CHAPTER 5

Sterilization
INTRODUCTION (ii) Sterilizing thc mediunn tu be employed.
(iii) Sterilizing the fermenter vessel.
(iv) Sterilizing all materials to be added to the

A FERMENTATION product is produced by the culture of

fermentation during the process.
(v) Maintaining aseptic conditions during the fer
a certain organism, or organisms, in a nutrient medium. mentation.
If the termentation is invaded by foreign micro
organism thcn thc following consequences may occur: The extent to which thesc proccdures aic adopted is
(i) Thec medium would Iave to support the determined by the likely probability of contamination
growth and the nature of its conscquences. Some fermenta
of both the production organism and the con tions are described as 'protected' that is, the medium
taminant, resulting in a loss of productivity. may bc utilizcd by only a very limited range of micro
(ii) If the fermentation is a continuous onc then
Lhe contaminant may 'outgrow' the production organisms, or the growth the process organism may
rcsult in the develupment of selective growth condi
organism and displace it from thc fermenta tions, such as a reduction in pH. Thc brcwing of beer
tion.
falls intu this categorv; hop resins tend to inhibit the
(iii) The foreign organism may contaminate the final
product, e.g. single-cell protein where the cells, growth many micro-orgunisms and the growtl1 of
separated from the bioth, constitute the brewing yeasts tends to decrease the pH of the medium.
Thus, brewing worts arc boiled, but uL necessarily
product. sterilized, and the fermenters are thoroughly cleaned
(iv) Tho contamnant nay produce compounds with disinfectant solution but are not necessarily ster
which make subsequent extraction of the final
product difficult. ile. Also, the precautions used in the development of
(v) The contaminant may degrade the desircd inoculum for brcwing are far less stringent than, for
product: this is common in bacterial contami example, in an antibiotic fermentation. Howcver, the
nation of antibiotic fermcntations where the vast majority vf lementations are not 'protected' and,
if contaminated, would suffer some of the conse
contaminant would have to be resistant to the
normal inhibitory cffccts of the antibiotic and quences previously listed. The approaches adopted to
avoid contamination will be discuSscd in moie delail,
degradation of the antibiotic is a common resis
apart from the development of aseptic inocula which
tance mcchanism, cg. tlie degradation of B
lactam antibiotics by considered in Chaptcr 6 and the aseptic operation and
bacteria.
B-lactamase-producing containment of fermentation vessels which are dis
(vi) cussed in Chaptcrs 6 and 7.
Contamination of a bacterial fermentation with
phage could result in the lysis of the culture.
MEDIUM STERILIZATION
Avoidance of contamination may be achieved by:
As poited out by Corbett (1985), media may be
() Using a pure inoculum to start the fermenta sterilized by filtration, radiation, ultrasonic trcal1lient
tion, as discussed in Chapter 6.
chemical treatment or heat. However, for practical
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 Sterllization

THE DESIGN OF BATCH STERILIZATION and cooling of the broth as well as during the period at
PROCESSES 121°C, thus, the overall Del factor may be represented
as:

Although a batch sterilization process is less success Voverall Vheating t Vholding t Voooling
ful in avoiding the destruction of nutrients than a Knowing the temperature-time profile for the heating
continuous one, the objective in designing a batch and cooling of the broth (prescribed by the characteris
process is still to achieve the required probability of tics of the available equipment) it is possible to de
obtaining sterility with the minimum loss of nutritive termine the contribution made to the overall Del factor
quality. The highest temperature which appears to be by these periods. Thus, knowing the Del factors con
feasible for batch sterilization is 121°Cso the proce- tributed by heating and cooling, the holding
dure should be designed such that exposure of the time may
be calculated to give the required overall Del factor.
medium to this temperature is kept to aa minimum. This
is achieved by taking into account the contribution
made to the sterilization by the heating and cooling Calculation of the Del factor during
periods of the batch treatment. Deindoerfer and Hum heating and cooling
phrey (1959) presented a method to assess the con
tribution made by the heating and cooling periods. The The relationship between Del factor, the tempera
following information must be available for the design ture and time is given by cquation (S.8):
of a batch sterilization process: V= A(-e-(E/RT)
However, during the heating and cooling periods the
(i) A profile of the increase and decrease in the temperature is not constant and, therefore, the calcula
temperature of the fermentation medium dur tion of V would require the integration of equation
ing the heating and cooling periods of the (5.8) for the time-temperature regime observed. Dein
sterilization cycle. doerfer and Humphrey (1959) produced integrated
(ii) The number of micro-organisms originally pre forms of the equation for a variety of temperature-time
sent in the medium.
profiles, including lincar, exponential and hyperbolic.
(iii) The thermal death characteristics of the de However, the regime observed in practice is frequently
sign' organism. As explained earlier this may difficult to classify, making the application of these
be Bacilus stearothermophilus or an alternative complex cquations problematical. Richards (1968) de
organism relevant to the particular fermenta monstrated the use of a graphical method of integra
tion. tion and this is illustrated in Fig. 5.7. The time axis is
divided into a number of cqual increments, t1, lz, ty+
Knowing the original number of organisms present in etc., Richards suggesting 30 as a reasonable number.
the fermenter and the risk of contamination considered
acceptable, the required Del factor may be calculated.
A frequently adopted risk of contamination is 1 in
1000, which indicates that N, should equal 10-3 of a
viable cell. It is worth reinforcing at this stage that we
are considering the total number of organisms present
in the medium and not the concentration. If a specific
case is considered where the unsterile broth was shown
to contain 10" viable organisms, then the Del factor Temperature
may be calculated, thus:

