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If the fermentation is contaminated
1. The medium would have to support the growth of
both producer and contaminant organism resulting
in loss of productivity.
2. In case of continuous cultivation the contaminant
may outgrow the producer organism.
3. In case of biomass as product, the contaminant will
result in total loss of product.
4. The contaminant may produce compounds, which
make downstream processing difficult.
5. The contaminant may degrade the product; this is
common in antibiotics fermentation eg. β
Lactamase producing organism degrade β-Lactum
6. Contaminant with phage could result in the lysis of
For avoiding contamination
1. Use pure inoculum to start the
2. Sterilizing the medium to be employed
3. Sterilizing the fermenter
4. Sterilizing all materials to be added
5. Maintaining the aseptic condition
Some fermentations are described as protected i.e
the medium may not be utilised by the other
organisms or the extreme pH are used for
Brewing of Beer falls into this category i.e Hop
resins used as the supplement in the medium
inhibit growth of most of the organism.
In such case of fermentations the medium can be
boiled ,the reactors can be cleaned with
disinfectants before fermentation not
But most of the fermentation are not protected
Medium SterilizationMedium Sterilization
Many cellular materials absorb ultraviolet light, leading
to DNA damage and consequently to cell death.
Wavelengths around 265 nm have the highest bacterial
However they have very little ability to penetrate. Use
is limited to clean chambers, operation theatre etc.,
X-rays are lethal and have penetration ability. However
due to expensive and safety concerns it is not used
Chemical agents are frequently used for disinfection.
Major antimicrobial chemical agents are :
phenol and compounds (phenol, cresol, orthophenylphenol)
halogens (iodine, hypochlorites, chloramines)
gaseous chemosterlizers (ethylene oxide, formaldehyde)
Chemical SterilizationChemical Sterilization
Destruction of microorganisms by steam is
represented by first order reaction
-dN/dt = kN ------------
Where N no of viable organisms present
t sterlization treatment time
k rate constant or Sp. Death rate
It is important to note that we are considering the
number of organisms and not the concentration –
the minimum number of organism required to
contaminate the batch is one regardless of the
Rearranging the eqn 
-dN/N = kdt
ln (Nt/No) = - kt-------
Nt/No = e-kt
Nt = No of organisms present after sterlization
No = No of organisms present before sterlization
Nt/No = e-kt
An infinite time is required to achieve sterile
condition i.e Nt=0
After certain period there will be less than
one viable cell present i.e Nt<1
ln (Nt/No) = - kt
Nt<1 is the probability of an organism
surviving the treatment
i.e Nt=0.1 is the probability that one organism
surviving ten treatments.
In other words one in ten batches may get
contaminated due to improper sterlization.
The value of k dependant on temperature,
species and its physiological form.
But your sterlization media contains mixture
Higher no of heat
Higher no of heat
As in any first order, The reaction rate
increases with increase in temperature i.e k
increases with increase in temperature.
This relation ship is given by Arrhenius eqn
E activation energy
R gas constant
T absolute temperature
A Arrhenius constant
Taking logarithm of eqn 4
Plot of ln k Vs 1/T will give a straight line
Intercept = ln A
Slope = -E/R
to etANN −
Combining eqn 2 and 4
Del factor is the sterilization criterion
( )ARTEt ∇+= lnln
Thus it is evident from the eqn that the
temperature and time are inversely proportional
and same degree of sterilization can be achieved
by varying time and temperature.
Also we have seen that the sterlization criterion
depend mainly on heat resistant organism.
Bacillus stereothermophilus is the known most
heat resistant organism
Assume all the contaminants are B.st and
calculate the temperature and time.
A=1 X 1036.2
Industrially accepted level of contamination is
0.001. i.e 1 in 1000 batches can get
Another common factor used in food industry is
decimal reduction time D. It is the time required
to reduce the cell population by 10 fold.
Decimal reduction timeDecimal reduction time
Plot ln No/Nt Vs Time for each temperature and find out
the slope. Slope will give k value.
