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SYSTEM SYSTEM SURROUNDING SURROUNDING BOUNDARY BOUNDARY

Batch, Fed-Batch, Continuous Cultivation

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Page 1: Batch, Fed-Batch, Continuous Cultivation

SYSTEMSYSTEM

SURROUNDINGSURROUNDING

BOUNDARYBOUNDARY

Page 2: Batch, Fed-Batch, Continuous Cultivation

BATCH CULTIVATION CONTINUOUS CULTIVATION1. The bacteria are inoculated into the

bioreactor (always stirred tank bioreactor).

2. Then, under certain conditions (temperature, pH, aeration, etc.) the bacteria go through all the growth phases (lag, exponential, stationary).

1. The fresh medium flows into the fermentor continuously, and part of the medium in the reactor is withdrawn from the fermenter at the same flow rate of the inlet flow.

2. The bacteria is grown under certain conditions (temperature, pH, aeration)

Advantages:• can be used for diff reactions every day. • Safe: can be properly sterilized.• Little risk of infection or strain mutation• Complete conversion of substrate is

possible

Advantages:• Works all the time: low labor cost, good

utilization of reactor• Often efficient: due to the autocatalytic

nature of microbial   reactions,.• the productivity can be high. • Automation may be very appealing.•  Constant product quality

Dis-advantages:•High labor cost•Much idle time – Sterilization, growth, cleaning•Safety – filling emptying, cleaning.

Dis-advantages:•promised continuous production for months fails due to a. infection. b. spontaneous mutation of microorganisms to non producing strain

Page 3: Batch, Fed-Batch, Continuous Cultivation

Batch cultivation is closed system Batch cultivation is closed system where there is no interaction between where there is no interaction between the system and the surrounding during the system and the surrounding during the process. Except air during the the process. Except air during the aerobic cultivation.aerobic cultivation.

In Batch cultivation we prepare In Batch cultivation we prepare medium, sterilize it and inoculate medium, sterilize it and inoculate the culture into the bioreactor.the culture into the bioreactor.

Allow the cells to grow and produce Allow the cells to grow and produce the product.the product.

Once the product formation reaches Once the product formation reaches maximum harvest the fermentation maximum harvest the fermentation broth.broth.

Page 4: Batch, Fed-Batch, Continuous Cultivation

How cells grow during How cells grow during Batch cultivationBatch cultivation

After inoculating the medium and After inoculating the medium and start measuring the biomass at start measuring the biomass at different time intervals, you may different time intervals, you may find six different phases. They arefind six different phases. They are

1.1. Lag phaseLag phase2.2. Accelerated growth phaseAccelerated growth phase3.3. Exponential growth phaseExponential growth phase4.4. Decelerated growth phaseDecelerated growth phase5.5. Stationary phaseStationary phase6.6. Death phase Death phase

Page 5: Batch, Fed-Batch, Continuous Cultivation

Ln X

X

µ

Page 6: Batch, Fed-Batch, Continuous Cultivation

Lag PhaseLag PhaseLag Phase is an initial period of cultivation Lag Phase is an initial period of cultivation during which the change of cell number is zero or during which the change of cell number is zero or negligible.negligible.

Eventhough the cell number doesnot increase, the Eventhough the cell number doesnot increase, the cells may grow in size during this period.cells may grow in size during this period.

The Lag phase results from several factors.The Lag phase results from several factors.

When cells are placed in the fresh medium, the When cells are placed in the fresh medium, the intracellular concentrations of cofactors, amino intracellular concentrations of cofactors, amino acids, ions will decrease and these have to be acids, ions will decrease and these have to be synthesized / transported first before cell synthesized / transported first before cell division to occur.division to occur.

When the cells are inoculated into medium When the cells are inoculated into medium containing different carbon source then the containing different carbon source then the enzymes for its metabolism have to be transported.enzymes for its metabolism have to be transported.

Page 7: Batch, Fed-Batch, Continuous Cultivation

When cell are placed in medium containing When cell are placed in medium containing several carbon sources then several lag several carbon sources then several lag phases may result. This is known as diauxic phases may result. This is known as diauxic growth. When glucose and lactose are present growth. When glucose and lactose are present then glucose will be utilized first then then glucose will be utilized first then lactose. Presence of glucose will have lactose. Presence of glucose will have catabolite repression on galactosidase enzyme catabolite repression on galactosidase enzyme which is required for lactose utilizationwhich is required for lactose utilization

Page 8: Batch, Fed-Batch, Continuous Cultivation

How to avoid lag phaseHow to avoid lag phaseLag phase is non productive period in Lag phase is non productive period in the industrial fermentations. Hence the industrial fermentations. Hence minimizing it is essential.minimizing it is essential.

