APPLIBD MICROBIOLOGY, Nov. 1967, p. 1284-1290Copyright @) 1967 American Society for Microbiology

Vol. 15, No. 6Printed in U.S.A

Effect of Growth Rate on the Synthesis of Penicillinby Penicillium chrysogenum in Batch and

Chemostat CulturesS. J. PIRT AND R. C. RIGHELATOl

Department of Microbiology, Queen Elizabeth College, University ofLondon, London, England

Received for publication 15 May 1967

The kinetics of penicillin production by Penicillium chrysogenum Wis 54-1255 ina glucose-limited chemostat and in batch cultures are reported. The specific produc-tion rate of penicillin, qpen (units per milligram of dry weight per hour) was inde-pendent of specific growth rate over the range 0.014 to 0.086 hr-'. Growth wasstopped by restricting the glucose supply to the "maintenance ration," that is, theglucose requirement of the organism at zero growth rate with all other nutrients inexcess. Under such conditions, the organism dry weight remained constant, but theqpen fell approximately linearly to zero at a rate inversely related to the previousgrowth rate. Glucose supplied in excess of the maintenance ration inhibited the decayof qpe,. At a critical growth rate between 0.009 and 0.014 hr-', the decay was com-pletely inhibited. Quantitative expressions for the qp1, of growing and nongrowingcultures were derived and used to predict the steady-state concentrations of penicillinaccumulating in one- and two-stage continuous processes. A rational explanationof the kinetics of penicillin accumulation in batch cultures is given, relating the rateof penicillin synthesis to growth rate. It is concluded that an important role of cornsteep liquor (CSL), a heterogeneous carbon and nitrogen source commonly usedin penicillin production media, is the provision of substrates which allow a highconcentration of mold to be reached before the growth rate falls below the criticalvalue. CSL had no significant effect on qpen.

In the chemostat type of continuous culture,control of the growth rate of the organism, andhence of many other metabolic rates, is possiblefor indefinite periods. Thus, if the chemostat is tobe used for a process such as penicillin produc-tion, a thorough understanding of the relation-ship between the process rate and growth rate isessential.The production of penicillin by Penicillium

spp., like the synthesis of other secondary metabo-lites, is thought to be dissociated from the growthof the organism. The antibiotic is produced at itsmaximal rate in batch cultures after an initialperiod of rapid growth of the mold. The initialperiod of rapid growth and a second phase inwhich little or no growth occurs are considerednecessary to the accumulation of high concentra-tions of penicillin. From an analysis of batchculture data, Jarvis and Johnson (4) concludedthat the specific rate of production of penicillin

'Present address: Vaccine Department, RijksInstituut voor de Volksgezonheid, Sterrenbos-1,Utrecht, The Netherlands.

(units of penicillin per milligram of mycelialnitrogen per hour) was at its highest whenthe organism growth rate was close to zero.Maxon (5) and Gaden (2) presented penicillinfermentation as an example of a class of mi-crobial processes in which growth and productformation are temporally separated. However, therestriction of penicillin synthesis to the periodafter the rapid growth of the organism may notreflect a dependency of penicillin biosynthesis onslow or no growth. In most research work, batchcultivation with a variety of carbon and nitrogensources is practiced. It is difficult to obtain mean-ingful information from such processes, becausenumerous, inter-related parameters such as pH,growth rate, and nutritional status vary simulta-neously. In contrast to the observations made onbatch cultures, penicillin is produced at a highrate in growing continuous cultures (1, 9, 10).

