Growth Streptomyces Continuous CultureGrowth of Streptomyces aureofaciens in Continuous Culture...

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Growth of Streptomyces aureofaciens in Continuous Culture BOHUMIL SIKYTA, JOSEF SLEZAK, AND MILO3 HEROLD Antibiotics Research Institute, Roztoky near Prague, Czechoslovakia Received for publication August 29, 1960 The technique of continuous cultivation of micro- organisms is in many respects superior to the traditional cultivation methods, and has therefore been used for many purposes. One of its great advantages over classi- cal methods is the possibility of holding a growing population at constant size over long periods of time. Another advantage lies in the fact that the concentra- tion of all chemical substances in the cultivation vessel remains constant. The theoretical basis for the continuous cultivation has been elaborated only recently, simultaneously by Monod (1950), and Novick and Szilard (1950), although the technique itself had been used before the solution of some theoretical and practical problems (Rogers and Whittier, 1930; Cleary, Beard, and Clifton, 1935; Mialek, 1943). Since that time, the theory has been further developed by a number of authors in some special questions. Most of the published work is con- cerned with the growth of bacteria in continuous culture, but the growth of yeasts, actinomycetes, algae, flagellata, and animal cells has been studied as well. Although lately a number of papers concerned with the continuous cultivation of actinomycetes have been published (Brown, 1959; Bartlett and Gerhardt, 1959; Sikyta, Doskocil, and Kasparova, 1959), it seems that an exact measuring of the growth of actinomycetes under various steady states has not so far been under- taken. It has been the aim of the present work to study the behaviour of the actinomycete Streptomyces aureofaciens as a representative of filamentous organisms, under exactly defined conditions of continuous growth, while using two nutrients as limiting factors, and to find out to what extent this behaviour is in accordance with the theory of continuous cultivation. MATERIALS AND METHODS Organism. S. aureofaciens strain BMK from the collection of the Antibiotics Research Institute in Roztoky near Prague was used. Medium. The composition of the synthetic fermenta- tion medium was as follows: sucrose, 2 per cent; ammo- nium sulphate, 0.5 per cent; potassium phosphate monobasic, 0.68 per cent; sodium hydroxide, 0.125 per cent; magnesium sulphate heptahydrate, 0.05 per cent; 1 All symbols used in this work were taken from the paper of Herbert Elsworth, and Telling (1956). sodium chloride, 0.2 per cent. The individual compo- nents of the medium were dissolved in distilled water and the medium was sterilized in a sterilization tank with mixing at 120 C for 20 min. After sterilization, the pH was 6.8 to 7.0. In this medium either sucrose (0.2 or 0.4 per cent) or ammonium sulphate (0.05 or 0.1 per cent) were used as growth limiting factors, the other components being present in excess. Continuous culture apparatus. The equipment for the continuous cultivation consisted of (a) a tank of 100-L working capacity in which the nutrient medium was prepared and sterilized; (b) a smaller tank of 20-L working capacity which was used for storing the nu- trient medium while another batch of the medium was being prepared in the sterilization tank; and (c) a fermentor which was of a design described in detail by Sikyta et al. (1958). This fermentor has an internal diameter of 24 cm. and a height of 49 cm; the working capacity is 10 L. It is equipped with an open-turbine- type agitator (six flat blades with a diameter of 12 cm), a sparger and four baffles. The agitating velocity was 400 rpm, the air flow 0.5 volume per volume of culture per min. The oxygen transfer rate, determined by the method of Cooper, Fernstrom, and Miller (1944) was 1.5 ml 02 per ml per hr. The feed-rate of the medium into the fermentor was adjusted by means of a peristal- tic pump and the level of the culture fluid in the vessel was maintained constant by an overflow tube. The fermented medium was collected in a collector. In all experiments described in this paper, the temperature in the culture vessel was 28 C. The sterile synthetic medium in the fermentor was invariably inoculated with 50 ml of a 24-hr-old inocu- lum, grown in flasks on a reciprocal shaking machine. The synthetic medium was used as inoculating medium in the flasks with the addition of 0.1 per cent corn steep liquor (solids); it had been demonstrated that experi- ments run in tanks inoculated with an inoculum grown in a synthetic medium without corn steep did not -furnish reproducible results. Analytical methods. Samples were taken every 3 hr and tested for pH value and dry weight of the myce- lium; samples were tested every 6 hr for ammonia- nitrogen content and sucrose concentration, and the chlortetracycline content was assayed biologically according to Hess (1955); the purity of the microbial species was examined in a sample taken every 12 hr. In 233 on February 20, 2020 by guest http://aem.asm.org/ Downloaded from

