UNIT V-

STERLIZATION
 Definition

• Sterility: the absence of detectable levels of viable organisms in a

culture medium or in a gas.

• Sterile means devoid of life.

• When a system has organisms but only those that are supposed to be
present, the situation is aseptic.

• After achieving sterility, asepsis must be maintained.

 Effects of contamination

• Medium would have to support the growth of both the

production organism as well as contaminating organism, resulting
in loss of productivity.

• In case of continuous process ,contaminant may outgrow the

production organism.

• Foreign organism may contaminate the end product.

• Contaminant may produce some compounds which may interfere

with the DSP.

• Contaminate may degrade the desired product.

 Avoidance of contamination

• Start the fermentation using a pure culture inoculum.

• Sterilization of medium.

• Sterilization of fermenter.

• Sterilization of ancillary equipment.

• Sterilization of all the materials to be added.

• Maintenance of aseptic conditions through out the process.

 Sterilization Agents
Thermal - preferred for economical large-scale sterilizations of
liquids and equipment.

Chemical - preferred for heat-sensitive equipment

• ethylene oxide (gas) for equipment
• 70% ethanol-water (pH=2) for equipment/surfaces
• 3% sodium hypo chlorite for equipment

Radiation - UV for surfaces, X-rays for liquids (costly/safety)

Filtration- membrane filters having uniform micro pores and

depth filters of glass wool.
 Medium sterilization
Moist heat (steam at high pressure) is used almost universally
for the sterilization of fermentation medium.

Kinetics of Thermal Sterilization

1. Not all organisms have identical death kinetics. (increasing
difficulty; vegetative cells < spores < virus)
2. Individuals within a population of the same organism may
respond differently.
3. Total number of viable organisms present in the volume of
medium to be sterilized are considered and not the
concentration.
4. Minimum number of viable organisms to contaminate a batch
is one, regardless of the volume.
 Kinetics of Thermal Sterilization
The destruction of microorganisms by steam may be described as a
first- order chemical reaction and may be represented as:

-dN / dt = kN

N = number of viable organisms present

t = time of sterilization treatment
k= reaction rate constant of the reaction or the specific death rate.

The value of k depends on the species as well as the physiological

form of the cell.
On integrating the above equation we get :

Nt / N0= e-kt

N0= Total number of viable organisms present at the start of

sterilization treatment.

Nt= number of viable organisms present after a treatment

period t.

On taking natural logarithms :

ln (Nt/N0) = -kt
 Graphical representation
• This kinetic description
makes two predictions:

1. An infinite time is required

to achieve sterile
conditions i.e. Nt=0.

2. After a certain time there

will be less than one viable
cell present.

Thus a value of Nt< 1 is

considered in terms of
probability of an organism
surviving the treatment.
 Relationship between k and absolute
temperature
• The reaction rate of thermal sterilization increases with increase in
temperature(as for all first order reactions),due to increase in the reaction
rate constant(specific death rate).

• Thus k is a true constant only under constant temperature conditions.

• Relationship between reaction rate constant and temperature is given by

Arrhenius Equation:
k = A e-E / RT
A = Arrhenius constant (time-1 )
R = gas constant
T = absolute temperature
E= activation energy for death (50 -150 kcal / g – mole for spores) & (2 - 20
kcal / g – mole for vitamins / growth factors).
• On taking natural logarithms :

ln k = ln A - E/RT

• A plot of ln k against the reciprocal of the absolute

temperature is called as Arrhenius plot and it will give a
straight line .

• This plot enables the calculation of the activation energy and

the prediction of reaction rate at any temperature.

• Combining above equations we get:

ln (Nt/N0) = A.t. e-E / RT

• The term ln (N0/Nt) is called as Del factor, Nabla factor or
sterilization criterion.

• It is represented as 

• a parameter which encompasses the contamination level

of the medium to be sterilized.

• It is defined as the fractional reduction in viable organism

count produced by a certain heat and time regime.

= ln (N0/Nt)
kt = A.t. e-E / RT

 = A.t. e-E / RT
On taking natural log and rearranging
above equation:

ln t = E/RT+ ln ( /A)

-A plot of the natural logarithm of the

time required to achieve a certain del
factor  value against the reciprocal of
the absolute temperature yield a
straight line, the slope of which is
dependent on the activation energy.

-Same degree of sterilization() may

be obtained over a wide range of time
and temperature regime.
 Nutrient loss during thermal sterilization
During heat sterilization there is always the loss of nutrient
quality.
Two types of reactions contribute to this:

1. Degradation of heat-labile components

2. Interaction between nutrient components of the medium:

Maillard-type browning reactions which cause discoloration
of the medium as well as loss of nutrient quality.