Because in a batch culture the organism is in aconstantly changing environment, behaviorexactly parallel to that observed in continuousculture cannot be expected. The adaptive response

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of an organism to a change in its environment isprobably always subject to a time lag. In batchcultures, therefore, it is expected that the cell iscontinually adapting to a past environment,whereas a steady-state chemostat culture containsthe fully adapted organism. A steady state rep-resents the state to which a cell tends under aparticular set of conditions, but in batch culturethe state may never be attained because the condi-tions are not sufficiently prolonged. This being so,observations on continuous cultures are probablyof greater value in the interpretation of nonsteady-state phenomena than the reverse. We have in-vestigated the influence of growth rate on severalaspects of penicillin synthesis in chemostat cultureand have used the results as the basis of a rationalexplanation of the kinetics of penicillin accumula-tion in batch cultures.

MATERIALS AND METHODSApparatus and aeration. Batch and chemostat

cultures of 1.5- to 2.0-liter volume were carried outin a vessel adapted for cultivation of filamentousmicroorganisms (13). Aeration was by vortex stirringwith air supplied at a few inches water gauge pressureabove atmospheric pressure. Measurements with anoxygen electrode revealed that, with the highestoxygen demand, the culture medium was 70% satu-rated with oxygen.

Penicillin production media. Synthetic mediumM4 consisted of (grams per liter): glucose, 2.3 Xmycelial dry weight required; sodium phenylacetate,0.05 X glucose concentration; K2HP04, 6.00;NaH1PO4, 1.86; MgSO4.7H20, 0.25; CaCl2, 0.05;ZnSO4.7H20, 0.02; MnSO4.4H10, 0.02; CuSO4-5H20, 0.005; FeSO4-7H20, 0.10; Na2SO4, 1.00;ethylenediaminetetraacetic acid, 0.60; (NH4)2S04,4.72. For corn steep liquor (CSL) medium, M5,filtered CSL replaced (NH4)2SO4 in M4 to give 1.5g/liter of CSL nitrogen. Glucose, sodium phenyl-acetate, and phosphates were each sterilized sepa-rately.

P. chrysogenum Wis 54-1255 was maintained onagar slopes of the sporulation medium of Moyer andCoghill (6). Spores were produced on moistenedbran cultures (14). Approximately 2 X 109 conidiawere inoculated into 200 ml of germination medium(4) and incubated on a rotary table shaker at 25 Cfor 32 hr. After inoculation of the fermentor, theculture volume was brought to 1.5 liters with freshmedium. Temperature was controlled at 25 C; pHwas controlled at 7.0 0.1 by automatic additionof 2 N NH4OH or 2 N H2S04. Foaming was inhibitedby the addition of 0.1 ml/liter medium of poly-propylene glycol (P2000; Dow Chemical Co. (UK)Ltd., London, England). The air flow was adjustedsuch that the carbon dioxide content of the effluentair was 1 to 3%. Glucose and phenylacetate weremetered into the culture indepenent of the rest ofthe medium. Glucose was the growth-limiting nu-trient in M4 and M5, up to at least 20 g/liter ofmycelium (dry weight). The organism concentra-

tions used were in the range of 4 to 14 g/liter (dryweight).

Corrections were made for changes in volumedue to addition of nutrients and control agents andfor evaporation.A steady state was considered to have been ob-

tained in continuous cultures when samples takenover at least two mean residence times showed notrend in the observed parameters. To avoid the straindegeneration observed by Pirt and Callow (10) onprolonged continuous cultivation, experiments wereterminated after throughputs of not more than 12culture volumes. No evidence of strain degenerationwas obtained in these short continuous cultures.

Analytical methods. The mycelium dry weightsin 10-ml samples of culture were measured afterfiltration and washing twice with 15 ml of distilledwater and drying at 105 to 110 C to constant weight.Penicillin was assayed by a cup method with Bacillussubtilis as the assay organism and sodium benzylpenicillin as standard.

RESULTS

To establish the relationships among glucoseutilization rate, growth rate, and penicillin pro-duction, a glucose-ammonia-salts medium (M4)with glucose as the growth-limiting nutrient wasused. The concentration of ammonium ion waskept constant by addition of ammonia as the pHcontrol agent.

