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In the Name of God

Presenter: Maryam Shahmansouri

Suprvisor: Dr.Reza Gheshlaghi

Selection, Scale up

and Operation of

Bioreactors

(Chapter 10 Shuler)

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Outline

types of Bioreactors

problems in large reactors

Scale-up

Scale-down

Sterilization

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Classification types of Bioreactors

Operation modes:

- batch: stirred tank.

- continuous:chemostat, fluidized-bed.

- modified types of the above modes:fed-batch, chemostat with recycle,

multi-stage continuous reactors.

Oxygen supply:

- aerobic: airlift.

- anaerobic

Application of energy:

-Mechanical (mixers)

-Pneumatic

-Hydraulic

Control of cell growth

- Chemostat

- Turbidostat

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Basic Reactor types

• Stirred-tank reactor

Reactors with internal mechanical agitation.

• Bubble columns

reactors that rely on gas sparging for agitation.

• Loop reactors

reactors that mixing and liquid circulation are induced by the motion of

an injected gas, by a mechanical pump, or by a combination of the two.

(Airlift, propeller loop, jet loop reactor)

Three-phase reactors are difficult to design

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Mechanical (stirred tank reactors with different stirrers)

marine impellers• Axial flow

• For low viscosity media

• For cellular systems with high levels

of shear sensitivity.

Disc impellers• Radial flow

• For high viscosity media

• Most commonly used bioreactor type(highly flexible, provide high heat & mass transfer)

• High energy consumption

supply air

supply air

deflectorplate

deflectorPlate(baffels)

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Pneumatic

supplyair

exhaustair

Bubble column

Advantages:

• Highly distributed gas bubbles

• Suitable for low viscosity Newtonian broths

• Provide a higher energy efficiency than STR

• Provide a low-shear environment.

• Absence of mechanical agitation reduces cost and

eliminates one potential entry point for contaminants.

Disadvantages:

• less vigorous mixing capabilities than STR

• Mixing may not be possible in highly viscous broths.

• Less flexible than STR.

• Work over a rather narrow range of gas flow rates

(foaming ,bubble coalescence ,nature of the broth)

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Advantages:

• low shear forces

• cultivation of animal ells

• Low energy consumption

• Can generally handle somewhat more viscous

fluids than bubble column

• Coalescence is not so much of problem

Disadvantages:

• The interchange of material between fluid

elements is small, so the transient time to

circulate is important.

exhaustair

supplyair

inner catalyst tube

loop reactor (airlift)

Pneumatic

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Main problems in large reactors

• The abilities of the design to provide an adequate supply of

oxygen.

• Remove metabolic heat efficiently.

• Foaming

• sterility

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Aeration(oxygen supply)

• For industrial-scale fermenters, oxygen supply and heat removal

are the key design limitations.

• Oxygen transfer from gas bubbles to cells is usually limited by

oxygen transfer through the liquid film surrounding the gas bubbles.

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The rate of oxygen transfer from the gas to liquid phase is given by:

KL: Oxygen transfer coefficient(cm/h)

a : gas-liquid interfacial area(Cm3/Cm2)

KLa : volumetric oxygen transfer coefficient(h-1)

C* :saturated DO concentration(mg/l)

CL : actual DO concentration(mg/l)

NO2: rate of oxygen transfer(mg/l.h)

OTR : oxygen transfer rate

Aeration

gas bubble

O2

cells

transmission of oxygen

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Aeration

The rate of oxygen uptake is denoted as OUR(oxygen uptake rate)

: yield coefficient on oxygen (g dw cells/gO2)

:cell concentration (g dw cells/l)

When oxygen transfer is rate-limiting step, the rate of oxygen Consumption is equal

to the rate of oxygen transfer. If the maintenance Requirement of O2 is negligible

compared to growth, then

: specific rate of oxygen consumption (mg O2 /g dw cells.h)

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The value of OUR:

In large-scale systems are 40 to 200 (mmol/l.h)

In most systems in the range of 40 to 60 (mmol/l.h)

Aeration

Demand side Supply side

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Aeration

A wide range of equations has been suggested for the estimation of Kla.

K:empirical constant

:Power requirement in an aerated bioreactor

:bioreactor volume

:superficial gas exit speed

N : rotational speed of agitator

K:constant based on reactor geometry

: power required in ungased fermenter

: impeller diameter

Q: aeration rate(volume of gas supplied per minute

divided by the liquid volume in the reactor)

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Main factors effect on KLa & C*

presence of salts and surfactants can significantly alter bubble size and

liquid film resistance around the gas bubble.

Temperature

Pressure

Vessel geometry

Operation

Fluid properties

Presence of biomass

Note1: On the supply side, CL should be maintained at a value above the critical

oxygen concentration but at a low enough value to provide good oxygen transfer.

Note2: one complication, in all methods is the value of C* to use. C* is proportional

to pO2 .at the sparger point, pO2 will be significantly higher than at the exit.

