Download - Media Sterilisation
If the fermentation is contaminated1. 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 antibiotics.
6. Contaminant with phage could result in the lysis of culture.
For avoiding contamination
1. Use pure inoculum to start the fermentation
2. Sterilizing the medium to be employed
3. Sterilizing the fermenter
4. Sterilizing all materials to be added during fermentation
5. Maintaining the aseptic condition during fermentation
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 cultivation etc.,
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 necessarily sterilized.
But most of the fermentation are not protected
Medium SterilizationMedium Sterilization Filtration
Radiation
Ultrasonic treatment
Chemical treatment
Heat
Many cellular materials absorb ultraviolet light, leading to DNA damage and consequently to cell death. Wavelengths around 265 nm have the highest bacterial efficiency.
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
RadiationRadiation
Chemical agents are frequently used for disinfection.
Major antimicrobial chemical agents are :
phenol and compounds (phenol, cresol, orthophenylphenol)
alcohol (ethyl,methyl)
halogens (iodine, hypochlorites, chloramines)
detergents,acids, alkali,
gaseous chemosterlizers (ethylene oxide, formaldehyde)
Chemical SterilizationChemical Sterilization
Destruction of microorganisms by steam is represented by first order reaction
-dN/dt = kN ------------[1]
Where N no of viable organisms presentt sterlization treatment timek 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 volume
Rearranging the eqn [1]
-dN/N = kdt
Upon Integration
ln (Nt/No) = - kt-------[2]
Nt/No = e-kt------------[3]
Nt = No of organisms present after sterlization
No = No of organisms present before sterlization
o
t
NN
Time Time
o
t
NN
ln
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 of microorganism.
E. coli B. stereothermophillus
Time
Ln N
Sensitive organism
Resistant organismWhole culture
Ln N
TimeHigher no of heat sensitive organism
Higher no of heat resistant organism
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 energyR gas constantT absolute temperatureA Arrhenius constant
RTEAek / [4]
Taking logarithm of eqn 4
[5]
Plot of ln k Vs 1/T will give a straight line
1/T
Ln kIntercept = ln ASlope = -E/R
RTEto etANN ln
to NNln
RTEetA
Combining eqn 2 and 4
[5]
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.E=67.7 kcal/moleA=1 X 1036.2 1/sec
Industrially accepted level of contamination is 0.001. i.e 1 in 1000 batches can get contaminated.
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.
D=2.303/k
Decimal reduction timeDecimal reduction time
ProblemProblem
Step 1
Plot ln No/Nt Vs Time for each temperature and find out the slope. Slope will give k value.
Step 2
Plot ln k Vs 1/T from the above values. Slope will give E/R values and Intercept will give you the A value
Step 3
From the above graph calculate the k value for 100 oC
Step 4
From the k value calculate time required to kill 99% spores
ktNN to ln
0.00E+00
5.00E-02
1.00E-01
1.50E-01
2.00E-01
2.50E-01
3.00E-01
3.50E-01
0 2 4 6 8 10Time [min]
lnN o
/Nt
Y=0.0122 X
Y=0.0326 X
0.00E+00
4.00E+00
8.00E+00
1.20E+01
1.60E+01
2.00E+01
0 2 4 6 8 10Time[min]
lnN o
/Nt
Y=1.5943 X
Y=9.5859 X
y = -26869x + 70.624
-5
-4
-3
-2
-1
0
1
2
3
0.0025 0.0026 0.0026 0.0027 0.0027 0.0028 0.0028 0.0029
Ln k
1/T
E/R = 26869 oK
R = 8.3144 J/K/gmol
E = 26869 X 8.3144 = 2.24 X 105J/gmol=224 KJ/gmol
A =4.7 X 1030 min-1
kd= 4.7 X 1030 e-26869/T
T=100 +273 =373 oK
kd=0.244 min-1
Ln(100/1)=0.244 t
t=18.9 min
ktNN to ln
Batch sterilization
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 jacket.
