Fermentation technology

1. 1
Chapter VII
Heat Transfer in Fermentation

2. 2
Introduction
Several important chemical engineering concepts in
Bioprocess Engineering are transport phenomena (fluid flow,
mixing, heat and mass transfer), unit operations, reaction
engineering, and bioreactor engineering.
Fluid flow, mixing, and reactor engineering are skipped in this
class. They are available more detail in several chemical
engineering books.
We start with the heat transfer in bioreactors

3. 3
Two types of common heat transfer application
in bioreactor operation
• In situ batch sterilization of liquid medium. In this process,
the fermenter vessel containing medium is heated using
steam and held at the sterilization temperature for a period
of time; cooling water is then used to bring the temperature
back to normal operating conditions
• Temperature control during reactor operation. Metabolic
activity of cells generates heat. Some microorganisms need
extreme temperature conditions (e.g. psycrophilic,
thermophilic microorganisms)
Heat transfer configurations for bioreactors: jacketed vessel,
external coil, internal helical coil, internal baffle-type coil,
and external heat exchanger.

4. 4
Pro’s and cons of the heat exchanger configurations
• External jacket and coil give low heat transfer area. Thus, they are
rarely used for industrial scale.
• Internal coils are frequently used in production vessel; the coils can be
operated with liquid velocity and give relatively large heat transfer
area. But the coil interfere with the mixing in the vessel and make
cleaning of the reactor difficult. Another problem is film growth of
cells on the heat transfer surface.
• External heat exchanger unit is independent of the reactor, easy to
scale up, and provide best heat transfer capability. However, conditions
of sterility must be met, the cells must be able to withstand the shear
forces imposed during pumping, and in aerobic fermentation, the
residence time in the heat exchanger must be small enough to ensure
the medium does not become depleted of oxygen.

5. 5
Heat exchangers in fermentation processes
• Double-pipe heat exchanger
• Shell and tube heat exchanger
• Plate heat exchanger
• Spiral heat exchanger
In bioprocess, the temperature difference is relatively small.
Thus, plate heat exchanger is almost never being used
The concepts and calculation for heat exchangers and their
configurations are available in the text book ( Pauline Doran,
Bioprocess Eng Principle, chapter 8)

6. 6
Chapter VIII
Mass Transfer in Fermentation

7. 7
Introduction
The Fick’s law of diffusion
Role of diffusion in Bioprocess
• Scale of mixing
Mixing on a molecular scale relies on diffusion as the final step in mixing
process because of the smallest eddy size
• Solid-phase reaction
The only mechanism for intra particle mass transfer is molecular diffusion
• Mass transfer across a phase boundary
Oxygen transfer to gas bubble to fermentation broth, penicillin recovery
from aqueous to organic liquid, glucose transfer liquid medium into mould
pellets are typical example.
dy
dC
D
J A
AB
A 


8. 8
Film theory
The two film theory is a useful model for mass transfer
between phase. Mass transfer of solute from one phase to
another involves transport from bulk of one phase to the
interface, and then from the interface to the bulk of the second
phase. This theory is based on idea that a fluid film or mass
transfer boundary layer forms whenever there is contact
between two phases. According to film theory, mass transfer
through the film is solely by molecular diffusion and is the
major resistance.
CA1i CA1 Bulk fluid 1
Bulk fluid 2 CA2i
CA2 Film 2 Film 1

9. 9
Convective mass transfer
 
 
AGi
AG
G
AG
AL
ALi
L
AL
C
C
a
k
N
C
C
a
k
N




It refers to mass transfer occurring in the presence of bulk
fluid motion
k: mass transfer coefficient [m/s]
a: area available for mass transfer [m2/m3]
CAo: concentration of A at bulk fluid
CAi: concentration of A at interface
For gas-liquid system, A from gas to liquid:
 
Ai
Ao
A C
C
ka
N 


10. 10
Overall mass transfer coefficient
Refers to the book Geankoplis (2003), Transport Processes and
Separation Process Principles, 4th ed, chapter 10.4.
Oxygen transport to fermentation broth can be modeled as
diffusion of A through stagnant or non-diffusing B.
If A is poorly soluble in the liquid, e.g. oxygen in aqueous
solution, the liquid-phase mass transfer resistance dominates
and kGa is much larger than kLa. Hence, KLa ≈ kLa.
 
