mycelium Yieldat 48 Hr

millons g/100 ml u/ml hr100 ml g/0

Vegetative.0.17 0.18 2,660 144Vegetative .1.7 0.70 2,540 144Vegetative.17 1.10 2,560 144Vegetative.170 1.10 2,430 144Spores .0.22 - 2,390 168Spores .2.2 - 2,800 144Spores .22 - 2,620 144Spores .220 - 2,420 144

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tration, either for making the vegetative seed or fordirect inoculation into the fermentation medium, wasdetermined for maximal penicillin yield. The resultsare shown in table 3. When only 0.17 million sporeswere used per 100 ml of seed medium for making thevegetative inoculum, very poor growth and formationof pellets (0.18 g per 100 ml dry weight) was obtained.However, for penicillin production, 10 ml of this inocu-lum were as effective as the thick inoculum (1 .1 g per100 ml dry weight) obtained with 100-fold increase inspore concentration; i.e., with 17 million spores per100 ml of seed medium. This indicated that in fermen-tation with 10 per cent vegetative inoculum, concen-tration of spores in the seed medium could be variedwithin wide limits without affecting the penicillin yield.Normally the seed medium is inoculated with approx-mately 10 to 20 million spores to give a thick mycelialgrowth.

For spore-inoculated fermentations the optimal con-centration of spores appeared to be between 2.2 and22 millions per 100 ml of medium under the conditionsof our experiments. At the highest spore concentrationused (220 million per 100 ml of medium) autolysistook place 24 hr earlier. It was thus established thatin shaken flasks the optimal concentration of sporesfor direct inoculation into the fermentation mediumwas of the same order as that necessary for preparingthe vegetative seed.

u 48 96 4.4

H u R.s

Figure 2. Biochemical changes in fermentations inoculatedwith spores or vegetative seed. 0 (i) = Fermentation me-

dium seeded with vegetative inoculum. X-----X = Fermenta-tion medium directly inoculated with spores.

Biochemical changes during penicillin production withvegetative and spore inoculum. The metabolic picture ofa penicillin fermentation with vegetative inoculumwas compared with that of a spore inoculated fermen-tation (figure 2).With vegetative inoculum the enzymes already

present in the seed mycelium utilized the amino acidsand peptides in the fermentation medium in preferenceto lactose as the carbon source during the growth phaseof fermentation. This resulted in a sharp rise in pHduring the first 48 hr due to liberation of NH3 whichwas later consumed as the source of nitrogen duringthe second phase of fermentation. Lactose was utilizedat a slow rate during the growth phase and at a rapidrate during the penicillin production phase. Whenlactose was exhausted autolysis set in, thereby termi-nating the penicillin production phase of fermentation.Autolysis was indicated by the rise in pH, release ofammonia, and a thinning of the mycelial suspension.With spores as inoculum the free ammonia nitrogen

present in the medium served as the nitrogen sourcefor germination and subsequent vegetative growthduring the first 24 hr of fermentation. Visually, a slightvegetative growth could be observed during this period.Utilization of the free ammonia nitrogen resulted inthe lowering of pH during this period. The myceliumformed by the end of 24 hr was able to utilize the aminoacids and peptides present in the medium as the carbonsource with liberation of NH3 and a corresponding risein pH. The maximal level of ammonia nitrogen releasedwhen vegetative inoculum was used was twice as muchas when spores were used. Mycelial growth was better(2.3 per cent maximum, dry weight) with vegetativeinoculum as compared to that with spore inoculum(1.96 per cent maximum, dry weight). The pH plateauof the spore-inoculated fermentation was slightly lowerthan that of the fermentation inoculated with vegeta-tive seed. There was practically no utilization of lactoseduring the first 48 hr in the spore-inoculated fermenta-tion. Later, lactose was utilized at about the same rateas that in the fermentation with vegetative inoculum.In this experiment higher penicillin yield was obtainedwith spore inoculum than with vegetative seed. How-ever, the study of several fermentations indicated thateither method of inoculation gave equally good yields.

Effect of media composition on penicillin yield withspores and vegetative inoculum. Penicillin yields obtainedwith different modifications of the control mediumusing spores or vegetative inoculum are shown intable 4.