The stage of culture from where the The stage of culture from where the inoculum is drawn is important. inoculum is drawn is important. Exponentially growing cells will Exponentially growing cells will have adequate concentrations of have adequate concentrations of intermediates and intracellular pool intermediates and intracellular pool of compounds. Hence if the inoculum of compounds. Hence if the inoculum is drawn from this stage they will is drawn from this stage they will not suffer dilution effect. not suffer dilution effect.

Page 9: Batch, Fed-Batch, Continuous Cultivation

Size of the inoculum – If the size Size of the inoculum – If the size of the inoculum is large then the of the inoculum is large then the lag phase can be minimized. lag phase can be minimized. Generally 10% are used for yeast Generally 10% are used for yeast and mold and 5% for bacteria.and mold and 5% for bacteria.

Medium of inoculum should be same Medium of inoculum should be same of that production medium.of that production medium.

In certain cases such as In certain cases such as recombinant recombinant E.coliE.coli cultivation to cultivation to minimize plasmid loss higher minimize plasmid loss higher percentage of inoculum will be percentage of inoculum will be used.used.

Page 10: Batch, Fed-Batch, Continuous Cultivation

Accelerated growth Accelerated growth phasephase

At the end of Lag At the end of Lag phase, when growth phase, when growth begins the begins the division rate division rate increase gradually increase gradually and reaches a and reaches a maximum value.maximum value.

The sp growth rate The sp growth rate increases to increases to maximum during maximum during this phase.this phase.

Page 11: Batch, Fed-Batch, Continuous Cultivation

Exponential growth Exponential growth phasephase

Cell division occurs in this phase.Cell division occurs in this phase. Often cell dry weight is used for Often cell dry weight is used for cell concentration. During cell concentration. During exponential phase we write as exponential phase we write as

Where µ - Specific growth rateWhere µ - Specific growth rateX- cell dry weightX- cell dry weight

XdtdX

Page 12: Batch, Fed-Batch, Continuous Cultivation

Rearranging and integrating the eqnRearranging and integrating the eqn

m2ln

d

μto

o

t0

XXo

t

0

X

Xo

teXX

μtXXln

tμlnX

dtμXdX

Page 13: Batch, Fed-Batch, Continuous Cultivation

Sometimes the doubling times for cell Sometimes the doubling times for cell number and cell dry weight may differ number and cell dry weight may differ as a result of non constant cell mass as a result of non constant cell mass per cell.per cell.

If the growth rates calculated based on If the growth rates calculated based on these are equal then the growth is these are equal then the growth is said to be balanced growth. In the said to be balanced growth. In the balanced growth the composition of the balanced growth the composition of the cell is equal. Balanced growth will cell is equal. Balanced growth will occur when adequate supply of all occur when adequate supply of all nutrients is there and no toxic by nutrients is there and no toxic by products are generated.products are generated.

In unbalanced growth the cell In unbalanced growth the cell composition varies (eg. Protein composition varies (eg. Protein content) etc., content) etc.,

Page 14: Batch, Fed-Batch, Continuous Cultivation

Other phases of growthOther phases of growth The end of the exponential phase occur The end of the exponential phase occur when any of the essential nutrients is when any of the essential nutrients is depleted or toxic metabolite accumulated depleted or toxic metabolite accumulated in the system. During this phase the in the system. During this phase the growth rate declines.growth rate declines.

Stationary phase will follow this phase. Stationary phase will follow this phase. The length of stationary phase may vary The length of stationary phase may vary with cell type, previous growth conditions with cell type, previous growth conditions etc., In certain cases the product etc., In certain cases the product formation will occur during this phaseformation will occur during this phase

Following this is the death phase where Following this is the death phase where the cells will start to lyse and the cell the cells will start to lyse and the cell density decreases. density decreases.

Page 15: Batch, Fed-Batch, Continuous Cultivation

Environmental conditions Environmental conditions affecting affecting cell growthcell growth

Substrate concentrationSubstrate concentration

TemperatureTemperature

pHpH

Dissolved oxygenDissolved oxygen

OthersOthers

Page 16: Batch, Fed-Batch, Continuous Cultivation

Substrate concentrationSubstrate concentrationThe effect of substrate concentration The effect of substrate concentration on the Specific growth rate is on the Specific growth rate is represented by monod equationrepresented by monod equation

µ - Specific growth rateµ - Specific growth rateµµmaxmax – Maximum specific growth rate – Maximum specific growth rateS – Residual substrate concentrationS – Residual substrate concentrationKKss- Substrate saturation constant- Substrate saturation constant

SKSμμs

max

Page 17: Batch, Fed-Batch, Continuous Cultivation

When S>>KsWhen S>>Ks µ = µµ = µmaxmax

When S=KsWhen S=Ks µ = µµ = µmaxmax/2/2

Thus the saturation constant Ks Thus the saturation constant Ks is numerically equivalent to the is numerically equivalent to the substrate concentration when its substrate concentration when its growth rate is half the maximum. growth rate is half the maximum.

