Batch, Fed-Batch, Continuous Cultivation, Growth Kinetics, Substation Utilisation, Product Formation, Luedeking Piret Model, Effect of pH, Effect of Temperature, Dissolved Oxygen Concentration

1. SYSTEMSYSTEM
SURROUNDINGSURROUNDING
BOUNDARYBOUNDARY

2. BATCH CULTIVATION CONTINUOUS CULTIVATION
1. 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medium in the reactor is withdrawn from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

3.  Batch cultivation is closed system whereBatch cultivation is closed system where
there is no interaction between the systemthere is no interaction between the system
and the surrounding during the process.and the surrounding during the process.
Except air during the aerobic cultivation.Except air during the aerobic cultivation.
 In Batch cultivation we prepare medium,In Batch cultivation we prepare medium,
sterilize it and inoculate the culture into thesterilize it and inoculate the culture into the
bioreactor.bioreactor.
 Allow the cells to grow and produce theAllow the cells to grow and produce the
product.product.
 Once the product formation reachesOnce the product formation reaches
maximum harvest the fermentation broth.maximum harvest the fermentation broth.

4. How cells grow duringHow cells grow during
Batch cultivationBatch cultivation
After inoculating the medium and startAfter inoculating the medium and start
measuring the biomass at different timemeasuring the biomass at different time
intervals, you may find six different phases.intervals, you may find six different phases.
They areThey are
1.1. Lag phaseLag phase
2.2. Accelerated growth phaseAccelerated growth phase
3.3. Exponential growth phaseExponential growth phase
4.4. Decelerated growth phaseDecelerated growth phase
5.5. Stationary phaseStationary phase
6.6. Death phaseDeath phase

5. Ln X
X
µ

6. Lag PhaseLag Phase
Lag Phase is an initial period of cultivation duringLag Phase is an initial period of cultivation during
which the change of cell number is zero or negligible.which the change of cell number is zero or negligible.
Eventhough the cell number doesnot increase, theEventhough 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, theWhen cells are placed in the fresh medium, the
intracellular concentrations of cofactors, amino acids,intracellular concentrations of cofactors, amino acids,
ions will decrease and these have to be synthesized /ions will decrease and these have to be synthesized /
transported first before cell division to occur.transported first before cell division to occur.
When the cells are inoculated into medium containingWhen the cells are inoculated into medium containing
different carbon source then the enzymes for itsdifferent carbon source then the enzymes for its
metabolism have to be transported.metabolism have to be transported.

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

8. How to avoid lag phaseHow to avoid lag phase
Lag phase is non productive period in theLag phase is non productive period in the
industrial fermentations. Henceindustrial fermentations. Hence
minimizing it is essential.minimizing it is essential.
 The stage of culture from where theThe stage of culture from where the
inoculum is drawn is important.inoculum is drawn is important.
Exponentially growing cells will haveExponentially growing cells will have
adequate concentrations of intermediatesadequate concentrations of intermediates
and intracellular pool of compounds.and intracellular pool of compounds.
Hence if the inoculum is drawn from thisHence if the inoculum is drawn from this
stage they will not suffer dilution effect.stage they will not suffer dilution effect.

9.  Size of the inoculum – If the size of theSize of the inoculum – If the size of the
inoculum is large then the lag phase caninoculum is large then the lag phase can
be minimized. Generally 10% are used forbe minimized. Generally 10% are used for
yeast and mold and 5% for bacteria.yeast and mold and 5% for bacteria.
 Medium of inoculum should be same ofMedium of inoculum should be same of
that production medium.that production medium.
 In certain cases such as recombinantIn certain cases such as recombinant
E.coliE.coli cultivation to minimize plasmid losscultivation to minimize plasmid loss
higher percentage of inoculum will behigher percentage of inoculum will be
used.used.

