Sterilization

Chapter 6
Introduction
 Most industrial fermentation are carried out as
pure cultures-selected strains are allowed to
grow.
 Foreign microorganisms exist in the medium or
any parts of the equipment, the production
organisms have to compete with the
contaminants (limited nutrients).
 Foreign microorganism can produce harmful
products-limit the growth of the production
organism.
 B4 starting fermentation, medium + all
fermentation equipment – free from any living
organism (completely sterilized).
 The aseptic condition - maintained
Sterilization Methods

 for fermentation media & equipment

 Heat (moist & dry)
 Chemical agents
 Radiation (ultraviolet/X rays)
 Mechanical (sonic/ultrasonic vibrations)
 Filtration
 Heat
 Widely used
 For liquid medium & heatable solid objects
 As dry or moist heat (steam)
 Moist heat-more effective
Note: Because the intrinsic heat resistance of
vegetative bacterial cells is greatly increased in a
completely dry state. The death rate-much lower
for the dry cells than for moist ones.
 Heat conduction in dry air-less rapid than in
steam
 Dry heat-for sterilization of glassware/heatable
solid materials
 By pressurizing a vessel, the steam
temperature increased significantly above
the boiling point of water
 Lab autoclave: steam pressure:30
psi,121°C (Bacterial spores rapidly killed
at 121°C).
 Chemical
 Kill microorganisms as the result of their oxidizing or
alkylating abilities
 Cannot be used for the sterilization of medium-the
residual chemical can inhibit the fermentation
organisms
 Implies the treatment to remove or reducethe risk
from pathogenic organisms
 Antimicrobial chemical:
 phenol & phenolic compounds (phenol, cresol)
 alcohol
 Halogens (iodine)
 Detergents
 Dyes
 Acids,alkalies
 Radiation (UV/X Rays)
 Many cellular materials absorb UV light-leading to
DNA damage & consequently to cell death.
 UV-very little ability to penetrate matter
 Limited to the reduction of microbial population in a
room (sterility needs to be maintained)
 Ex: hospital operating rooms, clean chambers in a
lab.
 X Rays-lethal to microorganisms & have penetration
ability
 Impractical as sterilization tools-expense & safety
concerns.
 Sonic/Ultrasonic

 Can disrupt & kill cells

 Usually employed in the disruption of cells
for the purpose of extracting intracellular
constituents rather than as a sterilization
technique.
 Filtration

 Most effectively-removal of
microorganisms from air or other gases.
 In the case of liquid solutions-used with
medium or products which easily
destroyed by heat: human & animal
serums, enzymes.
 Batch Sterilization
 Method:*
 steam sparging
 electrical heating
 steam heating (constant pressure condensing
steam).
• Sterilization cycles are composed of heating,
holding & cooling
• Total Del factor:
 Heating & cooling-the values by the methods used
for heating & cooling
 Holding- the values by the length of the controlled
holding period.
 Continuous Sterilization
 Advantages:
 simplifies production planning, thus allowing max
plant utilization & minimum delays
 provides reproducible conditions
 Can be operated at a high temperature (140°C
instead of 121°C in batch sterilization)-the
sterilization time can be shortened (holding time 1-2
min)
 requires less steam by recovering heat from the
sterilized medium.
 require less cooling water
 easier to automate the process – less labor
intensive
• Consist 3 main sections: heating, holding & cooling
 Heating section:
- 2 methods:
 direct steam injection
 indirect heating in the shell-and –tube or plate-
and-frame heat exchanger
- Direct heating more effective-no barrier
between the medium & heat source.
- the steam injection heats the medium to the
peak sterilization temperature quickly
(sterilization during heating period is negligible)
- Indirect heating- the plate-and-frame HE
more effective than shell-and-tube type for
heat transfer due to its larger heat transfer
area.
- the plate-and-frame favorable for the
sterilization of a high viscous system
Plate-and-frame
Shell-and-tube
 Holding Section:
- the heated medium passes through a holding
section, usually composed of a long tube.
- maintained in adiabatic conditions
- if the heat loss in the section is negligible, the
temperature can be assumed to be constant.
 Cooling Section:
- a quench cooler with adequate heat removal
capacity is effective.
- flash cooling- inject the hot medium through an
expansion valve into the vacuum chamber
- Both of these take a very short time
- therefore, the sterilization during the cooling
period can be assumed to be negligible.
- shell-and-tube & plate-and-frame HE can also
be employed for cooling.
 Air Sterilization
 For aerobic fermentations, air needs to be supplied
continuously
 Typical aeration rates for aerobic fermentation are 0.5-1.0
vvm (air volume per liquid vol per min).
 This requires an enormous amount of air
 Therefore, not only the medium but also the air must be
free from microbial contaminants.
 All of the sterilization technique for medium can be
employed for air.
 Sterilization of air by means of heat is economically
impractical & ineffective due to the low heat transfer
efficiency of air compared with liquids.
 Most effective technique for air sterilization- fibrous or
membrane filters
 The cotton plug, routinely used as a closure for tubes or
flask of sterile solution-good example of removal
microorganisms from air by a fibrous filter.
 A simple air filter can be made by packing cotton
into a column.
 With cotton filters the pressure drop is high and
wetting can be a breeding ground for the
contamination
 Glass fibers are favorable as filter medium- give a
lower pressure drop & less liable to wetting or
combustion
 Modern fibrous filter systems are cylinders made
from bonded borosilicate microfibers
 With fibrous filters, airborne particles are collected
by the mechanism of:
 Impaction
 Interception
 Diffusion
 Sterilization of Fermenter
 The fermenter has to be sterilized separately b4
the sterile medium is added to it.
 By heating the jackets or coils of fermenter with
steam and sparging steam into the vessel
through all entries, apart from the air outlet from
which steam is allowed to exit slowly.
 Steam pressure-15 psi for approximately 20 min
 Sterile air is sparged into the fermenter after
cycle is complete & +ve pressure is maintained.
 Sterilization of Feeds
 A variety of additive may be administered to a
fermentation during the process & it is essential
that these materials are sterile
 The sterilization method depends on the nature
of the additive, the volume and feed rate at
which it is administered.
 Ex: if fed in large quantities then continuous
sterilization may be desirable.
 Batch sterilization of feed liquids normally
involves steam injection into the material held in
storage vessels.
 Sterilization of Liquid Wastes
 Process organisms which have been engineered
to produce ‘foreign’ products & therefore contain
heterologous genes are subject to strict
containment regulations.
 Thus, waste biomass of such organisms must be
sterilized b4 disposal.
 By either batch or continuous.
 Batch sterilization- sparging the steam into
holding tanks
 Cont sterilization- employ the type of heat
exchangers