The Influence of Temperature on the Maximum Specific Growth Rate of

Klebsiella pneumoniae
INTRODUCTION

Temperature is an important environmental parameter for microbial growth. Microorganisms, unlike higher organisms like mammals, do not possess the capability of regulating
their internal temperature. In microbial cultures the cell temperature must become equal
to the environmental temperature. Therefore, all of the biochemical reactions taking place
in the cell are affected by the temperature. It has long been known that temperature influences the nature of metabolism, the nutritional requirements, and the biomass composition,
in addition to its primary effect: changing the reaction rates.'.'
If the maximal specific growth rate of microorganisms is limited by the reaction rate of
one specific enzymatic reaction in a complex sequence as postulated by the Monod model3
of growth, a mathematical relation between the absolute temperature and the maximal
growth rate can be sought after. This has already been studied by many authors and Arrhenius-type expressions have been derived but found to be applicable within only a limited

In this study the influence of the temperature on the maximum specific growth rate of the
bacterium Klebsiella pneurnoniae was studied in batch mode. Results were used for the
determination of the thermodynamic parameters in an Arrhenius-type model extended to
describe also the high temperature range where the maximum specific growth rate declines
with increasing temperature.
MODEL

Assuming that the bacterial growth is an end product of a number of enzymatic reactions
and that one specific reaction determines the rate of the overall reaction, the temperature
dependence of the maximum growth rate can be assumed to follow an Arrhenius relationship
of the following type:
r,,,,,

= A exp( - AHTIRT)EC,

(1)

where r, is the reaction rate, C , is the biomass concentration, E is the weight fraction of
the specific enzyme in biomass, AH? is the activation enthalpy of the rate limiting reaction,
A is a constant, R is the universal gas constant, and T is the absolute temperature. If the
maximum specific growth rate pmax
is defined in the usual way it follows from eq. (1):
pmax= A exp( - AHT/RT)E

(2)

This relationship has been shown to describe well the experimental observations in a limited
range, below the so-called optimal temperature.I At temperatures higher than optimum a
negative correlation has been observed between the temperature and pmax.This phenomenon can be allowed for in a generalized model if the influence of temperature on the activity
of the enzyme involved in the growth limiting reaction is taken into account.' Assuming
that this enzyme can exist in two possible configurations, an active and an inactive form
in equilibrium with each other, the effect of temperature on enzyme activity can be evaluated
by considering the activation-inactivation reaction. If the inactivation reaction is fast, the
following equilibrium relationship can be formulated:

f,
where

.fa

f a K e x p ( - AHzIRT)

(3)

and f, are the fractions of the total amount of enzyme being active and inactive,

Biotechnology and Bioengineering, Vol. XXIII, Pp. 1401-1405 (1981)

CCC OoO6-3592181/061401-05$0l.OO
0 1981 John Wiley & Sons, Inc.

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIII (1981)

1

Temp. (OC)

//

respectively, AH2 is the enthalpy change of the inactivation reaction, and K is a constant.
From eq. (31, since f, + fa = 1, one obtains:
fa = 1/[1

+ K exp(-AHZ/RT)]

(4)

Furthermore, if only the active fraction of the enzyme is engaged in the growth limiting
reaction, from eq. (1) it follows that:
r,,,,,

=A

exp( -AHf/RT)faEC,

(5)

then from eqs. (4) and ( 5 ) in combination with the definition of pmaxthe following can be
written:
pmax =

A ' exp( - AHTIRT)

+ Kexp(-AH2/RT)

MATERIALS AND METHODS

The organism, Klebsiella pneumoniae NCTC 418, was cultivated in synthetic medium as
described by Evans et aL9 Glycerol was used as the only carbon and energy source. It was
assayed enzymatically. Dry weights were determined in duplicates by the method of de
Vries and Stouthamer.'' Biomass was collected on a 0.2-pm pore diameter membrane filter
(Sartorius SM 11307), washed with distilled water, and dried to constant weight at 378 K.
Medium was sterilized by membrane filtration through a 0.2-pm membrane filter and inoculated with an inoculum actively growing at the experimental temperature to eliminate
any lag phase and the possibility of unbalanced growth.
Experiments were carried out in a 1 1 x lo-' m3 working volume fermentor. The pH was
controlled at 6.8 7 0.05. The air flow rate to the fermentor was controlled by a thermal
mass flow meter (Brooks 581 1) at about 0.77 kg dry air/hr.
The culture supernatant was checked for the presence of products other than biomass,
carbon dioxide, and water, but none could be detected at any significant level. All samples
were cooled during sampling to about 278 K by an on-line heat exchanger manufactured in
our workshop. The typical residence time in the exchanger was about 5 sec.

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COMMUNICATIONS TO T H E EDITOR

0.

-0

-1

6.15

3.20

I
3.25

3.30

I \

3.35

3.4

( 1 / ~ ) ~ 1 I0 ~ ~ 1

versus the reciprocal of absolute temperature.