These reactions are normally caused by carbonyl groups, usually

from reducing sugars, interacting with amino groups from amino
acids and proteins.
The thermal destruction of media components may be described
by the equations similar to those for the destruction of bacteria:

xt/x0 = e-kt
xt=concentration of nutrient after a heat treatment period t.
x0=original concentration of nutrient at the onset of sterilization.
k = reaction rate constant

The effect of temperature on rate of reaction is expressed by the

Arrhenius equation:

ln k =ln A –E/RT
- The slope of the plot is equal to –(E/R) i.e .determined by the
activation energy.
- as the temperature rises, the reaction rate rises more rapidly for
the reaction with higher activation energy.
 Batch Sterilization
• Objective- obtaining sterility with
the minimum loss of nutrients.

• Highest temperature is generally

1210C.

• Process is designed so as to
minimize the exposure of nutrients
to 1210C.

• Longer heat-up/cool down time

• The contribution of heating and

cooling period is also taken into
account.
 Design of Batch Sterilization
• Batch sterilization wastes energy and can overcook the
medium.

• The required information for designing the process:

1. A profile of increase and decrease in the temperature

during heating and cooling period.
2. Initial load of microorganisms(N0).
3. Thermal death characteristics of the design organism.
 STEPS INVOLVED
1.Calculate the desired Del factor from the original number of
organisms present & the acceptable risk of contamination
(generally 1 in 1000 is adopted risk of contamination i.e.Nt is 10-3).

2. The destruction also occur during heating and cooling period, so

the overall del factor may be represented as:
 overall=  heating+  holding+  cooling

Del factor during heating and cooling period is calculated by

different methods
METHOD 1
Integrating  = A.t. e-E / RT as T changes during heating and
cooling period.
METHOD 2

• Another way is the use of Richards’ graphical method of

integration.

• Time axis is divided into a number of equal increments,

t1,t2,etc. & the temperature corresponding to mid point is
recorded.

• The total Del factor of the heating period is equivalent to the

sum of the Del factors of the midpoint temperatures for each
time increment.
Design of Batch Sterilization

• The value of Del factor corresponding to each time

increment may be calculated as:
 1=k1t
 2=k2t etc.
• The value of k may be deduced from the Arrhenius
equation.

•The value for Del factor cooling period can be calculated

in the similar manner.
 Calculation of holding time at
constant temperature:
 holding =  overall – heating-  cooling

• Since the terms on R.H.S.are known the value of 

holding can be calculated.

• But  = kt

• From the data the value of k for can be known.

• Therefore holding time (t) = k / 

Scale up of Batch Sterilization process

• Del factor does not include a volume term, i.e. absolute number
of contaminants and survivors are considered.

• So on increasing the size of fermenter the value of N0 also

increases.

• If same Nt is to be achieved Del factor has to be increased.

 new= ln(Nt old x a) / N0

a= increase in dimensions.
 Methods of Batch Sterilization
1. Steam or direct firing to elevate the temperature, and
then cooling water stops the process and brings the
material back to room temperature.

2. The fermenter contents are heated by a jacket, coils,

and injection of live steam.

3. Injecting steam into the fermenter mantle or interior

coils - red sterilization.

4. Inject steam into the nutrient solution itself-condensate

accumulates in the fermenter and the volume of liquid
increases during the sterilization process.
 Methods of Batch Sterilization
• The cooling depends on heat transfer area & on driving
force which is best with very cold water.

• Some additional cooling can come from venting the steam

from the fermenter as the contents are above boiling
temperature.

• The ratio of killing to cooking becomes more favorable at

higher temperature.

• More time at lower temperature is bad because there is

cooking without the best amount of killing.
Disadvantages of Batch Sterilization

1. culture medium damage

2. high energy consumption

3. Incomplete mixing
Continuous Sterilization

1. Shorter time

2. Higher temperature

• Exponential relationship between death rate and temperature, makes the

time required for the sterilization shorter, if higher temperatures are used.

• While batch sterilization is carried out in 15-20 minutes at 121°C,continuous

sterilization is normally accomplished in 30-120 seconds at 140°C.
Methods of Continuous Sterilization
I. STEAM INJECTION
• One method of continuous
sterilization injects steam into
the medium (no heat
exchanger).

• The medium stays in a loop for a

predetermined holding time
until the entire medium is
sterile.

