Archives of
Microbiology
Arch. Microbiol. 107,41-47 (1976)
© by Springer-Verlag 1976
A Method for the Regulation of Microbial Population Density during Continuous Culture at High Growth Rates GLENN A. MARTIN* and WALTER P. HEMPFLING** Department of Biology, University of Rochester, River Station, Rochester, New York 14627, U.S.A.
Abstract. A method for the continuous culture of microorganisms is described which employs growthdependent pH changes to control the rate of addition of fresh medium to a culture vessel. The apparatus (the "phauxostat") supports, at constant pH, longterm continuous culture at rates near or at the maximum of which the organisms are capable. The buffering capacity of the inflowing medium determines the steady-state population density of the culture, but the rate of growth is independent of the buffering capacity. The fundamental theory of operation is tested and some basic parameters of growth are estimated using Escherichia coli B growing continuously in media containing glucose, glycerol or DL-lactate. Key words: Escherichia coli B - Continuous culture ,,Phauxostat" - Growth rate.
The chemostat is not particularly suitable for the purpose of the long term growth of microorganisms near their maximum growth rate. The rate of growth in the chemostat is determined by the rate of addition of culture medium containing a limiting amount of a substance required for growth (Herbert et al., 1956), and, at high dilution rates approaching # . . . . the population tends to wash out. The turbidostat, on the other hand, works most effectively at growth rates near/~ .... for it is in that range that the population density is most accurately controlled (Herbert, 1958). Wall growth has usually posed a problem to continuous culture devices and, since the turbidostat operates by setting the rate of medium input on the basis of photoelectric measure* Joseph C. Wilson Scholar. ** To whom offprint requests should be sent.
ment of population density, wall growth has an especially adverse effect (Anderson, 1953). To overcome this problem vessel walls have been coated with silicone derivatives and "windscreen washing devices" of various designs have been utilized (Anderson, 1956; Northrop, 1954). Besides the problem of wall growth the turbidostat cannot be vigorously aerated in such a way that might interfere with light detection, and experiments with chromophoric substances are also difficult (Bryson and Szybalski, 1952). To circumvent these problems Watson constructed a continuous culture device which employed the measurement of CO2 output to set the rate of medium addition by means of an infrared CO2 analyzer, control unit and medium pump (Watson, 1969). Unfortunately the expense of such analyzers is high enough to limit the number of units in operation in a laboratory. We describe in this paper a method of utilizing the pH change brought about by growth to set the rate of addition of medium. The device which has been developed allows extended continuous culture in the steady state but has none of the shortcomings of the turbidostat. We call the device the "phauxostat", since it functions by using the pH of the medium to maintain growth (auxo, from the Greek auxein, to increase) at a constant rate (stat, from the Greek -states, one that causes to stand). This is accomplished by allowing inflowing medium to return the pH value of the medium in the growth vessel to some preset value. Cells and spent medium exit from the growth vessel through an overflow portal. THEORY OF OPERATION During steady state growth changes of the proton concentration must be balanced by the inflow of the culture medium. If the metabolism of the culture is such as to produce protons, then the end point for
42
G.A. Martin and W. P. Hempfling
