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Design and Operation of Fermenters
Design and Operation of Fermenters
Design and Operation of Fermenters
Mahesh Bule
Basic Fermenter Design Criteria
• Microbiological and biochemical characteristics of the
cell system (microbial, mammalian, plant)
• Hydrodynamic characteristics of the bioreactor
• Mass and heat transfer characteristics of the bioreactor
• Kinetics of the cell growth and product formation
• Genetic stability characteristics of the cell system
• Aseptic equipment design
• Control of bioreactor environment (both macro and
micro-environment)
• Implications of bioreactor design on downstream
products separation
• Capital and operating costs of the bioreactor
• Potential for bioreactor scale-up
Requirements of Bioreactors
• There is no universal bioreactor.
• The general requirements of the bioreactor are as follows:
– The design and construction of bioreactors must keep sterility
from the start point to end of the process.
– Optimal mixing with low, uniform shear.
– Adequate mass transfer, oxygen.
– Clearly defined flow conditions.
– Feeding substrate with prevention of under or overdosing.
– Suspension of solids.
– Gentle heat transfer.
– Compliance with design requirements such as: ability to be
sterilized; simple construction; simple measuring, control,
regulating techniques; scale-up; flexibility; long term stability;
compatibility with up- downstream processes; antifoaming
measures.
Fermenter Design
• The basic points of consideration while designing a fermenter:
– Productivity and yield
– Fermenter operability and reliability
– Product purification
– Water management
– Energy requirements
– Waste treatment
• Other few significant things to be taken in account:
– Design in features so that process control will be possible over
reasonable ranges of process variables.
– Operation should be reliable
– Operation should be contamination free
Fermenter design
• To achieve these the fermenter should have:
– Heat and oxygen transfer configuration
– Sterilization procedures
– Foam control
– Fast and thorough cleaning system
– Proper monitoring and control system
• Traditional design is open cylindrical or rectangular vessels made
from wood or stone.
• Most fermentations are now performed in close system to avoid
contamination.
• It should be constructed from non-toxic, corrosion-resistant
materials.
• Small fermentation vessels of a few liters capacity are constructed
from glass and/or stainless steel.
Fermenter design
• Pilot scale and many production vessels are normally made of
stainless steel with polished internal surfaces
• Very large fermenters are often constructed from mild steel lined with
glass or plastic, in order to reduce the cost.
• If aseptic operation is required, all associated pipelines transporting
air, inoculum and nutrients for the fermentation need to be
sterilizable, usually by steam.
• Most vessel cleaning operations are now automated using spray jets,
and called cleaning in place (CIP). And located within the vessel.
• Associated pipe work must also be designed to reduce the risk of
microbial contamination. There should be no horizontal pipes or
unnecessary joints and dead stagnant spaces where material can
accumulate; otherwise this may lead to ineffective sterilization.
Fermenter design
• Normally, fermenters up to 1000 L capacity have an external jacket,
and larger vessels have internal coils.
• Pressure gauges and safety pressure valves must be incorporated,
(required during sterilization and operation).
• For transfer of media pumps are used. Centrifugal pumps (generate
high shear forces and path for easy contaminations), magnetically
coupled, jet and peristaltic pumps.
• Alternate methods of liquid transfer are gravity feeding or vessel
pressurization.
• In fermentations operating at high temperatures or containing
volatile compounds, a sterilizable condenser may be required to
prevent evaporation loss.
• Fermenters are often operated under positive pressure to prevent
entry of contaminants.
Summary of bioreactor systems
Bioreactor design Cell systems used Products
Air-lift bioreactors Bacteria, yeast and fungi SCP, Enzymes, Secondary
metabolites, Surfactants
Perforated
plate
D ow ncom er
Baffle
Impeller
Product
Air
2. Bubble column bioreactors
Broth
outlet
Sampling
nozzles
Jacket
Constant temp.
water bath Perforated
Pump
plate
Compressed
air Orifice
Sparger
Air filter
Peristaltic Medium
pump reservior
Flow
Medium meter
inlet
3. Air lift reactors
Mixing method: airlift
• Compared to bubble
column reactors, in an
airlift reactors, there
are two liquid steams:
up-flowing and down-
flowing steams. Liquid
circulates in an airlift
reactor as a result of
density difference
between riser and
downcomer.
ICI Deep shaft unit
EMLICHHEIM FLOWSHEET
CONDENSATE,
MAE-UP WATER, AND
CLARIFIER FLOCCULATING AGENT
WASH
WATER
SETTLEMENT RECYCLED
TANT WATER
SAND B RECYCLE SLUDGE
B
DECANTER
CENTRIFUGE FLOATATION
SOIL AND
SLUDGE
DEEP
SHAFT
AIR
COMPRESSOR
LAGOON
4. Packed bed bioreactor
Packed-bed
reactors are used
with immobilized
or particulate
biocatalysts.
