Introduction;
 Most industrial fermentations are carried out as pure
cultures in which only selected strains are allowed to
grow.
 If foreign microorganisms exist in the medium or any
parts of the equipment, the production organisms have
to compete with the contaminants for the limited
nutrients.
…cont
 The foreign microorganisms can produce harmful
products which can limit the growth of the production
organisms.
 Therefore, before starting fermentation, the medium
and all fermentation equipment have to be free from
any living organisms, in other words, they have to be
completely sterilized.
 Furthermore, the aseptic condition has to be
maintained.
STERI LIZATION METHODS
 Sterilization of fermentation media or equipment can
be accomplished by destroying all living organisms by
means of heat (moist or dry), chemical agents,
radiation (ultraviolet or X-rays), and mechanical
means (some or ultrasonic vibrations).
 Another approach is to remove the living organisms
by means of filtration or high-speed centrifugation.
…cont
 Heat is the most widely used means of sterilization,
which can be employed for both liquid medium and
heat able solid objects.
 It can be applied as dry or moist heat (steam).
 The moist heat is more effective than the dry heat,
because the intrinsic heat resistance of vegetative
bacterial cells is greatly increased in a completely dry
state.
…cont
 As a result the death rate is much lower for the dry
cells than for moist ones.
 The heat conduction in dry air is also less rapid than
in steam.
 Therefore, dry heat is used only for the sterilization of
glassware or heat able solid materials.
 By pressurizing a vessel, the steam temperature can be
increased significantly above the boiling point of
water.
…cont
 Laboratory autoclaves are commonly operated at a
steam pressure of about 30 psia, which corresponds to
121°C.
 Even bacterial spores are rapidly killed at 121 °C.
…cont
 Chemical agents can be used to kill microorganisms
as the result of their oxidizing or alkylating abilities.
 However, they cannot be used for the sterilization of
medium because the residual chemical can inhibit
the fermentation organisms.
…cont
 Among the techniques discussed, moist heat is the
most economical and efficient for the general
sterilization requirements of fermentation.
 Therefore, the following four sections describe cell
death kinetics and sterilization operations utilizing
moist heat.
THERMAL DEATH KINETICS
 Thermal death of microorganisms at a particular
temperature can be described by first-order kinetics:
 where kd is specific death rate, the value of which
depends not only on the type of species but also on the
physiological form of cells.
…cont
Integration of above equation gives
=
---
n =no exp ( ) …………..eqn (6.1)
 which shows the exponential decay of the cell
population.
 The temperature dependence of the specific death rate
kd can be assumed to follow the Arrhenius equation:
…cont
Which is
Kd=Kdo exp( )…………(6.2)
 where Ed is activation energy, which can be obtained
from the slope of the In(kd) versus 1/T plot.
DESIGN CRITERION
 From Eqs. (1) and (2), the design criterion for
sterilization
can be defined as
=
…..EQN(6.4)
 which is also known as the Del factor, a measure of the
size of the job to be accomplished.
 The Del factor increases as the final number of cells
decreases.
…cont
 For example, the Del factor to reduce the number of
cells in a fermenter from
is
viable organisms to one
= 10
 The reduction of the number of cells from
one seems to be impressive.
to
…cont
 However, even if one organism is left alive, the whole
fermenter may be contaminated.
 Therefore, all viable organisms have to be eliminated.
 The Del factor to reduce the number of cells to zero is
infinity, which means that it is theoretically impossible
to ensure the total destruction of the viable cells.
 Therefore, the final number of cells needs to be
expressed as the fraction of one, which is equal to the
probability of contamination.
BATCH STERI LIZATION
 Sterilization of the medium in a fermenter can be
carried out in batch mode by direct steam sparging, by
electrical heaters, or by circulating constant pressure
condensing steam through heating coil.
 The sterilization cycles are composed of heating,
holding, and cooling.
 Therefore, the total Del factor required should be
equal to the sum of the Del factor for heating, holding
and cooling as:
…cont
total =
heat + hold + cool ……..EQN(6.5)
 The values of
heat and cool are determined by the
methods used for the heating and cooling.
 The value of V hold is determined by the length of the
controlled holding period.
…cont
 The design procedure for the estimation of the holding
time is as follows:
1. Calculate the total sterilization criterion, total.
2. Measure the temperature versus time profile during
the heating, holding, and cooling cycles of
sterilization.
…cont
 If experimental measurements are not practical,
theoretical equations for heating and cooling can be
employed, which are of linear, exponential, or hyperbolic
form depending on the mode of heating and cooling.
 The suggested equations for different heating and cooling
processes are as follows:
A) For batch heating by direct steam sparging into the
medium, the hyperbolic form is used:
…..EQN(6.6)
…cont
b.For batch heating with a constant rate of heat flow
such as electrical heating, the
form is used:
……EQN(6.7)
c. For batch heating with a isothermal heat source such
as steam circulation through heating coil, the
l form is used:
...EQN(6.8)
…cont
d. For batch cooling using a continuous non isothermal
heat sink such as passing cooling water through
cooling coil, the exponential form is used:
.. ..EQN(6.9)
3. Plot the values of kd as a function of time.
4. Integrate the areas under the kd-versus-time curve for
the heating and the cooling periods to estimate heat
and
cool‘ respectively.
 If using theoretical equations, integrate Eq.
(6.4)numerically after substituting in the proper
temperature profiles. Then, the holding time can be
calculated from:
 thold=
total/kd =
heat+ cold +
cool /Kd……(6.10)
 Sterilization can be carried out in a continuous mode
rather than in batches.
 Continuous sterilization offers several advantages:
1. It simplifies production planning, thus allowing
maximum plant utilization and minimum delays.
2. It provides reproducible conditions.
3. It can be operated at a high temperature (140°C
instead of 121°C in batch sterilization); therefore, the
sterilization time can be shortened (holding time of 1
to 2 minutes).
4.It requires less steam by recovering heat from the
sterilized medium. As a result, it also requires
.
5. It is
to automate the process; thus, it is less
labor intensive.
A continuous sterilizer consists of three main sections:
heating, holding, and cooling.
 Methods of heating can be categorized into two types:
direct steam injection and indirect heating in shelland-tube or plate-and-frame heat exchanger.
 Direct heating is more effective than indirect heating
because there is no barrier between the medium and
the heat source.
 The steam injector heats the medium to the peak
sterilization temperature quickly.
Therefore, sterilization during the heating period is
negligible.
…cont
For indirect heating, the plate-and-frame heat
exchanger is generally more effective than the shelland-tube type for heat transfer due to its larger heattransfer area.
 However, the former is limited to lower pressures
(normally less than 20 atm) due to its weak structural
strength compared with the latter.
 The plate-and-frame type is also favorable for the
sterilization of a high viscous system
…cont
 The temperature change with respect to residence
time ('ῑ hold) as the medium passes through an
isothermal heat source can be
approximated as:
…cont