Steady-state glucose-limited chemostat cultureswere obtained at a number of growth rates be-tween 0.023 and 0.086 hr1. Over this range, thespecific production rate of penicillin (qpen; unitsper milligram of dry weight per hour) was approx-imately constant (Fig. 1). Thus, the steady-statepenicillin concentration in single-stage chemostats(p1; units/ml) rose as the reciprocal of the growthrate (j.t):

p, = X1 * qpen * (1)

growth rate h-1

FIG. 1. Relationship between the specific productionrate of penicillin (qpen) and specific growth rate inglucose-limited chemostat cultures of Penicilliumchrysogenum Wis 54-1255. Each point represents themean of one steady state. The mean value is 1.53 i0.32 (p = 0.95).

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0.05 1specific growth rate-h-'

FIG. 2. Relationship between specific growth rateand specifi utilization rate of glucose in glucose-limited chemostat cultures of Penicillium chrysogenumWis 54-1255. The intercept on the ordinate gives themaintenance requirement for glucose = 0.022 4z 0.005g of glucose per g (dry weight) per hr (p = 0.95).

where xl is the organism concentration (milli-grams of dry weight per milliliter).At a dilution rate of 0.095 hr-1, very slow wash-

out occurred, indicating that the maximal specificgrowth rate was a little below this value. Duringwashout, all substrates were in excess, and theqpen was 50 to 60% higher than under glucose-limited growth conditions.

Extrapolation of Fig. 1 suggests that no reduc-tion of qpen would occur as the growth rate ap-proaches zero. If the penicillin synthetic ratecould be maintained at the level observed in thesingle-stage chemostat cultures when the growthrate was reduced to zero, very high titers wouldbe obtained with the consumption of minimalquantities of substrates. Penicillin production bythe nongrowing organism was investigated bystopping the flow of medium to steady-statechemostat cultures and supplying only glucoseand sodium phenylacetate, the former as themaintenance ration. The maintenance coefficient(7) was found by extrapolation of the relationshipbetween the specific growth rate and the specificutilization rate of glucose to zero growth rate(Fig. 2). It represents the organism's requirementfor carbon and energy source for functions in-dependent of growth rate. The supply of glucoseonly for maintenance is here termed the main-tenance ration. As in the growing cultures, allother substrates were supplied in excess. Themycelial dry weight of cultures receiving themaintenance ration remained constant as long as

the experiments were continued. The penicillinproduction rate did not remain at the value ob-served in growing cultures but fell approximatelylinearly to zero. Figure 3 shows the organismconcentration and qpen of a typical culture duringcontinuous growth and during a subsequentmaintained, nongrowing phase. The individual

time h.

FIG. 3. Organism concentration (0) and specificproduction rate of penicillin, qp.. (0), of Penicilliumchrysogenum Wis 54-1255 in a glucose-limited chemo-stat at a growth rate of 0.023 hr-i and after cessationofgrowth by supplying the glucose maintenance ration.The arrow indicates the time at which growth wasstopped.

estimates of qpen during the nongrowing phasewere subject to considerable error owing to thedifficulty of measuring accurately small titerchanges at a high absolute penicillin concentra-tion. In the absence of evidence of nonlinearity, astraight line was fitted through the qpen values; itsslope is referred to as the "qpen decay rate." Wehave reported briefly that the growth rate of thecontinuous growth phase influenced the qpen de-cay rate in the subsequent nongrowing phase (11).This relationship is shown in Fig. 4; clearly, thehigher the previous growth rate, the slower is thedecay of penicillin synthetic activity. In these cul-tures, the qpen at any time (t) after the cessationof growth is given by

qpen(t) = qpen(o) - kt (2)

where qpen(o) is the value of the qpen in glucose-limited chemostats and k, the qpen decay rate, is aconstant dependent upon the previous growthrate of the mold.

