Sterilization

A fermentation product is produced by the culture of a certain organism, or

organisms in a nutrient medium. If the fermentation is invaded by a
foreign microorganism then the following consequences may occur:

Avoidance of contamination may

be achieved by:
1. Using a pure inoculums to start
the fermentation.
2. Sterilizing the me
dium to be
employed
3. Sterilizing the fermnter vessel
4. Sterilizing all material to be
added to the fermentation
during the process.
5. Maintaining aseptic condition
during the fermentation.
 Medium sterilization
Media may be sterilized by filtration, radiation ultrasonic treatment, chemical treatment or
heat. However, for practical reasons, STEAM is used universally.
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.
The destruction of micro organisms by steam may be described as a first order chemical
reaction and thus may by represented by the following equation:
The relationship in above Fig
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
depended on the
physiological form of the cell.
Eg. Endopsore of the genus
Bacillus are far more heat
 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 the true constant only under constant
temperature conditions.
The relationship b/w Temp & the reaction rate constant was
demonstrated by Arrhenius & may be represented by the
equation:
It is clear that the same degree of sterilization may be obtained
over a wide range of time & temp.
This kinetic description of bacterial death enables the design of
procedure for the sterilization of fermentation broths.
By choosing a value for Nt procedure may be designed having a
certain probability of achieving sterility, based upon the degree of
risk that is considered acceptable.
Only microbial contaminants present are spores of Bacillus
stearothermophilus (most heat resistant)
Bacillus macerans suspended in oil were ten times more resistant
to sterilization if they were dry than if they were wet.
 Time & temp may now be determined to achieve the
desired Del factor.
Deleterious reactions may occur in the medium during the
sterilization process, resulting in loss of nutritive quality.
Deleterious effect of increasing medium
sterilization time on the yield of product of
subsequent of fermentations.
Initial rise in yield is due to some
component of the medium being made
more available to the process micro-
organism by the Cooking Effect of a brief
sterilization period.
Two types of reactions contribute to the los
of nutrient quality during sterilization:
1. Interactions b/w nutrient components of
the medium
2. Degradation of heat liable components.
 The thermal destruction of essential media components conforms
approximately with first order reaction kinetics, may be describe
by equation similar to derived for destruction of bacteria:

Decline in conc. Of the nutrient components, along with decline in the

no of contaminants. The effect of temp on the reaction rate constant
may be expressed by the Arrhenius equations:

Therefore, a plot of natural logarithm of the reaction rate against 1/T

will give a straight line, slope - (E/R). As R (gas constant) is fixed, the
slope of the graph is determined by the value of the activation energy
(E). E.g the value of E for B. stearothermophilus spores has
67.7Kcal/mole, whereas the thermal destruction of the nutrients is 10
-30 kcal/mole (Richard 1968)
From this plot it may be seen that as temp increased, the reaction rate
rises more rapidly for the reaction with higher activation energy.
Thus, considering the difference b/w activation energies for spore
destruction & nutrient degradation, an increase in temp would
accelerate spore destruction more than medium de-naturation.
Del factor could be achieved over a range of temp/time regimes. So
high temp for a short time to achieve desired probability of sterility, with
minimum nutrient degradation.
 Advantages of continuous sterilization over batch
sterilization
Superior maintenance of medium quality
Ease of scale up
Easier automatic control
The reduction of surge capacity for steam.
Reduction of sterilization cycle time
Advantages
Under certain circumstances,
of batch the reduction of fermenter
sterilization overcorrosion.
continuous
sterilization
Lower capital cost
Lower risk of contamination continuous processes requires the
aseptic transfer of the sterile broth to the sterile vessel.
Easier manual control
Easier to use with media containing a high proportion of solid
matter

Early continuous sterilizers (plate heat exchangers), unsuitable

on two accounts
1. Failure of the gas kit b/w the plates resulting in mixing of
sterile & unsterile streams
2. Particulate components in the media would block the heat
exchangers.