Plot ln k Vs 1/T from the above values. Slope will give
E/R values and Intercept will give you the A value
From the above graph calculate the k value for 100 o
From the k value calculate time required to kill 99%
ktNN to =ln
0 2 4 6 8 10
0 2 4 6 8 10
E/R = 26869 o
R = 8.3144 J/K/gmol
E = 26869 X 8.3144 = 2.24 X 105
A =4.7 X 1030
kd= 4.7 X 1030
T=100 +273 =373 o
ktNN to =ln
Sterilization of the medium in a fermenter can
be carried out in batch mode by direct steam
sparging, by electrical heaters or by
circulating constant pressure condensing
steam through heating coil or jacket.
Cooling the medium is carried out by
sparging the steam through the cooling coil or
Heating CurveHeating Curve RTE
t1 t2 t3
The initial number of contaminants is 1014
and the decimal reduction value at 121o
1.5 min. Calculate the time required to
bring down the contaminants to industrially
D is the time required to reduce the
contaminants by 10 fold.
Del value is 1017
Time required = 17 X D = 17 X 1.5
Time required = 25.5 min
The initial concentration of
contaminants is 106
per ml and the
fermenter volume is 10000 litres. The
del factor contribution from heating is
9.8 and that of cooling is 10.1. Calculate
the holdup time assuming all the
contaminants are B.st having kd value at
C of 2.54 min-1
An autoclave is used for sterilizing 10 litre complex
medium containing 105
spores/ml. Due to the problem
in autoclave the temperature reached only 110o
same holdup time of 20 min is maintained what is the
probability of contamination. Compare that with of
regular autoclaving at 121o
E=2.83 X 105
R = 8.3144 J/K/gmol
Direct steam spargingDirect steam sparging
Steam sparging through the coil or jacketSteam sparging through the coil or jacket
Electrical HeatingElectrical Heating
Cooling coilsCooling coils
Batch sterlization is carriedout in 1 litre fermenter to
prevent contamination. The initial no of spores in
the medium is 1010
a) Calculate the holding time required to achieve
standard contamination levels neglecting heating
and cooling periods.
b) What is the probability of contamination if a 100
litre fermenter is heated for the same time.
c) Redo the calculation for part (a) assuming constant
temperature steam heating and constant
temperature cooling. Heat exchange coils are used.
For steam heating α and β are 0.05 sec-1
and that of cooling 0.065 sec-1
and 0.20. The heating
and cooling periods are 3 min and 10 min
d) Redo the calculation for part (a) using the
Temperature profile during sterlization
0 50 100 150
A steam sterilizer is used to sterilize
liquid medium for fermentation. The
initial concentration of contaminants is
per litre. How long 1 m3
treated if the temperature is 80o
Assume all the contaminants are B.st.
R = 8.3144 J/K/gmol
E=2.83 X 105
R = 8.3144 J/K/gmol
T -E/RT exp(-E/RT) A*exp(-E/RT)
353 -9.64E+01 1.33047E-42 2.10866E-06 272991.8
394 -8.64E+01 3.03152E-38 0.048046344 11.98106
413 -8.24E+01 1.61319E-36 2.556734665 0.225149
It is evident from theIt is evident from the
problem that a regime ofproblem that a regime of
time and temperature maytime and temperature may
now be determined tonow be determined to
achieve the desiredachieve the desired
However the fermentationHowever the fermentation
media is not an inertmedia is not an inert
Due to sterilizationDue to sterilization
deleterious reactions maydeleterious reactions may
occur resulting in loss ofoccur resulting in loss of
nutrient qualitynutrient quality
Duration of sterilizationProductyield
Loss of nutrient quality is due to two reasons
i) Interactions between nutrient components of the
Maillard type browning reaction – reactions of the
carbonyl group usually from reducing sugar with the
amino group of aminoacids, proteins.
Effect of sterilization time on availability of glucose
in CSL medium was investigated
Time Glucose remaining
60 min 35%
40 min 46%
30 min 64%
Problems of this type are normally resolved by
sterilizing sugar separately and adding to the medium
II. Degradation of heat labile components
Certain vitamins, amino acids and proteins may be
degraded during sterilization regime
In certain cases such as animal cell culture media
filtration is used
In other cases judicious choice of sterilization regime is
Nutrient destruction follows first order reaction
Activation energy for nutrient destruction
– 10 to 30 kcal/mole
Activation energy for B.st – 67.7 kcal/mole
From the graph it isFrom the graph it is
evident that sterilizingevident that sterilizing
the medium at higherthe medium at higher
temperature for lessertemperature for lesser
time will result in sporestime will result in spores
destruction with lesserdestruction with lesser
loss in nutrient quality.loss in nutrient quality.