Since spores used free ammonia nitrogen in theirinitial growth phase (figure 2), the effects of includingammonium nitrate and sucrose in the control mediumwas studied. The addition of the readily availablecarbon and nitrogen sources did neither increase the

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penicillin yield nor decrease the fermentation cycleas compared to the control.

It was found that when the medium contained pea-nut meal and lactose the pH plateau was maintainedat a high level (7.7) both with spores and vegetativeinoculum. Good growth with a lower pH plateau (7.3)was obtained with 6 per cent cornsteep in the medium.However, penicillin yields with either spore or vegeta-tive inoculum were very low in higher concentrationsof cornsteep medium as compared to the control. Sincebest results were obtained with the control medium,further studies were made with that medium only.

Effect of oilfeeding. Intermittent addition of oil duringfermentation has been reported to result in increasedpenicillin yields (Goldschmidt and Koffler, 1950; An-derson, Tornquist, and Peterson, 1956; Pan, Bonnanno,and Wagman, 1959). Di Accadia (1955) obtained equalpenicillin yields by substituting lactose with animalor vegetable oils as carbon source in a cornsteep me-dium. More recently Pan et al. (1959) reported thatunder the proper conditions of oil addition the fermen-tation can proceed at a rate 50 per cent higher than thelactose control, yielding a titre twice as much as thecontrol. All these studies have been made in fermenta-tions seeded with the usual vegetative inoculum. Westudied the effect of oil feeding on spore-inoculatedfermentations in control medium in which lactoseserved as the major carbon source and oil as extranutrient. The results of an experiment in which maxi-mal effect of oil was obtained is shown in figure 3.Groundnut oil, 0.1 per cent by volume, was fed every24 hr beginning from 48 hr of fermentation. The rateof penicillin production was the same with or withoutoil feeding until 144 hr; thereafter, in fermentationwithout feeding, mycelial autolysis set in with a cor-responding rise in pH and drop in penicillin titre. Withoil addition the maximal rate of penicillin productionwas maintained up to 192 hr without any mycelial

TABLE 4

Relative penicillin yield with vegetative and spore inoculum indifferent media

Relative PenicillinYield

Media Variation from Control MediumNo.

Vegetative Sporeinoculum inoculum

u/ml u/ml1 None 100 1002 Lactose, 3.5 per cent; sucrose, 0.5 - 94

per cent3 Medium 2, plus NH4NO3, 0.3 per - 83

cent4 Peanut meal, 4 per cent 96 845 Peanut meal, 4 per cent; lactose, 3.5 90 90

per cent; glucose, 0.5 per cent6 Cornsteep liquor, 6 per cent 65 50

* Maximal yield was obtained in 144 hr in all cases.

autolysis; pH was also maintained at a favourablerange. Maximal penicillin titre with oil feeding was

5,050 u per ml as compared to 3,150 u per ml in thecontrol with no oil addition. In the same set of experi-ments, oil feeding under identical conditions in fermen-tations seeded with vegetative inoculum gave a maxi-,mal penicillin yield of only 3,740 u per ml in 192 hrcompared to 2,970 u per ml in 144 hr in the control(data not shown in the figure). In general, the stimu-lating effect of oil was much more pronounced withspore-inoculated fermentations than with fermentationsseeded with vegetative inoculum.The results obtained when different indigenous oils

were tried in spore-inoculated fermentations are shownin table 5. Both groundnut oil and til oil (sesame oil) -

H O U R.S

Figure 3. Effect of feeding oil on spore inoculated fermentation. 0 0 = Groundnut oil was fed to the fermentation atthe rate of 0.1 per cent every 24 hr starting from 48 hr. X-----X= Control fermentation without oil addition.

TABLE 5Effect of different oils on penicillin yield with spore-inoculated

fermentation

Oil Added Maximal Penicillin pHYield Plateau*

u/ml hr

None (control)t ............... 2,760 138 7.4Groundnut .................. 3,620 162 7.4Linseed ................... 2,780 138 7.4Til ........................ 3,670 162 7.4Mustard ....... 3,470 162 7.4Cocoanut ...................... 3,220 162 7.35

Oil was fed at the rate of 0.1 per cent per day starting from48 hr.

* Average pH of the medium during penicillin productionphase.

t The fermentation medium was seeded with vegetativeinoculum.

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gave the maximal yields. Mustard oil and cocoanutoil gave slightly lower yields. With linseed oil, mycelialautolysis set in about the same time as in the controlwith no increase in penicillin yield.