For heating using a counter current
heat source of equal flow rate
and heat capacity,

Holding Section
 The heated medium passes through a holding
section, which is usually composed of a long tube.
 The holding section is maintained in adiabatic
conditions.
 If the heat loss in the section is negligible, the
temperature can be assumed to be constant.
 The average residence time in the holding section is:
…cont

Cooling Section
 For the cooling section, a quench cooler with
adequate heat removal capacity is effective.
 Another technique is to inject the hot medium
through an expansion valve into a vacuum chamber,
which is known as flash cooling.
 Both of these take a very
; therefore, the
sterilization during the
period can be
assumed to be negligible.
…cont
A
or a plate-and-frame heat exchanger
can also be employed for cooling.
 The
versus
relationship for cooling using an isothermal heat sink
is:

 For aerobic fermentations, air needs to be supplied
continuously.
 Typical aeration rates for aerobic fermentation are
(air volume per liquid volume per minute).
 This requires an enormous amount of air. Therefore,
not only the medium but also the air must be free of
microbial contaminants.
All of the sterilization techniques discussed for
medium can also be employed for air.
However, sterilization of air by means of heat is
economically impractical and is also ineffective due to
the low
efficiency of air compared
with those of liquids.
 The most effective technique for air sterilization is
using fibrous or membrane filters.
…cont
With fibrous filters, airborne particles are collected by
the mechanisms of impact on, interception, and
diffusion.
: When an air stream containing
particles flows around a
collector, the
particle will follow the streamlines until they diverge
around the collector.
…cont
 The particles because of their mass will have sufficient
momentum to continue to move toward the cylinder
and break through the streamlines, as shown in Figure
6.1.
 The collection efficiency by this inertial impaction
mechanism is the function of the Stokes and the
Reynolds number as

 The efficiency “ήimp” is defined as the
of
particles approaching the collector which impact.
 Various correlations are available in the literature.
 An empirical correlation for the efficiency developed
by Thorn is (Strauss, 1975):
…cont

…cont
The inertial impaction model assumed
particles had
, and hence
but no size.
 An interception mechanism is considered where the
particle has size, but no
, and so they can follow the
streamlines of the air around the collector.
 If a streamline which they are following passes close
enough to the surface of the fiber, the particles will contact
the fiber and be removed (Figure 8.4).
 The interception efficiency depends on the ratio of the
particle diameter to the cylindrical collector diameter (K =
dp/ Dc):

…cont


: Particles smaller than about 1 micron in diameter exhibit a
Brownian motion which is sufficiently intense to produce diffusion.
 If a streamline containing these particles is sufficiently close to the
collector, the particles may hit the collector and be removed.
 Contrary to the previous two mechanisms, the collection efficiency by
diffusion increases with decreasing particle
or
.
 The typical size of particles collected by this mechanism is less than
about 0.5 μm.
 The efficiency of collection by diffusion can be estimated by an equation
analogous to Langmuir'S equation,
…

…cont

Combined Mechanisms:
The total collection efficiency of a fibrous filter is
obtained from the combined effect of the preceding
three mechanisms.
One straightforward way to combine the collection
efficiencies of the different mechanisms is to add them
together, but this implies that a particle can be
collected more than once, which does not make sense.
 A better approach is to use the following correlation,
…cont
…cont

Effect of Multiple Layers and
Packing:
 All correlations for the collection efficiency discussed
so far are based on the ideal case of a single cylindrical
collector.
 Now, let's examine a filter unit consisting of
.
 Consider an area (A) of filter at a right angle to the gas
flow and with a depth dh.
 If the packing density α is defined as the
per unit
, the velocity
within the filter void space is equal to
…cont
…cont
…cont