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 transpoit and growth processes compete for essential energy
resources during growth on lactose.
INTRODUCTION
Proper reactor design and operation requires the development 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 maximize 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 presumably 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 accounted 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 growthlimiting 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 frequently 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
Medium
The process of lactose transport across the cellular membrane has been identified as the cause of observed growth
The carbon-free salts medium contained the components
inhibition.73’-’’This transport process is mediated by the
listed in Table I. Sodium-free medium was identical to that
lac permease and is known to occur by either a facilitated
listed in Table I minus the NaCl. The medium was prepared
diffusion mechanism or a secondary active transport mechain two parts: a concentrated solution of trace metals plus
nism14 which has been observed to interact with both comEDTA and a solution containing the remaining salts. Sugar
ponents of the protonmotive force, ApH and AY.739310315
solutions were prepared as concentrated stock solutions.
The chemiosmotic hypothesis as proposed by MitchellI6
Glucose and galactose were obtained from Sigma Chemical
identifies the protonmotive force as the driving force for
Company while lactose was obtained from Mallinckrodt.
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
Fermentor Description and Preparation
growth by the membrane-bound ATPase and secondary acAll experiments were carried out in a 2-L New Brunstive transport processes. l7
wick
fermentor operating in the batch mode. The carbonDuring the process of active transport of lactose across
free
salts
medium was added to the fermentor with the
the cellular membrane the coupling between the lac pernecessary
probes
(pH and dissolved 0,) in place, and the
mease and the protonmotive force can produce a signifientire
assembly
was
sterilized by autoclaving. The tracecant reduction or a collapse in ApH and AY.73’,103’5
The
metal
and
sugar
solutions
were sterilized separately. The
reduction of these gradients results in an accompanying
fermentor
was
placed
in
a
constant
temperature water bath
loss in driving force for ATP synthesis, and subsequently
and
allowed
to
equilibrate
to
37°C
and
to saturate with air.
the ATP pool within the cell is reduced.’.1° Observations
Just
prior
to
inoculation
the
appropriate
amounts of traceindicate a strong correlation between the reestablishment
metal
and
sugar
solutions
were
added
to
the
fermentor in an
of these gradients and the intracellular ATP pool with the
aseptic
manner.
Medium
pH
was
maintained
at 7.0 -+ 0.2
commencement of
Another factor is the intraunits
by
automatic
base
(KOH)
addition.
Air
was
supplied
cellular pH which is normally maintained at a constant
at
a
rate
of
approximately
1
L/min.
Culture
agitation
was
value of approximately 7.8 by respiratory and ion antiport
maintained
at
a
level
that
prevented
oxygen
limitation
from
processes in Escherichia coli. 18-20 Upon dissipation of the
occurring as confirmed by dissolved-oxygen measurements.
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
lnoculum Preparation
Other than cytoplasmic pH regulation there is not a direct
role for Na’ in growth. The theories of Skulachev,2”22
The organism was stored in small vials in a glycerol-rich
however, indicate that ion gradients (Na’, K’) may be
minimal-salts medium at -60°C. The inoculum was prepared
involved in processes other than osmotic regulation and
in 250-mL flasks containing 50 mL of the minimal-salts
nutrient transport. 18,23 He proposed that transmembrane gramedium with lactose at a concentration of 10 g/L and grown
dients of Na’ and K+ serve to maintain and buffer ApH and
to stationary phase at 37°C in a rotary shaker rotating in
A*. The presence of these gradients would act to prevent a
excess of 200 rpm. The organism was then transferred to a
rapid loss of membrane energy in unfavorable environments.
second flask containing the identical medium and grown
In the following investigation a wild-type strain of Klebagain to stationary phase. Approximately 10 mL of this
siellu oxytoca has experienced lactose inhibition growing
culture was subsequently injected into the fermentor. All
aerobically in batch culture in a minimal-salts medium. Incell transfers were made in an aseptic manner.
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
Table I. Composition of PA medium
order to preinduce the lac operon. Results indicate that the
Component
Concentration (g/L)
degree of inhibition is a function of the lactose concentration, the preferred substrate in multiple-substrate environK2HP04 3H20
6.85
ments, and the ionic composition of the medium.
KHIPO,
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~~~
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.