Since the maintenance ration of glucose did notmaintain the qpen at the value observed in growingcultures, higher glucose supply rates were investi-gated (Fig. 5). As glucose supplied at rates abovethe maintenance ration allowed growth to occur,frequent adjust,ment of the glucose feed pumpswas necessary to keep the specific supply rate(grams of glucose per gram of dry weight perhour) constant. The highest glucose supply rate,0.056 g of glucose per g of dry weight per hr,allowed a specific growth rate of 0.014 hr-' andcompletely inhibited the decay of qpen. Thus, therange over which the qpen was about 1.5 can beextended to this lower limit. When the glucosesupply rate was 0.038 g of glucose per g of dryweight per hr (specific growth rate = 0.009 hr-'),the qpen fell.

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S 2.01'la

",1.0

l=0 0.05

previous growth rate h-0.10

FIG. 4. Effect of the specific growth rate of theglucose-imited chemostat growth phase of Penicilliumchrysogenum Wis 54-1255 on the decay of the specificpenicillin production rate in cultures whose growthwas stopped by supplying the glucose maintenanceration. The 95% confidence limits of each point areincluded. Symbols: *, synthetic medium M4; 0,

CSL medium M5.

40

2.

2.0-

A

I. ...........A1

1.0 Io - ,

C \. o \ v-_ 0 N. A

0~~~~N1000 50

time h

FIG. 5. Effect of the specific utilization rate ofglucose on the specific rate of penicillin production,qpen, of Penicillium chrysogenum Wis 54-1255 afterglucose-limited chemostat growth at a specific growthrate of 0.051 + 0.002 hr-1. Glucose supply (in grainsof glucose per gram of dry weight per hour): 0 (@);0.022 (0); 0.038 (A); and 0.056 (A).

Thus, it appears that a critical specific growthrate exists for steady-state penicillin synthesis,given by a glucose supply rate of between 0.038and 0.056 g of glucose per g of dry weight per hr.Below the critical value, the qpen falls at a ratedepending on the glucose supply and the previousgrowth rate of the organism; above it, the peni-cillin synthetic activity of the mold is independentof glucose supply and growth rate, providedglucose is the growth-limiting substrate.The experimental system used here was unlike

most penicillin fermentations, not only in the use

of the chemostat principle, but also in the sim-plicity of the medium. The media most commonlyemployed for penicillin production contain CSL.

a heterogeneous carbon and nitrogen source con-taining a variety of amino and other organic acids,sugars, trace elements, and other inorganic ions.Its adoption as a major medium component was asignificant step in the history of penicillin produc-tion, giving considerably higher yields of theantibiotic. Part of the effect of CSL may beascribed to its ability to buffer the fermentationaround a neutral pH. In addition, the carbon andenergy sources supplement others in the medium,allowing higher mold concentrations to develop.Our observations on feimentations carried outwith glucose and lactose as the main carbohy-drates have shown that CSL is particularly effec-tive in reducing the diauxic lag between the utiliza-tion of the two sugars, presumably by supplyingenergy sources enabling more rapid synthesis ofthe new enzyme system.The presense in CSL of numerous metabolic

intermediates may enable it to modify metabolismin favor of penicillin synthesis. To test this hy-pothesis, the qpen of the mold was measured underthe controlled conditions of the chemostat usingthe CSL medium (M5) and comparing it with theqpen observed on the synthetic medium (M4). Nosignificant difference was observed in the qpen oforganisms grown at 0.033 and 0.077 hr-, norwere the qpen decay rates observed after growthhad stopped significantly different from those ofsynthetic medium-grown cultures (Fig. 4). Itappears that CSL does not have a specific effect onthe synthesis of penicillin by the mold. This con-clusion is supported by the observations on batchcultures presented below.The kinetics of penicillin accumulation in batch