AL
AL
L
A
L
G
G
C
C
a
K
N
a
k
m
a
k
a
K




*
'
1
1

11. 11
Oxygen transfer from gas bubble to cell
Eight steps involved:
i. Transfer from the interior of the bubble to the gas-liquid interface
ii. Movement across the gas-liquid interface
iii. Diffusion through the relatively stagnant liquid film surrounding the
bubble
iv. Transport through the bulk liquid
v. Diffusion through the relatively stagnant liquid film surrounding the
cells
vi. Movement across the liquid-cell interface
vii. If the cells are in floc, clump or solid particle, diffusion through the
solid of the individual cell
viii. Transport through the cytoplasm to the site of reaction.

12. 12
Analyzes for most bioreactors in each step involved
i. Transfer through the bulk phase in the bubble is relatively fast
ii. The gas-liquid interface itself contributes negligible resistance
iii. The liquid film around the bubble is a major resistance to oxygen
transfer
iv. In a well mixed fermenter, concentration gradients in the bulk liquid
are minimized and mass transfer resistance in this region is small,
except for viscous liquid.
v. The size of single cell <<< gas bubble, thus the liquid film around
cell is thinner than that around the bubble. The mass transfer
resistance is negligible, except the cells form large clumps.
vi. Resistance at the cell-liquid interface is generally neglected
vii. The mass transfer resistance is small, except the cells form large
clumps or flocs.
viii. Intracellular oxygen transfer resistance is negligible because of the
small distance involved

13. 13
Chapter IX
Unit Operations in Fermentation
(introduction to downstream processing)

14. 14
Downstream processing, what and why
Downstream processing is any treatment of culture broth after fermentation
to concentrate and purify products. It follows a general sequence of steps:
1.Cell removal (filtration, centrifugation)
2.Primary isolation to remove components with properties significantly
different from those of the products (adsorption, liquid extraction,
precipitation). Large volume, relatively non selective
3.Purification. Highly selective (chromatography, ultra filtration, fractional
precipitation)
4.Final isolation (crystallization, followed by centrifugation or filtration
and drying). Typical for high-quality products such as pharmaceuticals.
Downstream processing mostly contributes 40-90 % of total cost.

15. 15
Filtration
Type of filtration unit:
• Plate and frame filter. For small fermentation batches
• Rotary-drum vacuum filter. Continuous filtration that is widely used in the
fermentation industry. A horizontal drum 0.5-3 m in diameter is covered
with filter cloth and rotated slowly at 0.1-2 rpm.
The filtration theory and equation are not explained here since they are
available in the course “Unit Operations of Chemical Engineering I”.

16. 16
Centrifugation
Centrifugation is used to separate materials of different density when a
force greater than gravity is desired
The type of industrial centrifugation unit:
• Tubular bowl centrifuge (Narrow tubular bowl centrifuge or
ultracentrifuge, decanter centrifuge, etc). Simple and widely applied in food
and pharmaceutical industry. Operates at 13000-16000 G, 105-106 G for
ultracentrifuge
• Disc-stack bowl centrifuge. This type is common in bioprocess. The
developed forces is 5000-15000 G with minimal density difference between
solid and liquid is 0.01-0.03 kg/m3. The minimum particle diameter is 5 µm

17. 17
Centrifugation (dry solid decanter centrifuge)

18. 18
The centrifugation theory
g
D
u p
f
p
g
2
18

 

The terminal velocity during gravity settling of a small spherical particle in
dilute suspension is given by Stoke’s law:
Where ug is sedimentation velocity under gravity, ρp is particle density, ρf
is liquid density, µ is liquid viscosity, Dp is diameter of the particle, and g
is gravitational acceleration.
In the centrifuge:
uc is particle velocity in the centrifuge, ω is angular velocity in rad/s, and r
is radius of the centrifuge drum.
r
D
u p
f
p
c
2
2
18



 


19. 19
The centrifugation theory
g
r
Z
2


g
u
Q
2


The ratio of velocity in the centrifuge to velocity under gravity is called the
centrifuge effect or G-number.
Industrial Z factors: 300-16 000, small laboratory centrifuge may up to 500 000.
The parameter for centrifuge performance is called Sigma factor
Q is volumetric feed rate. The Sigma factor explain cross sectional area of a gravity
settler with the same sedimentation characteristics as the centrifuge. If two
centrifuge perform with equal effectiveness
2
2
1
1



Q
Q

20. 20
The centrifugation theory
  
3
1
3
2
2
tan
3
1
2
r
r
g
N






Disc-stack bowl centrifuge
N is number of disc, θ is half-cone angle of the disc.
The r1 and r2 are inner and outer radius of the disc, respectively.
Tubular-bowl centrifuge  
2
1
2
2
2
3
2
r
r
g
b




b is length of the bowl, r1 and r2 are inner and outer radius of the wall of the
bowl.

