bacterial growth on lactose: an experimental investigationdrops/ramkipapers/092... · ·...

Bacterial Growth on Lactose: An Experimental Investigation

Jeffrey V. Straight and D. Ramkrishna" School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907

Satish J. Parulekar School of Chemical Engineering, Illinois Institute of Technology, Chicago, Illinois 60616

Norman B. Jansen The Upjohn Company, 1450-89- 1, Kalamazoo, Michigan 49001

Accepted for publication November 18, 7988

A wild-type strain of Klebsiella oxytoca growing aerobically in batch culture has exhibited intermittent or oscillatory growth while growing on lactose at concentrations on the order of 1 g/L or less. In two-substrate experiments, preferred growth on glucose followed by growth on lactose also produced oscillatory growth behavior during the lactose growth phase at lactose concentrations of 1 g/L or less. Only oscillations in cell density have currently been observed. Alkalinization of the medium during growth on lactose indicated the presence of lactose active transport. The observed intermittent growth was reduced or removed during growth on lactose after preferred growth on galactose or in a medium containing 50mM NaCI. Results suggested that the presence of an intracellular energy source or a sufficient ApH buffer may alleviate growth inhibition when trans- poit and growth processes compete for essential energy resources during growth on lactose.

INTRODUCTION

Proper reactor design and operation requires the devel- opment of a model that adequately describes the range of expected system behavior. With regards to bioreactors the prediction of microbial growth in response to varying environmental conditions is a subject of primary concern. Within a fluctuating environment a microorganism adapts by utilizing the processes of metabolic regulation in order to maxi- mize the probability of species survival. Ramkrishna and co-workers''2 have developed a framework that accounts for metabolic regulation and predicts bacterial growth under single- and multiple-substrate environments which support both low and high specific growth rates. This cybernetic framework describes the outcome of metabolic regulation as being consistent with some goal which could presum- ably be an optimality criterion. These modeling efforts"' typically assumed that cellular growth on a single limiting

* To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 34, Pp. 705-716 (1989) 0 1989 John Wiley & Sons, Inc.

substrate can be represented by the actions of a single key enzyme. Previ~usly,~ single-substrate lactose experiments in batch culture have indicated a more complicated system than that described by a simple structured model which ac- counted for a single key enzyme. A multiple-key-enzyme system during microbial growth on lactose has been suggested by the results of Postma4 and Kennedy,' who identified the significance of the cellular membrane as a site of metabolic regulation and lactose transport as the growth- limiting process, respectively. The expansion of the cybernetic framework'.' to describe nutrient transport processes and ultimately metabolic regulation during nutrient transport has therefore been initiated here with an experimental investigation of the effect of lactose on bacterial growth in batch culture.

Inhibition of bacterial growth by lactose has been known since Hofsten6 observed growth inhibition when lactose was added to lac constitutive cells growing on succinate. He attributed the inhibition to the accumulation of intracellular galactose and the associated osmotic conditions. Since Hofsten6 several other investigators have observed growth inhibition upon the addition of lactose to growing cell^,^-'^ upon plating cells on lactose minimal medium," and upon addition of lactose to a culture starved in nonnutrient buffer, a phenomenon known as substrate-accelerated death. 1'2'3 The cells exhibiting growth inhibition are fre- quently constitutive with respect to the lac per on.^.^*^-'' However, inducible wild-type cells also experience inhibition if the lac operon is preind~ced.~,"

Observation of lactose inhibition in the presence of lac2 mutants""' indicated that catabolite repression by glucose and other metabolites derived from lactose hydrolysis and subsequent metabolism was not the apparent cause of the inhibition. Furthermore, the accumulation of galactose, lactose, and phosphorylated intermediates as well as osmotic effects have been discounted as possible causes of the inhibition."."