Page 18: Batch, Fed-Batch, Continuous Cultivation

Effect of TemperatureEffect of TemperatureTemperature is an important factor Temperature is an important factor affecting growth of cells. According affecting growth of cells. According to temperature optima of the growth to temperature optima of the growth the cells can be classified into the cells can be classified into three groupsthree groups

Psychrophiles < 20Psychrophiles < 2000CCMesophiles – 20-50Mesophiles – 20-50ooCCThermophiles > 50Thermophiles > 5000CC

Page 19: Batch, Fed-Batch, Continuous Cultivation

Above or below the Above or below the optimum temperature optimum temperature range the growth rate range the growth rate decreases.decreases.

Temperature growth rate Temperature growth rate relationship is relationship is explained by arrhenius explained by arrhenius equationequation

µ = A eµ = A e-E/RT-E/RT

Temperature also affects Temperature also affects the product formation.the product formation.

Temperature may also Temperature may also affect the rate limiting affect the rate limiting step eg. at higher step eg. at higher temperatures diffusion temperatures diffusion may become rate limitingmay become rate limiting

Page 20: Batch, Fed-Batch, Continuous Cultivation

Effect of pH Effect of pH Hydrogen ion concentration affects the Hydrogen ion concentration affects the activity of enzymes and therefore the activity of enzymes and therefore the microbial growth rate.microbial growth rate.

The optimal pH for growth and product The optimal pH for growth and product formation may be different eg. Citric acid formation may be different eg. Citric acid fermentation.fermentation.

pH optimum for growth ofpH optimum for growth of

Bacteria – 3 to 8; Yeast – 3 to 6; Molds – 3 Bacteria – 3 to 8; Yeast – 3 to 6; Molds – 3 to 7to 7

Plant cells – 5 to 6; Animal cells – 6.5 to Plant cells – 5 to 6; Animal cells – 6.5 to 7.5 7.5

Page 21: Batch, Fed-Batch, Continuous Cultivation

Many organisms have mechanism to Many organisms have mechanism to maintain intracellular pH at a maintain intracellular pH at a relatively constant level in the relatively constant level in the presence of fluctuations in presence of fluctuations in environmental pH.environmental pH.

When pH differs from optimal value When pH differs from optimal value the maintenance energy requirement the maintenance energy requirement increases.increases.

In most fermentations pH can vary In most fermentations pH can vary substantially. Often the nature of substantially. Often the nature of nitrogen source can be important.nitrogen source can be important.

Page 22: Batch, Fed-Batch, Continuous Cultivation
Page 23: Batch, Fed-Batch, Continuous Cultivation

Dissolved oxygen Dissolved oxygen concentrationconcentration

Dissolved oxygen concentration is an Dissolved oxygen concentration is an important substrate in aerobic concentration important substrate in aerobic concentration and may be limiting in certain cases. and may be limiting in certain cases.

Oxygen is sparingly soluble in water and the Oxygen is sparingly soluble in water and the cells will uptake oxygen from the medium cells will uptake oxygen from the medium only.only.

The growth of microorganisms will be The growth of microorganisms will be dependant on dissolved oxygen concentration dependant on dissolved oxygen concentration upto certain concentration known as critical upto certain concentration known as critical oxygen concentration and above which the oxygen concentration and above which the growth will not be dependant on the DO growth will not be dependant on the DO concentration.concentration.

Page 24: Batch, Fed-Batch, Continuous Cultivation

DO concentration should be above 20 % DO concentration should be above 20 % for bacteria and yeast and it is 40% for bacteria and yeast and it is 40% for molds depending on the size.for molds depending on the size.

DODO

µµ

Critical DO concentration

Page 25: Batch, Fed-Batch, Continuous Cultivation

Redox potentialRedox potential

Ionic concentrationsIonic concentrations

Dissolved Carbondioxide Dissolved Carbondioxide concentrationconcentration

Other factorsOther factors

Page 26: Batch, Fed-Batch, Continuous Cultivation

Product formationProduct formation

Page 27: Batch, Fed-Batch, Continuous Cultivation

Luedeking–Piret ModelLuedeking–Piret Model Growth-associated products are produced Growth-associated products are produced simultaneously with microbial growth. The simultaneously with microbial growth. The specific rate of product formation is specific rate of product formation is proportional to the specific growth rate µproportional to the specific growth rate µgg

Eg: production of a constitutive enzymeEg: production of a constitutive enzyme Nongrowth-associated product formation: @ Nongrowth-associated product formation: @ stationary phase when growth rate is zero. stationary phase when growth rate is zero. The specific rate of product formation is The specific rate of product formation is constant. constant.