21. 21
Cell disruption
Mechanical cell disruption methods
•French press (pressure cell) and high-pressure homogenizers. In these
devices, the cell suspension is drawn through a check valve into a pump
cylinder. At this point, it is forced under pressure (up to 1500 bar) through a
very narrow annulus or discharge valve, over which the pressure drops to
atmospheric. Cell disruption is primary achieved by high liquid shear in the
orifice and the sudden pressure drop upon discharge causes explosion of the
cells.
•Ultrasonic disruption. It is performed by ultrasonic vibrators that produce a
high-frequency sound with a wave density of approximately 20 kHz/s. A
transducer convert the waves into mechanical oscillations via a titanium
probe immersed in the concentrated cell suspension. For small scale

22. 22
Cell disruption

23. 23
The equation for Manton-Gaulin homogenizer

kNp
R
R
R
m
m 









ln
Rm: maximum amount protein available for release
R: amount of protein release after N passes through the
homogenizer
k: temperature-dependent rate constant
p: operating pressure drop
: resistance parameter of the cells, for S. cerevisiae is 2.9

24. 24
Cell disruption
Non mechanical cell disruption methods
Autolysis, use microbe own enzyme for cell disruption
Osmotic shock. Equilibrating the cells in 20% w/v buffered sucrose, then
rapidly harvesting and resuspending in water at 4oC.
Addition of chemicals (EDTA, Triton X-100), enzymes (hydrolyses, b-
glucanases), antibiotics (penicillin, cycloserine)

25. 25
Chromatography
Chromatographic techniques usually employed for high value products.
These methods, normally involving columns of chromatographic media
(stationary phase), are used for desalting, concentration and purification
of protein preparations. Several important aspects are molecular weight,
isoelectric point, hydrophobicity and biological affinity. The methods are:
1.Adsorption chromatography
2.Affinity chromatography
3.Gel filtration chromatography
4.High performance liquid chromatography
5.Hydrophobic chromatography
6.Metal chelate chromatography

26. 26
Finishing steps (final isolation)
Crystallization
Product crystallization may be achieved by evaporation, low-temperature
treatment or the addition of a chemical reactive with the solute. The product’s
solubility can be reduced by adding solvents, salts, polymers, and
polyelectrolytes, or by altering pH.
Drying
Drying involves the transfer of heat to the wet material and removal of the
moisture as water vapor. Usually, this must be performed in such a way as to
retain the biological activity of the product. The equipment could be rotary
drum drier, vacuum tray drier, or freeze-drier.

27. 27
Chapter X
Bioreactor

28. 28
Bioreactor configurations
Stirred tank bioreactor
Similar to CSTR; this requires a relatively high input of energy per unit
volume. Baffles are used to reduce vortexing. A wide variety of impeller sizes
and shapes is available to produce different flow patterns inside the vessel; in
tall fermenters, installation of multiple impellers improves mixing.
Typically, only 70-80 % of the volume of stirred reactors is filled with liquid;
this allows adequate headspace for disengagement of droplets from exhaust
gas and to accommodate any foam which may develop. Foam breaker may be
necessary if foaming is a problem. It is preferred than chemical antifoam
because the chemicals reduce the rate of oxygen transfer.
The aspect ratio (H/D) of stirred vessels vary over a wide range. When
aeration is required, the aspect ratio is usually increased. This provides for
longer contact times between the rising bubbles and liquid and produces a
greater hydrostatic pressure at the bottom of the vessel.
Care is required with particular catalysts or cells which may be damaged or
destroyed by the impeller at high speeds.

