BY Dr. Prasanna V Dharani S. P. T Science College Godhra Shri Govind Guru University

BSC C 505C UNIT 2
B. Sterilization of media: Use of high-pressure steam: Principle, batch and

continuous sterilization process
BY
Dr. PRASANNA V DHARANI
S. P. T SCIENCE COLLEGE GODHRA
SHRI GOVIND GURU UNIVERSITY
Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

Wednesday, June 29, 2022 1
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INTRODUCTION
• A fermentation product is produced by the culture of an organism in nutrient
media
• If the fermentation media is contaminated then
• The media would have to support the growth of process organism and
contaminate causing loss in productivity
• The contaminant may displace the process organism in continuous fermentation
• The contaminant may be present even in the final product e.g. single cell protein
i.e. biomass
• Contaminant may produce compound which makes down stream process difficult.
• The contaminant may degrade the final product. For example degradation of beta
lactam antibiotics by beta lactamase producing contaminant.
• Phage contamination may cause lysis of bacterial process organism.

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Conatamination can be avoided by :
• Using pure inoculums to start fermentation
• Sterile media and sterile fermenters must be used
• Sterilizing all the additives to be added during the process
• Maintaining aseptic conditions during fermentation

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Protected fermentation:
• Some of the fermentations are protected

• The media may be used by limited range of microbes.
• Otherwise the growth of the process organism may develop selectie
growth conditions e.g. reduced pH as in brewing fermentation
• Hop resins that are used as media component inhibit growth of many
microbes
• Brewing yeast reduces pH of the media
• So brewing worts are boiled but not sterilized.
• The fermenters are cleaned and disinfected and not sterilized.
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Media may be sterilized by
1) Filtration,
2) Radiation,
3) Ultrasonic treatment,
4) Chemical treatment
5) Heat
• Heat or steam is the widely used method for the sterilization of fermentation
media.
• It involves a cycle of heating up, holding time and cooling period
Factors influencing the efficiency of heat sterilization
• The number and types of microorganisms present
• The composition of the culture medium
• The pH value and the size of the suspended particle
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Medium sterilization
 Filtration is used for the sterilization of medium which is exception for the medium containing
heat labile components
Time for the sterilization is dependent on the type of the population.
If the sensitive organisms are more in number than whole culture sterilization will be equal to
that of the sensitive culture.
But if the number of the resistant organisms is more than the sterilization of whole culture is
equal to that of the sterilization of the resistant organisms.
Contaminant may be by not a single type of organism but by different types of organisms.
Sterilization of media require destruction of all types of organisms.
The destruction of organisms in sterilization process is given by the factor called Del factor.
Deindoerfer and Humphrey (1959) used the term In No / Nt as a design criterion for
sterilization, which has been variously called the Del factor, Nabla factor and sterilization
criterion represented by the term ▼
Thus, the Del factor gives idea about fractional reduction in viable organism count produced by
a definite heat in particular time
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During sterilization reaction causes the decrease in nutrient value because of :
Interactions between nutrient components of the medium

• Maillard-type browning reaction discoloration of the medium as well as deterioration of
nutrient value caused by the reaction of carbonyl groups and amino groups from reducing
sugars, and amino acids and proteins respectively
Degradation of heat labile components
• Certain vitamins, amino acids and proteins may be degraded during a steam sterilization regime
• Thus heat labile compounds can be sterilized by filtration
• However, for the vast majority of fermentations these problems may be resolved by the
judicious choice of steam sterilization regime
• The activation energy for thermal destruction of Bacillus steareothermophilus spores is more
than for thermal destruction of nutrient.
• Thus it would be advantageous to employ high temperature for shorter period of time to
achieve desired sterility causing minimum degradation of nutrients.
• Batch sterilization is not possible as high temperature cannot be kept for short period of time by
this method
• So the solution to this problem is continuous stream sterilization
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The Design of Batch Sterilization Processes
• Main aim of batch sterilization process is sterilization with the least change in
nutrient value of the medium
• Continuous sterilization process is better than batch sterilization process in
avoiding the damage of nutrients than a continuous sterilization process
• The maximum temperature in batch sterilization is 121.6°C
• High temperature and short time sterilization is attained by taking into
consideration the heating and cooling time of the batch sterilization
• The following point should taken into consideration for a batch sterilization
process
• How much temperature of the fermentation medium is increased during
heating or decreased during cooling periods of the batch sterilization
• The initially number of micro-organisms in the medium
• The thermal death rate of the selected organism
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Batch sterilization Methods
• Most nutrient media are sterilized in batch in bioreactors at 121.6oC