CCC 0006-3592/89/050705 12$04.00

The process of lactose transport across the cellular membrane has been identified as the cause of observed growth inhibition.73’-’’ This transport process is mediated by the lac permease and is known to occur by either a facilitated diffusion mechanism or a secondary active transport mecha- nism14 which has been observed to interact with both components of the protonmotive force, ApH and AY.739310315 The chemiosmotic hypothesis as proposed by MitchellI6 identifies the protonmotive force as the driving force for many processes in energy-coupling membranes. Two primary processes coupled to the energy gradients across the cellular membrane are the synthesis of ATP during aerobic growth by the membrane-bound ATPase and secondary active transport processes. l7

During the process of active transport of lactose across the cellular membrane the coupling between the lac permease and the protonmotive force can produce a significant reduction or a collapse in ApH and AY.73’,103’5 The reduction of these gradients results in an accompanying loss in driving force for ATP synthesis, and subsequently the ATP pool within the cell is reduced.’.1° Observations indicate a strong correlation between the reestablishment of these gradients and the intracellular ATP pool with the commencement of Another factor is the intracellular pH which is normally maintained at a constant value of approximately 7.8 by respiratory and ion antiport processes in Escherichia coli. 18-20 Upon dissipation of the pH gradient the cytoplasmic pH is disrupted and key growth processes may be prevented. Such a correlation between intracellular pH and growth has also been observed.20

Other than cytoplasmic pH regulation there is not a direct role for Na’ in growth. The theories of Skulachev,2”22 however, indicate that ion gradients (Na’, K’) may be involved in processes other than osmotic regulation and nutrient transport. 18,23 He proposed that transmembrane gradients of Na’ and K+ serve to maintain and buffer ApH and A*. The presence of these gradients would act to prevent a rapid loss of membrane energy in unfavorable environments.

In the following investigation a wild-type strain of Kleb- siellu oxytoca has experienced lactose inhibition growing aerobically in batch culture in a minimal-salts medium. In- hibition is evident in the cell density profiles and appears as an oscillatory growth behavior. Single-substrate (lactose) and two-substrate (glucose-lactose, galactose-lactose) experiments are presented with preculturing on lactose in order to preinduce the lac operon. Results indicate that the degree of inhibition is a function of the lactose concentration, the preferred substrate in multiple-substrate environments, and the ionic composition of the medium.

MATERIALS AND METHODS

Organism

Klebsiella oxytocu B 199 (ATCC 8724) obtained from the U.S. Department of Agriculture (Peoria, IL) was used in all experiments.

Medium

The carbon-free salts medium contained the components listed in Table I. Sodium-free medium was identical to that listed in Table I minus the NaCl. The medium was prepared in two parts: a concentrated solution of trace metals plus EDTA and a solution containing the remaining salts. Sugar solutions were prepared as concentrated stock solutions. Glucose and galactose were obtained from Sigma Chemical Company while lactose was obtained from Mallinckrodt.

Fermentor Description and Preparation

All experiments were carried out in a 2-L New Bruns- wick fermentor operating in the batch mode. The carbon- free salts medium was added to the fermentor with the necessary probes (pH and dissolved 0,) in place, and the entire assembly was sterilized by autoclaving. The trace- metal and sugar solutions were sterilized separately. The fermentor was placed in a constant temperature water bath and allowed to equilibrate to 37°C and to saturate with air. Just prior to inoculation the appropriate amounts of trace- metal and sugar solutions were added to the fermentor in an aseptic manner. Medium pH was maintained at 7.0 -+ 0.2 units by automatic base (KOH) addition. Air was supplied at a rate of approximately 1 L/min. Culture agitation was maintained at a level that prevented oxygen limitation from occurring as confirmed by dissolved-oxygen measurements.

lnoculum Preparation

The organism was stored in small vials in a glycerol-rich minimal-salts medium at - 60°C. The inoculum was prepared in 250-mL flasks containing 50 mL of the minimal-salts medium with lactose at a concentration of 10 g/L and grown to stationary phase at 37°C in a rotary shaker rotating in excess of 200 rpm. The organism was then transferred to a second flask containing the identical medium and grown again to stationary phase. Approximately 10 mL of this culture was subsequently injected into the fermentor. All cell transfers were made in an aseptic manner.