Heating the huge reactorsHeating the huge reactors
for higher temperaturefor higher temperature
and cooling down in shortand cooling down in short
duration is impossibleduration is impossible
Alternately continuousAlternately continuous
sterilizer can be usedsterilizer can be used
Advantages of continuousAdvantages of continuous
Superior maintenance ofSuperior maintenance of
medium qualitymedium quality
Ease of scale upEase of scale up
Easier automatic controlEasier automatic control
Reduction in fermenterReduction in fermenter
Advantages of BatchAdvantages of Batch
Lower capitalLower capital
equipment costequipment cost
Lower risk ofLower risk of
Easier to use withEasier to use with
media containing solidmedia containing solid
suspended matter.suspended matter.
Performance ofPerformance of
continuous sterilizercontinuous sterilizer
depends on the nature ofdepends on the nature of
fluid flow in the systemfluid flow in the system
Plug flow is an ideal flowPlug flow is an ideal flow
where no mixing orwhere no mixing or
change in velocity occurchange in velocity occur
Deviation from plug flowDeviation from plug flow
is characterized by axialis characterized by axial
dispersion the degree todispersion the degree to
which mixing occurswhich mixing occurs
along the length of thealong the length of the
Axial dispersion is theAxial dispersion is the
critical factor in designcritical factor in design
of continuous sterilizer.of continuous sterilizer.
Axial dispersion and flow through the pipe isAxial dispersion and flow through the pipe is
characterized by dimensionless variable PECLETcharacterized by dimensionless variable PECLET
u – linear velocityu – linear velocity
L- Length of the pipeL- Length of the pipe
DDzz- axial dispersion- axial dispersion
For perfect plug flow axial dispersion is zero and NFor perfect plug flow axial dispersion is zero and NPePe isis
NNPePe between 3 – 600 is typical.between 3 – 600 is typical.
Once the NOnce the NPePe is known the extent of cell destruction andis known the extent of cell destruction and
another dimension less variable Damkohler number cananother dimension less variable Damkohler number can
be calculatedbe calculated
kkdd- specific death rate- specific death rate
Soln to Problem 1
N1= 1.44 X 1016
N2/N1=6.9X 10 -17
Damkholer number for Pe 368 and N2/N1 6.9 X 10 -17
42 (From graph)
For perfect plug flow Dz is zero.
Hence Pe number is infinity.
Dilution rate = Feed rate/reactor volume
For first part calculate Nt/No.
From the graph for this Nt/No value and Pe infinity
calculate the Da Number.
Calculate Kd with the given value of Arrhenius
constant, activation energy and temperature 130o
Calculate velocity u from the feed rate and diameter
of the steriliser.
Length of the sterliser holding section can be
calculated from the above.
From the graph calculate Dz/uD
Calculate Pe number using the length
calculated in the first part.
Now using the Pe number and Nt/No value
calculate Da number.
From this calculate length.
For third part
Using the length of perfect plug flow calculate
For this Da number and Peclet number
calculated in the second part, Calculate Nt/No
value from the graph.
From this calculate No and find the
contamination rate . i.e 1 organism in how
A continuous steirliser with a steam injector and a flash cooler will beA continuous steirliser with a steam injector and a flash cooler will be
employed to sterlize medium continuously with the flow rate of 2 memployed to sterlize medium continuously with the flow rate of 2 m33
/h. The/h. The
time for heating and cooling is negligible with this type of steriliser. Thetime for heating and cooling is negligible with this type of steriliser. The
typical bacterial count of the medium is about 5 X 10typical bacterial count of the medium is about 5 X 101212
, which needs to be, which needs to be
reduced to such an extent that only one organism can survive during tworeduced to such an extent that only one organism can survive during two
months of operation. The heat reesistant bacterial spores in the mediummonths of operation. The heat reesistant bacterial spores in the medium
can be characterized by A of 5.7 X 10can be characterized by A of 5.7 X 103939
1/h and E of 2.834X101/h and E of 2.834X1055
KJ/Kmol. TheKJ/Kmol. The
steriliser will be constructed with the pipe with an inner diameter of 0.102m.steriliser will be constructed with the pipe with an inner diameter of 0.102m.