cultures on the synthetic and the CSL mediumwere investigated. Glucose was metered into thesecultures at a constant rate and became limitingwithin a few hours of inoculation. The growth ofthe mold, the accumulation of penicillin, andqpen in one of each of these types of cultures isshown in Fig. 6. On the synthetic medium, theorganism concentration increased in proportionto the glucose supply. Eventually, the growth rate(dx/dt) fell off as a greater proportion of theglucose was diverted to maintenance functions.The presence of CSL in the medium allowed morerapid initial growth, although glucose was sup-plied at the same rate to both cultures. This was afunction of the carbon and energy sources presentin the CSL; when they were exhausted, the growthrate fell to that supported by the glucose supply.Initially, the penicillin accumulation rate (dp/dt)increased until it reached a maximal value; then itremained constant for a time. The maximal linearpenicillin accumulation rate was reached when thespecific growth rate fell to about 0.03 hr-1 (Table

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time h.

FIG. 6. Mold concentration (0), penicillin con-centration (0), and specific penicillin production rate,qpen (broken line), in batch cultures of Penicilliwnchrysogenum Wis 54-1255 supplied glucose at 0.33g per liter per hr. (a) Synthetic medium M4; (b) CSLmedium M5.

TABLE 1. Relationship between the linearaccumulation of penicillin in batch cultures andthe organism concentration and growth rateat its onset

At onset of linearaccumulation rate

LinearGlucose supply rate accumulation(g per liter per hr) rate of penicillin My- Specific qpen

(units ml/hr) celium growth (units/dry wt rate mg dry(g/liter) (hrl) wt/hr)

Syntheticmedium

0.34 10.5 4.8 0.03 2.20.67 14 6.3 0.03 2.1

CSL medium0.33 24 11.4 0.02 2.10.67 21 9.5 0.03 2.2

1). Eventually, the penicillin accumulation ratefell to zero. The fall is explained by the decay ofthe penicillin synthetic system in the mold andnot by inhibition by the medium. The differencebetween the initial rates of decay of the qpen in thesynthetic and CSL media are largely accounted forby the effect of the previous growth rate on theqpen decay rate.

DIscussIoNThree important relationships between myce-

lial growth and penicillin synthesis have beenobserved. (i) In glucose-limited chemostat cul-tures, the specific production rate of penicillin,qpen, is independent of specific growth rate fromjust below the maximum to a very low growthrate (about one-eighth ofthemaximal growthrate).(ii) When the specific growth rate is decreased tobelow about one-eighth of the maximal value, thepenicillin synthetic activity of the mold cannot bemaintained. (iii) When the specific growth rate isdecreased to below one-eighth of the maximalvalue, then the qpen falls at a rate inversely relatedto the previous specific growth rate.The relationship between growth rate and

qpen in glucose-limited chemostat cultures (equa-tion 1) and between growth rate and qpen decayafter growth has stopped (equation 2) can beused to predict the steady-state concentrations ofpenicillin in single-stage and multistage chemo-stats (8). It is assumed that the behavior of thewhole culture in the phases of the experiments inwhich growth was stopped by restricting the glu-cose supply to the maintenance ration was thesame as that of a single element of culture passedfrom a growing first stage to a maintained secondstage. Then, with the relationships described byequations 1 and 2, and by use of the expressionfor the distribution of residence times in a chemo-stat culture, the penicillin titer in a maintained,nongrowing second stage can be calculated.The quantity of organism with a residence time

of t hours = x2 e-D2t, where D2 is the dilutionrate of the second stage and x2 = the organismconcentration.The qpen of the organism t hours after entry into

the nongrowing stage is given by equation 2(qpen(o) - kt). By summation from t = 0 to t =q (4 being the age of the mold at which qpenbecomes zero), the penicillin titer increments inthe second stage become

X2- eD2t(qp.n(o) - kt) * dt (3)

= [X2 - D2t(-qp..(o) + kt+ kID2)] (4)

Figure 7 shows the predicted penicillin con-centrations for the observed values of qpen decayrate (Fig. 4) for an organism concentration of1 g/liter dry weighs and second-stage mean resi-dence times 0 to 200 hr. The intercept on thepenicillin concentration axis indicates the titerobtained in the first stage. A test of these calcula-