10. Accelerated growth phaseAccelerated growth phase
 At the end of LagAt the end of Lag
phase, when growthphase, when growth
begins the divisionbegins the division
rate increaserate increase
gradually and reachesgradually and reaches
a maximum value.a maximum value.
 The sp growth rateThe sp growth rate
increases toincreases to
maximum during thismaximum during this
phase.phase.

11. Exponential growth phaseExponential growth phase
 Cell division occurs in this phase.Cell division occurs in this phase.
 Often cell dry weight is used for cellOften cell dry weight is used for cell
concentration. During exponential phase we writeconcentration. During exponential phase we write
asas
Where µ - Specific growth rateWhere µ - Specific growth rate
X- cell dry weightX- cell dry weight
X
dt
dX µ=

12. Rearranging and integrating the eqnRearranging and integrating the eqn
mµ
2ln=
=
=





=
= ∫∫
d
μt
o
o
t
0
X
Xo
t
0
X
Xo
t
eXX
μt
X
Xln
tμlnX
dtμ
X
dX

13. Sometimes the doubling times for cellSometimes the doubling times for cell
number and cell dry weight may differ asnumber and cell dry weight may differ as
a result of non constant cell mass pera result of non constant cell mass per
cell.cell.
If the growth rates calculated based onIf the growth rates calculated based on
these are equal then the growth is saidthese are equal then the growth is said
to be balanced growth. In the balancedto be balanced growth. In the balanced
growth the composition of the cell isgrowth the composition of the cell is
equal. Balanced growth will occur whenequal. Balanced growth will occur when
adequate supply of all nutrients is thereadequate supply of all nutrients is there
and no toxic by products are generated.and no toxic by products are generated.
In unbalanced growth the cell compositionIn unbalanced growth the cell composition
varies (eg. Protein content) etc.,varies (eg. Protein content) etc.,

14. Other phases of growthOther phases of growth
 The end of the exponential phase occur whenThe end of the exponential phase occur when
any of the essential nutrients is depleted or toxicany of the essential nutrients is depleted or toxic
metabolite accumulated in the system. Duringmetabolite accumulated in the system. During
this phase the growth rate declines.this phase the growth rate declines.
 Stationary phase will follow this phase. TheStationary phase will follow this phase. The
length of stationary phase may vary with celllength of stationary phase may vary with cell
type, previous growth conditions etc., In certaintype, previous growth conditions etc., In certain
cases the product formation will occur during thiscases the product formation will occur during this
phasephase
 Following this is the death phase where the cellsFollowing this is the death phase where the cells
will start to lyse and the cell density decreases.will start to lyse and the cell density decreases.

15. Environmental conditions affectingEnvironmental conditions affecting
cell growthcell growth
 Substrate concentrationSubstrate concentration
 TemperatureTemperature
 pHpH
 Dissolved oxygenDissolved oxygen
 OthersOthers

16. Substrate concentrationSubstrate concentration
The effect of substrate concentration on theThe effect of substrate concentration on the
Specific growth rate is represented bySpecific growth rate is represented by
monod equationmonod equation
µ - Specific growth rateµ - Specific growth rate
µµmaxmax – Maximum specific growth rate– Maximum specific growth rate
S – Residual substrate concentrationS – Residual substrate concentration
KKss- Substrate saturation constant- Substrate saturation constant
SK
S
μμ
s
max
+
=

17. When S>>KsWhen S>>Ks
µ = µµ = µmaxmax
When S=KsWhen S=Ks
µ = µµ = µmaxmax/2/2
Thus the saturation constant Ks isThus the saturation constant Ks is
numerically equivalent to the substratenumerically equivalent to the substrate
concentration when its growth rate is halfconcentration when its growth rate is half
the maximum.the maximum.