Table I. Composition of PA medium ~~~

Component Concentration (g/L)

K2HP04 3H20 6.85 KHIPO, 1 .o (NHdzHP04 1.65 (NH&SO, 3.3 MgSO, . 7HZ0 0.125 FeS04 . 7H,O 0.025 ZnSO, . 7H,O 0.0005 MnSO, . H 2 0 0.0005 CaCI, . 2H,O 0.005 NaCl 2.9 EDTA 0.025

706 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 34, AUGUST 1989

Growth Measurement

The cell dry weight was estimated from absorbance measurements at a wavelength of 540 nm. Due to secondary light scattering the proportionality between absorbance and cell dry weight changes at absorbance values above 0.2 units. Dense cultures were diluted to give an absorbance below 0.3 units, and the following correlation was used to correct the absorbance such that it was linearly related to cell dry weight24:

abs, = abs + 0 .325(ab~)~ .~

One unit of optical density has been determined to be equivalent to 0.35 g dw biomass/L for K . oxytoca under conditions which support both low and high specific growth

Sugar Analysis

Sugar concentrations were determined by high- performance liquid chromatography (HPLC) analysis and a Beckman glucose analyzer. The HPLC analysis was per- formed with a Bio-Rad HPX-87c column in tandem with an in-line deashing column for salt removal.

RESULTS

Single-Substrate Experiments

Batch culture experiments utilizing monosaccharides such as glucose, fructose, or xylose have exhibited normal phases of growth: lag phase, exponential phase, and stationary phase.3 However, batch experiments utilizing lactose, a disaccharide consisting of a glucose and a galactose resi- due, have exhibited some unusual growth behavior.

Cell growth on initial lactose concentrations of 0.54 and 1 . 1 g/L also exhibits multiphasic growth behavior, as shown in Figures 1 and 2, respectively. The first growth phase appears to be a very long lag phase consisting of growth

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separated by periods of nongrowth or extremely slow growth. Periods of nongrowth typically last between 15 and 30 min before growth resumes. This oscillatory behavior lasts approximately 12 h in the presence of 0.54 g/L lactose and 9 h in the presence of 1 . 1 g/L lactose. The second phase of growth is continuous and accelerated with respect to the first phase. Growth appears to be exponential with a specific growth rate of approximately 0.4 h-' in both cases and continues until lactose is exhausted (i.e., stationary phase). Cell yields are 0.46 and 0.45 g dw/g on 0.54 and 1 . 1 g/L lactose, respectively.

Dissolved oxygen (DO) within the culture was monitored and found to remain at or above 65% air saturation; therefore, oxygen was not a limiting factor. At 0.54 and 1 . 1 g/L lactose the dissolved-oxygen level within the culture de- creased throughout the experiment even during periods of nongrowth and approached an exponential decrease only during the second phase of growth (Figs. 1 and 2).

The character of cell growth as the initial lactose concentration was increased to 2.5 or 5.0 g/L changed considerably (Figs. 3 and 4). In comparison with results at low lactose concentrations (51 g/L) cell growth at these concentrations exhibits little if any initial lag period and no intermittent growth behavior. Exponential phase specific growth rates and cell yields are 0.44 h-' and 0.40 g dw/g on 2.5 g/L lactose and 0.76 h-' and 0.44 g dw/g on 5.0 g/L lactose.

Closer inspection of Figures 3 and 4 reveals that the specific growth rate decreases to 0.35 h-' at t = 7.5 h and 0.55 h-' at t = 5.5 h on 2.5 and 5.0 g/L lactose, respectively. Since the concentration of lactose remaining in the medium is still a significant fraction of the initial value and the level of DO is decreasing at a significant rate in both cultures (Figs. 3 and 4), the reduction in specific growth rate does not appear to be a result of the cultures entering stationary phase. Inspection of the DO profiles shows that a metabolic transition appears to be occurring in both cultures. At 2.5 g/L lactose the DO level decreases to a plateau

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STRAIGHT ET AL.: BACTERIAL GROWTH ON LACTOSE 707

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at t = 8.0 h and then continues to decrease at t = 8.5 h until the stationary phase. At 5.0 g/L lactose the DO level continually decreases until the stationary phase with a sharp increase in the rate of 0, consumption occurring at t = 5.5 h. Transitions in DO levels have previously indicated a change in cellular metabolism from one carbon source to a n ~ t h e r . ~ However, HPLC analysis of the medium indicated only the presence of lactose in all single-substrate experiments. In contrast to other investigator^,*^-*^ no external glucose or galactose was detected, and a cellular preference for glucose or galactose was not apparent unless it was an intracellular preference.

