Sterilization

Adama Science &
Technology University
Department of Chemical
Engineering
Chapter Six
06. Sterilization
Saturday, April 17, 2021 Slide By Eba- Adapted from James Lee 2nd Ed 1
Course Contents
1. Biotechnology and Biochemical
2. Enzyme Kinetics
3. Immobilized Enzyme
4. Industrial Applications of Enzymes
5. Cell Kinetics and Fermenter Design
6. Sterilization
7. Agitation and Aeration
8.Downstream Processing
6. Sterilization
 Sterilization is the process designed to produce a sterile
state.
Absolute condition of total destruction or elimination of
all living
microorganisms.
 Aseptic condition indicates a controlled process or
condition in which the level of microbial contamination is
reduced to the degree that microorganisms can be
excluded from a product during processing.
It describes an "apparently“ sterile state.
 Sterilization of all medium and fermentation equipment
should be done to avoid growth of un needed organisms.
Disinfection refers to the process of eliminating or reducing
harmful microorganisms from inanimate objects or surfaces
Sterilization Methods
Sterilization can be done by the following
methods.
 Thermal Sterilization
 Sterilization By Chemical Agents
 Sterilization by Radiation
 Sterilization By Mechanical Means
 Sterile Filtration
 High Speed Centrifugation
Thermal sterilization
• Involves the use of either moist or dry heat.
• Moist –heat sterilization is the most widely used and reliable
sterilization method.
• Dry – heat sterilization is appropriate for materials that cannot
withstand moist – heat sterilization
In Moist heat sterilizer,
• Microorganism are destroyed by cellular protein coagulation .
• The objects to be sterilized are exposed to saturated steam under 1
atmosphere pressure at a minimum temperature of 121°C for at least 20-
60 minutes.
• Because it does not require as high a temperature, moist – heat sterilization
cause less product and equipment damage compared to dry – heat
sterilization
Dry – heat sterilization is appropriate for materials that cannot withstand moist
– heat sterilization (e.g., oily materials and powders) .
• temperature of at least 160 °C for 120 minutes ( if higher temperatures
can be used , less exposure time is required).
Chemical Sterilization
Is used to sterilize surfaces and porous materials
( e.g., surgical dressings ) that other sterilization
methods may damage
Residual gas must be allowed to dissipate after
sterilization and before use of the sterile product .
This method is generally used for disinfection( reduction of the
risk of contamination)
1. Phenol and phenolic cpds( phenol, cresol,orthophenylphenol)
2. Alcohols (ethyl, methyl)
3. Halogens(iodine, hypochlorites, chloramines)
4. Detergents. Dyes
5. Quaternary ammonium compounds
6. Gaseous chemosterilizers( ethylene oxide, Formaldehayde)
Saturday, April Slide By Eba- Adapted from James Lee 2nd Ed 6
Radioactive Sterilization
 Is suitable for the industrial sterilization of contents in sealed
packages that cannot be exposed to heat ( e.g. prepackaged surgical
components , some ophthalmic ointments )
 Uv Disinfection
- damage DNA resulting in cell death
- wavelength 265nm is of the highest bactericidal efficiency
- used to reduce microbial population
 X- rays: lethal to most microorganism but not recommended due to
safety concerns.
 Sonic/Ultrasonic Waves: can disrupt and kill cells
 used for disruption of cells for the purpose of extracting
intracellular constitutents
Sterile Filtration
• Removes but does not destroy microorganisms
and clarifies solutions by eliminating particulate
matter.
• For solutions rendered unstable by thermal,
chemical, or radiation sterilization, filtration is the
preferred method .
• A depth filter or screen filter may be used .
• Depth filter usually consist of fritted glass or
unglazed porcelain (i.e., substances that trap particles
in channels ).
• Screen (membrane) filters are films measuring
1-200 mm thick made of cellulose esters,
microfilament, polycarbonate , synthetics polymers,
silver, or stainless steel.
Thermal Death Kinetics
• The thermal death of microorganisms at a particular temperature is given by
1st order kinetics as below.
where, Kd is specific death rate.
n is number of viable microorganism
Specific death rate = f( types of species, physiological form of cells)

= 1/min for bacteria spores @ 121 ℃
= 10- 1010 /min for vegetative cell
Integration of the above equation yields which shows

the exponential decay of the cell population.
Temperature dependence of specific death rate is given by Arrhenius equation:
where Ed is activation energy, R is gas constant
Design Criterion
 Design criterion for sterilization can be described
interms of Del factor 𝛻
Del factor, 𝛻 is give as
Del factor is a measure of the size of the job to be
accomplished.
Del factor increases as the final number of cells decreases
 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 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.
Based on the sterilization criterion calculated we can
design the sterilization unit.
Batch Sterilization
 Sterilization can be done batch wise or continuously.
 In batch sterilization, the entire volume of media is sterilized at
once through the use of different sterilization methods. Batch
sterilization is done using the following major methods
1. Direct steam sparging
2. By electrical heaters
3. By circulating constant pressure condensing steam through
heating coil
Total sterilization cycles = Heating + holding + Cooling
Total Del factor = 𝛻 + 𝛻 +𝛻
heating holding cooling
Del factor for heating and cooling are determined by the methods
used for heating and cooling
Del factor for holding is determined by the length of the
controlled holding period
Estimation of Holding Time
1. Calculate the total sterilization criterion, 𝛻 total.
2. Measure the temperature versus time profile during the heating, holding,
and cooling cycles of sterilization. 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.
a. For batch heating by direct steam sparging into the medium, the hyperbolic
𝐻𝑚𝑠𝑡
form is used:T= 𝑇𝑜 +
𝑐 𝑀+𝑚𝑠𝑡
b. For batch heating with a constant rate of heat flow such as electrical heating,
𝑞𝑇𝑡
the linear form is used: 𝑇 = 𝑇𝑜 +
𝑐𝑀
c. For batch heating with a isothermal heat source such as steam circulation
through heating coil, the exponential form is used:
𝑈𝐴𝑡
𝑇 = 𝑇ℎ + 𝑇𝑜 − 𝑇ℎ exp −
𝑐𝑀
Estimation of Holding Time…
3. For batch cooling using a continuous non-isothermal heat sink
such as passing cooling water through cooling coil, the exponential
form is used:
𝑈𝐴 𝑚𝑐𝑡
𝑇 = 𝑇𝑐𝑜 + 𝑇𝑜 − 𝑇𝑐𝑜 exp{− 1 − exp − }
𝑚𝑐𝐶 𝑀
4. Plot the values of Kd as a function of time.
5. Integrate the areas under Kd Vs time curve for heating and