18. Effect of TemperatureEffect of Temperature
Temperature is an important factor affectingTemperature is an important factor affecting
growth of cells. According to temperature optimagrowth of cells. According to temperature optima
of the growth the cells can be classified intoof the growth the cells can be classified into
three groupsthree groups
Psychrophiles < 20Psychrophiles < 2000
CC
Mesophiles – 20-50Mesophiles – 20-50oo
CC
Thermophiles > 50Thermophiles > 5000
CC

19.  Above or below the optimumAbove or below the optimum
temperature range thetemperature range the
growth rate decreases.growth rate decreases.
 Temperature growth rateTemperature growth rate
relationship is explained byrelationship is explained by
arrhenius equationarrhenius equation
µ = A eµ = A e-E/RT-E/RT
 Temperature also affects theTemperature also affects the
product formation.product formation.
 Temperature may also affectTemperature may also affect
the rate limiting step eg. atthe rate limiting step eg. at
higher temperatureshigher temperatures
diffusion may become ratediffusion may become rate
limitinglimiting

20. Effect of pHEffect of pH
 Hydrogen ion concentration affects theHydrogen ion concentration affects the
activity of enzymes and therefore theactivity of enzymes and therefore the
microbial growth rate.microbial growth rate.
 The optimal pH for growth and productThe optimal pH for growth and product
formation may be different eg. Citric acidformation 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 toBacteria – 3 to 8; Yeast – 3 to 6; Molds – 3 to
77
Plant cells – 5 to 6; Animal cells – 6.5 to 7.5Plant cells – 5 to 6; Animal cells – 6.5 to 7.5

21.  Many organisms have mechanismMany organisms have mechanism
to maintain intracellular pH at ato maintain intracellular pH at a
relatively constant level in therelatively constant level in the
presence of fluctuations inpresence of fluctuations in
environmental pH.environmental pH.
 When pH differs from optimal valueWhen pH differs from optimal value
the maintenance energythe maintenance energy
requirement increases.requirement increases.
 In most fermentations pH can varyIn most fermentations pH can vary
substantially. Often the nature ofsubstantially. Often the nature of
nitrogen source can be important.nitrogen source can be important.

23. Dissolved oxygen concentrationDissolved oxygen concentration
 Dissolved oxygen concentration is an importantDissolved oxygen concentration is an important
substrate in aerobic concentration and may besubstrate in aerobic concentration and may be
limiting in certain cases.limiting in certain cases.
 Oxygen is sparingly soluble in water and theOxygen is sparingly soluble in water and the
cells will uptake oxygen from the medium only.cells will uptake oxygen from the medium only.
 The growth of microorganisms will be dependantThe growth of microorganisms will be dependant
on dissolved oxygen concentration upto certainon dissolved oxygen concentration upto certain
concentration known as critical oxygenconcentration known as critical oxygen
concentration and above which the growth willconcentration and above which the growth will
not be dependant on the DO concentration.not be dependant on the DO concentration.

24.  DO concentration should be above 20 % forDO concentration should be above 20 % for
bacteria and yeast and it is 40% for moldsbacteria and yeast and it is 40% for molds
depending on the size.depending on the size.
DODO
µµ
Critical DO concentration

25.  Redox potentialRedox potential
 Ionic concentrationsIonic concentrations
 Dissolved Carbondioxide concentrationDissolved Carbondioxide concentration
Other factorsOther factors

26. Product formationProduct formation

27. Luedeking–Piret ModelLuedeking–Piret Model
 Growth-associated products are producedGrowth-associated products are produced
simultaneously with microbial growth. Thesimultaneously with microbial growth. The
specific rate of product formation is proportionalspecific rate of product formation is proportional
to the specific growth rate µto 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. Thestationary phase when growth rate is zero. The
specific rate of product formation is constant.specific rate of product formation is constant.
Eg: antibioticsEg: antibiotics
 Mixed: takes place during slow growth and inMixed: takes place during slow growth and in
stationary phases.stationary phases.
Eg: Lactic acid fermentation, xantham gumEg: Lactic acid fermentation, xantham gum