706
(NHdzHP04
(NH&SO,
MgSO, . 7HZ0
FeS04 . 7H,O
ZnSO, . 7H,O
MnSO, . H 2 0
CaCI, . 2H,O
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EDTA
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 34, AUGUST 1989
1.65
3.3
0.125
0.025
0.0005
0.0005
0.005
2.9
0.025
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 highperformance liquid chromatography (HPLC) analysis and
a Beckman glucose analyzer. The HPLC analysis was performed 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 residue, 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 decreased 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 ( 5 1 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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Figure 1. Batch growth data for K. oxytocu on 0.54 g/L lactose: (0)
cell density, (0)
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STRAIGHT ET AL.: BACTERIAL GROWTH ON LACTOSE
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Figure 2. Batch growth data for K. oxytoca on 1.1 g/L lactose: (0)cell density, (0)lactose concentration, (--) D.O.
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Figure 3. Batch growth data for K. oxytoca on 2.5 g/L lactose: (0)
cell density, (0)lactose concentration, (--) D.O.
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Figure 4. Batch growth data for K. oxytoca on 5.0 g/L lactose: (0)
cell density, (0)lactose concentration, (--) D.O.
708
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 34, AUGUST 1989
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. Singlesubstrate 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,
lo1
1.5
which may persist at different frequencies, are most apparent at 1 g/L lactose. (Compare Figs. 6 and 15). Dissolvedoxygen 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. Increasing 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. However, 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 inhibited 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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Figure 5. Batch growth data for K. oxytoca on 0.5 g/L lactose and 1 g/L glucose:
(0)
cell density, (0)
lactose concentration, (A) glucose concentration, (--) D.O.
STRAIGHT ET AL.: BACTERIAL GROWTH ON LACTOSE
709
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Figure 6. Batch growth data for K. oxyrocu on 1 g/L lactose and 1 g/L glucose: (0)
density, (0)
lactose concentration, (A) glucose concentration, (--) D.O.
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Figure 7. Batch growth data for K. oxytocu on 2 g/L lactose and 1 g/L glucose: (0)
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Figure 8. Batch growth data for K. oxyrocu on 3 g/L lactose and 1 g/L glucose: (0)
density, (0)
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710
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 34, AUGUST 1989
fore, the presence of galactose apparently permitted the
accumulation of a significant level of intracellular lactose
before the exhaustion of galactose (Figs. 9-12). This may
explain the absence of an appreciable lag phase before
exponential growth on lactose begins. The cell yield on
galactose and DO profiles indicate that galactose is primarily consumed during the first exponential growth phase
while lactose is primarily consumed during the second exponential growth phase. These results suggest that intracellular repression of the lac operon alone is present during
growth on galactose while both lac operon repression and
lactose transport inhibition are present during growth on
glucose. The reduced lactose growth rates in the presence
of glucose may therefore be partly due to the greater repressive nature of glucose.
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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Figure 9. Batch growth data for K. oxyroca on 0.5 g/L lactose and 1 g/L galactose:
(0)
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STRAIGHT ET AL.: BACTERIAL GROWTH ON LACTOSE
711
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Figure 11. Batch growth data for K. oxytocu on 2 g/L lactose and 1 g/L galactose:
(0)
cell density, (0)lactose concentration, (A) galactose concentration, (--) D.O.
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Figure 12. Batch growth data for K. oxytocn on 3 g/L lactose and 1 g/L galactose:
(0)
cell density, (0)lactose concentration, (A) galactose concentration, (--) D.O.
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
712
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 expelled 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,2'322the 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-
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 34, AUGUST 1989
1.25
I
1
n
7.50
7.25
,
II
B
f
I
1.00
d
bo
W
0.75
h
5m
d
0.50
Q)
n
0.25
0)
u
0.0
0
2
4
6
8
TIME
10
12
14
16
(HRS)
TIME (HRS)
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.
h
t
;
10-1
0)
n
d
d
Q)
u
0
2
4
6
8
1 0 12 1 4 1 6 18
TIME (HRS)
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.
Figure 15. Comparison of K. oxyroca growth on 1 g/L lactose and
1 g/L glucose (-)
in the presence of 50mM NaCl and (--) in the absence
of 50mM NaCI. Data points have been removed to enhance comparison,
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 identification 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.30Inducible 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 KnorreZ9used a synthetic medium containing
3 g/L Na' (130mM) in his culturing procedures as decribed 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-
STRAIGHT ET AL.: BACTERIAL GROWTH ON LACTOSE
713
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 illumination, 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.34.35The lowest initial lactose concentration investigated
in this study was 0.54 g/L. With observed saturation constants 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 becomes 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 a c t o ~ eThis
. ~ form
of control, as demonstrated experimentally (Figs. 5 4 , effectively 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 uptake, and upon galactose exhaustion the lactose present in-
714
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 O k t ~ a b r ’ s k i ito~ ~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-
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 34, AUGUST 1989
,*’
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 establishment 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 multiplesubstrate 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. Therefore, 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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