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