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Page 1: Growth Streptomyces Continuous CultureGrowth of Streptomyces aureofaciens in Continuous Culture BOHUMIL SIKYTA, JOSEF SLEZAK, AND MILO3 HEROLD Antibiotics Research Institute, Roztoky

Growth of Streptomyces aureofaciens in Continuous CultureBOHUMIL SIKYTA, JOSEF SLEZAK, AND MILO3 HEROLD

Antibiotics Research Institute, Roztoky near Prague, Czechoslovakia

Received for publication August 29, 1960

The technique of continuous cultivation of micro-organisms is in many respects superior to the traditionalcultivation methods, and has therefore been used formany purposes. One of its great advantages over classi-cal methods is the possibility of holding a growingpopulation at constant size over long periods of time.Another advantage lies in the fact that the concentra-tion of all chemical substances in the cultivation vesselremains constant.The theoretical basis for the continuous cultivation

has been elaborated only recently, simultaneously byMonod (1950), and Novick and Szilard (1950), althoughthe technique itself had been used before the solution ofsome theoretical and practical problems (Rogers andWhittier, 1930; Cleary, Beard, and Clifton, 1935;Mialek, 1943). Since that time, the theory has beenfurther developed by a number of authors in somespecial questions. Most of the published work is con-cerned with the growth of bacteria in continuousculture, but the growth of yeasts, actinomycetes, algae,flagellata, and animal cells has been studied as well.Although lately a number of papers concerned with thecontinuous cultivation of actinomycetes have beenpublished (Brown, 1959; Bartlett and Gerhardt, 1959;Sikyta, Doskocil, and Kasparova, 1959), it seems thatan exact measuring of the growth of actinomycetesunder various steady states has not so far been under-taken.

It has been the aim of the present work to study thebehaviour of the actinomycete Streptomyces aureofaciensas a representative of filamentous organisms, underexactly defined conditions of continuous growth, whileusing two nutrients as limiting factors, and to find outto what extent this behaviour is in accordance with thetheory of continuous cultivation.

MATERIALS AND METHODS

Organism. S. aureofaciens strain BMK from thecollection of the Antibiotics Research Institute inRoztoky near Prague was used.Medium. The composition of the synthetic fermenta-

tion medium was as follows: sucrose, 2 per cent; ammo-nium sulphate, 0.5 per cent; potassium phosphatemonobasic, 0.68 per cent; sodium hydroxide, 0.125 percent; magnesium sulphate heptahydrate, 0.05 per cent;

1 All symbols used in this work were taken from the paperof Herbert Elsworth, and Telling (1956).

sodium chloride, 0.2 per cent. The individual compo-nents of the medium were dissolved in distilled waterand the medium was sterilized in a sterilization tankwith mixing at 120 C for 20 min. After sterilization, thepH was 6.8 to 7.0. In this medium either sucrose (0.2or 0.4 per cent) or ammonium sulphate (0.05 or 0.1 percent) were used as growth limiting factors, the othercomponents being present in excess.

Continuous culture apparatus. The equipment for thecontinuous cultivation consisted of (a) a tank of 100-Lworking capacity in which the nutrient medium wasprepared and sterilized; (b) a smaller tank of 20-Lworking capacity which was used for storing the nu-trient medium while another batch of the medium wasbeing prepared in the sterilization tank; and (c) afermentor which was of a design described in detail bySikyta et al. (1958). This fermentor has an internaldiameter of 24 cm. and a height of 49 cm; the workingcapacity is 10 L. It is equipped with an open-turbine-type agitator (six flat blades with a diameter of 12 cm),a sparger and four baffles. The agitating velocity was400 rpm, the air flow 0.5 volume per volume of cultureper min. The oxygen transfer rate, determined by themethod of Cooper, Fernstrom, and Miller (1944) was1.5 ml 02 per ml per hr. The feed-rate of the mediuminto the fermentor was adjusted by means of a peristal-tic pump and the level of the culture fluid in the vesselwas maintained constant by an overflow tube. Thefermented medium was collected in a collector. In allexperiments described in this paper, the temperature inthe culture vessel was 28 C.The sterile synthetic medium in the fermentor was

