This document discusses sterilization methods for large-scale fermentation processes. It focuses on heat sterilization of liquids in batch and continuous systems. For batch sterilization, the liquid is heated to the sterilization temperature and held for a period of time before being cooled. Continuous sterilization allows for faster and more economical sterilization through rapid heating, exposure, and cooling. The key factors in sterilization include temperature, exposure time, and achieving plug flow to ensure all fluid is uniformly sterilized.

1. S t e r i l i s a t i o n
• Commercial fermentations typically require
thousands of litres of liquid medium and millions of
litres of air.
• For processes operated with axenic cultures, these
raw materials must be provided free from
contaminating organisms.
• Of all the methods available for sterilisation
including chemical treatment, exposure to
ultraviolet, gamma and X-ray radiation, sonication,
filtration and heating, only the last two are used in
large-scale operations.

2. Batch Heat S t e r i l i s a t i o n o f L i q u i d s
• Liquid medium is most commonly sterilised in batch in
the vessel where it will be used.
• The liquid is heated to sterilisation temperature by
introducing steam into the coils or jacket of the vessel;
• alternatively, steam is bubbled directly into the
medium, or
• the vessel is heated electrically.
• If direct steam injection is used, allowance must be
made for dilution of the medium by condensate which
typically adds 10-20% to the liquid volume;
• quality of the steam must also be sufficiently high to
avoid contamination of the medium by metal ions or
organics.

3. • A typical temperature-time profile for batch
sterilisation is shown in Figure 13.35(a).
• Depending on the rate of heat transfer from the
steam or electrical element, raising the temperature
of the medium in large fermenters can take a
significant period of time.
• Once the holding or sterilisation temperature is
reached, the temperature is held constant for a
period of time thd.
• Cooling water in the coils or jacket of the fermenter
is then used to reduce the medium temperature to
the required value.

5. • For operation of batch sterilisation systems, we
must be able to estimate the holding time required
to achieve the desired level of cell destruction.
• As well as destroying contaminant organisms, heat
sterilisation also destroys nutrients in the medium.
• To minimise this loss, holding times at the highest
sterilisation temperature should be kept as short as
possible.
• Cell death occurs at all times during batch
sterilisation, including the heating-up and cooling-
down periods.
• The holding time thd can be minimised by taking into
account cell destruction during these periods.

6. • Rate of heat sterilisation is governed by the
equations for thermal for first-order death kinetics,
in a batch vessel.
• where cell death is the only process affecting the
number of viable cells:
• where
N is number of viable cells,
t is time and
– k d is the specific death constant.
• Eq. (13.95) applies to each stage of the batch
sterilisation cycle: heating, holding and cooling.

7. • where thd is the holding time,
• N 1 is the number of viable cells at the start of
holding, and
• N 2 is the number of viable cells at the end of
holding,
• k d is evaluated as a function of temperature using
the Arrhenius equation:

8. • where
• A is the Arrhenius constant or frequency factor,
• E d is the activation energy for the thermal cell
death,
• R is the ideal gas constant and
• T is absolute temperature.
• N1 and N2 are determined by considering the extent
of cell death during the heating and cooling periods
when the temperature is not constant. Combining
Eqs (13.95) and (11.46) gives:

9. • Integration of Eq. (13.98) gives for the heating period:
• where
• t 1 is the time at the end of heating,
• t 2 is the time at the end of holding and
• t f is the time at the end of cooling.
• We cannot complete integration of these equations
until we know how the temperature varies with time
during heating and cooling.

11. Continuous Heat Sterilisation of Liquids
• Continuous sterilisation, particularly a high-
temperature, short-exposure-time process, can
significantly reduce damage to medium ingredients
while achieving high levels of cell destruction.
• Other advantages include improved steam economy
and more reliable scale-up.
• The amount of steam needed for continuous
sterilisation is 20-25% that used in batch processes;
• the time required is also significantly reduced
because heating and cooling are virtually
instantaneous.

13. • Typical equipment configurations for continuous sterilisation
are shown in Figure 13.37.
• In Figure 13.37(a), raw medium entering the system is first
pre-heated by hot, sterile medium in a heat exchanger;
• this economises on steam requirements for heating and cools
the sterile medium.
• Steam is then injected directly into the medium as it flows
through a pipe;
• The liquid temperature rises almost instantaneously to the
desired sterilisation temperature.
• The time of exposure to this temperature depends on the
length of pipe in the holding section of the steriliser.
• After sterilisation, the medium is cooled instantly by passing it
through an expansion valve into a vacuum chamber; further
cooling takes place in the heat exchanger where residual heat
is used to pre-heat incoming medium.

14. • Figure 13.37(b) shows an alternative sterilisation scheme
based on heat exchange between steam and medium.
• Raw medium is pre-heated with hot, sterile medium in a heat
exchanger then brought to the sterilisation temperature by
further heat exchange with steam.
• The sterilisation temperature is maintained in the holding
section before being reduced to the fermentation
temperature by heat exchange with incoming medium.
• Heat-exchange systems are more expensive to construct than
injection devices;
• fouling of the internal surfaces also reduces the efficiency of
heat transfer between cleanings.
• On the other hand, a disadvantage associated with steam
injection is dilution of the medium by condensate;
• foaming from direct steam injection can also cause problems
with operation of the flash cooler.

15. • As indicated in Figure 13.38, rates of heating and cooling in
continuous sterilisation are much more rapid than in batch;
• accordingly, in design of continuous sterilisers, contributions
to cell death outside of the holding period are generally
ignored.
• An important variable affecting performance of continuous
sterilisers is the nature of fluid flow in the system.
• Ideally, all fluid entering the equipment at a particular instant
should spend the same time in the steriliser and exit the
system at the same time;
• unless this occurs we cannot fully control the time spent in
the steriliser by all fluid elements.
• No mixing should occur in the tubes; if fluid nearer the
entrance of the pipe mixes with fluid ahead of it, there is a
risk that contaminants will be transferred to the outlet of the
steriliser.

16. • The type of flow in pipes where there is neither
mixing nor variation in fluid velocity is called plug
flow
• Plug flow is an ideal flow pattern; in reality, fluid
elements in pipes have a range of different
velocities.