TimeTime
Temperature
Temperature
Heating Holdup Cooling
coolingholdupheatingoverall
tooverall NNln
heating cooling
coolingheatingoverallholdup
kt holdup
Heating CurveHeating Curve RTEetA
111
RTEetA
222
RTEetA
333
RTEetA
TimeTime
Temperature
Temperature
t1 t2
t3
T3
T2
T1
321heating
The initial number of contaminants is 1014 and the decimal reduction value at 121oC is 1.5 min. Calculate the time required to bring down the contaminants to industrially acceptable limits.
ProblemProblem
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 121oC of 2.54 min-1
ProblemProblem
coolingholdupheatingoverall
1.10cooling8.9heating
8.36)ln( tooverall NNNNoo = 10 = 1066X1000X10000 =10X1000X10000 =101313 NNtt=0.001=0.001
9.161.108.98.36 holdup
coolingheatingoverallholdup
kt holdup
min65.654.2/9.16 t
An autoclave is used for sterilizing 10 litre
complex medium containing 105 spores/ml. Due to
the problem in autoclave the temperature reached
only 110oC. If same holdup time of 20 min is
maintained what is the probability of
contamination. Compare that with of regular
autoclaving at 121oC.
ProblemProblem
E=2.83 X 105 J/gmol
A=1036.2sec-1
R = 8.3144 J/K/gmol
Direct steam spargingDirect steam sparging
TTo
th SMCp
UATH
QC’p
WTco
Steam sparging through the coil or Steam sparging through the coil or jacketjacket
TTo
th SMCp
UATH
QC’p
WTco
Electrical HeatingElectrical Heating
TTo
th SMCp
UATH
QC’p
WTco
Cooling coilsCooling coils
TTo
th SMCp
UATH
QC’p
WTco
Batch sterlization is carriedout in 1 litre fermenter to prevent contamination. The initial no of spores in the medium is 1010 spores/litre.
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 and β are 0.05 sec-1 and -0.25 and that of cooling 0.065 sec-1 and 0.20. The heating and cooling periods are 3 min and 10 min respectively.
d) Redo the calculation for part (a) using the temperature profile
Temperature profile during sterlization
020406080
100120140
0 50 100 150Time
Tem
p
HEATING CURVETime Temp0 305 3510 5015 6420 8625 9630 10435 11140 11645 121
COOLING CURVETime Temp0 1217 11014 9521 8728 7535 6442 5449 4556 3964 37
A steam sterilizer is used to sterilize liquid medium for fermentation. The initial concentration of contaminants is 108 per litre. How long 1 m3 medium be treated if the temperature is 80oC, 121oC, 140oC
Assume all the contaminants are B.st.
E=283 KJ/gmol
A=1036.2sec-1
R = 8.3144 J/K/gmol
ProblemProblem
RTEetA E=2.83 X 105 J/gmol
A=1036.2sec-1
R = 8.3144 J/K/gmol
No=1011
Nt=0.001
Ln(No/Nt)=34.54-E/R=-3.4X104 oK
T -E/RT exp(-E/RT) A*exp(-E/RT)time (min)
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 the It is evident from the problem that a regime of problem that a regime of time and temperature may time and temperature may now be determined to now be determined to achieve the desired achieve the desired sterilization.sterilization.
However the fermentation However the fermentation media is not an inert media is not an inert mixture.mixture.
Due to sterilization Due to sterilization deleterious reactions may deleterious reactions may occur resulting in loss occur resulting in loss of nutrient qualityof nutrient quality
Duration of sterilization
Prod
uct
yiel
d
Loss of nutrient quality is due to two reasonsi)Interactions between nutrient components of the medium
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 remaining60 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 selected.
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
kt
o
t exx
Ln k
1/T
Spores destruction
Nutrient destruction
From the graph it is From the graph it is evident that sterilizing evident that sterilizing the medium at higher the medium at higher temperature for lesser temperature for lesser time will result in time will result in spores destruction with spores destruction with lesser loss in nutrient lesser loss in nutrient quality.quality.
