Kinetics of cell death

The kinetics of cell death is an important consideration in design of sterilisation processes and in
analysis of fermentations where substantial viability loss is expected. In a lethal environment, all
cells in a population do not die at once. Deactivation of the culture occurs over a finite period of
time depending on the initial number of viable cells and the severity of the conditions imposed.
Loss of cell viability can be described mathematically in the same way as enzyme deactivation.
The destruction of micro-organisms by steam (moist heat) at specific temperature can be
described as a first-order chemical reaction provided if we considerer loss of viability not
destruction.

= k.N

Where, r is rate of cell death,

N is number of viable cells, and
k is the specific death constant.

x,

If
is the cell concentration then rate of cell death can be expressed using cell concentration
rather than cell number as:

= k.X

Where, k is the specific death constant based on cell concentration and

X is the concentration of viable cells.
In a closed system, cell death is the only process that affecting viable cell concentration, the rate
of cell death is equal to the rate of decrease in cell number. Therefore, from Eq. (1):

-dN/dt = k . N

If k is constant, then we can integrate Eq. (3) to derive an expression for N as a function of time
as :

Nt/N0 =e

-kt

Nt = N0 .e

-kt

...4

Where, NO is the initial number of viable cells at time zero.

Taking natural logarithms of both sides of Eq. (4) we have :

ln Nt = lnN0 - kt

ln (Nt/N0) =

-k t

..5

According to Eq. (5), if first-order death kinetics apply, then a plot of In (Nt/N0 ) versus t gives a
straight line with slope -kd.
Fig 1 Plots of the proportion of survivors and the natural logarithm of the proportion of survivors
in a population of microorganisms subjected to a lethal temperature over a time period.
The relationship displayed in Fig. 1 would be observed only with the sterilization of a pure
culture in one physiological form, under ideal sterilization conditions.
The value of k is not only species dependent, but dependent on the physiological form of the cell;
for example, the endospores of the genus Bacillus are far more heat resistant than the vegetative
cells.
The relationship of Eq. (5); have confirmed by the experimental measurements for many
vegetative cells. As an example, data for thermal death of Escherichia coli at various
temperatures are shown in Figure 2 given below.

Figure 2 Relationship between temperature and rate of thermal death for vegetative Escherichia
coli cells. (From S. Aiba, A.E. Humphrey and N.F. Millis, 1965, Biochemical Engineering,
Academic Press, New York.)
As we know that for any first-order reaction, the reaction rate increases with increase in
temperature due to an increase in the reaction rate constant. Similarly in the case of the
destruction of micro-organisms, is the specific death rate (k) is same as reaction rate constant.
Therefore , k is a true constant only under constant temperature conditions. The relationship
between temperature and the reaction rate constant was demonstrated by Arrhenius and may be
represented by the equation:
d/dT .lnK = Ea/RT2..6
Where Ea is the activation energy
R is the gas constant
T is the absolute temperature
On integrating eq. (6) we have

k = A.e Ea/RT

..7

by taking natural logarithm in eq. (7) we have

lnk = lnA-Ea/RT

Typical Ea values for thermal destruction of microorganisms are high, and will be generally 250290 kJ gmo1-1. Therefore, small increases in temperature have a significant effect on k and rate
of death.
From equation (8) it may be seen that a plot of Ink against the reciprocal of the absolute
temperature will give a straight line. Such a plot is termed an Arrhenius plot which helps in the
calculation of the activation energy and the prediction of the reaction rate for any temperature.
By combining together equations (5) and (7), the following expression may be derived for
the heat sterilization of a pure culture at a constant temperature:
ln N0/Nt = A.t.e Ea/RT

.9

Deindoerfer and Humphrey (1959) used the term ln(N0/Nt) as a design criterion for
sterilization, which has been also called Del factor or Nabla factor and sterilization criterion
represented by the term V'.
Therefore we can say that the Del factor is a measure of the fractional reduction in viable
organism count produced by a certain heat and time regime.
Therefore:

10
On rearranging, and taking natural logarithm of equation (10) we have

..11
Thus, a plot of the natural logarithm of the time required to achieve a certain V' value against the
reciprocal of the absolute temperature will yield a straight line, the slope of which is dependent
on the activation energy, as shown in Fig. 4 below.

From Fig. 4 it is clear that the same degree of sterilization (V') may be obtained over a wide
range of time and temperature regimes; that is, the same degree of sterilization may result from
treatment at a high temperature for a short time as from a low temperature for a long time.
This kinetic description of bacterial death give the design of procedures by giving certain value
of V factors for the sterilization of fermentation broths.

Questiion 1. The number of viable spores of a new strain of Bacillus subtilis is measured as a
function of time at various temperatures.

(a) Determine the activation energy for thermal death of B. subtilis spores.
(b) What is the specific death constant at 100~
(c) Estimate the time required to kill 99% of spores in a sample at 100~