invariably inoculated with 50 ml of a 24-hr-old inocu-lum, grown in flasks on a reciprocal shaking machine.The synthetic medium was used as inoculating mediumin the flasks with the addition of 0.1 per cent corn steepliquor (solids); it had been demonstrated that experi-ments run in tanks inoculated with an inoculum grownin a synthetic medium without corn steep did not-furnish reproducible results.

Analytical methods. Samples were taken every 3 hrand tested for pH value and dry weight of the myce-lium; samples were tested every 6 hr for ammonia-nitrogen content and sucrose concentration, and thechlortetracycline content was assayed biologicallyaccording to Hess (1955); the purity of the microbialspecies was examined in a sample taken every 12 hr. In

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B. SIKYTA, J. SLEZAK, AND M. HEROLD

some cases, the amino-nitrogen content (Schroeder,Kay, and Mills, 1950) of the medium and the nitrogenin the dry weight of the mycelium was determined.

For analytical assays 100 ml of the sample were with-drawn to flasks containing 1 ml of a 50 per cent solutionof mercuric chloride.

RESULTS

Growth in batch culture. To insure that the limitingsubstrate used was in fact the only limiting substrate,experiments were run in fermentors using a syntheticmedium with the following concentrations of sucrose:0, 0.2, and 0.4 per cent, and the following concentrationsof ammonium sulphate: 0, 0.05, and 0.1 per cent. Whenzero concentrations of the limiting substrates wereused, no growth occurred; with the remaining concen-trations the termination of the logarithmic phase (infact, practically the termination of growth altogether,for the logarithmic phases ended rather abruptly) cor-responded well with the complete depletion of thelimiting factor.The values of the dry weight of mycelium, obtained

at 3-hr intervals, were used for determining the maxi-mal growth rate (,Mm).' For the logarithmic phases ofthe growth curves (natural logarithms of the dry weightof mycelium plotted against time) which took in indi-vidual experiments 15 to 21 hr, the slopes of the straightlines were determined by calculating their regressioncoefficients; this was done for both concentrations ofthe two limiting factors.From the values obtained, the average value of the

slope, 0.18, was calculated, i.e., the average value ofthe maximal, attainable growth rate of the strain S.aureofaciens in the synthetic medium used. This valuedetermines at the same time the maximal, theoreticaldilution rate, which can be used for the cultivation ofthis microorganism without having it washed out fromcultivation vessel.

Growth in continuous culture. The experiments wereperformed by allowing the cultivation (after inoculatingthe nutrient broth in the tank) to proceed batchwiseuntil a concentration of the organisms reached 80 to 90per cent of the expected value. This concentrationusually takes place between 18 to 24 hr. Using lowerdilution rates (lower than 0.08 hr-1) the steady statebecame settled after exchanging approximately onevolume of the nutrient broth; whereas, using dilutionrates over 0.08 hr-1, the steady-state was achieved afterexchanging approximately two volumes of the nutrientbroth in the fermentor. Therefore, measurements wereperformed only after exchanging the respective vol-umes. We proceeded in this way not only when startingthe first run of an experiment, but also when changingone steady state for another, i.e., when alternatingbetween a higher and a lower dilution rate. The actualmeasuring of the values at a given dilution rate were

performed for a period of 27 to 65 hr, after which thedilution rate was changed. This was continued until anincrease occurred in the dry weight of the mycelium,due to growth of the microorganisms on the walls of thevessel, or until contamination by another microorgan-ism or some mechanical defect made it necessary tointerrupt the experiment.The dry weight of mycelium remainied at a given

dilution rate (and within the margin of experimentalerror) constant for the entire duration of the measuring.As growth limiting factors, sucrose in 0.2 and 0.4 percent concentration and ammonium sulphate in 0.05and 0.1 per cent concentration were used.