• The sterilization time depends on nature of media, size of fermenter, fittings,
valves, electrodes
• The sterilization time is long
• The sterilization procedure must be designed so that the exposure of medium to
high temperature must be minimum
• To design the batch sterilization process the information required are:
• Profile of increase and decrease in temperature of fermentation media during
heating and cooling periods of sterilization cycles
• Number of microbes present in the media
• Thermal death character of design microbe i.e B sterethermophilus
• By knowing the initial number of organisms in the medium and the danger of
contamination. Accepted threat of contamination is 1 in 1000, which means
number of living organisms after time t is 0.001
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• For example if any unsterile broth contain 1011 number of cells then Del factor for that situation is 32.2
• The killing of cells take place during both the heating period and cooling period of the sterilization
process in addition to during holding period at 121.6°C
• So, Del factor can be
• ▼overall = ▼heating + ▼holding + ▼cooling
• Knowing the temperature and time required to reach that temperature during heating period and
cooling period of sterilization process it is possible to determine the overall Del factor by these periods
• Thus, from the Del factors contributed by heating and cooling periods, it is possible to estimate the
holding time that may be required for overall Del factor
• One method of sterilization is to inject steam into fermentor mantle or interior coils. i.e indirect steam
sterilization
• Another method is to inject steam into the nutrient broth i.e direct method , in which pure steam
which is free of chemicals must be used
• Condensate accumulate in fermentor and the volume of liquid increases during sterilization
• The batch sterilization of the medium for a fermentation may be achieved by
• in the fermentation vessel
• in a separate mash cooker

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Advantages of a separate medium sterilization vessel/mash cooker
• The medium may be sterilized in a cooker in a more concentrated form than would be used
in the fermentation
• It is then diluted in the fermenter with sterile water prior to inoculation.
• This would allow the construction of smaller cookers
• One cooker may be used to serve several fermenters
• The medium may be sterilized as the fermenters are being cleaned and prepared for the
next fermentation
• This saves time between fermentations
• Some of the fermentation medium is viscous during sterilization
• So the power requirement for agitation is not alleviated (lessen) by aeration.
• Fermenter equipped with a powerful motor providez sterile medium for several fermenters
• The fermenter are less likely to corrosion which may occur with medium at high
temperature.
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Advantages of batch sterilization over continuous sterilization
• Lower capital equipment costs
• Lesser assets apparatus expenditure
• Less chance of contamination
• The processes require the aseptic inoculums transfer of the sterile broth to the sterile vessel
• Easier manual control
• Easier to use with media having a high amount of solid material
Disadvantages of a separate medium sterilization vessel/cooker
• The cost of constructing a batch medium sterilizer is much the same as that for the fermenter
• If a cooker serves a large number of fermenters complex pipe work would be necessary to
transport the sterile medium
• So there is a risk of contamination
• Mechanical failure in a cooker supplying medium to several fermenters may cause all the
fermenters temporarily redundant (unneeded).
• The provision of contingency equipment may be costly

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Continuous sterilization of media:
• Continuous sterilization includes heating period, holding time at desired

temperatures and cooling period to reach fermentation temperatures
• The medium is heated to reach to the sterilization temperature (121 oC), holding
this temperature to particular period of time and then cooling the medium to
reach to the temperature of the fermentation process
• The temperature of the medium is increased in a continuous heat exchanger and
is maintained for the holding time in an shielding serpentine winding holding coil
• The period of the holding time is decided by the coil length and the medium
stream speed
• The medium after holding time is cooled to the temperature required for
fermentation using two sequential heat exchangers
• The first used the coming medium as the cooling source and
• The second uses cooling water
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Advantages of continuous sterilization over batch sterilization
• Media quality can be maintained

• Scale-up is easy
• Automatic control is easier
• The decrease of flow ability for steam
• Sterilization cycle time is shorter
• Under certain conditions, corrosion of fermentor is lesser
• In continuous process high temperature is used which reduce the holding time and
nutrient loss
• The necessary Del factor required may be attained by the proper temperature and
holding time which decrease the amount of nutrient loss
• Continuous process engage heating of small amount of medium and cooling of small
amount of medium which is very less in contrast with batch system
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There are two types of continuous sterilizer:
•The indirect heat exchanger

•The direct heat exchanger (steam injector)
•Indirect Heat Exchanger
•There are different types of heat exchangers:
Double spiral type of heat exchangers
• It consists of two sheets of high-grade stainless steel (SS)
•They are mould around central axis in such a way that they form a double spiral
•For sterilization steam is passed through one spiral and media through the other in counter
current i.e opposite direction
•Spiral heat exchangers are also used to cool the media after passing the holding coil
•Incoming unsterile media is used as cooling agent in the first cooler
•So the incoming media is preheated before it reaches the sterilizer so heat is conserved
•
Advantages of spiral heat exchangers:
•The two streams i.e media and steam or media and cooling liquid are separated by
continuous stainless barrier so cross contamination of two streams is unlikely
•The spiral route traversed by the media allows sufficient clearance so it is self cleaning
•This reduces the risk of sedimentation, fouling and burning of media particles
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Plate heat exchangers:
• It consists of alternating plates through which countercurrent streams are circulated

• The plates are separated by gasket
• Failures of gaskets can cause cross contamination between two stream
• Suspended solids in media may block exchamger so it is useful in sterilizing soluble
media
• There are more adjustable as extra plates may be added to increase the capacity.