Fig. 2. Natural logarithm of pmax
COMPUTATIONS

The maximum specific growth rate pmax

was determined from the dry weight data collected
during the exponential phase. The upper boundary of the exponential phase was determined
by plotting the oxygen uptake rate data and evaluating the time at which the maximum is
was determined by performing nonlinear regression based on Marquarts
reached. Then pmax
algorithm." The same computer program was used for evaluating the parameters of the
temperature-p,,,
model, i.e., eq. (6).
RESULTS AND METHODS

Experimental results are shown in Figure 1 together with the 95% confidence levels. The
continuous line represents the model evaluated with the parameters given in Table I . In
Figure 2 the natural logarithm of kmax
is plotted against the reciprocal of temperature. Since
there are no abrupt changes in the slope of this bell-shaped curve, one can conclude that
there has been no significant changes in the cell metabolism and that the same enzymatic

TABLE I
Calculated and Reported Model Parameters
95% Confidence
levels
AH?
A H2
A'
K

86.40
287.78
5.69 x I O l 4
1.38 x lo4'

kJ/mol
kJ/mol
I/hr
(dimensionless)

(44.19-128.64)
(188.33-387.23)

Range

A H2
a

343a

kJ/mol

From Morowitz, average for 20 proteins.

min
147

max
828

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIII (1981)

Fig. 3. Active fraction of the enzyme taking part in the growth limiting reaction as a
function of temperature. Computed according to model eq. (4).

reaction remained the rate limiting one throughout the experimental range. A supporting
observation for this was the absence of metabolic products at all temperatures.
From Figure 2 it is evident that a simple Arrhenius-type expression can only describe a
part of the experimental observations. If such a simple expression is assumed to hold up
to the optimal temperature for fastest growth, the magnitude of the activation enthalpy,
AHT, may be underestimated significantly. This is why workers who did not allow for
thermal enzyme inactivation process have reported low AH? values.'*
From eq. (6) the optimal cultivation temperature (if the fastest growth is the only consideration) can be shown to be given by:

AH2/R In[K(AH2/AHT - l)]

(7)

A result which is obtained by setting the first derivative of pmaxwith respect to T equal to
zero.
Inserting the parameters obtained one gets 36.9"C as the optimal temperature for this
system. An inflection point of 31.7"C was calculated by evaluating the temperature at which
)
zero. Up to this point increase in pmaxis mainly
the second derivative ( d 2 ~ , / d T 2becomes
determined by the simple Arrhenius type of expression, i.e., the nominator of eq. (6). At
higher temperatures the contribution of the thermal enzyme inactivation process becomes
significant. This can best be visualized from Figure 3 where the active fraction of the enzyme
is plotted as a function of the growth temperature. As can be seen from this plot, at low
temperatures almost all of the enzyme remains active while after about 32C a dramatic
decrease in the activity is calculated. In the range 33 =s T 38C. pmaxchanges relatively
little as the contributions of the two processes balance each other. Therefore the optimal
operation temperature for an industrial process can be selected in this range for fast biomass
production.
Morowitz has compiled a list of ethalpy change values for the thermal inactivation of
some 20 protein^.'^ The value of AH2 calculated in this study, 287 kJ/mol, compares well
with his average of 343 kJ/mol (Table I).

COMMUNICATIONS TO T H E EDITOR

1405

These results indicate that the model can be used as a useful approximation to describe
the temperature-reaction-rate relationship of microbial growth.
Nomenclature

constants (I/hr)
concentration (kg/m3)
weight fraction of the specific enzyme in biomass (dimensionless)
active fraction of the specific enzyme
activation enthalpy for the growth limiting enzymatic reaction (kJ/mol)
enthalpy change for enzyme inactivation reaction (kJ/mol)
a constant (dimensionless)
rate of reaction (kg/m31hr)
gas constant (kJ/kg.mol)
temperature (K)
maximum specific growth rate (lihr)
References
1. S. J. Pirt, Principles ofMicrobe and Cell Cultivation (Blackwell, London, 1975).
2. D. W. Tempest, in Microbial Growth, 19th Symposium of the Society ofGeneral
Microbiology (Cambridge U . P., Cambridge, 1969). p. 87.
3. J. Monod, Recherches sur la Croissance des Cultures Bacteriennes (Hermann, Pans,
1942).
4. H. Topiwala and C. G. Sinclair, Biotechnol. Bioeng., 13, 795 (1971).
5. F. Watanabe and S. Okada, J . Cell. Biol., 32, 309 (1967).
6. A. C. R. Dean and C. Hinshelwood, Growth Function and Regulation in Bacterial
Cells (Clarendon, Oxford, 1966).
7. A. Prokop and A. E. Humphrey, Kinetics of Disinfection, in Disinfection, M. A.
Benarde, Ed. (Marcel Dekker, New York, 1970).
8. H. Eyring and D. W. Urry, Thermodynamics and Chemical Kinetics, in Theoretical
and Mathematical Biology, T. H. Waterman and H. J. Morowitz, Eds. (Blaindell, New
York, 1965).
9. C. G. T. Evans, D. Herbert, and D. W. Tempest, in Methods in Microbiology, J. R.
Norris and W. W. Robbins, Eds. (Academic, London, 1970), Vol. 2, p. 313.
10. De Vries and A. H. Stouthamer, J . Bacteriol., 96, 472 (1968).
11. D. W. Marquardt, J . Soc. Ind. Appl. Math., 2, 431 (1963).
12. J. H. Lee, D. Williamson, and P. L. Rogers, Biotechnol. Lett., 2(4), 83 (1980).
13. H. J. Morowitz, Energy N o w in Biology (Academic, New York, 1968), p. 114.

A. A. ESENER
J . A. ROELS
N. W. F. KOSSEN
Biotechnology Group, Department of Chemical Engineering
Delft University of Technology
Jaffalan 9, T.H., P.O. Box 5029, 2600, GA Delft, The Netherlands
Accepted for Publication November 26, 1980