Sucrose limitation. It can be seen from figures 1 and 2that the values of the dry weight of mycelium remainconstant within a certain range of dilution rate (D =

0.052 to 0.17 hr-1). When the DM value is exceeded, adecrease of the dry weight of mycelium takes place.Complete washing out of the organisms occurs at a dilu-tion rate about 0.2 hr-1, this value, obtained by extra-polation, is denoted Dc in the figure. A drop in the dryweight of mycelium was observed even at dilution rateslower than 0.052 hr-'. The nitrogen content in the dryweight of the mycelium is approximately constant at alldilution rates examined, about 11.5 per cent (figure 3).The output of the dry weight of mycelium increaseswith increasing dilution rate up to DM, after which itdrops abruptly. In the range of dilution rates, 0.02 to0.052 hr-', the output is smaller in accordance with thelower weight of dry mycelium. The yield constant has aconstant value 0.43 in the range of dilution rates, 0.052to 0.17 hr-1; at dilution rates below 0.052 hr-' and above0.17 hr-1, it decreases (figure 4). The substrate concen-tration is practically nil up to the dilution rate 0.17

Dilution rateFigure 1. Steady-state relationships in continuous culture

of Streptomyces aureofaciens using sucrose as the limitingfactor in the inflowing medium. Media: O;4 per cent sucrose and(e) dry weight mycelium, (0) sucrose concentration, (03) out-put of dry weight mycelium; 0.2 per cent sucrose and (A) dryweight mycelium, (A) sucrose concentration, (A) output ofdry weight mycelium.

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CONTINUOUS CULTURE OF S. AUREOFACIENS

hr-1, whereupon the substrate begins to appear in themedium.Very interesting is the correlation between the pro-

duction of the antibiotic and the dilution rate (figure 2).At low dilution rates (lower than about 0.05 hr-1) noantibiotic is produced. At higher dilution rates (over

Dilution roteFigure 2. Steady-state relationships in continuous culture

of Streptomyces aureofaciens using sucrose as the limitingfactor in the inflowing medium. Media: 0.4 per cent sucrose and(*) chlortetracycline concentration, (0) chlortetracycline/dryweight mycelium, (0) output of chlortetracycline; 0.2 per centsucrose and (A) chlortetracycline concentration, (A) chlorte-tracycline/dry weight mycelium, (A) output of chlortetracy-cline.

-t5

-.5

IC2t

sucrose /itaiition* ammoniumn sw/phate liati1on

o 0o5 61 615 02Dilution rate

Figure 3. The influence of the type of limiting factor usedon the N content in the dry weight of mycelium at differentdilution rates.

" Q3-

wQ2

a1

0 0*

* 0

* sucro5s 02peicento sucrose a4/#p.ctten

o Q05 OI 015 0.2Dilution rate

Figure 4. Relationship between the yield constant valuesand dilution rate in sucrose limitation.

0.052 hr-1 up to DM), the concentration of the antibioticin the broth is held at a constant value. Because thedry weight of mycelium (in the said range of dilutionrate) remains constant as well, the ratio chlortetra-cycline to 1 g dry weight of mycelium is also constant.At higher values of dilution rates (when D = 0.17 hr-'is exceeded) the content of the antibiotic in the nutrientbroth varies considerably and is therefore not listed inthe figures.

Similar to the production of the antibiotic, theproduction of yellow pigments is also dependent on thedilution rate. Exact determinations of their concentra-tion, e.g. colorimetric, would be rather difficult: whenattempts were made to isolate them, they were shownto be composed of a complex of a least 6 compoundsof different colors (unpublished experiments); there-fore, these pigments were merely estimated by visualobservation. At those dilution rates where no antibioticis produced, pigments are not excreted to the medium,and the medium is whitish. At dilution rates above 0.05hr-', excretion of pigments to the medium startssimultaneously with the production of the antibiotic,and the medium becomes yellow. Strong pigmentationof the medium and mycelium occurs at dilution rateshigher than 0.08 hr-1. It follows from these observa-tions that there is a close relationship between theproduction of the antibiotic and that of the pigments. Anonpigmented mycelium, producing no antibiotic, ap-pears at dilution rates lower than 0.05 hr-' after 40 to 80hr of continuous growth. The time necessary for thisdepends on the dilution rate in such a way, e.g., with adiminishing value of the dilution rate that the timenecessary for the disappearance of the antibiotic andpigments at both concentrations of the limiting factorsgrows shorter.WVhen the dilution rate is altered repeatedly during