Medium can be
fed either at the
top or bottom and
forms a
continuous liquid
phase.
Packed bed perfusion bioreactor for
mammalian cell culture
5. Trickle-bed bioreactor
The trickle-bed
reactor is another
variation of the
packed bed
reactors.
Liquid is sprayed
onto the top of the
packing and
trickles down
through the bed in
small rivulets.
Trickle bed reactor for treating waste
water
6. Fluidized bed reactor
28.40
21.30 26.43
6.895
2.876
0.1
0.953
Miscellaneous reactors
• Vacuum fermenter
• Tray fermenter
• Stirred cascade reactor
Vacuum Fermenter
Chilled
water
Condenser
Vacuum
Level pump
control
Vacuum
control
Receiving
tank
(product)
Dry ice
bath
Heating Fermenter
water
Filter
Medium
reservoir
Metering Receiving
Rheostat pump tank
Filter
(bleed)
Air or O2
Tray fermenter
Stirred cascade reactor
Bioreactor Operation Modes
1. Batch Operation
•A foam breaker may be installed to disperse foam
A batch bioreactor is
normally equipped
with an agitator to
mix the reactant, and dCs rmax CS
the pH of the reactant
r
dt K m CS
is maintained by
employing either
buffer solution or a
Batch operation
pH controller
with stirring
Cs 0
Change of Cs
K m ln Cs 0 Cs rmax t
with time, t
Cs
Bioreactor Operation Modes
-2. Plug-flow mode
An ideal plug-flow reactor can
approximate the long tube,
In a plug-flow packed-bed and hollow fiber or
reactor, the substrate multistaged reactor
enters one end of a
cylindrical tube with F, Cs0 V F, Cs
is packed with
immobilized enzyme V Residence
and the product t=0 time
steam leaves at the
F
Continuous operation
other end.
without stirring
Cs 0
K m ln Cs 0 C s rmax t
Cs
Bioreactor Operation Modes
3. Continuous stirred-tank
A continuous stirred-
F, Cs0
tank reactor (CSTR)
is an ideal reactor
which is based on the
F, Cs
assumption that the V
reactants are well
mixed.
Continuous
operation with
stirring
Bioreactor Operation Modes
3. Continuous stirred-tank reactor-Con.
Mass balance of substrate:
F, Cs0
Input - Output Consumption Accumulation
F, Cs
V dCs
FCs 0 FCs rsV V
dt
dCs
Steady state: 0
dt
Michaelis- rmaxC S
r
Menten rate: K m CS
rmax Cs
FCs 0 FCs V 0
K m Cs
Bioreactor Operation Modes
3. Continuous stirred-tank reactor-Con.
Mass balance of substrate:
F, Cs0
rmax Cs
FCs 0 FC s V 0
F, Cs K m Cs
V
F rmaxCs
V Cs 0 Cs K m Cs
F 1
V
rmax Cs
Cs K m
Cs 0 Cs
Aeration and oxygen transfer in
bioreactor systems
• Bio-oxidation of substrate with molecular oxygen as
the final electron acceptor
Oxygen transfer in fermentation
system
GAS FILM
GAS-LIQUD
INTERFACE
O2
CELL-LIQUD
O2 INTERFACE INTERNAL
CELL
AIR BUBBLE RESISTANCE
O2O2 CELL
LIQUID FILM
Dissolved O2
O2 in liquid phase,
nutrients
(medium mostly Electron
Transport
water)
System +
TCA cycle
LIQUID FILM enzymes
The oxygen transport path to the microorganism. Generalized path of oxygen from the gas
bubble to the microorganism suspended in a liquid is shown. The various regions where a
transport resistance may be encountered are as indicated
Oxygen transfer
Q O2 8
6
2
Q O 2 max /2
QO
2
KO2 C L CRIT.
0
0 2 4 6 8 10 12 14 16 18 20
Oxygen CONC. (C L)
a
UNIT LIQUID
VOLUME
AIR BUBBLE
O2 CL
O2 TRANSFER CELLS
(CONC. X)
kL OXYGEN
C *L BULK
(CONC. C )
L
LIQUID
LIQUID FILM
PHASE
Schematic diagram of the mass balance of oxygen transfer in unit liquid volume
Mass balance of oxygen in unit liquid
volume
Mass balance of oxygen in unit liquid
volume (cont’d)
Mass balance of oxygen in unit liquid
volume (cont’d)
2-
F, SO
3
In flu e n t
A ir o u tle t
M o to r
A ir flo w , r a te
rp m
W a te r o u t
W a te r in
-2
● The reaction with [SO3 ] is extremely
fast.
● As a result, the O2 gas molecules are
consumed as soon as they diffuse into
the liquid phase.
● Therefore, the D.O. concentration in the
liquid phase, CL 0.