Two-Substrate Experiments

Two-substrate experiments typically exhibit a diauxic growth character that reflects a cellular preference for a particular carbon source. The sugars present within the lactose system (i.e., glucose, galactose, and lactose) were investigated in a set of two-substrate experiments where glucose-lactose and galactose-lactose were the pairs of sugars available for metabolism. In all cases lactose was the less preferred sugar, and the initial concentrations of glucose and galactose were approximately 1 g/L. Glucose and galactose exhibited average exponential phase specific growth rates of 1.04 and 1.02 h-I, respectively. Single- substrate batch experiments with glucose or galactose indicated that both sugars have essentially equivalent maximum specific growth rates of 1.08 h-' with cell yields of 0.52 g dw/g on glucose' and 0.507 g dw/g on galactose in minimal-salts medium (data not shown).

Examination of the glucose-lactose experiments (Figs. 5-8) shows lactose growth behavior that is similar to behavior observed when lactose was the sole carbon source. Growth on lactose after the consumption of glucose again exhibits oscillatory behavior when lactose is present at 0.5 and 1 g/L (Figs. 5 and 6). The oscillations,

which may persist at different frequencies, are most apparent at 1 g/L lactose. (Compare Figs. 6 and 15). Dissolved- oxygen profiles (Figs. 5-8) show the expected exponential decay due to growth on glucose; a rapid rise in DO follows after consumption of glucose and indicates a switch to lactose metabolism. Unlike the single-substrate results, the DO profile when 1 g/L lactose is metabolized after consumption of glucose shows an oscillatory behavior with a period similar to the period of the cell density profile. In- creasing the lactose concentration removes the intermittent growth behavior and continuous growth is observed. At an initial lactose concentration of 2 g/L the specific growth rate is 0.19 h-'. At an initial lactose concentration of 3 g/L growth is continuous as well, but inspection of the DO profile (Fig. 8) shows a behavior that is similar to behavior observed in the single-substrate lactose experiments. Upon transition to lactose metabolism the DO level decreases to a plateau at t = 6.5 h. At c = 7 h the DO level continues to decrease until the onset of stationary phase. The specific growth rate is 0.44 h-' before the plateau and 0.29 h-' after the plateau. This growth behavior suggests an environment containing multiple limiting substrates, each serving as both carbon and energy sources, in which the organism grows sequentially on the available substrates and consumes first the substrate that maximizes the specific growth rate. How- ever, sugar analysis indicated only a single limiting carbon and energy source consisting of lactose within the medium. Figures 5-8 also show the control that glucose exerts over lactose metabolism; lactose transport is completely inhib- ited until growth on glucose has completed. Finally, cell yields on lactose after consumption of glucose are as follows: 0.51 g dw/g on 0.5 g/L lactose, 0.45 g dw/g on 1 g/L lactose, 0.35 g dw/g on 2 g /L lactose, and 0.46 g dw/g on 3 g/L lactose. Inspection of the cell yield values on lactose when present as the sole carbon source or after consumption of glucose indicates that the cell yield approaches a minimum value at a lactose concentration

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near 2 g/L. The cell yield on 2 g/L lactose was determined to be 0.35 g dw/g lactose when lactose was the sole carbon source (data not shown).

Examination of the galactose-lactose experiments (Figs. 9-12) shows no oscillatory growth behavior. Cell density and DO profiles indicate only exponential growth after short or no lag periods at all lactose concentrations. Exponential phase specific growth rates and cell yields on lactose are observed to increase with increasing lactose concentration: 0.17 h-' and 0.33 g dw/g on 0.5 g/L lactose, 0.26 h-' and 0.33 g dw/g on 1 g/L lactose, 0.54 h-' and 0.38 g dw/g on 2 g/L lactose, and 0.76 h-' and 0.45 g dw/g on 3 g/L lactose.