Heating the huge Heating the huge reactors for higher reactors for higher temperature and cooling temperature and cooling down in short duration down in short duration is impossibleis impossible
Alternately continuous Alternately continuous sterilizer can be usedsterilizer can be used
Advantages of continuous Advantages of continuous sterilizationsterilization
Superior maintenance of Superior maintenance of medium qualitymedium quality
Ease of scale upEase of scale up
Easier automatic Easier automatic controlcontrol
Reduction in fermenter Reduction in fermenter corrosion corrosion
Advantages of Batch Advantages of Batch sterilizationsterilization
Lower capital Lower capital equipment costequipment cost
Lower risk of Lower risk of contaminationcontamination
Easier to use with Easier to use with media containing media containing solid suspended solid suspended matter.matter.
Performance of Performance of continuous sterilizer continuous sterilizer depends on the nature of depends on the nature of fluid flow in the systemfluid flow in the system
Plug flow is an ideal Plug flow is an ideal flow where no mixing or flow where no mixing or change in velocity occurchange in velocity occur
Deviation from plug flow Deviation from plug flow is characterized by is characterized by axial dispersion the axial dispersion the degree to which mixing degree to which mixing occurs along the length occurs along the length of the pipe.of the pipe.
Axial dispersion is the Axial dispersion is the critical factor in critical factor in design of continuous design of continuous sterilizer. sterilizer.
Axial dispersion and flow through the pipe is Axial dispersion and flow through the pipe is characterized by dimensionless variable PECLET Numbercharacterized by dimensionless variable PECLET Number
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 is is infiniteinfinite
NNPe Pe between 3 – 600 is typical.between 3 – 600 is typical.
Once the NOnce the NPePe is known the extent of cell destruction and is known the extent of cell destruction and another dimension less variable Damkohler number can another dimension less variable Damkohler number can be calculated be calculated
kkdd- specific death rate- specific death rate
zDuLPe
uLkDa d
Soln to Problem 1
N1= 1.44 X 1016
N2/N1=6.9X 10 -17
u= 254.6m/h Re=7.07X103
0.650.65
From graph Dz/uD=0.65From graph Dz/uD=0.65Dz=16.6 sq.m/hDz=16.6 sq.m/hPe=368Pe=368
Damkholer number for Pe 368 and N2/N1 6.9 X 10 -17 is 42 (From graph)
k=445.6 1/h
T=-(E/R)/ln(k/A) T=398.4 K
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 130oC Calculate velocity u from the feed rate and diameter
of the steriliser. Length of the sterliser holding section can be
calculated from the above.
Second part: Calculate NRe. From the graph calculate Dz/uD Calculate Dz 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
Da number. 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 many days.