Steam is available at a temperature of 125Steam is available at a temperature of 125oo
C. The physical properties of theC. The physical properties of the
medium aremedium are ρρ=1000kg/m=1000kg/m33
andand μμ=4 Kg/m/h R=8.3144 J/K/g mol=4 Kg/m/h R=8.3144 J/K/g mol
What length should the pipe be in the sterliser if you assume ideal plugWhat length should the pipe be in the sterliser if you assume ideal plug
What length should the pipe be in the sterliser if the effect of axialWhat length should the pipe be in the sterliser if the effect of axial
dispersion is considered.dispersion is considered.
The following equations can be used for the
Dz/uD=2 X 107
for NRe < 10000
for NRe > 10000
ln(Nt/No)= -Da + (Da2
Solution to ax2
C.s.area 0.008174571 sq.m
Vol. flow rate 2 cu.m/h
Velocity 244.6611443 m/h
Dia 0.102 m
density 1000 Kg/cu.m
Viscosity 4 Kg/m/h
Reynolds no 6238.85918
Dz 19.67636897 sq.m/h
A 5E+39 1/h
E 283400 J/gmol
R 8.3144 J/K/gmol
T 398 K
K 320.0387736 1/h
L=DaU/K 28.43841221 m
L=DaU/K 32.3031373 m
Filter sterilizationFilter sterilization
Suspended solids may be separated from aSuspended solids may be separated from a
fluid during sterilization by the followingfluid during sterilization by the following
1.1. Inertial ImpactionInertial Impaction
3.3. Electrostatic attractionElectrostatic attraction
Inertial ImpactionInertial Impaction
The fluid will tend to move through the leastThe fluid will tend to move through the least
resistance path during filtration.resistance path during filtration.
Suspended particles in the fluid stream haveSuspended particles in the fluid stream have
Due to this they tend to move straight andDue to this they tend to move straight and
therefore impact on the filtration fibres wheretherefore impact on the filtration fibres where
they may be retained.they may be retained.
This mechanism is more prominent in filtration ofThis mechanism is more prominent in filtration of
gases than liquids.gases than liquids.
Extremely small particles suspended in the fluidExtremely small particles suspended in the fluid
are subjected to Brownian movement which isare subjected to Brownian movement which is
random movement due to collision of particles.random movement due to collision of particles.
Thus due to this the fluid particles deviate fromThus due to this the fluid particles deviate from
the path of movement and impact on the filterthe path of movement and impact on the filter
Diffusion is more significant in the filtration ofDiffusion is more significant in the filtration of
gases than liquids.gases than liquids.
Electrostatic attractionElectrostatic attraction
Charged particles are attracted byCharged particles are attracted by
opposite charges on the surface of theopposite charges on the surface of the
filtration mediumfiltration medium
The fibres comprising filter are interwoven to definite openingThe fibres comprising filter are interwoven to definite opening
of different sizes eg: 0.22 µm etc.,of different sizes eg: 0.22 µm etc.,
Particles which are larger than these pore sizes are removed byParticles which are larger than these pore sizes are removed by
direct interception.direct interception.
Also significant number of smaller size particles are alsoAlso significant number of smaller size particles are also
retained due to:retained due to:
More than one particle arriving at a time to the pore.More than one particle arriving at a time to the pore.
An irregularly shaped particle may bridge the pore.An irregularly shaped particle may bridge the pore.
Interception is the equally important mechanism in both liquidsInterception is the equally important mechanism in both liquids
and gases.and gases.
Filters are classified into two typesFilters are classified into two types
Depth filter Or Non fixed pore filterDepth filter Or Non fixed pore filter
In depth filter the pore sizes are larger than theIn depth filter the pore sizes are larger than the
particle to be removedparticle to be removed
In these filters particles are removed more byIn these filters particles are removed more by
impaction, diffusion and electrostatic attractionimpaction, diffusion and electrostatic attraction
rather than interceptionrather than interception
Absolute filter Or Fixed pore filterAbsolute filter Or Fixed pore filter
In this the pore sizes are smaller than the particle toIn this the pore sizes are smaller than the particle to
be removed.be removed.