Sugar analysis indicated that the presence of galactose did not completely inhibit the transport of lactose; there-

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Sodium Experiments

Two-substrate experiments utilizing glucose or galactose confirmed .that the absence or presence of Na' did not significantly alter the nature of cellular growth when these substrates limited growth (first growth phase in Fig. 15 for glucose). Lactose, however, is transported by a mechanism that utilizes the energy present in certain cellular gradients, ApH and A q , that may be maintained by Na' and K+ gradients.

An indication that active transport of lactose was occurring is shown in Figure 13. The pH profiles during cellular growth on glucose show the typical decrease in pH as growth proceeds and the subsequent control action of the pH controller. Upon exhaustion of glucose after approximately 3 h of growth, metabolism switches to lactose (0.5

and 1 g/L) and the pH of the medium begins to rise. In the absence of active transport the medium pH would be expected to decrease in the same manner as that seen when growth occurs on glucose or galactose. The rise in pH suggests that the culture is removing protons from the medium, via symport with lactose, faster than they are being ex- pelled by respiratory processes and a dissipation of ApH occurs. The medium pH begins to decrease only after growth begins to increase significantly. According to Skulachev,2'322 the dissipation of the pH gradient can be buffered by a sufficient Na' gradient. Normal "PA" medium contains a small amount of Na' in the form of EDTA disodium salt. Medium containing 50mM NaCl reduced the degree of oscillatory behavior considerably in the absence or presence of glucose; however, a control experi-


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Figure 13. pH profiles during growth of K. oxyroca. Sugar concentrations are (-) 0.5 g/L lactose and 1 g/L glucose, (--) 1 g/L lactose and 1 g/L glucose.

ment utilizing the same inoculum but without NaCl present in the medium continued to show a high level of intermittent growth behavior (Fig. 14). Sodium also produced a similar effect on lactose growth after growth on glucose (Fig. 15). Finally, presence of an increased level of Na' resulted in an observed increase in cell yield of 0.45 to 0.50 g dw/g on 1 g/L lactose without a significant change in growth rate (Fig. 14). The effects of Na' and K' on the buffering of ApH and A", respectively, were confirmed by the experimental results of Ahmed and Booth.' Our experimental results in the presence of Na' do not confirm the theories of Skulachev,2'322 but the change in growth character due to the presence or absence of Na' does suggest a causal relationship.

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Figure 14. Comparison of K. oxyroca growth on 1 g/L lactose (--) in the presence of 50mM NaCI and (--) in the absence of 50mM NaCI. Data points have been removed to enhance comparison.

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DISCUSSION

Growth inhibition due to the presence of lactose has been clearly documented in the literature. In this study a phenomenon has been observed which suggests that lactose inhibition is present under certain experimental conditions. Several investigators10-" have determined with the aid of lacZ mutants that the cause of observed inhibition was due to an interaction between the lactose permease and essential cellular energy gradients during active lactose transport. Without the aid of specific mutations the identifi- cation of the cause for the oscillatory behavior observed in this study requires the consideration of several phenomena that can or may demonstrate intermittent growth behavior.

Periodic enzyme synthesis has been observed in several systems.'* Knorre29x30 has studied the lac enzyme system specifically and has found the dynamics of P-galactosidase activity to be oscillatory in nature. He attributed the oscillatory behavior to an interaction at the lac operon between glucose as a repressor and allolactose as an inducer.30 In- ducible and constitutive strains of E. coli were grown on glucose, washed, and regrown on lactose. Enzyme synthesis was synchronous under these conditions while cell division was observed to be asynchronous. It is interesting to note that KnorreZ9 used a synthetic medium containing 3 g/L Na' (130mM) in his culturing procedures as de- cribed above. Pih and Dhurjati3' also observed oscillations in P-galactosidase activity during glucose perturbation experiments in batch culture. Oscillations in cell division were not observed for inducible and constitutive strains of E. coli growing in a supplemented M9 medium also containing 2 g/L Na' (87mM). Since oscillations in enzyme activity were only present during the glucose perturbations and not during cell growth on lactose alone, it is difficult to compare the results of Pih and Dhurjati3' with those of Kn01-1-e~~ or the results presented here where the oscilla-


tions occur in the absence of glucose or after exhaustion of glucose. It remains to be determined whether any periodic enzyme synthesis is present within this experimental system. If periodic enzyme synthesis is present, is it the cause or the result of the observed oscillations in cell density?