29. 29
Bioreactor configurations

30. 30
Bioreactor configurations
Bubble column
In bubble-column reactors, aeration and mixing are achieved by gas sparging;
this requires less energy than mechanical stirring. Bubble columns are applied
industrially for production of bakers’ yeast, beer and vinegar, and for
treatment of wastewater.
A height-to-diameter ration of 3:1 is common in bakers’ yeast production; for
other applications, towers with H/D of 6:1 have been used. The advantages
are low capital cost, lack of moving parts, and satisfactory heat and mass
transfer performance. Foaming can be problem.
Homogeneous flow: all bubbles rise with the same upward velocity and there
is no back-mixing of the gas phase.
Heterogeneous flow: At higher gas velocity. Bubbles and liquid tend to rise up
in the center of the column while a corresponding down flow of liquid occurs
near the walls.

31. 31
Bioreactor configurations
Airlift reactor
Airlift reactors are often chosen for culture of plant and animal cells and
immobilized catalyst because shear level are low. Gas is sparged into only part
of the vessel cross section called the riser. Gas hold-up and decreased liquid
fluid density cause liquid in the riser to move upwards. Gas disengages at the
top of the vessel leaving heavier bubble-free liquid to recirculate through the
downcomer. Airlift reactors configurations are internal-loop vessels and
external-loop vessels. In the internal-loop vessels, the riser and downcomer
are separated by an internal baffle or draft tube. Air may be sparged into either
the draft tube or the annulus. In the external-loop vessels, separated vertical
tubes are connected by short horizontal section at the top and bottom. Because
the riser and downcomer are further apart in external-loop vessels, gas
disengagement is more effective than in internal-loop devices. Fewer bubbles
are carried into the downcomer, the density difference between fluids in the
riser and downcomer is greater, and circulation of liquid in the vessel is faster.
Accordingly, mixing is usually better in external-loop than internal-loop
reactors.

32. 32
Bioreactor configurations

33. 33
Stirred and air-driven reactors: comparison of
operating characteristic
For low-viscosity fluids, adequate mixing and mass transfer can be achieved in
stirred tanks, bubble columns and airlift vessels. When a large fermenter (50-
500 m3) is required for low-viscosity culture, a bubble column is an attractive
choice because it is simple and cheap to install and operate. Mechanical-
agitated reactors are impractical at volumes greater than about 500 m3 as the
power required to achieve adequate mixing becomes extremely high.
Stirred reactor is chosen for high-viscosity culture. Nevertheless, mass transfer
rates decline sharply in stirred vessels at viscosities > 50-100 cP.
Mechanical-agitation generates much more heat than sparging of compressed
gas. When the heat of reaction is high, such as in production of single cells
protein from methanol, removal of frictional stirrer heat can be problem so that
air-driven reactors may be preferred.
Stirred-tank and air-driven vessels account for the vast majority of bioreactor
configurations used for aerobic culture. However, other reactor configurations
may be used in particular processes

34. 34
Other bioreactors
Packed bed
Used with immobilized or particulate biocatalysts, for example during the
production of aspartate and fumarate, conversion of penicillin to 6-
aminopenicillanic acid, and resolution of amino acid isomers. Damaged due
to particle attrition is minimal in packed beds compared with stirred reactors.
Mass transfer between the liquid medium and solid catalyst is facilitated at
high liquid flow rate through the bed. To achieve this, packed are often
operated with liquid recycle. The catalyst is prevented from leaving the
columns by screens at the liquid exit. Aeration is generally accomplished in a
separated vessel because if air is sparged directly into the bed, bubble
coalescence produces gas pockets and flow channeling or misdistribution.
Packed beds are unsuitable for processes which produce large quantities of
carbon dioxide or other gases which can become trapped in the packing.

35. 35
Other bioreactors
Fluidized bed
To overcome the disadvantages of packed bed, fluidized bed may be preferred.
Because particles are in constant motion, channeling and clogging of the bed
are avoided and air can be introduced directly into the column. Fluidized bed
reactors are used in waste water treatment with sand or similar material
supporting mixed microbial populations, and with flocculating organisms in
brewing and production of vinegar.
Trickle bed
Is another variation of the packed bed. Liquid is sprayed onto top of the
packing and trickles down through the bed in small rivulets. Air may be
introduced at the base; because the liquid phase is not continuous throughout
the column, air and other gases move with relative ease around the packing.
Trickle-bed reactors are used widely for aerobic wastewater treatment.

36. 36
Other bioreactors