F= In (1o"/10-)
t.
V= In l04
-32.2, Time

FIG. 5.7. The graphical integration mcthod applicd to the increase

Therefore, the overall Del factor required is 32,2. How in temperature over a time period. , 2, ctc. represent cqual time
ever, the destruction of cells occurs during the heating intervals (Richards, 1968).

129
Principles of Fermentation Technology, 2nd Edn.

For each increment, the temperature corresponding the cycle a considerable reduction in exposure time is
to the mid-point time is recorded. It may now be achieved
approximated that the total Del factor of the hecating-up
period is equivalent to the sum of the Del factors of Richards' rapid method for
the mid-point temperatures for each time increment. the design of sterilization cycles
The value of the specific death rate of B. stearother
mophilus spores at each mid-point temperature may be
deduced from the Arrhenius equation using the ther Richards (1968) proposed a rapid method for the
mal death characteristic published by Deindoerfer and design of sterilization cycles avoiding the time-consum
Humphrey (1959). The value of the Del factor corre ing graphical integrations. The method assumes that all
sponding to each time increment may then be calcu spore destruction occurs at temperatures above 100C
lated from the equations: and that those parts of the heating and cooling cycle
above 100° are linear. Both these assumptions appear
V,=k, reasonably valid and the technique loses very little in
accuracy and gains considerably in simplicity. Further
more, based on these assumptions, Richards has pre-.
V,= kst,
sented a table of Del factors for B. stearothermophilus
etc. spores which would be obtained in hcating and cooling
The sum of the Del factors for all the increments will a broth up to (and down from) holding temperatures of
then equal the Del factor for the heating-up period. 101-130°C, based on a temperature change of 1C per
The Del factor for the cooling-down period may be minute. This information is presented in Table 5.2,
calculated in a similar fashion. together with the specific death rates for B. stearother.
mophilus spores over the temperature range. If the rate
Calculation of the holding time of temperature change is 1° per minute, the Del factors
at constant temperature for heating and cooling may be read directly from the
table; if the temperature change deviates from 1° per
minute, the Del factors may be altered by simple
From the previous calculations the overall Del fac proportion. For example, if a fermentation broth were
tor, as well as the Del factors of the heating and heated from 100° to 121°C in 30 minutes and cooled
cooling parts of the cycle, have been determincd. from 121° to 100° in 17 minutes, the Del factors for the
Therefore, the Del factor to be achieved during the heating and cooling cycles may be determined as fol
holding time may be calculated by difference: lows:
Vholding Voverall -Vheating - Vcooling From Table 5.2, if the change in temperature had
Using our example where the overall Del factor is 32.2
been 1° per minute, the Del factor for both the heating
and cooling cycles would be 12.549. But the tempera
and if it is taken that the heating Del factor was 9.8 ture change in the heating cycle was 21° in 30 minutes;
and the cooling Del factor 10.1, the holding Del factor therefore,
may be calculated:
Vholdinw =32.2 - 9.8- 10.1,
Delheating (12.549 x 30)/21 = 17.93
Vholding = 12.3. and the temperature change in the cooling cycle was
21° in 17 minutes, therefore,
But =kt, and from the data of Deindoerfer and
Humphrey (1961) the specific death rate of B. Del cogling = (12.549 X 17)/21 = 10.16.
stearothermophilus spores at 121°C is 2.54 min
Having calculated the Del factors for the heating and
Therefore,t = V/k or t = 12.3/2.54 = 4.84 min. cooling periods the holding time at the constant tem
If the contribution made by the heating and cooling perature may be calculated as bcfore.
parts of the cycle were ignored then the holding time
would be given by the equation: The scale up of batch sterilization processes
t-Voeal/k = 32.2/2.54 = 12.68 min.
Thus, by considering the contribution made to the The use of the Del factor in the scale up of batch
sterilization process by the heating and cooling parts of sterilization processes has been discussed by Banks
130