However, modern continuous sterilizers use double spiral heat

exchangers in which two streams are separated by a
 DESIGN OF BATCH STERILIZATION PROCESS
It is less successful in avoiding the destruction of nutrients than a
continuous one. But still used with minimum loss of nutritive quality.
Highest temp 121C, but procedure should be designed for the exposure
of medium to this temp kept minimum.
This is achieved by heating and cooling periods. Following information
must be available for the design of batch sterilization process:

Knowing the original no of organisms present in the fermenter &

the risk of contamination considered acceptable, the required Del
Factor may be calculated.
A frequently adopted risk of contamination is 1 in 1000 which
indicate that Nt should equal to 10-3 of the viable cells.
e.g: if a specific case in which unsterile broth was shown to contain
1011 viable organism then the Del factor may be calculated, thus:
 Therefore the overall Dell Factor required is 32.2. however, the destruction of
cells occurs during the heating and cooling of the broth as well as during the
period of 121C, thus the over all Del Factor may re presented as:

Method of batch sterilization: (advantages of separate

sterilization vessels)
1. Cooker is used to sterilized the medium
2. Sterilized in a cooler in more concentrated form than used in
the fermentation, then diluted in the fermenter by sterile
water
3. If medium is viscous then require more power in fermenter,
thus if requirement for agitation is removed it need less
power, but sterilization kettle would have with power motor.
4. Fermenter would be spared the corriosn at high temperature
Disadvantage of a separate sterilization vessels:
5. Cost of constructing a batch medium sterilizer is much the
same as that of the fermenter
6. If cooker server a large no of fermenter complex pipe work
would be necessary to transport the sterile medium with
inherent danger of contamination.
7. Mechanical failure in a cooker will stop all the process, it
 THE DESIGN OF CONTINUOUS STERILIZATION
PROCESS
The design of continuous sterilization cycles may be approached in exactly
the same way as for batch sterilization system.
The continuous system includes a time period during which the medium is
heated to the sterilization temp, a holding time at the desired temp & a
cooling period to restore the medium to the fermentation temp.
The length of the holding period is dictated by the length of the coil & the
flow rate of the medium. First it maintain the temp for heating up the
incoming medium & second using cooling water.
Advantage:
A much higher temp may be utilized, thus reducing the holding time and
reducing the degree of nutrient degradation.
The required Del Factor may be achieved by the combination of temp &
holding time which gives an acceptably small degree of nutrient decay.
Furthermore, b/c of continuous process involves treating small increments of
medium the heating up and cooling down periods are very small compared
with those in a batch system.
There are two types of continuous sterilizer which is used for the treatment
of fermentation media:
1. indirect heat exchanger
2. direct heat exchanger (steam)
 The most suitable indirect heat exchanger are of double spiral-type
which consist of two sheets of high grade stainless steel which have
been curved around a central axis to form a double spiral.
 To achieve sterilization temp steam is passed
through one spiral & medium through the
other in countercurrent stream.
Spiral heat is also used to cool the medium
after passing through the holding coil.
Incoming unsterile medium is used as the
cooling agent in the first cooler so that the
incoming medium is partially heated before it
reaches the sterilizer & thus, heat is
conserved.
Advantages of spiral heat:
1. avoid contamination.
2. Clearances self cleaning reduce the risk of
sedimentation, fouling & burning on.
 Indirect plate heat exchanger consist of
alternating plates through which the
countercurrent streams are circulated.
The plates are separated by gas kit & failure of
these gaskets can cause cross contamination
b/w two streams.
Clearances b/w the plates i.e suspended solids
in the medium may block the exchanger & thus
system is only useful in sterilizing completely
soluble media
However, the plate exchanger is more
adaptable than the spiral system in that extra
plates may be added to increase its capacity.
 The continuous steam injector injects steam directly into the unsterile broth.
Advantage:
1. Very short heating up time.
2. May be used for suspended solids
3. Low capital cost
4. Easy cleaning & maintenance.
5. High steam utilization efficiency.
Disadvantages:
6. Foaming occur during heating
7. Direct contact of the medium with steam are requires, condense dilution & requires clean steam,
free from anticorrosion additives.

In some cases a combination of direct & indirect heat exchangers

may be used.
 The most widely used continuous sterilizations system is that based on the spiral
heat exchangers.
Plant is sterilize by circulating a hot (sterilized) water through the plant in a closed
circuit.
Heat conservation is achieved by cooling the sterile medium against cold, incoming
unsterile medium which will then be partially heated before it reaches the sterilizer.
Del factor to be achieved in a continuous sterilization process has to be increased
with an increase in scale eg. If the volume to be sterilized is increased from
1000dm3 to 10,000 dm3 & the risk of failure is to remain at 1 in 1000 then Del factor
must be increased from 34.5 to 36.8.
Advantage of the continuous process is that temp may be used as variable in
scaling up continuous process so that the increased Del factor may be achieved,
maintaining the nutrient quality constant.