t i t r a t i o n is set so t h a t the buffering a c t i o n o f the inflowing m e d i u m decreases the p r o t o n c o n c e n t r a t i o n . S h o u l d cellular m e t a b o l i s m r e m o v e p r o t o n s f r o m the m e d i u m in the c u l t u r e vessel, then the end p o i n t o f t i t r a t i o n is set so t h a t inflowing m e d i u m increases the p r o t o n c o n c e n t r a t i o n . In a d d i t i o n to the buffering effect o f fresh m e d i u m , the c o n c e n t r a t i o n o f p r o t o n s will t e n d to c h a n g e in the vessel in p r o p o r t i o n to the p H difference b e t w e e n the m e d i u m in the vessel a n d the m e d i u m in the reservoir. Since the p H o f the m e d i u m in the vessel r e m a i n s c o n s t a n t d u r i n g the s t e a d y state, the a d d i t i o n o r subt r a c t i o n o f p r o t o n s b y a n y o t h e r event is negligible. T h e rate o f c h a n g e o f p r o t o n c o n c e n t r a t i o n in the growth medium under conditions of acid-producing m e t a b o l i s m m a y be expressed as follows:
dH+/dt=btxh+D[H~]-D[Hv]-D(BCR),
(1)
where # is the specific g r o w t h rate ( h - l ) ; x is the p o p u l a t i o n d e n s i t y [lag (dry weight) p e r ml]; h is the s t o i c h i o m e t r y o f p r o t o n p r o d u c t i o n related to growth, or H+/x; D is the d i l u t i o n rate, o r ml i n p u t o f m e d i u m p e r h o u r d i v i d e d b y the v o l u m e o f m e d i u m in m l in the culture vessel ( h - l ) ; [ H i ] is the p r o t o n c o n c e n t r a t i o n in the m e d i u m c o n t a i n e d in the reserv o i r ; [ H +] is the p r o t o n c o n c e n t r a t i o n in the m e d i u m c o n t a i n e d in the c u l t u r e vessel, a n d BCR is the buffering c a p a c i t y o f the m e d i u m in the reservoir. W e define the buffering c a p a c i t y as the a m o u n t o f acid or alkali r e q u i r e d to c h a n g e the p H o f i 1 o f the m e d i u m in the reservoir to the p H o f the m e d i u m in the g r o w t h vessel. E q u a t i o n (1) can be simplified to
d H + / d t = # x h - D ( [ H ~ ] - [H~I)-D(BCR)
(2)
w h e r e the t e r m D ( [ H v ] - [ H i ]) represents the effect on p H o f a d d i n g m e d i u m f r o m the reservoir to the culture vessel. Since the p r o t o n c o n c e n t r a t i o n s o f the m e d i u m in the vessel a n d the reservoir are u s u a l l y very n e a r to each other, this t e r m reduces to a very small value a n d m a y be i g n o r e d , except in cases w h e n the difference in c o n c e n t r a t i o n s is large. Since such a c o n f i g u r a t i o n is n o t c o n s i d e r e d here, the expression t h e r e f o r e reduces to
dH+/dt = #xh - D (BCR).
(3)
Since, b y definition, in the s t e a d y state d H +/dt = zero, then
# xh = D (BCI¢).
(4)
BCR is c o n s t a n t at a given p H value in the vessel, a n d there is no r e a s o n to expect t h a t the value o f h will c h a n g e at c o n s t a n t p H , I n the s t e a d y state, x is constant, a n d # = D, so Eq. (4) m a y be further simplified to x h = (BCR).
(S)
I f h is i n d e p e n d e n t o f (BCR) then x can be v a r i e d by c h a n g i n g (BCR). F u r t h e r m o r e the g r o w t h rate in the steady state s h o u l d be i n d e p e n d e n t o f the buffering c a p a c i t y o f the inflowing m e d i u m , unless the buffer exerts a n effect on the cells in a d d i t i o n to simple r e a c t i o n with p r o t o n s in the m e d i u m . In the following a c c o u n t o f the characteristics o f g r o w t h in the p h a u x o s t a t we d e m o n s t r a t e t h a t r e a s o n able a g r e e m e n t is o b s e r v e d b e t w e e n the p r e d i c t i o n s o f the f o r e g o i n g d e s c r i p t i o n o f t h e o r y a n d the experim e n t a l results.
