D Value

Organism D value min @ 121.1˚C

• Bacillus Stearothermophilus 4-5
• C. thermosaccharolyticum 3-4
• Desulfotomaculum nigrificancs 2-3
• Clostridium botulinum type A & B 0.1-0.25
• C. sporogenes (P.A. 3679) 0.1 - 1.5
• B. coagulans 0.01 - 0.07
 Thermal Death Time Curve
• Thermal death time (TDT) time in minutes required to
inactivate an arbitory chosen number of spores of a given
bacteria at a specified temperature.
• Thermal death time are plotted on log scale against
corresponding temperature in linear scale.
• Logarithms of death times can be plotted against corresponding
temperature, both on linear scale.
• A straight line graphs_ Thermal Death Time Curve (TDT curve)
TDT Curve
 Z Value
• The slope of the TDT curve is defined as “Z” which is equal to
the number of degrees on the temperature scale when the curve
traverse one log cycle.
• Z is the change in temperature necessary to cause a ten fold
change in D-value.
• The value of Z for C. botulinum is 10˚C
• Every 10˚C change in temperature there is a ten fold change
In its death rate.
• B. subtilis has Z value of 6.5˚
 F Value
• Sterilizing value_ time in minutes required to kill an organism
in a specific medium at 121.1˚C (TDT).
• When the Z value of the process is 10˚C, F is denoted as Fo.
• The unit of sterilization is Fo. Fo can be defined as the
integrated heating effect received by all points inside the can.
• Fo value of 1 is equivalent to holding the product at 121.1˚C for
one minutes.
 F Value
• Do for B. Stearothermophilus to be 5 min. & initial number (No) in the
container to be 10,000, if it was required to reduce this number to one
(Nt) in the heat process, four decimal reduction would be needed.
The time at 121.1℃ would be 4 x Do = 4 x 5 = 20 min. the number of
decimal reductions required is given by

• log No/Nt = logNo-logNt = 4-0 = 4

• This log No/Nt some times refereed as “order of process” factor of

“m” & the value of the product of m & Do is called “Process value”
or “F value” i.e. Fo=mDo
 Sterilization of
industrial medium

9
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.
10
Contamination 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

11
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
selective 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.
12
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
13
 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 14
 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 15
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
16
 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
17
• 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
18
• in a separate mash cooker
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 provides sterile medium for several
fermenters
• The fermenter are less likely to corrosion which may occur with medium at high 19
temperature.
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
20
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 (121oC),
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
21
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
22
 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 23
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.

24
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
25
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
26
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

27
FILTER STERILIZATION OF MEDIA AND AIR
• Suspended solids can be separated from fluid and gas by filteration
• This is due to
Inertial impactionDiffusion 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
•
28
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.

29
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.
30
 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. 31
• 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.

32
Sterilization of air by filters (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

33
• 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.

34
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.
35
Questions
• Explain ‘protected fermentation’.
• Why is sterilization in fermentation
process important?
• Types of heat exchanges/steam
sterilization methods
• Types of filters used?
• How is air/exhaust air/ animal cell
culture media sterilized?
• Describe sterilization cycle?
• Del factors??
• Factors affecting sterilization
process??
THANK YOU
• Effect of media due to sterilization???
Reference:
• Stanbury P F, Whitaker A, And Hall S J, (1995).
Principles Of Fermentation technology, 2nd Edn,
Pergamon Press, London, Uk
• Waites M J, And Morgan N L,(2002). Industrial
Microbiology: An Introduction
• Blackwell Science
• Crueger W And Crueger A, (2000),
Biotechnology: A Text Book Of Industrial
• Microbiology, 2nd Edn, Panama Publishing
Corporation, New Delhi, India
• Trevan M D, Boffey S, Goulding K H, And
Standury S, (Eds), (1987), Biotechnology: The
Biological Principles, Tata Mcgraw-Hill, New
Delhi, India
• Casida L E, Jr. (1968). Industrial Microbiology,
Wiley Eastern Ltd, New Delhi, India
36