The inducement of synchronous growth in a culture typically requires environmental manipulation such as temperature cycling, nutrient pulsing, intermittent illumina- tion, e t ~ . ~ ’ , ’ ~ Since no cyclic variation in such parameters is occurring in this study, it is unlikely that synchronous growth can explain the observed oscillatory behavior.

Intermittent growth has also been observed in continuous cultures under conditions of carbon deficiency. Growth rates were typically below 6% of the maximum specific growth rate.34.35 The lowest initial lactose concentration investigated in this study was 0.54 g/L. With observed saturation con- stants of 0.01 and 0.012 g/L for glucose’ and galactose (data not shown), respectively, it seems unlikely that a carbon deficiency exists within the cell once lactose is hydrolyzed. A more important aspect to consider is the diffusion limitation of lactose transport across the outer membrane. This subject has been addressed by several investigators. 36237

Passive lactose diffusion across the outer cell wall be- comes growth limiting when the medium concentration approaches a concentration on the order of 100-200pM. This corresponds to an external lactose concentration in the range of 0.05 g/L. Therefore, the initial levels of lactose in this study were always one or two orders of magnitude above this critical range, and the observed oscillatory behavior does not appear to be a result of a lactose diffusion limitation.

Control of the transport of nutrients across the cellular membrane is often a significant form of regulation. When PTS and non-PTS sugars interact the presence of a PTS, sugar such as glucose exerts a form of control called “inducer exclusion” over non-PTS sugars such as l ac to~e .~ This form of control, as demonstrated experimentally (Figs. 5 4 , ef- fectively blocks the transport of lactose across the cell membrane. In the absence of extracellular glucose during lactose growth phases it is therefore unlikely that the oscillatory behavior is due to an interaction between glucose-associated growth and the lactose transport system. However, the presence of oscillations in the cell density profile during growth on lactose after consumption of glucose but not after consumption of galactose may be explained by the difference in cellular control over lactose transport by glucose and galactose. Since glucose completely inhibits the accumulation of intracellular lactose, there is little energy source available internally for the cell to support the active transport of lactose. The cell subsequently experiences an initial energy deficit due to the energy required for active transport, and growth may be intermittent until sufficient energy is available to support both active transport and growth. In the case of galactose, a non-PTS sugar in enteric bacteria, complete inhibition of lactose transport is not observed and the two sugars are transported simultaneously. Catabolism of galactose may therefore support lactose up- take, and upon galactose exhaustion the lactose present in-

tracellularly supports further lactose transport. Therefore, the existence of an intercellular energy source or an energy source that does not rely on ApH or A? for transport may supply a sufficient amount of respiratory substrate to prevent a collapse in the protonmotive force during lactose active transport, and growth proceeds uninterrupted. A similar explanation has been given by Ahmed and Booth7 when a cell culture is presented with glycerol and lactose.

When glucose enters the cell in the form of a disaccharide such as lactose, melibiose, or maltose, does the intracellular glucose produced by the hydrolysis of these disaccharides produce a significant level of catabolite repression? Wilson et al.9 measured near maximal levels of /3-galactosidase in the presence of maltose and melibiose. Likewise, Adhya and Echols3’ measured significant levels of gal enzymes in the presence of lactose and extracellular glucose. The high activity of galactose enzymes in the presence of glucose is further supported by the presence of two promoter sites on the gal operon: one induced by cAMP and one repressed by CAMP.^^,^' Apparently the gal operon is transcribed even under severe cAMP repression. Finally, Jobe and Bourgeois4’ observed that repressed lac enzyme levels in E . coli cells growing on lactose were not derepressed upon addition of CAMP, which is a test that normally indicates the presence of the “glucose” effect. This evidence suggests that glucose derived from intracellular hydrolysis does not significantly repress inducible operons such as the lac and gal operons, and the observed oscillations in cell density cannot be completely explained by the phenomenon of catabolite repression. Under these conditions it is also unlikely that a strong preference of glucose over galactose exists intracellularly, and the observed oscillatory behavior is not due to an interaction between glucose and galactose derived from lactose hydrolysis.