MATERIALS
AND METHODS
Organism EseherichiacoliB was maintained and cultivated prior to inoculation into the phauxostat as described (Hempfting and Mainzer, 1975). Growth Media The minimal medium employed, described elsewhere (Hempfling, 1970), was prepared in 3 1batches and sterilized for 40 min at 121° C. Potassium phosphate buffer, pH 7.0, and glucose, both at concentrations of I M, were autoclaved separately and added to the sterile minimal base after cooling. The final concentration of glucose was 10 mM and phosphate was varied from 5 mM to 50 mM. When additional buffering capacity was required 1 M bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl) methane (Bis-Tris), pH 7.2, was sterilized by autoclaving and then added to the sterile minimal base. If the medium was supplemented with amino acids Nutritional Biochemical Company's Casein Hydrolysate was added to the minimal base at a concentration of 0.5 ~ (w/v) before autoclaving.
Batch Culture Growth in batch culture was accomplished using a New Brunswick Microferm Fermentor with 2 1 culture volume in a 5 1 vessel. The culture was aerated at 2 1of air/min and was stirred at 400- 600 rpm. The temperature was maintained at 37°C. The values of growth rates reported were obtained during the logarithmic period of growth. Dry weight measurements were carried out as described (Hempfling and Mainzer, 1975).
The Phauxostat The culture vessel employed was a Buchner funnel flask with tubulature (which served as the portal for effluent medium and gas) at such a height so as to give a culture volume of 125 ml. Growth medium was added through a stainless steel catheter inserted through a silicone rubber stopper which closed the top of the flask. Apertures for the pH electrode, for air entry and a glass tube sealed at the bottom to serve as a well for a thermistor probe were also placed in the stopper. The flow of medium into the vessel was regulated by a Radiometer magnetic valve (Type MNV lc) controlled by a pH meter (Radiometer Model priM 28) and a titrator unit (Radiometer Model TTT lib). The combination pH electrode used was the Radiometer Model GK23J50; it was sterilized as described (Hempfling and Mainzer, 1975).
Continuous Culture at High Growth Rates The culture medium was mixed by a Teflon-covered magnetic stirring bar driven by a magnetic stirrer placed directly under the vessel. Air passed through a Teflon tube of I mm diameter into the culture medium was from the laboratory compressed air supply delivered through a pressure reducing valve (The Matheson Co., Model 70) and monitored with a Matheson flowmeter (No. 601). Aeration occurred at 1.2-1.5 l/h, and the air was sterilized as described (Hempfling and Mainzer, 1975). When anaerobic conditions were required, both the culture vessel and the reservoir received a continuous flow of N 2 (Air Products) through suitable submerged tubes. No effort was made to remove the last traces of oxygen from the nitrogen preparation. The temperature in the culture vessel was measured and regulated at 37°C as described (Hempfling and Mainzer, 1975). Spent medium and organisms overflowed through the tubulature provided in the side of the vessel. The tube was extended by attaching a 20 cm length of silicone rubber tubing (6 mm I.D.) terminating in a filling bell; the tubing was bent at 90° downward about 5 cm from the vessel. Before inoculation the entire apparatus (without the pH electrode) was assembled and sterilized by autoclaving.
Growth in Continuous Culture After being filled with medium the phauxostat was inoculated with about 5 ml of a suspension of growing E. coli B (Hempfling and Mainzer, 1975) and the sterilized combination pH electrode was aseptically inserted. Growth was allowed to proceed until the pH of the culture had changed significantly from the desired value, whereupon the pH control circuit was activated and the flow of fresh medium began. The signal from the combination pH electrode tended to drift for about 6 h but then generally became stable; daily calibration (Hempfting and Mainzer, 1975) thereafter showed drift of no more than 0.1 pH unit per 24 h during the first several days of operation, becoming less than 0.05 pH unit per 24 h thereafter. The throughput of 5 culture volumes of medium was more than adequate to assure that a steady state was reached when variation about the mean flow rate did not exceed ± 7 ~ (Hempfling and Mainzer, 1975). Samples of the effluent medium were collected as described and the determination of the flow rate of medium through the vessel was also accomplished as before (Hempfling and Mainzer, 1975). In order to measure residual glucose and acetate formed in the culture, about 10 ml of effluent medium was allowed to pass through a filter of 0.8 gm porosity (Millipore Corp.) in order to remove the bacteria. The spent medium, freed of bacteria, was used for the determination of residual glucose and of acetate by means of the Glucostat (Worthington Biochemical Corp.) and the acetate kinase methods, respectively (Hempfling and Mainzer, 1975). The population density within the culture vessel was measured at 650 nm with Pyrex cuvettes of 1.00 cm light path using a Hitachi-Coleman Perkin Elmer Model 124 Dual wavelength Spectrophotometer. The absorbance of a very dilute suspension of cells (growing at g = 0.9/h) at 420 nm versus that at 600 nm (A42o/A6oo) was found to be 2.18 (Koch, 1970). The relations between dry weight and optical density were determined to be O.D. = 0.336 for a population density of 100 gg (dry weight) per ml at ~t = 0.9 h -I and O.D. = 0.290 for a population density of 100 gg (dry weight) p e r m l a t g = 0.6h -1.
