BIOREACTOR ENGINEERING

MODULE – 1- Sterilisation

By
J.Kanimozhi
P130087CH
 Sterilisation
• Thermal death kinetics of microorganisms,
• batch and continuous heat-sterilization of
liquid media,
• filter sterilization of liquid media and air
sterilization,
• radiation and chemical sterilization,
sterilization equipments – batch and
continuous.
Thermal death kinetics of microorganisms
Sterilization can be defined as any process that effectively kills or eliminates
transmissible agents (such as fungi, bacteria, viruses and prions) from a surface,
equipment, foods, medications, or biological culture medium.

Importance of Sterilisation
Loss of productivity
Contaminate final product
Troublesome during downstream processing
Contaminant may cause degradation of product
Cause lysis of culture e.g bacteriophages

Steps to avoid contamination

Use pure inoculums
Media sterilization
Fermenter sterilization
Sterilize all raw materials
Maintain aseptic conditions during process
 Methods of Sterilization
• The various methods of sterilization are:
1. Physical Method
a. Thermal (Heat) methods
Dry Heat
1. Incineration
2. Red heat
3. Flaming
4. Hot air oven
Moist Heat-
1. Dry saturated steam – Autoclaving
2. Boiling water/ steam at atmospheric pressure
3. Hot water below boiling point

b. Radiation method
c. Filtration method

2. Chemical Method
a. Gaseous method
 BASIC KINETICS OF DESTRUCTION (/GROWTH)
The destruction of microorganisms by steam may be described as a first
order reaction
first-order reaction— A chemical reaction involving only one chemical
species, in which the rate of decrease of the concentration of the
reactant is directly proportional to its concentration.

Represented by the following equation -dN / dt = k .N

where N = number of viable organisms present

t = time of the sterilisation treatment
k = reaction rate constant
 BASIC KINETICS

Integration gives Nt / N0 = e -kt

Where
N0 = initial number of viable microorganisms,
Nt = no. of viable m-organisms present after treatment period t
t = time,
k = destruction coefficient (B. stearothermophilus)

On taking natural logs, equation is reduced to

ln (Nt / N0) = -kt

Del factor ( V ) = In (No/Nt),

where No is the number of organisms at the start of sterilisation
and Nt is the number remaining after time t.

•Therefore, the Del factor is a measure of the fractional reduction in viable

organism count produced by a certain heat and time regime.
Graphical representation of previous equations

Nt Nt Slope = -k
ln
No No

Time Time

Plots of the proportion of survivors and the natural logarithm of the

proportion of survivors in a population of microorganisms subjected to a
lethal temperature over a period of time
 Terms relating to heat sterilization used in
fermentation industries
Thermal Death Time (TDT) is the shortest time required to kill all
microorganisms in a sample at a specific temperature and under
defined conditions.
Decimal reduction time (D-value) is the time required to kill 90%
of the microorganisms in a sample at a specific temperature
D=2.303/kd

F-value is the time in minutes at a specific temperature (usually

250oF or 121.1oC) necessary to kill a population of cells or spores
 TECHNOLOGY OF MEDIA STERILISATION
(a) METHODS; MECHANICAL / PHYSICAL METHODS;
• Centrifugation, adsorption to ion exchange, adsorption to activated carbon, or
filtration are possible.
• Filtration is the only method in practical use. Filter sterilization is often used for
components of nutrient solutions which are heat -sensitive .Deep filters (plate filters)
are some time used to filter complex nutrient solutions.
• The disadvantages of filtration are:
• 1) certain components of the nutrient solution may be
• adsorbed on the filter material, and
• 2) high pressures must be used (up to 5 bar), which are undesirable in industrial
practice.
 HEAT PROCESSING;
EXPENSIVE PROCESS
Two criteria;
 Achieve required probability of sterility
 Minimize loss of nutrients
 MAIN HEATING METHODS 1. BATCH 2. CONTINUOUS - less destruction
Difference lies in the TEMPERATURE - TIME PROFILE
Continuous Sterilization:
- Simplifies production planning

- Therefore gives maximum plant utilization and minimum delays

- Provides reproducible conditions

- Can be operated at high temperature (1400C)

- Therefore sterilization time can be reduced (2 to 3 min)

- Requires less steam and less cooling water

- Suitable when the capacity of operation is high

- High initial capital investment (use of aseptic transfer system for the sterile broth to be
transported to a sterile vessel)