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Indirect heat exchanger for continuous sterilization of media:
• Advantages Indirect heat exchanger

(i) Immediate heating up times
(ii) Media containing solids can be
sterilized by this exchanger
(iii) Less investment
(iv) Easy to maintain and clean
(v) Efficient in using steam
• Disadvantages Indirect heat exchanger

(i) Heating may cause foams
(ii) Steam is in direct contact with
medium, so medium should be enough
concentrated and steam should be free
from any agent responsible for
anticorrosion
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The direct heat exchanger (steam injector)
• The steam is injected directly in the unsterile broth
• The heating up of media is almost instantaneously
• It could be used for media containing suspended solids
• This is cheaper
• Steam is used efficiently
• Cleaning and maintenance is easy
• Steam injection may cause foaming of media during heating
• Condensate may dilute the media
• The steam used must be free from particles and anticorrosion additives
• The injection system is combined with flash cooling
• The sterilized media is cooled by passing it through the expansion valve in a
vacuum chamber so cooking is almost instant
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Advantages of continuous steam injector
1. It requires very short heating up time
2. It may be used for media containing suspended solids
3. It needs low capital cost
4. It is easy to clean and maintain
5. It has high seam utilization efficiency
Disadvantages of continuous seam injector
6. Foaming may occur during heating
7. Direct contact of medium with steam require that allowance be made for condensate dilution and require ‘clean’
steam, free from anticorrosion additives
 For starch containing broths preheating is done by steam injection
 The plant is sterilized, before media sterilization by circulating hot wate through closed circuit
 Fermenters and pipelines are also steam sterilized
Heat is conserved by using incoming media which will cool sterile medium, which in turn get preheated before
reaching the sterilizer
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FILTER STERILIZATION OF MIDIA AND AIR
•Suspended solids can be separated from fluid and gas by filteration

•This is due to
Inertial impaction Diffusion Electrostatic attraction Interception
Intertial impaction:
•Suspended particle that have momentum travel in straight line and become impacted upon the fibres
where they remain
Diffusion:
•Very small particles in fluid have Brownien motion
•So the particles change their direction from the fluid flow and get impacted on filter fibres.
Electrostatic attraction:
•Charged particles are attracted by opposite charge on surface of filters
Interception:
•The fiber of filter are woven to give pores of various sizes
•Particles that are larger than the filter pores are removed by direct interception
•Smaller particles can also be removed by interception because:
•More than one particle arrives at the pore simultaneously
•Irregular shaped particles may bridge the pore
•Pores that trap a particle may trap smaller particles
•
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Types of filters:
• There are two types of filters:

Absolute filters or fixed pore size filters:
• These filters have pore size smaller than the particle
which is removed
• These filters may be 100 percent efficient in removing microbes
• In fixed pore filters the pore size is controlled during manufacturing so that
the absolute rating can be maintained as mentioned for the filter
• The removal of particular size particles is guaranteed by interception,
diffusion , inertial impaction and attraction.
• Fixed pore filters are superior.
• Modern fixed pore filters are of cartridge and may be pleated to give large
surface area and minimize pressure drop across filter.
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Depth filters or nonfixed pore size filters:
• It consists of felts, yarn, asbestos pads, cotton, loosely packed fiber glass or glass wool
• Particles are removed by inertial impact, diffusion and electrostatic attraction
• There is a possibility that an organism may pass through the filtes as the fibers are not
cemented in position
• So when the pressure increases the material may move producing channels through the filter.
• Increased pressure may also displace trap particles
Filters should be
• Steam sterilized before and after use.
• The material must be stable at sterilization temperature.
• Should not adsorb protein as it gets fouled
• Should be hydrophilic

Steam used for filters sterilization must be filtered through stainless steel mesh filters of 1
micrometer.
Nylon/polyester filters will minimize proteins adsorption and 0.2 micrometer absolute rating.