Effect of aeration on spore-inoculated fermentation.Rolinson (1952) found that mycelium grown undervarying conditions of aeration showed metabolic differ-ences as indicated by their different respiratory quo-tient values. Rolinson and Lumb (1953) reported thatat high aeration rates in submerged culture lard oilmay be utilized as a source of carbon in preference tocarbohydrate. At low aeration rates, carbohydratesserved as the principal carbon source.The effect of aeration on a spore-inoculated penicillin

fermentation was studied using different volumes ofmedium in 500-ml Erlenmeyer flasks. Groundnut oilwas added to the fermentations at the rate of 0.1 mlper 100 ml of medium every 24 hr. The results are shownin figure 4. It was found that the maximal rate of peni-cillin production of the 50-ml fermentation (64 u per hr)was twice that of the 100-ml fermentation (31 u per hr),although mycelial growth was practically the samein both cases.Ammonia nitrogen released into the medium in the

50-ml fermentation was much higher than that ob-tained with the 100-ml fermentation. This was reflectedin a comparatively higher pH level obtained with the50-ml as compared to the 100-ml fermentation. How-

ever, the pH values of both fermentations were main-tained within favourable range during the penicillinproduction phase.The oxidative metabolism of the mycelium indicated

by the oxygen uptake rate (Qo2) and the respiratoryquotient (RQ) were the same in both fermentations.The Qo2 values steadily decreased from 18.5 at 44 hrto 10.5 at 138 hr. The RQ values decreased from 0.8at 44 hr to 0.62 at 138 hr.The rate of lactose utilization of the 50-ml fermenta-

tion was slightly higher than that of the 100-ml fermen-tation.The only observable metabolic difference between

the 50- and 100-ml fermentations was in the liberationand subsequent utilization of the ammonia nitrogen.The utilization of a larger amount of ammonia nitrogenby the mycelium of the 50-ml fermentation might haveresulted in a more proteinaceous and active mycelium,which might account for the high rate of penicillinproduction. Since the medium contained insolublenitrogenous materials (peanut meal) the mycelialnitrogen was not determined. Experiments with me-dium containing soluble nitrogenous material are neces-sary to determine the variation, if any, in the mycelialnitrogen content.

Spore-inoculated fermentation in 2,200-L fermentor.Two trial fermentations were run in a 2,200-L fermen-tor with direct inoculation of spores into the fermenta-tion medium. The details of these experiments and theresults obtained are given below.The fermentor was of a conventional design, fully

baffled and fitted with motor agitator and perforatedcircular air ring sparger at the bottom.

co

=-

-j

0

I 4

z z

x a)

x4

I .o 'k- - - I0 30 60 90 120 1I0 180 210

HOU RS

Figure 4. Effect of aeration on penicillin fermentation withspores. 0* 0 Media volume 50 ml in 500-ml flask. 0 -----0Media volume 100 ml in 500-ml flask. Groundnut oil was fed atthe rate of 0.1 per cent every 24 hours.

1OLZ S9

Figure 5. Spore-inoculated fermentation in a 2,200-L fer-mentor.

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Experimenit 1.Inoculum: 30 billioni spores (approximately).Fermentation medium: same as control medium;

pH after sterilization, 6.6; volume, 1,600 L.Aeration: 0.4 vol/vol/min. up to 48 hr and 0.7 vol/

vol/min thereafter.Antifoam an-d oil addition: 3 per cent octadecanol

in groundnut oil, 0.075 per cent by volume every 12hr beginning from 48 hr.The result of this experiment is shown in figure 5.

Because of excessive foaming, practically no agitationwas possible during the first 48 hr. As a result, germina-tion of spores was delayed and there was very littlemycelial growth during this period; fermentation wasin effect delayed by about 48 hr. Maximal penicillinyield was 3,100 u per ml in 144 hr. With vegetativeinoculum there is practically no foaming problem duringthe growth phase and the same yield of penicillin wasnormally obtained in about 100 hr.

If allowance is made of the period of inactivity dur-ing the first 48 hr or so, the biochemical changes (figure5), with respect to lactose utilization, pH, utilizationand liberation of ammonia-N during spore germination,mycelial growth, and penicillin production, were notmuch different from that obtained in shaken flaskfermentation with spore (figure 2).