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mean residence time h

FIG. 7. Steady-state penicillin concentrations inthe second stage of two-stage processes as a functionof the mean residence time of the second stage sup-plied the maintenance glucose ration. Titers werecalculated by the method given in the text by use ofthe q1,z(,) and qpcn decay rates observedfor Penicilliumchrysogenum Wis 54-1255 (Fig. I and 4) and an or-ganism concentration of I glliter o0 dry weight. As-suming a q.,, decay rate = 0.033 qp.. units/hr, firststage growth rate = 0.086 hr-1 (curve 1); = 0.053 hr-1(curve 2); = 0.038 hr-I (curve 3); = 0.023 hr-1(curve 4); = 0.015 hr-1 (curve 5).

tions is afforded by the results of Pirt and Callow(10) for penicillin production by P. chrysogenumWis 54-1255 in a chain of two chemostats. Theqpen predicted from the data presented here forthe second stage, when the maintenance ration ofglucose is supplied to the second stage, is 0.64;Pirt and Callow (10) observed values of 0.6 and0.7 at glucose supply rates a little above and alittle below the maintenance ration, respectively.At a growth rate close to the critical minimum

for steady-state penicillin synthesis, high titers andmaximal productivity can be obtained in a single-stage chemostat. However, to avoid strain de-generation, i.e., loss of penicillin synthetic abilityobserved on prolonged continuous cultivation, itmay be necessary to use multistage cultures (8).

In the chemostat, there is constant selection forvariants capable of utilizing the limiting substratemore efficiently, i.e., with a higher growth yield(weight of organism produced/weight of substrateconsumed) and with a higher affinity for the sub-strate. Research may show that under some condi-tions loss of the ability to produce penicillin doesnot occur. These conditions may not be those thatare optimal for the expression of the character.Pirt, Thackeray, and Harris-Smith (12) foundthat Pasteurella pestis produced large amounts ofcertain antigens at 37 C but rapidly lost the ability.

However, at 28 C, although small quantities ofantigen were produced, the ability to producethem was not selected against. This may be acommon phenomenon, for when a gene is notexpressed it will not affect the growth yield, andso the continuous culture selection mechanismwill not be operative against it. It may be desir.able, then, in the production of antibiotics andother compounds inessential to the growth of theorganism, to operate a small first stage underconditions unfavorable to the expression of thedesired character. Production would be in asecond and subsequent stage operated underconditions optimal for the accumulation of theproduct.

In the case of penicillin production, the firststage might be sulfur or nitrogen-limited, and thesecond sugar-limited with a growth rate just abovethe minimum for steady-state penicillin synthesis.

Entry into the phase of linear increase in peni-cillin titer in batch cultures may be correlatedwith the minimal growth rate required for steady-state penicillin synthesis. A linear accumulationrate would be observed if the synthetic systemceased to be produced once the specific growthrate had fallen below a critical value.The accumulation rate will depend on the mold

concentration reached before the growth rate fallsbelow the critical value. To achieve the maximalaccumulation rate, most of the growth shouldoccur at a rate above the critical value. The resultsshow that the decay of the qpen of nongrowingmold from chemostat cultures was slowest aftergrowth at high rates and that the decay was in-hibited by glucose supplied a little in excess of themaintenance ration. Therefore, in an optimalbatch fermentation, an essential (though not suf-ficient) condition should be an initial fast growthphase to give a high organism concentration,followed by a phase of slow growth to minimizethe qp.n decay rate. This type of fermentation wasdeveloped empirically early in the history of peni-cillin production by the adoption of CSL as amajor medium component. In addition to itspH buffering capacity, CSL contains rapidlyutilizable carbon and nitrogen sources whichallow considerable growth at a high rate. Hence,an important criterion of the suitability of a batchof CSL for the penicillin fermentation should beits ability to support a high growth rate of themold. The slow growth of the second phase wasa result of the slow utilization of a substrate suchas lactose and later an empirically chosen slowsugar feed rate (3).The concept of "maintenance energy," or more

generally the "maintenance ration" of a nutrient.