-It is the only option if the medium is to be exposed to high temperatures for a short time
(HTST process) to avoid denaturation of proteins or to avoid destruction of some of the
enzymes,

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 TECHNOLOGY OF CONTINUOUS STERILIZATION
(a) STEAM INJECTION TYPE;
ADVANTAGES
higher steam utilization efficiency
low capital cost
easy cleaning and maintenance
shorter heating and cooling cycle

DISADVANTAGES
foaming of media
media and steam have direct contact - chemical contamination

(b) CONTINUOUS PLATE EXCHANGER

Has longer heating and cooling period
plate-and-frame heat exchanger is favourable with high viscous system
Continuous Sterilization:
Continuous Injection Type
Steam
Raw
medium
Expansion
vacuum
valve

Holding section
(where most of the
sterilization takes place) Flash
cooler
- Direct steam injection for heating (relatively a rapid
process)
- Flash cooling (rapid process)
Sterile
medium

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Continuous Sterilization:
Sterile Continuous Heat Exchanger Type
medium

Steam

Holding section

Indirect steam heating in

plate-and-frame (or shell-and-
tube)
Raw medium
Cooling water heat exchanger

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Time Versus temperature in continuous sterilisation
Continuous Sterilization:
Most of the sterilization in the continuous sterilization process may occur in the holding
section.

Therefore

no
 hold
= ln
nt
= kd λhold = kd0 exp(-Ed/RT) λhold

Since the holding section is a long pipe,

length of the pipe

L
λ hold = uav

average fluid velocity

in the pipe

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Continuous Sterilization:
The ratio of average velocity to maximum velocity

uav
= 0.5 for laminar flow of Newtonian fluids through a smooth
umax round pipe

= 0.75 for turbulent flow

= 0.87 for turbulent flow with the Reynolds Number of 1000,000

Therefore, using the average velocity to calculate the length of the pipe
required for sterilization may leave some portion of the medium
understerilized, which may cause contamination problem

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 TECHNOLOGY OF BATCH STERILIZATION
Can be carried out in ;
FERMENTATION VESSEL (in situ medium sterilization)
SEPARATE MASH COOKER
Advantages of separate medium sterilization;
• medium sterilized while fermentor are cleaned - less downtime
• design conditions for sterilization more severe than fermentation
Disadvantages;
• Cost
• More transfers - more pipe work, more contamination risk
• All fermentor would depend on cooker - fault render plant redundant
METHOD OF HEATING;
 Steam sparging; direct through heating coils
 Electric element
 Heating jacket
Time and Temperature profile in batch sterilisation
 (b) COMPARISON OF CONTINUOUS & BATCH
ADVANTAGES OF CONTINUOUS;
• Reduction of sterilization cycle time
• Ease of scale-up
• Superior maintenance of medium quality (less destruction)
• Reduced surge capacity for steam (more efficient plant
use)

ADVANTAGES OF BATCH;
• Lower capital cost (fermentor used as autoclave)
• Lower contamination risk (less transfers of liquids)
• Presence of solids (particles) less of a problem
Filtration:
Sterilize solutions that may be damaged or denatured by high temperatures
or chemical agents.
The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-
0.45 μm
The pore size for filtering viruses and some large proteins is in the range of
0.01 μm

(a) Depth filters: Consist of fibrous or granular materials so packed as to

form twisted channels of minute dimensions. They are made of
diatomaceous earth, unglazed porcelain filter, sintered glass or asbestos.

(b) Membrane filters: These are porous membrane about 0.1 mm thick,
made of cellulose acetate, cellulose nitrate, polycarbonate, and
polyvinylidene fluoride, or some other synthetic material. The membranes
are supported on a frame and held in special holders. Fluids are made to
transverse membranes by positive or negative pressure or by centrifugation.

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Concerns in air sterilization:

Pressure drop is critical in a filter.

Energy input for compressed air for a commercial-

scale process is significant.

Air treatment can account for 25% of total production

costs.

Design engineer has to balance the assurance sterility

against the pressure drop.

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Figure 3.2: Installation of an air filter system in a fermenter
 Chemical Sterilisation
• Applicable only for surface sterilisation
To maintain Aseptic conditions
Formaldehyde
Ethanol
Phenol
To sterilise Disposable and non autoclavable items
Ethylene Oxide
UV Radiation
Gamma Radiation
Thank You