Experiment 2.To minimize foaming difficulties, media volume was

reduced to 1,350 L and initial addition of oil beforesterilization was omitted. Spore concentration wasalso increased to approximately 130 billion for an earlybuild up of mycelia.

During the first 72 hr antifoam oil addition was madein small quantities as needed to control foaming. Regu-lar 12-hr feeding started from 72 hr. With the aboveinodification it was possible to run the agitator con-tinuously and the maximal penicillin yield obtainedwas 3,500 u per ml. in 148 hr. Although the yield washigher in this experiment, spore germination and my-celial growth was not much faster than in the previousexperiment.The results of these two fermentor trials indicated

the possibility of using spore-inoculated fermentationseven in commercial scale. Further work is in progressto determine the optimal conditions for agitated andaerated fermentors with respect to spore concentration,aeration, and oil feed rate.

SUMMARY

A comparative study of penicillin fermentation inshaken flasks was made by inoculating the fermenta-

tion medium directly with spores and with vegetativeseed. Practically identical penicillin yields were ob-tained under both of these fermentation conditions.The optimal conditions in spore-inoculated fermenta-tions were defined with respect to initial pH of fermen-tation medium and spore concentration.

Intermittent oil feeding gave much higher penicillinyield in spore-inoculated fermentation than in fermen-tation seeded with vegetative inoculum.

Pilot scale fermentations in 2,200-L fermentors in-dicated the possibility of employing spore-inoculatedfermentation in commercial practice.

REFERENCESALICINO, J. F. 1946 lodometric method for the assay of peni-

cillin. Ind. Eng. Chem. (Anal. Ed.), 18, 619.ANDERSON, R. F., TORNQUIST, E., AND PETERSON, W. H. 1956

Penicillin production, effect of oil in pilot plant fermenta-tions. J. Agr. Food Chem., 4, 556-559. 4

BACKUS, M. P. AND STAUFFER, J. F. 1955 The productionand selection of a family of strains in Penicillium chry-sogenum. Mycologia, 47, 429-463.

CRAWFORD, K., HEATLEY, N. G., BOYD, P. F., HALE, C. W.,KELLY, B. K., MILLER, G. A., AND SMITH, N. 1952 Anti-biotics production by a species of Cephalosporium. J.Gen. Microbiol., 6, 47-59.

Di ACCADIA, F. D. 1955 Abstracts, Third International Con-gress of Biochemistry, Brussels, p. 150.

FOSTER, J. W., WOODRUFF, H. B., AND MCDANIEL, L. E. 1946Microbiological aspects of penicillin. IV. Production ofpenicillin in submerged cultures of Penicillium notatum.J. Bacteriol., 51, 465-478.

GAILEY, F. B., STEFANIAK, J. J., OLSON, B. H., AND JOHNSON,M. J. 1946 A comparison of penicillin producing strainsof Penicillium notaturn-chrysogenum. J. Bacteriol., 52,129-140.

GOLDSCHMIDT, M. C. AND KOFFLER, H. 1950 Effect of surfaceactive agents on penicillin yield. Ind. Eng. Chem., 42,1819-1823.

PAN, S. C., BONNANNO, S., AND WAGMAN, G. H. 1959 Effi-cient utilization of fatty oils as energy source in penicillinfermentation. Appl. Microbiol., 7, 176-180.

ROLINSON, G. N. 1952 Respiration of Penicillium chrysoge-num in penicillin fermentation. J. Gen. Microbiol., 6,336-343.

ROLINSON, G. N. AND LUMB, M. 1953 The effect of aerationon the utilization of respiratory substrates by Penicilliumchrysogenum in submerged culture. J. Gen. Microbiol., 8,265-272.

SHAFFER, P. A. AND SOMOGYI, M. 1933 Copper iodometricreagents for sugar determination. J. Biol. Chem., 100,695-713.

THIRUMALACHAR, M. J. AND GOPALKRISHNAN, K. S. 1958Factors influencing pellet formation in shake flask fermen-tation. Antibiotics symposium, pp. 80-82. Council of Sci-entific and Industrial Research, New Delhi, India.

VINZE, V. L. AND GHOSH, D. 1959 Respiratory metabolismof Penicilliumn chrysogenum during commercial penicillinfermentation. J. Sci. and Ind. Research, 18C, 73-76.

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