Aeration

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Aeration

Although KLa is difficult to predict, it is measurable parameter.

Methods of Measurement of Kla in a Bioreactor

Two basic methods for measuring Kla:

Chemical methods(no cell present)

Physical methods(with/without cells)

Four approaches are commonly used :unsteady state,

steady state, dynamic, sulfite test.

Sodium sulfite oxidation method

Absorption of CO2

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Aeration

Methods of Kla Measurement:

Steady-state method (best way)

a) Whole reactor is used as a respirometer.

b) uses a gaseous oxygen analyzer to measure the oxygen concentration

both in the inlet and the outlet gas stream of the bioreactor

c) uses a probe for measuring the dissolved oxygen concentration

in the liquid

An oxygen mass balance under steady-state conditions yields:

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Aeration (Unsteady-state method)

Unsteady-state method (without cell)

a) Measured C*accurately.

b) Oxygen is removed from the system by

sparging with N2.

c) Air is introduced and the change in DO is

monitored until the solution is nearly

saturated.

integration

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Aeration

Sulfite method

a) In the presence of Cu2+ The sulfur in sulfite (SO32- ) is oxidized to sulfate (SO4

2- )

in a zero order reaction.

b) This reaction is very rapid and consequently CL approaches zero.

c) Rate of sulfate formation is monitored and is proportional to the rate of oxygen

consumption (1/2 mol of O2 is consumed to product 1 mol of SO42- .

desadvantages:

the sulfite method probably overestimates Kla.

physicochemical properties are very different from those of fermentation broths.

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Dynamic method

a) this method shares similarities with the steady-state method in that it uses a

fermenter with active cells.

b) It is simpler in that is requires only a dissolved oxygen(DO).

c) It is requires that the air supply be shut off for a short period (eq<5 min)and then

turned back on.

The governing equation for DO levels is:

There is no gas bubble when the gas is off

The lowest value of CL must be above the critical oxygen concentration.

Advantage

Aeration

KLa can be estimated under actual fermentation conditions.

If qO2 is known, the value of OUR can be used to estimate X.

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The slope of the descending curve will give the OUR or .

When air sparsing is resumed, the ascending curve can be used to calculate KLa.

A plot of versus result in a line with a slope of KLa

Aerator off

Aerator on

TIME (MIN)

DO

2 C

ON

C. C

L (

mM

O2/L

)

AIR-OFF

AIR-ONCL,CRIT

3 - 5

CL STEADY-STATE

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Heat removal

In aerobic fermentation ,since oxygen is the final electron acceptor,the rate of metabolic heat evolution can roughly be correlated to therate of oxygen uptake.

The total amount of cooling surface (either jacket or coil) requiredcan be calculated by:

given the temperature of the cooling water

the maximum flow rates allowable

the desired temperature differential between the exiting coolant and thereactor

and the overall heat transfer coefficient.

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Scale-up

What is Scale-up?

• Study of problems associated with the transfer of

experimental data from laboratory and pilot-plant equipment

to large scale industrial equipment.

• No actual data or correlation exist for scale-up.

• Stages

– Bench Scale ( 2 – 20 L)

– Pilot Scale (100 – 500 L)

– Plant Scale (500 – 20,000 L)

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• scale-up the box below

• The same type of bioreactors of different size may be:

Geometrically similar

Geometrically non-similar

The scale-related volume and surface area are fundamental physical

parameters that can not be maintained at a constant state.

As a result of scaling up, whether geometrically similar or not, the scale

related surface area per unit volume will be decrease.

Scale-up

3

3 6

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To make a box twice as big, just

multiply the dimensions by 2 and

you have the scaled dimensions.

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Geometric Scale-up of Bioreactors

Preservation of Geometrical Similarity:

HL1/Dt1 = HL2/Dt2 =….. = HL3/Dt3….=2/1 or 3/1

• Unlike a vessel’s dimensions, manufacturing process parameters should

not be scaled linearly. Linear scaling of process parameters would

produce undesired results and can greatly affect cell growth.

DT2 DT3

HL3

HL2HL1

DT1

LAB SCALE PILOT SCALE COMMERCIAL SCALE

Scale-up

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Problems in scale-up

Surface-to-volume ratio decreases dramatically during

scale up

Wall growth in bacterial and fungal fermentation.

Physical conditions in a large fermenter can never exactly

duplicate those in a smaller fermenter if geometric

similarity is maintained.

Scale-up

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• Scale up rules can be used to establish which

parameters will be varied and how?

• Rules

o Constant power input(P0/V)

implies constant OTR

o Constant impeller rotation number(N)

give constant mixing time.

o Constant impeller tip speed(NDi)

give constant shear.

o Constant Reynolds number( )

implies geometrically similar flow pattern.

Scale-up

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Scale-up

Important depended variables used in scale-up.