one single run, the nonpigmenting mycelium grown ata dilution rate lower than 0.05 hr-1 is found to startproducing the pigments on increasing the dilution rateover 0.05 hr-1, and the production of the pigmentsceases again upon decreasing the dilution rate below0.05 hr-'; for that reason the process may be assumed tobe reversible. Simultaneously with these changes inpigmentation the concentration of the antibioticchanges correspondingly.These observations applv to both concentrations of

the limiting substrate.Ammonium sulphate limitation. Figures 5 and 6 sum-

marize the results obtained during continuous growthof a culture of S. aureofaciens on a synthetic mediumwith the limiting concentrations of 0.05 and 0.1 percent of ammonium sulphate. The relationship betweenthe values of the dry weight of mycelium and thedilution rate (figure 5) is the same as in similar experi-ments with sucrose limitation. At very low dilutionrates, the values of the dry weight of mycelium decrease

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for both concentrations of ammoniuin sulphate used.The shape of the output curve of the dry weight ofmycelium is analogous to sucrose limitation. The nitro-gen content in the dry weight of mycelium (figure 3)decreases at lower dilution rates from an average valueof 11 per cent to as low as 8 to 9 per cent. This decrease,and also a generally lower nitrogen content in the dryweight of mycelium grown at all dilution rates used,differentiates the ammonium sulphate limitation fromsucrose limitation. The substrate first appears at thedilution rate 0.16 hr-1, i.e., somewhat sooner than isthe case with sucrose being used as the limiting factor.

D 4z

ZI 2~~~~~~~~~~~~2J-1 ~ -

L.01

0 W5 0.1 05 Q0 bDilution rare Pc

Figure 5. Steady-state relationships in continuous cultureof Streptomyces aureofaciens using (NH4)2SO4 as the limitingfactor in the inflowing medium. Media: 0.1 per cent (NH4)2SO4and (-) dry weight mycelium, (O) NH3-N concentration, ((i))output of dry weight mycelium; 0.05 per cent (NH4)2SO4 and(A) dry weight mycelium, (A\) NH3-N concentration, (A) out-put of dry weight mycelium.

o ct

43

%Jo

a-la

0)

t~~~~ -

0 00.5 0.1 015 CDilution rate

12t

444:

2Z'S

Figure 6. Steady-state relationships in continuous cultureof Streptomyces aureofaciens using (NH4)2SO4 as the limitingfactor in the inflowing medium. Media: 0.1 per cent (NH4)2SO4and (0) chlortetracycline concentration; (0) chlortetracy-cline/dry weight mycelium, ((0) output of chlortetracycline;0.05 per cent (NH4)2SO4 and (A) chlortetracycline concentra-tion, (A) chlortetracycline/dry weight mycelium, (A) outputof chlortetracycline.

The dry weight of mycelium was proportional to theconcentration of the limiting substrate in the feed.

Principally, the ammonium sulphate limitation differsfrom sucrose limitation in antibiotic and pigmentformation. With nitrogen limitation, both the antibioticand the pigments are produced even at very low dilutionrates (figure 6). It is possible that at still lower dilutionrates (slower than 0.02 hr-', which was the lowest dilu-tion rate used) the antibiotic and the pigments woulddisappear, but this could not be verified for technicalreasons. At any rate, the loss of the ability to producethe antibiotic and the pigments is, with nitrogen limita-tion, shifted to extremely low values of dilution rates.In trial experiments run at the dilution rate 0.022 hr-1,the production of the antibiotic and the pigments didnot stop even after 150 hr of growth. With nitrogenlimitation, the amount of the antibiotic produced issomewhat less than with sucrose limitation, althoughthe values of the dry weight of mycelium are higher withnitrogen limitation than in corresponding runs withsucrose limitation. This is why the total output of theantibiotic and the amount of antibiotic per unit of thedry weight of mycelium is lower, as compared withsucrose limitation.