A continuous steirliser with a steam injector and a flash cooler will be A 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. The time 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 m m-3-3, which needs to , which needs to
be reduced to such an extent that only one organism can survive during be reduced to such an extent that only one organism can survive during
two months of operation. The heat reesistant bacterial spores in the two months of operation. The heat reesistant bacterial spores in the
medium can be characterized by A of 5.7 X 10medium can be characterized by A of 5.7 X 103939 1/h and E of 1/h and E of
2.834X102.834X1055KJ/Kmol. The steriliser will be constructed with the pipe with an KJ/Kmol. The steriliser will be constructed with the pipe with an
inner diameter of 0.102m. Steam is available at a temperature of 125inner diameter of 0.102m. Steam is available at a temperature of 125ooC. The C. The
physical properties of the medium are physical properties of the medium are ρρ=1000kg/m=1000kg/m33and and μμ=4 Kg/m/h =4 Kg/m/h
R=8.3144 J/K/g molR=8.3144 J/K/g mol
What length should the pipe be in the sterliser if you assume ideal plug flowWhat length should the pipe be in the sterliser if you assume ideal plug flow
What length should the pipe be in the sterliser if the effect of axial What 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 design:
Dz/uD=2 X 107(NRe) -1.951 for NRe < 10000
Dz/uD=1.6317(NRe) –0.1505 for NRe > 10000
ln(Nt/No)= -Da + (Da2/Pe)
Solution to ax2+bx+c=0 is
aacbbx
242
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/UD 0.788460214
Dz 19.67636897 sq.m/h
Nt 1
No 1.44E+16
Nt/No 6.94444E-17
ln(Nt/No) -37.2060046
A 5E+39 1/h
E 283400 J/gmol
R 8.3144 J/K/gmol
T 398 K
K 320.0387736 1/h
Peclet infinity
Da=KL/U 37.2
L=DaU/K 28.43841221 m
Peclet=UL/Dz 353.6106933
a 1
b -353.61
c 13156.41529
Da 42.25540789
L=DaU/K 32.3031373 m
Filter sterilizationFilter sterilization
Suspended solids may be separated from a Suspended solids may be separated from a fluid during sterilization by the following fluid during sterilization by the following mechanismsmechanisms
1.1. Inertial ImpactionInertial Impaction2.2. DiffusionDiffusion3.3. Electrostatic attractionElectrostatic attraction4.4. InterceptionInterception
Inertial ImpactionInertial Impaction The fluid will tend to move through the least The fluid will tend to move through the least
resistance path during filtration. resistance path during filtration.
Suspended particles in the fluid stream have Suspended particles in the fluid stream have momentum. momentum.
Due to this they tend to move straight and Due to this they tend to move straight and therefore impact on the filtration fibres where therefore impact on the filtration fibres where they may be retained.they may be retained.
This mechanism is more prominent in filtration of This mechanism is more prominent in filtration of gases than liquids.gases than liquids.
DiffusionDiffusion Extremely small particles suspended in the fluid Extremely small particles suspended in the fluid
are subjected to Brownian movement which is are 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 from Thus due to this the fluid particles deviate from the path of movement and impact on the filter the path of movement and impact on the filter fibres.fibres.
Diffusion is more significant in the filtration of Diffusion is more significant in the filtration of gases than liquids.gases than liquids.
Electrostatic attractionElectrostatic attraction Charged particles are attracted by Charged particles are attracted by
opposite charges on the surface of the opposite charges on the surface of the filtration mediumfiltration medium
InterceptionInterception The fibres comprising filter are interwoven to definite opening The 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 by Particles which are larger than these pore sizes are removed by direct interception.direct interception.
Also significant number of smaller size particles are also Also 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 liquids Interception 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 the In depth filter the pore sizes are larger than the particle to be removedparticle to be removed
In these filters particles are removed more by In these filters particles are removed more by impaction, diffusion and electrostatic attraction impaction, 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 to In 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 a In 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 microorganism Thus always there is probability that microorganism can pass through the filter immaterial of the depth of can 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 material Hence 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 the Also 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 following During 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 the Minimal adsorption of the proteins from the medium during filtrationmedium during filtration
Medium should be free of viruses and Medium should be free of viruses and endotoxins. endotoxins.
Absolute filters are used.Absolute filters are used.
Pressure drop in these filters are high hence the Pressure 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 controlled Pore sizes of these membranes are well controlled during manufacture.during manufacture.
To extend the life of the filter prefilters of bigger pore To 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 microbial This requires continuous and large amount of air free of microbial
contaminants.contaminants. 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 example The 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 for However the pressure drop is huge and wet cotton may become source for
contamination.contamination. 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 (PTFE In the case of absolute filters the filter should be hydrophobic (PTFE
membranes).membranes).
Air FilterAir Filter
Theory of Depth filters To reduce the population entering into the filter and exiting out,Expression given as,dN/dx = - KNWhere, N is the concentration of particles in the air at a depth x in the filter& K is a constant,On integrating above expression, we get:N/No = e-kt
Where, 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 filter,
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