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was originally developed to account for thekinetics of carbon and energy source utilized bymicrobes. No real function of the maintenancerequirement was demonstrated. The present workshows that the carbon and energy source main-tenance ration is essential to stabilize the penicillinsynthetic system and prevent its rapid decay inthe absence of growth. Elsewhere (Righelato et al.in press), it is shown that the maintenance ration isan aid to the organism during conidiation.

ACKNOWLEDGMENTSWe are grateful to Imperial Chemical Industries

(Pharmaceuticals Division) for the gift of materialsand to K. D. MacDonald, Microbiological ResearchEstablishment, Porton, Wiltshire, England, for a giftof the strain of Penicillium and the penicillin assayorganism.A research studentship for one of us (R. C. R.) and

an equipment grant from the Science ResearchCouncil are gratefully acknowledged.

LITERATURE CITE

1. BARTLETF, M. C., AND P. GERHARDT. 1959. Con-tinuous antibiotic fermentation. Biotech. Bio-eng. 1:359-377.

2. GADEN, E. L. JR. 1959. Fermentation processkinetics. Biotech. Bioeng. 1:413-429.

3. HOsLER, P., AND M. J. JOHNSON. 1953. Penicillinfrom chemically defined media. Ind. Eng.Chem. 45:871-874.

4. JARVIS, F. G., AND M. J. JOHNSON. 1947. The roleof the constituents of synthetic media for peni-cillin production. J. Am. Chem. Soc. 69:3010-3017.

5. MAXON, W. D. 1955. Microbiological processreport. Continuous fermentation. A discussion

of its principles and applications. Appl. Micro-biol. 3:110-122.

6. MOYER, A. J., AND R. D. COGHILL. 1946. Penicil-lin. VIII. Production of penicillin in surfaceculture. J. Bacteriol. 51:57-78.

7. PIRT, S. J. 1965. The maintenance energy of bac-teria in growing cultures. Proc. Roy. Soc.(London) Ser. B 163:224-321.

8. PIRT, S. J. 1967. Steady state conditions for syn-thesis of exocellular products, p. 162-172.In E. 0. Powell, C. G. T. Evans, R. E. Strange,and D. W. Tempest [ed.], Microbial physiologyand continuous culture. Her Majesty's Sta-tionery Office, London.

9. PIRT, S. J., AND D. S. CALLOW. 1960. Studies onthe growth of Penicillium chrysogenum in con-tinuous-flow culture with reference to penicillinproduction. J. Appl. Bacteriol. 23:87-98.

10. PIRT, S. J., AND D. S. CALLow. 1961. The produc-tion of penicillin by continuous flow fermenta-tion. Sci. Rept. Ist. Super. Sanita (Engl. ed) 1:250-259.

11. PIRT, S. J., AND R. C. RIGHELATO. 1966. Theeffect of growth rate on the maintenance ofpenicillin synthesis and morphology of Penicil-lium chrysogenum. J. Gen. Microbiol. 45:vii.

12. PIRT, S. J., E. J. THACKERAY, AND R. HARRIS-SMITH. 1961. The influence of environment onantigen production by Pasteurella pestis studiedby means of the continuous-flow technique. J.Gen. Microbiol. 25:119-130.

13. RIGHELATO, R. C., AND S. J. PIRT. 1967. Improvedcontrol of organism concentration in continu-ous cultures of filamentous micro-organisms. J.Appl. Bacteriol. 30:246-250.

14. WHIFFEN, A. J., AND G. M. SAVAGE. 1947. Therelation of natural variation in Penicilliumnotatum to the yield of penicillin in surfaceculture. J. Bacteriol. 53:231-240.

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