Eg: antibiotics Eg: antibiotics Mixed: takes place during slow growth and Mixed: takes place during slow growth and in stationary phases. in stationary phases.

Eg: Lactic acid fermentation, xantham gumEg: Lactic acid fermentation, xantham gum

Page 28: Batch, Fed-Batch, Continuous Cultivation

Product formation for growth Product formation for growth associatedassociated

Product formation for mixed growth Product formation for mixed growth associatedassociated

Product formation for non growth Product formation for non growth associatedassociated

XPp YdtdP

Xq /

1

tconsqp tan

pq

Page 29: Batch, Fed-Batch, Continuous Cultivation
Page 30: Batch, Fed-Batch, Continuous Cultivation

Continuous cultivationContinuous cultivation

SYSTEMSYSTEM

SURROUNDINGSURROUNDING

BOUNDARYBOUNDARY

Open systemOpen system

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Page 35: Batch, Fed-Batch, Continuous Cultivation

Steady state : steady state is the condition Steady state : steady state is the condition at which there is no change in the at which there is no change in the concentrations of biomass, substrate, concentrations of biomass, substrate, product or anythingproduct or anything

How will you say that the culture has How will you say that the culture has attained steady stateattained steady state

When there is no change for two volume When there is no change for two volume change of medium then the culture is change of medium then the culture is assumed to be in steady stateassumed to be in steady state

What is meant by dilution rateWhat is meant by dilution rate

Dilution rate is the ratio of feed rate to Dilution rate is the ratio of feed rate to working volume working volume

D= F/V D= F/V

Page 36: Batch, Fed-Batch, Continuous Cultivation

x-D diagram is the plot of dilution rate Vs x-D diagram is the plot of dilution rate Vs steady state biomass concentration at steady state biomass concentration at that dilution rate.that dilution rate.

Productivity in continuous cultivation is Productivity in continuous cultivation is the product of dilution rate with steady the product of dilution rate with steady state biomass at that dilution rate.state biomass at that dilution rate.

Upto what dilution rate you can run the Upto what dilution rate you can run the continuous cultivation?continuous cultivation?

In continuous cultivation the dilution rate In continuous cultivation the dilution rate will be equal to the growth rate of the will be equal to the growth rate of the microorganism. You can increase the microorganism. You can increase the dilution rate to the max. growth rate of dilution rate to the max. growth rate of the organism. Beyond which washout will the organism. Beyond which washout will occuroccur

Page 37: Batch, Fed-Batch, Continuous Cultivation

Medium optimization by Medium optimization by continuous cultivationcontinuous cultivation

First construct x-D diagramFirst construct x-D diagram Pulse various compounds to the Pulse various compounds to the medium during steady state and medium during steady state and identify the positive nutrients.identify the positive nutrients.

Shift the inlet medium with Shift the inlet medium with positive nutrients.positive nutrients.

Continue this process till you Continue this process till you have completed with all the have completed with all the positive nutrientspositive nutrients

Final x-D diagram and evaluate Final x-D diagram and evaluate kinetic constantskinetic constants

Page 38: Batch, Fed-Batch, Continuous Cultivation

Productivity in batch and Productivity in batch and continuouscontinuous

Batch cultivationBatch cultivation Continuous Continuous cultivationcultivation

batch

oSX

turnom

b

tSY

P

tXXlnμ

1t

oSXm SYμPDXP

turnmo

turnmo

oSXm

oSXm tXX

tXX

SY

SYBatch

Chemostat

ln

ln

Page 39: Batch, Fed-Batch, Continuous Cultivation

Secondary metabolites are produced Secondary metabolites are produced only during the stationary phase. only during the stationary phase. Hence the continuous cultivation Hence the continuous cultivation is not suitable for them.is not suitable for them.

Maintenance of sterility is very Maintenance of sterility is very difficult.difficult.

High infrastructure cost is High infrastructure cost is required.required.

Why continuous cultivation Why continuous cultivation is not used in the is not used in the

industriesindustries

Page 40: Batch, Fed-Batch, Continuous Cultivation

Fed Batch cultivationFed Batch cultivation Batch cultivation have low productivityBatch cultivation have low productivity

Though continuous cultivation Though continuous cultivation productivity is higher it cannot be productivity is higher it cannot be used for secondary metabolitesused for secondary metabolites

Fed batch cultivation is semi open Fed batch cultivation is semi open systemsystem

Here nutrients are fed slowly into Here nutrients are fed slowly into medium either continuously or medium either continuously or discontinuously in stages.discontinuously in stages.

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