28. Product formation for growth associatedProduct formation for growth associated
Product formation for mixed growth associatedProduct formation for mixed growth associated
Product formation for non growth associatedProduct formation for non growth associated
µXPp Y
dt
dP
X
q /
1
==
tconsqp tan== β
βαµ +=pq

30. Continuous cultivationContinuous cultivation
SYSTEMSYSTEM
SURROUNDINGSURROUNDING
BOUNDARYBOUNDARY
Open systemOpen system

35. Steady state : steady state is the condition atSteady state : steady state is the condition at
which there is no change in the concentrationswhich there is no change in the concentrations
of biomass, substrate, product or anythingof biomass, substrate, product or anything
How will you say that the culture has attainedHow will you say that the culture has attained
steady statesteady state
When there is no change for two volumeWhen there is no change for two volume
change of medium then the culture is assumedchange of medium then the culture is assumed
to be in steady stateto be in steady state
What is meant by dilution rateWhat is meant by dilution rate
Dilution rate is the ratio of feed rate to workingDilution rate is the ratio of feed rate to working
volumevolume

36. x-D diagram is the plot of dilution rate Vsx-D diagram is the plot of dilution rate Vs
steady state biomass concentration at thatsteady state biomass concentration at that
dilution rate.dilution rate.
Productivity in continuous cultivation is theProductivity in continuous cultivation is the
product of dilution rate with steady stateproduct of dilution rate with steady state
biomass at that dilution rate.biomass at that dilution rate.
Upto what dilution rate you can run theUpto what dilution rate you can run the
continuous cultivation?continuous cultivation?
In continuous cultivation the dilution rate will beIn continuous cultivation the dilution rate will be
equal to the growth rate of the microorganism.equal to the growth rate of the microorganism.
You can increase the dilution rate to the max.You can increase the dilution rate to the max.
growth rate of the organism. Beyond whichgrowth rate of the organism. Beyond which
washout will occurwashout will occur

37. Medium optimization by continuousMedium optimization by continuous
cultivationcultivation
 First construct x-D diagramFirst construct x-D diagram
 Pulse various compounds to the mediumPulse various compounds to the medium
during steady state and identify theduring steady state and identify the
positive nutrients.positive nutrients.
 Shift the inlet medium with positiveShift the inlet medium with positive
nutrients.nutrients.
 Continue this process till you haveContinue this process till you have
completed with all the positive nutrientscompleted with all the positive nutrients
 Final x-D diagram and evaluate kineticFinal x-D diagram and evaluate kinetic
constantsconstants

38. Productivity in batch and continuousProductivity in batch and continuous
Batch cultivationBatch cultivation Continuous cultivationContinuous cultivation
batch
o
S
X
turn
om
b
t
SY
P
t
X
X
ln
μ
1
t
=
+





=
oSXm SYμP
DXP
=
=
turnm
o
turnm
o
oSXm
oSXm
t
X
X
t
X
X
SY
SY
Batch
Chemostat
µ
µ
µ
µ
+=










+
= ln
ln

39.  Secondary metabolites are produced onlySecondary metabolites are produced only
during the stationary phase. Hence theduring the stationary phase. Hence the
continuous cultivation is not suitable forcontinuous cultivation is not suitable for
them.them.
 Maintenance of sterility is very difficult.Maintenance of sterility is very difficult.
 High infrastructure cost is required.High infrastructure cost is required.
Why continuous cultivation is notWhy continuous cultivation is not
used in the industriesused in the industries

40. Fed Batch cultivationFed Batch cultivation
 Batch cultivation have low productivityBatch cultivation have low productivity
 Though continuous cultivationThough continuous cultivation
productivity is higher it cannot be usedproductivity is higher it cannot be used
for secondary metabolitesfor secondary metabolites
 Fed batch cultivation is semi openFed batch cultivation is semi open
systemsystem
 Here nutrients are fed slowly intoHere nutrients are fed slowly into
medium either continuously ormedium either continuously or
discontinuously in stages.discontinuously in stages.