Determination of Buffering Capacity Buffering capacity of the medium in the reservoir was determined at 37° using an Instrumentation Laboratories pH meter (Model 245)
43 in the expanded scale mode and a Markson combination pH electrode (Model 1885). The pH of the reservoir medium was determined and then the volume of 0.10N HC1 or 0.10N NaOH necessary to bring 100 ml of that medium to the pH of the culture was measured.
RESULTS
Effects of Variation of Buffering Capacity on Population Density and Growth Rate The foregoing theory predicts that the population density during steady-state continuous culture in the phauxostat should be dependent on the buffering capacity of the medium in the reservoir and the growth rate should be independent of the buffering capacity. In order to test this prediction, the buffering capacity was varied by changing the concentration of phosphate from 5 - 5 0 mM and by adding Bis-Tris buffer ( 4 - 2 8 mM) to medium which already contained 10 mM phosphate. The pH value of the medium in the reservoir in the experiment in which phosphate was varied remained at 6.85 ( _+ 0.03), but the pH value increased from 6.75 to 6.91 when Bis-Tris was added. The population density and growth rate in the steady state were measured at each value of buffering capacity and the results are shown in Figure 1. The population density increases linearly up to a buffering capacity of about 1.7 milliequivalents of H ÷/1 (meq/1), which corresponds to a phosphate concentration of 15 mM. The function appears not to pass through the origin, but this may be the result of a small error in the determination of the pH of the culture medium. Dependency of population density upon buffering capacity decreases at about 2 meq/1 to a new linear function. The specific growth rate varies from about 0.96/h to 0.90/h over the range of buffering capacity of 0 . 5 - 7 . 6 meq/1. Using an extinction coefficient we calculate that h equals 41.7 meq of H ÷/g (dry weight) at buffering capacities below 1.7 meq/1 and that h equals 128 meq of H+/g (dry weight) at buffering capacities from 2 - 7 . 6 meq/1. Apparently the stoichiometry of acid production per g of cell mass is augmented by increased phosphate concentrations without a concomitant effect on the rate of growth. Since all products of glucose oxidation were not accounted for, we cannot suggest the reason for the change of the value of h. At least part of the theory is confirmed, however, in that the specific growth rate is independent of buffering capacity. When Bis-Tris is used to vary the buffering capacity an increase of h with increasing buffering capacity is less apparent, since h equals 71 meq of H+/g (dry weight) at buffering capacities above 2 meq/1. The specific growth rate, although higher than that ob-
44
G.A. Martin and W. P. Hempfling
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Fig. 1. Population density and specific growth rate of Escherichia coliB as a function of buffering capacity (steady-state pH of culture: 6.70 in phosphate experiment,6.60 in Bis-Trisexperiment) Fig.2. Population density, specific growth rate and glucose metabolism of Escherichia coliB as a function of the steady-state pH of the culture. Reservoir mediumheld at pH 6.85 Fig.3. Population density as a function of buffering capacity. Data from the experiment of Figure 2 served when phosphate serves as the principal buffer, is still relatively constant over an interval of buffering capacities of 3 - 1 1 meq/1. The reason for the increase of growth rateconsequent upon the addition of BisTris is not known.