cooling periods to estimate 𝛻 heating and 𝛻 cooling respectively.
t = 𝛻total/ Kd= [ 𝛻
hold
Saturday, April 17, 2021
heating +𝛻 holding
Slide By Eba- Adapted from James Lee 2nd Ed
+𝛻 cooling ]/ Kd 13
Continuous Sterilization
Continuous mode offers several advantages over batch sterilization.
I. It simplifies production planning, thus allowing maximum plant
utilization and minimum delays.
II. It provides reproducible conditions.
III. 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).
IV. It requires less steam by recovering heat from the sterilized
medium.As a result, it also requires less cooling water.
V. It is easier to automate the process; thus, it is less labor intensive.
A continuous sterilizer consists of three main sections: heating,
holding, and cooling.
Heating Section
Heating can be done by direct
steam injection or indirect
heating(PHE and STHE)
Direct heating heats the media to
the peak sterilization temperature
quickly
PHE is more effective than STHE
due to its high surface area.  For heating using a
countercurrent heat source
Temperature change w.r.t of equal flowrate and heat
residence time as the medium capacity
passes through an isothermal heat ∆𝑇𝑈𝐴𝜏ℎ𝑒𝑎𝑡
source is 𝑇𝑐2 = 𝑇𝑐1 −
𝑐𝑊
𝑇𝑐2
𝑈𝐴𝜏ℎ𝑒𝑎𝑡
= 𝑇ℎ − 𝑇ℎ − 𝑇𝑐1 exp(− )
𝑐𝑊
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:
𝜏ℎ𝑜𝑙𝑑 = 𝐿/𝑈
Del factor can be estimated as
Where n is the number of cells at the beginning of the holding
o
sections
For ideal plug flow, residence time will be the same for all
sections. Thus , uniform degree of sterilization is assumed to be
achieved.
Saturday, April 17, 2021 16
Holding Section….
The flow is not ideal plug flow due to the following factors.
 slippage due to viscous nature of the fluid
 the friction of the pipe wall
 Turbulent eddies of the flowing fluid
Velocity profile is maximum at the centerline and minimum at the
vicinity of the pipe wall.
For laminar flow:Average velocity/Max velocity: u/u = 0.5 max
For turbulent flow: u/u = 0.5 – 0.75

max
For Re>1000,000 : u/u = 0.87

max
As a result, if we use the mean velocity u in calculating the m
required residence time for sterilization, some portion of the

medium will be under sterilized which may cause a serious
contamination problem. See(Example 8.2)
Cooling Section
• For the cooling section, a quench cooler with
adequate heat removal capacity is effective.
• Inject the hot medium through an expansion
valve into a vacuum chamber, which is known as
flash cooling. Both of these take a very short time;
therefore, the sterilization during the cooling
period can be assumed to be negligible.
• The temperature versus residence time
relationship for cooling using isothermal
𝑈𝐴𝜏𝑐𝑜𝑜𝑙
heat sink
is 𝑇ℎ2 = 𝑇𝑐 − 𝑇𝑐 − 𝑇ℎ1 exp(− )
𝑐𝑊
• For a cooling using a countercurrent heat sink of
equal equal flow rate and capacity.
𝑇ℎ2 = 𝑇ℎ1 − ∆𝑇𝑈𝐴𝜏𝑐𝑜𝑜𝑙/𝑐𝑊
Air Sterilization
 Aerobic fermentation requires air to proceed.
Typical air flowrates is 0.5 -1.0vvm( air volume
per liquid volume per minute).
• Sterilization of air by heat is economically
impractical and ineffective due to the low heat
transfer efficiency of air compared with those of
liquid.
• The most effective techniques are:
Filtration using fibrous(membrane filters) eg.
Cotton plug (closure of tubes/flasks)
 glass fibers
 With fibrous filters, airborne particles are
collected by the mechanisms of impaction,
interception and diffusion
Saturday, April 17, 2021 19
Air Sterilization…
 Impaction: When air stream containing particles flows
around cylindrical collector, the particle will follow the
streamlines until they diverge at the collector.
 Interception: This is considered where the particle has
size , but no mass, so they can follow the streamlines of
the air around the collection.
 Diffusion: Particles smaller than about 1micron in
diameter exhibit a Brownian motion which is
sufficiently intense to produce diffusion.
Collection efficiency increases as the particle size
decreases.
 Total collection efficiency of a fibrous filter is obtained
from the combined effect of interception, impaction
and diffusion.
THANK YOU!

Sterilization

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Adama Science &

Specific death rate = f( types of species, physiological form of cells)

Integration of the above equation yields which shows

4. Plot the values of Kd as a function of time.

5. Integrate the areas under Kd Vs time curve for heating and

For turbulent flow: u/u = 0.5 – 0.75

For Re>1000,000 : u/u = 0.87

As a result, if we use the mean velocity u in calculating the m

required residence time for sterilization, some portion of the

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