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Filter sterilization of fermentation media:
•Animal cell media cannot be sterilized by steam as it has heat labile proteins
•So the media is sterilized by filtration and absolute filters are preferred
•The media to be filtered must be free of fungal, bacteria and mycoplasma contaminations.
•Adsorption of protein to filter surface must be minimum
•Filtered media must also be free of endotoxins
•Absolute filters are generally used
•They are made of membranes that are steam sterilizable and hydrophilic.
•The membranes are in form of cartridge which are fitted in stainless steel steam sterilizable modules
•It is difficult t construct on filters that can remove all forms of microbes and toxins.
So series of filters are used.
•The first filter is positively charged polypropylene prefilters with an absolute rating of 5 micrometer to
remove coarse materials, clots, etc
•The second filter is also positively charged polypropelene and its absolute rating is 0.5 microns. It can
remove microbes, gels, endotoxins etc.
•The third filter is a single layered nylon or polyester filter which has positive charge and absolute rating of
0.1 microns. It can remove microbes and endotoxins.
•The fourth filter is of 0.1 microns with double layer nylon/polyester layer with a positive charge. It removes
mycoplasma and endotoxins.
•To remove virus a fifth filters of 0.04 microns made of nylon or polyester could be used.
Wednesday, June 29, 2022 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU
23
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• Filters can also be used for down stream process to
separate cell and cell debris for the fermented broth
and for purification of desired products.
• Filters of 0.1 microns rating and of polypropelene and
the second filter if used is of hydroxyl modified is used
to remove cell debris from animal cell broth.

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Sterilization of air by filtes (air sterilization filters)

Sterile air is required during fermentation for:

• Aerobic fermentation
• For cooling of sterile fermenter and pipelines
• For maintaining positive pressure in sterile fermenter and during the
process
• For transferring of sterile additives into fermenters during the process
• For sterilization of exhaust air

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• Air can be sterilized by heat but it is costly.
• Fixed pore filters with absolute rating are used.
• The filters consist of pleated membrane cartridges and placed in
stainless steel modules.
• Material used for making filters are of polytetrafloroethelene (PTFE)
• It is hydrophobic so is resistant to wetting.
• It can be steam sterilized.
• Sometimes ammonia is injected into air stream for pH control, so
these filters are also resistant to ammonia.
• Pre filters can be used prior to filters to remove dust, oil, carbon,
pipeline scales, rust, moisture etc.

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Sterilization of fermenter exhaust air
• In traditional fermentation exhaust gas from fermenter

is vented out without sterilization
• Since the use of genetically modified microbes i.e. GMM
or recombinant microbes, emissions of allergens,
the containment of exhaust air has become important.
• Fixed pore membrane that could sterilize water saturated
air of comparatively high temperature and having high level of contaminants are used.
• Their pore size are 0.2 microns.
• Foam may also enter the filters, when the fermenter overflows, so pre filters or pre
treatment of exhaust air can be done.
• For pre treatment hydrophobic pre filters or mechanical separator to remove
moisture particles and foam can be used.

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Questions
Reference:
• Explain ‘protected fermentation’. • Stanbury P F, Whitaker A, And Hall S J, (1995).
Principles Of Fermentation technology, 2nd Edn,
• Why is sterilization in fermentation Pergamon Press, London, Uk
process important? • Waites M J, And Morgan N L,(2002). Industrial
Microbiology: An Introduction
• Types of heat exchanges/steam
• Blackwell Science
sterilization methods
• Crueger W And Crueger A, (2000), Biotechnology: A
• Types of filters used? Text Book Of Industrial
• Microbiology, 2nd Edn, Panama Publishing
• How is air/exhaust air/ animal cell Corporation, New Delhi, India
culture media sterilized? • Trevan M D, Boffey S, Goulding K H, And Standury S,
• Describe sterilization cycle? (Eds), (1987), Biotechnology: The Biological
Principles, Tata Mcgraw-Hill, New Delhi, India
• Del factors?? • Casida L E, Jr. (1968). Industrial Microbiology, Wiley
• Factors affecting sterilization process?? Eastern Ltd, New Delhi, India
THANK YOU
• Effect of media due to sterilization???
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BSC C 505C UNIT 2

B. Sterilization of media: Use of high-pressure steam: Principle, batch and

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

• Some of the fermentations are protected

Interactions between nutrient components of the medium

• Most nutrient media are sterilized in batch in bioreactors at 121.6oC

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

• Continuous sterilization includes heating period, holding time at desired

• Media quality can be maintained

•The indirect heat exchanger

• It consists of alternating plates through which countercurrent streams are circulated

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

• Advantages Indirect heat exchanger

• Disadvantages Indirect heat exchanger

•Suspended solids can be separated from fluid and gas by filteration

• There are two types of filters:

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

Sterile air is required during fermentation for:

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

• In traditional fermentation exhaust gas from fermenter

Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU

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