Particles are removed by interception.Particles are removed by interception.
In depth filters the removal of microorganism is aIn depth filters the removal of microorganism is a
probability and it cannot be absolute.probability and it cannot be absolute.
Thus always there is probability that microorganismThus always there is probability that microorganism
can pass through the filter immaterial of the depth ofcan pass through the filter immaterial of the depth of
the filter.the filter.
Fibres are just tightly packed and not fixed in the filter.Fibres are just tightly packed and not fixed in the filter.
Hence due to larger pressure movement of materialHence due to larger pressure movement of material
and creation of larger channels is possible.and creation of larger channels is possible.
Also increased pressure may sometime remove theAlso increased pressure may sometime remove the
previously trapped particles.previously trapped particles.
Filter sterilization of mediumFilter sterilization of medium
During Animal cell culture media filtration followingDuring Animal cell culture media filtration following
precautions have to be taken.precautions have to be taken.
Filtered medium should be free of fungal,Filtered medium should be free of fungal,
bacterial and mycoplasma contamination.bacterial and mycoplasma contamination.
Minimal adsorption of the proteins from theMinimal adsorption of the proteins from the
medium during filtrationmedium during filtration
Medium should be free of viruses andMedium should be free of viruses and
Absolute filters are used.Absolute filters are used.
Pressure drop in these filters are high hence thePressure drop in these filters are high hence the
pleated structure membranes are used.pleated structure membranes are used.
Pore sizes of these membranes are well controlledPore sizes of these membranes are well controlled
during manufacture.during manufacture.
To extend the life of the filter prefilters of bigger poreTo extend the life of the filter prefilters of bigger pore
sizes are used.sizes are used.
Generally polypropylene or nylon membranes are usedGenerally polypropylene or nylon membranes are used
Filter should be steam sterilizable in between batches.Filter should be steam sterilizable in between batches.
Filter sterilization of airFilter sterilization of air
For aerobic fermentations air needs to be supplied continuously.For aerobic fermentations air needs to be supplied continuously.
Typical aeration rates are 0.5 to 2 vvm.Typical aeration rates are 0.5 to 2 vvm.
This requires continuous and large amount of air free of microbialThis requires continuous and large amount of air free of microbial
Sterilization of air by means of heat is economically not viable.Sterilization of air by means of heat is economically not viable.
Most effective technique is filtration by fibrous or membrane filtersMost effective technique is filtration by fibrous or membrane filters
The cotton plug used as closure for flasks during cultivation is good exampleThe cotton plug used as closure for flasks during cultivation is good example
of fibrous filter.of fibrous filter.
Simple air filter can be made by packing cotton in the tube.Simple air filter can be made by packing cotton in the tube.
However the pressure drop is huge and wet cotton may become source forHowever the pressure drop is huge and wet cotton may become source for
Therefore glass wool is favourable filtration medium.Therefore glass wool is favourable filtration medium.
In the case of absolute filters the filter should be hydrophobic (PTFEIn the case of absolute filters the filter should be hydrophobic (PTFE
Theory of Depth filters
To reduce the population entering into the filter and exiting out,
Expression given as,
dN/dx = - KN
Where, N is the concentration of particles in the air at a depth x in the
& K is a constant,
On integrating above expression, we get:
N/No = e-kt
No is the no. of particles entering the filter
N is the no. of particles leaving the filter
Efficiency of filter is given by E = (No – N) / No
But, (No – N) / No = 1 – (N/No)
Thus, (No – N) / No = 1 – e-kt
The log penetration relationship has been used by
Humphery and Garden (1955) in the filter design, by
using the concept X90 the depth of filter required to
remove 90% of the total no. of particles entering the
Thus If, No were 10 and x were X90, then N would be 1,
ln (1/10) = - K X90
2.303 log10 (1/10) = - K X90
2.303 (-1) = - K X90
X90 = 2.303 / K