Effects of Variation of the p H of the Culture Enough deviations from the predictions of the theory of operation were seen in the experiments of Figure 1 to prompt us to seek ways of varying the buffering capacity of the medium in the reservoir without changing the concentration of the buffer. Since preliminary experiments in batch culture had demonstrated that the rate of growth during the logarithmic phase was independent of pH over the range pH 6 . 0 7.0, the pH of the medium was varied over the range pH 6 . 1 - 6 . 7 while holding the phosphate concentration in the reservoir at 15 m M (pH 6.85). In this way the buffering capacity could be varied from about 1 to nearly 10 meq/1 without changing the phosphate concentration. The effects of changing the pH of the culture are shown in Figure 2. As the pH is decreased from 6.7 to 6.1, the optical density of the culture increases in a nearly linear fashion but the specific growth rate remains at about 0.88/h. As expected, the total amount of glucose metabolized by the culture increases with decreasing pH. The. relation between population density and buffering capacity in the experiment of Figure 2 is given in Figure 3. A value of h of 37 meq of H +/g
(dry weight) is calculated from the slope of the linear function, which is directly comparable to that obtained in the experiment of Figure I when the concentration of phosphate was 15 mM or less. The results of this experiment bear out the predictions of the theory. Discrepancies between the theory's predictions and the results of the experiments of Figure i may be explained by effects of the increased buffer concentrations upon the metabolic apparatus of proton production. These observations deserve further investigation.
Effects of Shifts in the Condition of Growth In order to test the responsiveness of the population in the phauxostat to changes in a culture condition which ought to bring about a different rate of growth, a steady state was established in the presence of oxygen and then oxygen was removed by flushing the reservoir medium and the medium in the growth vessel with N 2. After a steady state had been established in the absence of Oz, air was again admitted to the vessel. As shown in Figure 4, within a few minutes after cessation of aeration, the population density fails and the rate of medium addition increases. After 2.5 h the population density reaches a steady level which is maintained for the remainder of the anaerobic period. After 5 h the rate of medium flow becomes stable for a period of 2 h (1.6 generations) at a specific growth rate of 0.56/h. At that point (7 h after the onset of anaerobiosis) air is again admitted, and after an initial decrease in the rate of medium flow, the population
Continuous Culture at High Growth Rates
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Table 1. Comparison of continuous culture in the phauxostat with growth in batch culture
4
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Fig. 4. Population density and medium flow rate in the presence and absence of oxygen (steady-state pH of culture: 6.50. Phosphate concentration 10 mM)
density and the flow rate increase until both the previous values of population density and specific growth rate have been re-established. The results demonstrate that the phauxostat allows smooth transitions from one growth rate to another and that reproducible values of both growth rate and population density can be obtained. We calculate a value for h of anaerobically growing cells in this experiment of 109 meq of H+/g (dry weight). The value of h of the aerobic culture in the experiment of Figure 4 is 32 meq of H÷/g (dry weight).