1. Energy input

2. Energy input/volume P/V N3 Di2

3. Pump rate of impeller Q N Di3

4. Pump rate of impeller/volume Q/V N

5. Impeller Tip Velocity Vt = (2R)(N) = (Di)(N)

6. Reynolds Number NRe = NDi2/.

Scale-up criterion in general are a function of independent variables N, Di.

P N3Di5

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the choice of scale-up criterion depends

On two considerations:

a) Nature of the fermentation and morphology of the

microorganism.

b) During scale-up, what is the objective parameter of

fermentation we wish to optimize (maximize).

Scale-up

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Volumetric scale-up ratio = V2/V1 = 10,000/80 = 125

Impeller diameter scale-up ratio = Di2/Di1= 5

SCALE-UP

80 L 10,000 L

i1N

Ni2

Scale-up Example

geometric similarity was applied.

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Independence of scale up parameters

Constant,

Re

0.2

0.0016

0.04

5.0

5.0

0.04

0.2

1.0

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• Traditional scale up is highly empirical and make sense

only if there is no change in the controlling regime during

scale up.

• In empirical scale up operating parameters for the large

scale are often determined experimentally (i.e. trial and

error).

• Example: if constant DO is desirable, then the setpoint value for DO

is maintained at the large scale, and other parameters (agitation speed,

aeration rate,...) are varied to ensure the setpoint is achieved.

Scale-up

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What is Scale-down?• The basic concept is to provide at a smaller scale an

experimental system that duplicates exactly the same

heterogeneity in environment that exists at the large scale.

Advantages:• In many case scale-up will require using existing

production facilities, but scale-down dose not.

• At the smaller scale many parameters can be tested more

quickly and inexpensively than at the production scale.

• A small-scale system can be used to evaluate proposed

process changes for an existing operating process.

Scale-down

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• Sterility: means the absence of any detectable viable organism. Sterility is

an absolute concept ;a system is never partially or almost sterile.

• Pure culture: means that only the desired organism is detectably present.

• Disinfection: means reduce the number of viable organisms, often specific

type of organisms, to a low, but none zero value.

• Death: means the failure of the cell, spores, or virus to reproduce or

germinate when placed in a favorable environment.

Sterilization

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Sterilization

Reasons for sterilization

1. Economic penalty for contamination is high.

2. Many fermentation must be absolutely devoid of foreign

organisms.

3. Recombinant DNA fermentations-exit streams must be

sterilized.

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Sterilization

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Sterilization

E.coliSpores of bacillus stearothermophilus

N = number of viable spores or cells at any time, N0 = original number of viable spores or cells. 38

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Sterilization

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Dependence of the specific death rate on temperature is given

by Arrhnius equation:

Sterilization

R:gas constant

T:absolute temperature

E0d :activation energy for the death of the organism(50-150 kcal/g-mol)

Spores of bacillus stearothermophilus E0d =70( kcal/g-mol)

E.coli E0d =127( kcal/g-mol)

Vitamins and growth factors in many media E0d =2-20( kcal/g-mol)

Most thermal sterilizations take place at 121° c .the values for kd in

such situations are very high for vegetative cells(often> 1010 min-1 ).

For spores the values of kd typically range from 0.5-5.0 min-1

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The main factors in any sterilization protocol are:

Temperature and pH of environment

Time of exposure

Initial number of organisms that must be killed

Nature of microbes in the population

Presence of solvents, organic matter, or inhibitors

Sterilization

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Sterilization

Problems of sterilization increase with scale up

Example: Consider the probability of an unsuccessful sterilization in a1L

and 1000L reactor, where each contains the same identical solution , if

kdt=15 and n0( concentration of particles)= 104 spores/l then:

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Sterilization

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Sterilization

Example:1-P0(t)=0.001, N0=108 , kd=1 min-1 @121°c

From the chart Kdt=26 t=26min

Use of sterilization chart:

1. Spesify1-P0(t) which is acceptable

2. Determine N0 in the system

3. Read kdt from the chart

4. With knowing kd for spores or cells , obtained requred time,t.

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Steam sterilization can be accomplished batch wise, often in

situ in the fermentation vessel, or in a continuous apparatus.

Sterilization

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Batch sterilization

Disadvantages:

Thermal lags

Incomplete mixing

Time required to heat (121̊C)and to cool it back(37 ̊C) is often

much longer than the time of exposure to the desired temperature.

For most spores Kd falls very rapidly with temperature so heat-up

and cool-down periods do little to augment spore killing.

Elevated temperature during Heat-up and cool-down can be very

damaging to vitamins and proteins.

Sterilization

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Continuous sterilization

Advantages:

Particularly a high-temperature, short exposure time, can achieve

complete sterilization.

Both the heat-up and cool-down period are very rapid

Easier to control and reduce downtime in the fermenters.

Disadvantages:

Dilution of medium with steam injection

Foaming

Sterilization

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Sterilization

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