The active transport of lactose has been well d~cumented.’~ The presence of lactose active transport in this study is indicated by the pH profiles obtained in the glucose-lactose experiments. Hydrogen ion concentration profiles have been utilized by West4’ to verify the active transport of lactose and by Smirnova and Ok t~abr ’ sk i i~~ to demonstrate the penetration of acetate into the cell as a weak acid. Such a coupling between active transport and cellular energy resources may result in the allocation of a significant amount of the cell’s energy-producing capacity to support a nongrowth yet vital cellular process, nutrient transport. Upon transfer of a stationary phase culture (state of the inoculum) into a medium containing lactose as the only carbon and energy source, the energy requirement of active transport may overload a cell’s capacity to maintain the energy gradients, ApH and A*, due to an initial lack of intracellular energy source within a stationary phase culture. Under these conditions the culture may or may not be capable of growing continuously until the respiration rate has attained a level capable of correcting the intracellular pH and/or meeting the energetic demands of growth and active transport. The priority of maintaining ApH over growth has been observed by Zilberstein et al. ,*’ who observed the es-


tablishment of a transmembrane pH gradient before growth resumed in a wild-type strain of E . coli after a shift in medium pH. Likewise, Wilson et aL9.’’ have observed the es- tablishment of a transmembrane pH gradient and the ATP pool before growth resumed significantly after a pulse of lactose in E . coli cells having constitutive or inducible lac operons. In addition to inhibition of growth and reduction in A* and ApH, Ahmed and Booth7 have observed a reduction in respiration rate upon the addition of lactose to a lac constitutive strain of E . coli. The inhibition of respiratory processes by lactose in the absence of a sufficient energy buffering system ii.e., sufficient K+ and Na’ gradients) and in the presence of a reduced level of respiratory substrate (i.e., low to medium lactose concentrations) may lengthen the recovery time during which growth may be intermittent.

CONCLUSIONS

The cybernetic framework as developed by Ramknshna and co-workers’.* has successfully described a wide range of bacterial growth behaviors by describing the outcome of metabolic regulation by an appropriate set of control or cybernetic variables which direct growth along an optimal path in the presence of alternative reaction pathways. The regulation of cellular resources among alternative reaction pathways forms the basis of the cybernetic framework. However, the description of a particular alternative reaction pathway within the cybernetic framework has in the past only considered the existence of a single key enzyme rather than a system of key enzymes which may act in series and/or parallel.

In this article the initial experimental work necessary to incorporate general nutrient transport processes, in addition to growth processes, within the cybernetic framework has been presented. The specific system chosen to initiate this effort is the lactose system. Batch experiments utilizing lactose as the sole carbon and energy source not only indicate that lactose transport limits growth but also indicate that the coupling between lactose active transport and key energy resources competes with growth processes for these same energy resources. Such competition may produce an energy limitation within the cell; cellular growth occurs intermittently while growth and maintenance processes alternate until the cell can maintain a favorable intracellular environment. The presence of an appropriate energy buffering system appears to be able to reduce the level of apparent growth inhibition. An expansion of the cybernetic framework in a companion paper will therefore extend its description of metabolic regulation to describe the utilization and regulation of explicit cellular resources that are associated with energy metabolism and to describe a multiple key enzyme system, which includes nutrient transport enzymes, for the utilization of a single substrate.

As demonstrated by the two-substrate experiments presented within this article a description of the regulation of substrate utilization by a bacterial culture in an extracellular multiple-substrate environment requires the consideration

of regulation at the cellular membrane. However, since lactose hydrolysis produces only an intracellular multiple- substrate environment, the need to consider the regulation between substrates at the cell membrane is not necessary in a description of bacterial growth on lactose alone. There- fore, once intracellular lactose has been hydrolyzed, the original cybernetic framework’ can be utilized to describe the regulation of resources between growth on glucose and galactose, lactose hydrolysis products.

Support from the National Science Foundation through CPE-8405 138 and CBT-8609293 is gratefully acknowledged.

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