Effects oJ" Variation of the Source and Direction of pH Change To determine whether or not the phauxostat was capable of supporting growth when the product of metabolism of a neutral carbon source was only CO2, Escherichia coli B was cultivated continuously using glycerol as sole carbon source. Since only negligible production of acetate occurred, the major reaction giving rise to protons was the formation of bicarbonate from water and CO2. A steady state was established and the characteristics of growth are reported in Table 1. A change o f p H opposite in direction to that previously studied is given by growth at the expense of DL-lactate as sole carbon source. Even though appreciable amounts of acetate are formed during lactate oxidation, the major change of pH is due to the formation of CO2, and since the pK' value of the couple HzO + CO2/HCO 3 is higher than that of the couple lactic acid/lactate anion, the pH of the culture increases during lactate metabolism. A steady state was established by setting the pH of the culture above the pH of the reservoir medium and the results are given in Table 1. Even faster growth rates than those previously observed are obtained by supplementing glucoseminimal medium with Casein Hydrolysate. The steadystate growth rate obtained using this medium in the phauxostat is shown in Table 1. Table i also contains
-
a The pH of the culture medium refers to that in the phauxostat in which the pH was controlled. The pH of the culture medium in_ batch culture varied from pH 6.2 (glucose + casein hydrolysate) to pH 7.2 (lactate). b Milliequivalents of H +/g (dry weight) during continuous culture in the phauxostat. c Mean and percent standard deviation in parentheses. a 10 mM glucose and 10-15 mM phosphate buffer (h is calculated from the mean of the values reported for the experiments of Figures 1, 3, and 4). e 20 mM glycerol and 10 mM phosphate buffer (pH 6.74). f 20 mM DL-lactate and 10 mM phosphate buffer (pH 6.78). g Negative sign indicates consumption of protons.
the specific growth rates observed in batch culture. It can be seen that growth rates obtained in the phauxostat are characteristic of each substrate but are uniformly somewhat greater than in batch culture. Growth in a complex medium (glucose plus Casein Hydrolysate) in the phauxostat is 25 ~ faster than in batch culture.
DISCUSSION
Continuous Culture We may consider two distinct types of continuous culture devices (auxostat): that which limits the rate of growth of a microbial population by external control of the rate of input of a growth-limiting concentration of a nutrient (the chemostat) and that in which the rate of medium input is determined by the growth rate of the culture in response to the change of some growth-dependent parameter (light scattering in the turbidostat, CO2 production in the auxostat developed by Watson (Watson, 1969) or change of pH in the phauxostat). Even though the two kinds of auxostats maintain populations in steady-state growth according to the same quantitative expressions (Herbert, 1958), the nutritional state of the two cultures differs between the two types. Hence the physiological properties of a given organism in a chemostat may be different from the same organism growing in the same medium in a phauxostat. This difference may be especially
46 apparent in cases where repressive effects on enzyme biosynthesis may be exerted by a carbon source in the phauxostat but reduced or not apparent when that carbon source limits the growth rate in the chemostat. For a comprehensive study of the determinants of maintenance metabolism, growth yield and growth rate, iris essential that both kinds of auxostats be available to the investigator. The rate of growth may be externally controlled by means of nutrient limitation in any auxostat. For example, the phauxostat may function as a nutrientlimited auxostat (chemostat) by lowering the concentration of say, the source of carbon, to a level which limits the rate of change of proton concentration in the growth medium. The buffering capacity of the reservoir medium must also be lowered so as to permit an adequate change of pH in the vessel. The phauxostat is also effective when it is desired to limit the rate of growth by lowering the concentration of a gaseous nutrient such as oxygen. We have successfully cultivated Escherichia coli B continuously under conditions of oxygen limitation over a range of specific growth rate of 0.09/h to 0.61/h with DL-lactate as carbon source by varying the rate at which air flows into the phauxostat vessel. Under these circumstances the rate of maintenance respiration (Hempfting and Mainzer, ]975) appears to be the same as when lactate limits the rate of growth in the chemostat in the presence of excess oxygen (W. P. Hempfling, unpublished observations). It further seems likely that the phauxostat will be useful for the light-limited continuous culture of photosynthetic microorganisms, and for the cultivation of non-photosynthetic microorganisms at very high population densities.
Apparatus and Operation We set out to construct the phauxostat in the simplest and most inexpensive way available to us. The glassware and tubing used in the culture vessel are available in any laboratory, although we have constructed more elaborate vessels. The most expensive component is, of course, the pH meter and associated control circuitry. The Radiometer TTT 11 titrator unit is particularly useful because of its "proportional band" feature. This circuit allows variation of the frequency of operation of the magnetic valve for medium addition according to the magnitude of difference between the pH of the culture medium and the preset pH value. Higher frequency operation at more narrow intervals and supplementary addition of medium through a peristaltic pump connected in parallel is suitable for cultivating organisms at rapid growth rates.
