## Medium Sterilization

As pointed out by Corbett (1985), media may be sterilized by filtration, radiation, ultrasonic treatment, chemical treatment or heat. However, for practical reasons, steam is used almost universally for the sterilization of fermentation media. The major exception is the use of filtration for the sterilization of media for animal-cell culture — such media are completely soluble and contain heat labile components making filtration the method of choice. Filtration techniques will be considered later in this chapter. Before the techniques which are used for the steam sterilization of culture media are discussed it is necessary to discuss the kinetics of sterilization. The destruction of micro-organisms by steam (moist heat) may be described as a first-order chemical reaction and, thus, may be represented by the following equation:

where N is the number of viable organisms present, t is the time of the sterilization treatment, k is the reaction rate constant of the reaction, or the specific death rate.

It is important at this stage to appreciate that we are considering the total number of organisms present in the volume of medium to be sterilized, not the concentration — the minimum number of organisms to contaminate a batch is one, regardless of the volume of the batch. On integration of equation (5.1) the following expression is obtained:

where N0 is the number of viable organisms present at the start of the sterilization treatment, Nt is the number of viable organisms present after a treatment period, t. On taking natural logarithms, equation (5.2) is reduced to:

The graphical representations of equations (5.1) and (5.3) are illustrated in Fig. 5.1, from which it may be seen that viable organism number declines exponentially over the treatment period. A plot of the natural logarithm of Nt\N0 against time yields a straight line, the slope of which equals - k. This kinetic description makes two predictions which appear anomalous:

(i) An infinite time is required to achieve sterile conditions (i.e. Nt = 0).

(ii) After a certain time there will be less than one viable cell present.

Thus, in this context, a value of Nt of less than one is considered in terms of the probability of an organism surviving the treatment. For example, if it were pre-

Fio. 5.1. 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.

dieted that a particular treatment period reduced the population to 0.1 of a viable organism, this implies thai the probability of one organism surviving the treatmenl is one in ten. This may be better expressed in practical terms as a risk of one batch in ten becoming contaminated. This aspect of contamination will be considered later.

The relationship displayed in Fig. 5.1 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. Richards (1968) produced a series of graphs illustrating the deviation from theory which may be experienced in practice. Figures 5.2a, 5.2b and 5.2c illustrate the effect of the time of heat treatment on the survival of a popula tion of bacterial endospores. The deviation from an immediate exponential decline in viable spore number is due to the heat activation of the spores, that is the induction of spore germination by the heat and moisture of the initial period of the sterilization process. In Fig. 5.2a the activation of spores is significantly more than their destruction during the early stages of the process and, therefore, viable numbers increase before the observation of exponential decline. In Fig. 5.2b activation is balanced by spore death and in Fig. 5.2c activation is less than spore death.

Figures 5.3a and 5.3b illustrate typical results of the sterilization of mixed cultures containing two species with different heat sensitivities. In Fig. 5.3a the population consists mainly of the less-resistant type where the

Fig. 5.2a. Initial population increase resulting from the heat activation of spores in the early stages of a sterilization process (Richards, 1968).

Fig. 5.2b. An initial stationary period observed during a sterilization treatment due to the death of spores being completly compensated by the heat activation of spores (Richards, 1968).

Fig. 5.2c. Initial population decline at a sub-maximum rate during a sterilization treatment due to the death of spores being compensated by the heat activation of spores (Richards, 1968).

initial decline is due principally to the destruction of the less-resistant cell population and the later, less rapid decline, is due principally to the destruction of the more resistant cell population. Figure 5.3b represents the reverse situation where the more resistant type predominates and its presence disguises the de-

- Sensitive organism

--Resistant organism

Fig. 5.3a. The effect of a sterilization treatment on a mixed culture consisting of a high proportion of a very sensitive organism (Richards, 1968).

Fig. 5.3a. The effect of a sterilization treatment on a mixed culture consisting of a high proportion of a very sensitive organism (Richards, 1968).

----Whole culture

-Sensitive organism

--Resistant organism

Fig. 5.3b. The effect of a sterilization treatment on a mixed culture consisting of a high proportion of a relatively resistant organism (Richards, 1968).

crease in the number of the less resistant type.

As with any first-order reaction, the reaction rate increases with increase in temperature due to an increase in the reaction rate constant, which, in the case of the destruction of micro-organisms, is the specific death rate (k). Thus, 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:

where E is the activation energy, R is the gas constant, T is the absolute temperature. On integration equation (5.4) gives:

where A is the Arrhenius constant.