• Sterilization with steam

injection is done by injecting
steam into the nutrient solution.
• The temperature is raised
quickly to 140°C and is
maintained for 30-120 seconds.
Methods of Continuous Sterilization
II. USE OF HEAT EXCHANGER

In the continuous process using heat exchanger the nutrient solution in the
first heat exchanger is preheated to 90-120°C within 20-30 seconds by the
exiting previously sterilized nutrient solution.

• Then in the second heat exchanger, it is heated indirectly with steam to

140°C.

• This temperature is maintained for 30-120 seconds in a holding pipe before it

is placed in the first exchanger for preliminary cooling and then in a third
exchanger for cooling to the temperature of the fermenter.

• The cooling phase is only 20-30 seconds.

• In the process using heat exchangers, 90% of the energy, input is recovered.
Spiral heat exchanger
•A heat exchanger utilizes the
fact that heat transfer occurs
when there is a difference in
temperature.
•In a heat exchanger, there is a
cold stream and a hot stream.
•The two streams are separated
by a thin, solid wall.
•The wall must be thin and
conductive in order for heat
exchange to occur.
• Yet the wall must be strong
enough to withstand any
pressure by he fluid.
•Copper/Stainless steel- most
common choice for construction.
 Spiral heat exchanger
Flow diagram showing how heat transfers in a heat exchanger:

This flow arrangement is called co-current. If the direction of one of the

stream is reversed, the arrangement is called counter-current flow.
 Disadvantages of spiral heat exchanger

1. with some nutrient solutions, insoluble salts (e.g., calcium oxalate) are
formed and crusts a the first heat exchanger, due to the temperature
differences between the nutrient solution and the incoming cold water.

2. Starch-containing solutions which turn viscous when heated are difficult

to use in continuous sterilization processes.
Plate heat exchanger
Spiral heat Plate heat
exchanger exchanger
Advantages of continuous sterilization

1. Increase of productivity since the short period of exposure to heat

minimizes damage to media constituents.

2. Better control of quality.

3. Leveling of the demand for process steam.

4. Suitability for automatic control.

Scale up of continuous Sterilization process
Del factor is scale dependent so it has to be increased with
increase in scale .
Calculations are similar to that of batch sterilization.
But in continuous sterilization the advantage is that temperature
may be used as a variable for scale up rather than time.
So the increased Del factor may be achieved without
compromising on nutrient quality.
However nutrient quality criterion Q is not scale dependent, so
that by changing the temperature-time regime to achieve new Del
factor nutrient quality may be adversely affected.
 Filter sterilization
• Filter sterilization is often used for all components of nutrient solutions
which are heat sensitive.

• Sugars, vitamins, antibiotics or blood components are examples of heat-

labile components which must be sterilized by filtration.

• Empty glassware that are used to hold media must be sterilized in an

autoclave before filter sterilization.

• Filters themselves must be steam sterilized before and after the

operation.

• So the material must be stable at high temperature.

• Steam itself must be free of particulate matter.

 Filter sterilization mechanisms

• Suspended solids are separated from a fluid during filtration by

the following mechanisms :

1. Inertial impaction
2. Diffusion
3. Electrostatic attraction
4. Interception.
Types of filters
1. Absolute filters(fixed pore size filters): are those filters in which the
pore size is smaller than the particles to be removed.

• Pore size is controlled so that an absolute rating can be given to the

filter.

• Interception is the major mechanism for removal of particles.

• Since this kind of filter is also having a depth so filtration can also take
place through inertial impaction,diffusion and electrostatic attraction.

• Are less susceptible to pressure changes .

• Chances of release of trapped particles are less.

Types of filters(cont.)
2. Depth filters (non-fixed pore size filters) : are those filters in which the
pore size is larger than the particles to be removed.

• made up of woven yarns, asbestos pads or loosely packed fiber glass.

• Removal of particles is through inertial impaction, diffusion and

electrostatic attraction rather than interception.

• Since fibers are not cemented into a fixed position, movement of material
may occur on applying high pressure.

• This produces large channels from which the trapped material can be
released.
1.STERILIZATION OF FERMENTER
2.STERILIZATION OF FEEDS
3.STERILIZATION OF LIQUID WASTES
4.FILTER STERILIZATION OF AIR
Most industrial fermentations are operated under vigorous
aeration and the air supplied to the fermenter must be sterilized.

To prevent contamination of either the fermentation by airborne

microorganisms or the environment by aerosols generated within
the fermenter, both air input and air exhaust ports have air filters
attached.

These filters are designed to trap and contain microorganisms.

Filters are made of glass fiber, mineral fibers, poly tetra-
fluoroethylene (PTFE) or polyvinyl chloride (PVC), and these
have to be steam sterilizable and easily changed.