G.A. Martin and W. P. Hempfling The stability of the population density and the medium flow rate with time has been demonstrated. Transitions to new values of buffering capacity usually result in damped oscillations of both population density and flow rate, but these variations become inconsequential well within the period required for the flow of 5 culture volumes of medium which we customarily allow to elapse when approaching a new steady state. Equipping the apparatus with both peristaltic pump and pH-controlled medium addition connections allow its operation as either a chemostat or a phauxostat, thereby permitting growth over the full range of growth rates of which the culture is capable.
Confirmation of Theory of Operation As shown in the experiments of Figures 1 and 2 the prediction that the rate of growth in the phauxostat should be independent of the buffering capacity of the medium in the reservoir is borne out well enough to accept that feature of the theory of operation. The relation between buffering capacity and population density in those experiments is confused, however, by the effects of buffer concentration on the value of h [amount of protons produced/g (dry weight)]. Nevertheless, when the buffer concentration is held at the same level and the buffering capacity is varied by changing the difference between the pH of the medium in the reservoir and the steady-state pH of the culture medium (Fig. 3), the predicted linear relation between population density and buffering capacity is observed. It ought to be possible to reach the same result by holding constant the pH of the culture medium and varying the pH of the medium in the reservoir. Unanticipated inconsistencies remain, probably arising from interaction of the buffers employed with the metabolic apparatus. We cannot explain, for example, the positive values of population density at zero buffering concentrations in Figure 1. It may be that higher phosphate or Bis-Tris concentrations elicit growth-linked reactions which bring about partial alkalinization. Such reactions would account for both the positive value of population density obtained by extrapolation to zero buffering capacity and for the decrease of the values of h at higher buffer concentrations. The quantitative significance of the values of h observed will only be discovered after full analysis of the products of metabolism.
Acknowledgements.We are gratefulto TheresaA. Kurtzfor her excellenttechnicalassistance. This work was supported by a grant from the National ScienceFoundation(No. GB-25582).
Continuous Culture at High Growth Rates
REFERENCES Anderson, P. A. : Automatic recording of the growth rates of continuously cultured microorganisms. J. gen. Physiol. 36, 733 - 737 (1953) Anderson, P. A.: Continuous recording of the growth of microorganisms under turbidostatic and chemostatic control. Rev. Scient. Instrum. 27, 4 8 - 5 1 (1956) Bryson, V., Szybalski, W. : Microbial selection. Science 116, 4 5 51 (1952) Hempfling, W.P.: Repression of oxidative phosphorylation in Escherichia coli B by growth in glucose and other carbohydrates. Biochem. biophys. Res. Commun. 41, 9 - 15 (1970) Hempfling, W. P., Mainzer, S. E.: Effects of varying the carbon source limiting growth on yield and maintenance characteristics of Escheriehia coli B in continuous culture. J. Bact. 123, 10761087 (i975)
47 Herbert, D.: Some principles of continuous culture. In: Recent progress in microbiology, G. Tunevall, ed., pp. 381 - 396. Springfield, Ill. : Thomas 1958 Herbert, D., Ellsworth, R., Telling, R. D. : The continuous culture of bacteria: A theoretical and experimental study. J. gen. Microbiol. 14, 601-622 (1956) Koch, A. L. : Turbidity measurements of bacterial cultures in some available commercial instruments. Analyt. Biochem. 38, 2 5 2 259 (1970) Northrop, J. H.: Apparatus for maintaining bacterial cultures in the steady state. J. gen. Physiol. 38, 105-115 (1954) Watson, T. G. : Steady-state operation of a continuous culture at maximum growth rate by control of carbon dioxide production. J. gen. Microbiol. 59, 8 3 - 8 9 (1969) Received September 25, 1975