Chemical Methods of KLa Measurement (Cont’d)
Chemical Methods of KLa Measurement (Cont’d)
80
70 d[SO -2]-2 ] [SO3-2]
SLOPE
SLOPE= - = dt- 3 ~-
d[ SO -2 ] 3[SO 3 ~ -
60 dt t t
50
]
2
40
-
3
SO
30
[
20
10
0
0 2 4 6 8
TIME, t, (min)
Concentration of SO3-2 as a function of oxidation time
Chemical Methods of KLa Measurement (Cont’d)
In Situ Measurement of KLa, QO2, and CL* During
Cell Growth in a Bioreactor
Where:
HO2 = Henry’s Constant for O2 in Water
CL = D.O. Concentration In the Liquid
Phase (Mass of O2/L)
In Situ Measurement of KLa, QO2, and CL*
During Cell Growth in a Bioreactor (Cont’d)
15 10
1. Feed
DO2 pH rpm 2. Flow meter
3. Ring sparger
5 4. Impeller
Alkali Acid 11 12 13 5. Motor
6
6. Shaft
7. pH probe
Water out 8. D.O. probe
2
7 8 9. Baffle
15 10. To Condenser
11. D.O. meter
1 12. pH meter
14
30 deg. 13. Speed controller
4 9
water in 14. Water Jacket
3 16 15. Thermometer
16. Chart recorder
Set up of a Stirred tank Bioreactor with Dissolved Oxygen Probe, pH probe and
In Situ Measurement of KLa, QO2, and CL*
During Cell Growth in a Bioreactor (Cont’d)
AIR-OFF
C L STEADY-STATE
DO2 CONC. C L (mM O 2/L)
C L,CRIT AIR-ON
SLOPE = - Q O2 X
3
CL (mMO2/L)
0
0 1 2 3 4 5 6 7 8 9 10
Time, t (min)
dCL
● By plotting CL vs. dt + QO2X at a given fermentation time, t,
- 1
we can get the slope which is equal to KLa
In Situ Measurement of KLa, QO2, and CL* During
Cell Growth in a Bioreactor (Cont’d)
● and therefore, the value of KLa is found, and the intercept
also gives the value of
PyO2
CL* = H
4.4
*
Intercept = C L
3.8
CL (mgO2/L)
3.2
SLOPE = -1/k La
2.6
2.0
1.4
0.8
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
[dC L /dt+Q O2 X]
Dynamic air-on/air-off data during Poly(glutamic acid (PGA) production by Bacillus subtilis IFO 3335 (fermentation
time = 26 h). Dissolved oxygen concentration CL () as a function of time.
Taken from A. Richard and A. Margaritis, “Rheology, Oxygen Transfer, and Molecular Weight Characteristics of
Poly(glutamic acid) Fermentation by Bacillus subtilis”, Biotechnology and Bioengineering, Vol. 82, No. 3, p. 299-305
(2003).
Agitation requirement in fermentation systems
Where:
Dt = tank diameter,
HL = liquid height
Di = impeller diameter
Hb = impeller distance from bottom of vessel
Wb = baffle width
Li = impeller blade length
Wi = impeller blade height
Different Impeller Types. (a) Marine-type propellers; (b) Flat-blade turbine, Wi
= Di/5. © Disk flat-blade turbine, Wi = Di/5, Di = 2Dt/3, Li = Di/4; (d) Curved-
blade turbine, Wi = Di/3; (e) Pitched-blade turbine, Wi = Di/8; and (f) Shrouded
turbine, Wi = Di/8.
Mixing Patterns for Flat-Blade Turbine Impeller. Effect of Baffles. Liquid agitation in
presence of a gas-liquid interface, with and without wail baffles: (a) Marine impeller and
(b) Disk flat-blade turbines; (c) in full vessels without a gas-liquid interface (continuous
flow) and without baffles.
Mixing and Power Requirements for Newtonian
Fluids in a Stirred Tank
NP vs. NRe; the power characteristics are shown by the power number, NP, and the modified
Reynolds number, NRe, of single impellers on a shaft. [Adopted from S. Aiba, A.E.
Humphrey and N.F. Millis. “Bubble Aeration and Mechanical Agitation”. In Biochemical
Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 174].
Last slides figure shows relationship between NP
and
NRe at three different flow regimes:
● Laminar
● Transient
● Fully Turbulent
for three different impeller types:
● Six-bladed flat blade turbine
● Paddle impeller
● Marine Propeller
The power number is given by
NP = Pgc/n3Di5
Where:
NRe = dimensionless Reynolds number
NP = dimensionless Power number
P = Un-gassed power for liquid (no air), W
gc = 1, for SI units system
n = Impeller rotational speed, revolutions per
sec., (s-1)
Di = Impeller diameter, m
= Density of liquid, kg/m3
= Viscosity of liquid, (N.m)/(s)
NP = 6 = Pgc/n3Di5
or P = (6)(n3Di5)/(gc)