DISCUSSIONComparison of Behaviour of Filamentous with

Nonfilamentous Organisms in ContinuousCulture

The theory of continuous cultivation, elaborated byMonod (1950) and Novick and Szilard (1950), wasextensively verified, especially by Herbert, Elsworth,and Telling (1956), for nonfilamentous organisms. Asit has not been so far ascertained exactly to whatextent the behaviour of filamentous organisms incontinuous culture is in agreement with the laws derivedfor nonfilamentous organisms, we tried to verify thistheory for filamentous organisms.From the curves in figures 1, 4, and 5, it can be seen

that S. aureofaciens behaves in continuous culture inaccordance with assumptions derived by Monod, exceptfor the decrease of dry weight of mycelium at low dilu-tion rates and at those which are higher than a maximalgrowth rate. These variations are of the same characteras those of nonfilamentous microorganisms and thepossible cause shall be discussed later. According to thetheory of the continuous cultivation, the behaviour oforganisms in continuous culture with substrate limita-tion is characterized by three constants: the maximalgrowth rate (ju), the yield constant (Y), and the satura-tion constant (K8).Maximal growth rate (Mllm). The maximal growth rate of

S. aureofaciens in batch cultivation is about 0.18 hr-1,irrespective of the type or concentration of the limitingfactor used. As compared with the maximal growth rateof bacteria and yeasts, this value is about five times

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CONTINUOUS CULTURE OF S. AUREOFACIENS

smaller (MIonod, 1950; Herbert et al., 1956; Holme,1957). It is well known that the value of the maximalgrowth rate is dependent on the composition of themedium; from that it may appear that the low maximalgrowth rate is the result of the use of a synthetic me-dium. In our case, however, this is not so, for even anatural medium containing corn steep liquor failed toyield a higher maximal growth rate (unpublished data).As can be seen from figures 1 and 5, the critical dilutionrate, Dc (at this rate the microorganisms would be,under ideal conditions, completely washed out from thecultivation vessel), is invariably higher than the maxi-mal growth rate determined in batch cultivation (figure1). This fact, often quoted by other authors for non-filamentous organisms, is largely explained by thegrowth of microorganisms on the walls of the fermenta-tion vessel or by imperfect mixing (Herbert et al., 1956).Pirt (1957) states that this phenomenon is caused by abetter adaptation of microorganisms to the cultivationconditions during a longer continuous cultivation.

Yield constant (Y). It proved impossible to determinereliably the yield constant from batch cultivation databecause the termination of the logarithmic phase ofgrowth was usually followed by a rapid drop in thedry weight of mycelium as a result of autolysis. In acontinuous culture with sucrose limitation, the yieldconstant remained unchanged in the range of dilutionrates from 0.08 to 0.15 hr-1 and equalled 0.43; this valuedecreased at dilution rates lower than 0.08 hr-' or higherthan 0.15 hr-1 to 0.32 (figure 4). The shape of the curveshowing the relationship between the values of theyield constant and the dilution rate was the same forboth sucrose concentrations used. A similar shape isusual in nonfilamentous organisms. Herbert et al. (1956)and Maxon and Johnson (1953) explain the decrease ofthe yield constant at low dilution rates by presumingthat, thanks to endogenous respiration, more carbonfromn the carbon source is incorporated into carbon di-oxide than into the cell matter. The decrease of the yieldconstant at high dilution rates is explained by the pro-duction of different compounds which are not formed atlow dilution rates (Maxon and Johnson, 1953; Pirt,19.57).

Saturation constant (K8). The relationship betweenthe growth rate and the concentration of the limitingfactor (Monod, 1950)

SJ = Mil/m *KJ+s

certainly is too simple to express accurately the compli-cated process of growth (recently other, more compli-cated relationships have been postulated) but is quitehelpful for such purposes where it is not necessary toexpress directly the reaction kinetics. This constantwas not calculated from batch data, as in batch cultiva-tion the logarithmic phase ends too abruptly. It cannot

be calculated from data obtained in continuous cultiva-tion at low dilution rates, for the concentration of thelimiting substrate is too low and lies beyond the accur-acy limit of the assay method. We saw little purposein calculating it from values obtained at high dilutionrates, respective from the DM value (Herbert et al.,1956), as these values are probably already influencedby the "apparatus effect" (wall growth and imperfectmixing). It can be said with certainty, however, that,as with bacteria, the K8 is very low because the log-arithmic phase terminates very abruptly in batch culti-vation and, in continuous cultivation, a very steep de-cline in the dry weight of mycelium occurs as soon asDM is exceeded.