On taking natural logarithms, equation (5.5) becomes:

From equation (5.6) it may be seen that a plot of In k against the reciprocal of the absolute temperature will give a straight line. Such a plot is termed an Arrhenius plot and enables the calculation of the activation energy and the prediction of the reaction rate for any temperature. By combining together equations (5.3) and (5.5), the following expression may be derived for the heat sterilization of a pure culture at a constant temperature:

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

V = In (N0/Nt) but In (N0/Nt) = kt and kt=A-t-t~(-E/RT)

On rearranging, equation (5.8) becomes:

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. 5.4. From Fig. 5.4 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 enables the design of procedures (giving certain V factors) for the sterilization of fermentation broths. By choosing a value for Nt, procedures may be designed having a in sterilization time

125 120 115 110 105 100 Temperature (°Cj

Fig. 5.4. The effect of sterilization and temperature on the Del factor achieved in the process. The figures on the graph indicate the Del factors for each straight line (modified after Richards, 1966).

certain probability of achieving sterility, based upon the degree of risk that is considered acceptable. According to Deindoerfer and Humphrey (1959), Richards (1968), Banks (1979) and Corbett (1985) a risk factor of one batch in a thousand being contaminated is frequently used in the fermentation industry — that is, the final microbial count in the medium after sterilization should be 10 ~3 viable cells. However, to apply these kinetics it is necessary to know the thermal death characteristics of all the taxa contaminating the fermenter and unsterile medium. This is an impossibility and, therefore, the assumption may be made that the only microbial contaminants present are spores of Bacillus stearothermophilus — that is, one of the most heat-resistant microbial types known. Thus, by adopting B. stearothermophilus as the design organism a considerable safety factor should be built into the calculations. It should be remembered that B. stearothermophilus is not always adopted as the design organism. If the most heat-resistant organism contaminating the medium ingredients is known, then it may be advantageous to base the sterilization process on this organism. Deindoerfer and Humphrey (1959) determined the thermal death characteristics of B. stearothermophilus spores as:

Activation energy = 67.7 kcal mole-1 Arrhenius constant = 1 X 1036 2 second-1

in sterilization time

125 120 115 110 105 100 Temperature (°Cj

Fig. 5.4. The effect of sterilization and temperature on the Del factor achieved in the process. The figures on the graph indicate the Del factors for each straight line (modified after Richards, 1966).

However, it should be remembered that these kinetic values will vary according to the medium in which the spores are suspended, and this is particularly relevant when considering the sterilization of fats and oils (which are common fermentation substrates) where the relative humidity may be quite low. Bader et al. (1984) demonstrated that spores of Bacillus macerans suspended in oil were ten times more resistant to sterilization if they were dry than if they were wet.

A regime of time and temperature may now be determined to achieve the desired Del factor. However, a fermentation medium is not an inert mixture of components, and deleterious reactions may occur in the medium during the sterilization process, resulting in a loss of nutritive quality. Thus, the choice of regime is dictated by the requirement to achieve the desired reduction in microbial content with the least detrimental effect on the medium. Figure 5.5 illustrates the deleterious effect of increasing medium sterilization time on the yield of product of subsequent fermentations. The initial rise in yield is due to some components of the medium being made more available to the process micro-organism by the 'cooking effect' of a brief sterilization period (Richards, 1966).

Two types of reaction contribute to the loss of nutrient quality during sterilization:

(i) Interactions between nutrient components of the medium. A common occurrence during sterilization is the Maillard-type browning reaction which results in discoloration of the medium as well as loss of nutrient quality. These reactions are normally caused by the reaction of carbonyl groups, usually from reducing sugars, with the amino groups of amino acids and proteins. An example of the effect of sterilization time on the availability of glucose in a corn-steep liquor medium is shown in Table 5.1 (Corbett, 1985). Problems of this type are normally resolved by sterilizing the sugar separately from the rest of the medium and recombining the two after cooling.

(ii) Degradation of heat labile components. Certain vitamins, amino acids and proteins may be degraded during a steam sterilization regime. In extreme cases, such as the preparation of media for animal-cell culture, filtration may be used and this aspect will be discussed later in the chapter. However, for the vast majority of fermentations these problems may be resolved by the judicious choice of steam sterilization regime.