Influence of Type of Limiting Substrate and DilutionRate on Nitrogen Content in Dry Weight of MyceliumThe data concerning changes of the nitrogen content

in the dry weight of mycelium are in accordance withsimilar studies performed on bacteria by Holme (1957)and Formal, Baron and Spilman (1956). When con-sidering the decrease of nitrogen content in the dryweight of mycelium in nitrogen limitation, accom-panying the lowering of the dilution rate, it must beemphasized that, in our experience, this phenomenoncannot be explained by the production of nitrogen-free compounds in the mycelium (e.g., glycogen (Holme,1957)); there is, simultaneously with the decrease ofnitrogen content in the dry weight of mycelium, a dropin the dry weight of mycelium itself. Instead, it seemsprobable that nitrogenous compounds are produced andreleased to the medium; this possibility is corroboratedby the observed increase in concentration of amino-nitrogen at low dilution rates in nitrogen limitation.

Production of Chlortetracycline and Pigments inContinuous Culture of S. aureofaciens

In previously published papers, the continuous bio-synthesis of antibiotics was aimed at the practicalutilization of the continuous method. We have there-fore in our experiments followed the production of anantibiotic under well-defined conditions in dependenceon the dilution rate, and type and concentration of thelimiting factor, even though the synthetic medium wasnot favourable to the antibiotic production. The concen-trations of the antibiotic obtained are in fact very low,yet still exactly measurable.From the above results, the sudden disappearance of

the antibiotic and pigments at low dilution rates insucrose limitation, and the gradual disappearance ofthe antibiotic and pigments in nitrogen limitation, arethe most interesting. A similar observation was de-scribed by Bartlett and Gerhardt (1959) during thecontinuous biosynthesis of chloroamphenicol (although,in their case, no substrate limitation was concerned),namely a decrease of the antibiotic content in the myce-

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lium resulting from a decrease of the dilution rate. Thephenomenon seems therefore to be of a more generalnature. Incidentally, it has been pointed out that evenin the biosynthesis of simple products such productsas are produced at high dilution rates often cease to beproduced at low dilution rates (Maxon and Johnson,1953; Pirt, 1957).In our experience, two possible explanations present

themselves for the loss of the antibiotic and pigmentproducing ability. At low dilution rates, when the con-centration of the substrate in the cultivation vessel islow, the enzyme surfaces related to the production ofthe antibiotic and pigments are not saturated, so thatneither the antibiotic nor the pigments are produced.Another explanation may be found in the selecting of acertain part of the heterogeneous population. A non-pigmented part of population, producing no antibiotic,might have a higher growth rate at low concentrationsof the limiting factor (at low dilution rates), whereas apigmenting and antibiotic-producing part of populationmight have a higher growth rate at a higher concentra-tion of the limiting substrate (at a higher dilution rate).The second alternative is supported by the fact that theantibiotic and pigment production is not resumed imme-diately upon increasing the substrate concentration inthe cultivation vessel. Undoubtedly, other explanationscould be found, e.g., the difference in the "physiologicalstate" of the culture at different dilution rates, asstressed by Malek (1958). To solve this question and toexplain the differences between nitrogen and sucroselimitation, further research is necessary.

ACKNOWLEDGMENT

The authors wish to thank V. Brynda and V. Kra-tochvil for technical assistance.

SUMMARY

The behaviour of a strain of Streptomyces aureofa-ciens, as representative of filamentous microorganisms,was studied during continuous growth in synthetic me-dium. It was found that under substrate limitation thebehaviour of filamentous microorganisms is the same asnonfilamentous microorganisms. In both cases, the con-formity with the theory is only fair and the discrepan-cies from the theory are of the same character. The pro-duction of chlortetracycline and pigment formationwere also studied with regard to dependence on the type

and concentration of the limiting substrate and the di-lution rate.

REFERENCESBARTLETT, M. C. AND GERHARDT, P. 1959 Continuous anti-

biotic fermentation. Design of a 20 litre, single-stage pilotplant and trials with two contrasting processes. J. Bio-chem. Microbiol. Technol. Eng., 1, 359-377.

BROWN, W. E. 1959 Recent progress in microbiology, pp. 416-417. Edited by G. Tunevall. Almqvist and Wiksell, Stock-holm, Sweden.

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