Student Guid - Part 3

Topic 3
Bioreactor Engineering
Contents
3.1 Bioreactors for Cell Culture 61

3.2 Mixing 63
3.3 Oxygen Transfer 66
3.4 Oxygen Consumption in Bioreactors 69
3.4.1 Oxygen Transfer in Bioreactors 70
3.4.2 Oxygen Transfer Rate & the Kla 71
3.4.3 Measurement of Oxygen Concentrations 72
3.4.4 Measurement of the Oxygen Transfer Rate Kla 73
3.5 Scaling Up & Scaling Down 76
3.5.1 Hierchy of Scaling 77
3.5.2 Scaling Up of Bioreactors 78
Appendix 1: Determination of Dimensionless Numbers 83
©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

TOPIC 3. BIOREACTOR ENGINEERING 60
Learning Objectives
By the end of this topic, you should be able to:
• Suggest a suitable reactor system for a given biological agent.
• Explain the application of different bioreactors.
• Determine the oxygen transfer in bioreactors.

3.1 Bioreactors for Cell Culture

Bioreactors for cell culture can vary significantly in size. They can range from
microbioreactors (up to a few ml) to hundreds of cubic metres. Most bioreactors,
however, have a number of things in common:
• They have to provide an environment in which cells are active; in most

cases that means grow
• They usually have to be sterilisable, and be able to maintain sterility.
• During operation transport of heat from or to the reactor must be gentle
• There must be adequate mixing
• Solids must remain suspended
• There must be adequate mass transfer, particularly with regard to oxygen
A variety of other factors may also be important, e.g. no foaming.
Industrial scale cell culture is typically performed in suspension culture. Such

cultures are usually done in stirred tank reactors or air-driven reactors such as airlift
fermenters and bubble columns, although in some cases the gases produced
during fermentation are sufficient to keep the cells suspended (this is the case for
example during beer production).
Some production processes use immobilised cells. Actual culture of immobilised

cells is rare; in most cases the cells to be immobilised are grown separately as
suspension culture, and then immobilised in an immobilising agents such as
alginate. The cells are then used as catalysts. For immobilised cells fluidised bed
reactors and packed bed bioreactors are most often used. Figure 3.1 shows a
variety of bioreactors commonly used for cell culture.
Figure 3.1, from left to right: Stirred tank bioreactor, airlift bioreactor, fluidised bed
bioreactor, packed bed bioreactor.

Stirred tanks are usually the fermenter of choice; a sketch of a small stirred tank
bioreactor is shown in Figure 3.2. The stirred tank may or may not be aerated,
depending on the type of cells grown.
Air Filter
Air Filter
Condensor
Baffle
Rushton
turbine
Mantle
for
Sparger heating/c
ooling
Figure 3.2, Stirred tank bioreactor. Not shown: medium input/output, sampling
port, sensors.

3.2 Mixing
Stirred reactors
The impeller used in stirred tank reactors is typically a Rushton turbine or a

propeller, although other types are also used e.g. anchor or helical types for
viscous fluids. Rushton turbines are able to put a lot of power/volume in, giving
intense mixing, but the streaming pattern from propellers is more efficient and
allows mixing to occur with less power. To improve mixing efficiency and stop fluids
from rotating in the fermenter stirred tanks have baffles.
Figure 3.3, Left: Flow pattern in tank with Rushton turbine. Right: Flow pattern in
tank with propeller.
Mixing in fermenters involves three consecutive steps:
• Distribution over the vessel;
• Dispersion of the material by eddies formed by the impeller;
• Homogeneity at the smallest scale (smaller than the smallest eddy) is

achieved by diffusion.
For Rushton turbines, the mixing time tm at high Reynolds number can be estimated
from:
1.54𝑉𝑉
𝑡𝑡𝑚𝑚 =
𝑁𝑁𝑖𝑖 𝐷𝐷𝑖𝑖3
In which V is the reactor volume, Ni the rotational speed of the stirrer, and Di the
diameter of the impeller. A typical mixing time is 30 seconds for a 1 m3 bioreactor.
Mixing causes mechanical stresses in the fluid which can break shear sensitive
cells such as animal cells, plant cells and some filamentous organisms. The
average amount of shear produced is roughly proportional to the stirrer speed; low
stirrer speeds therefore need to be used with shear sensitive cells.

Suspension of solids in stirred tanks: the Zwietering equation
Many fermenters contain solid particles of substrate or cells clumps, and during
the fermentation these particles have to kept in suspension.
A useful correlation for determining the minimum stirrer speed Nmin for keeping
particles just suspended is the Zwietering equation.
The Zwietering equation is:
S ν L d 0.2 [ g∆ρ / ρ L ]0.45 X 0.13

0.1
N min =
Di0.85
In which
Nmin is minimum stirrer speed
S is a dimensionless constant. Its value depends on the stirrer type, the size of the
stirrer relative to tank size, and the distance of the stirrer to the bottom of the tank.
νL is the liquid kinetic viscosity
d is the particle diameter
g is the gravitational constant
∆ρ is the difference in the density of the particle and the fluid
ρ L is the density of the liquid
X is the weight percentage of particles

Di is the diameter of the stirrer
Table 1. Values of S for a selection of impellers.
Impeller Dimpeller/Dtank Himpeller/Dtank S

Rushton 0.25 0.25 12
Rushton 0.33 0.17 5.8
Rushton 0.33 0.25 6.7
Rushton 0.33 0.5 8
Rushton 0.5 0.25 4.25
Rushton 0.5 0.17 3.9
Propeller 0.33 0.25 6.6

Air driven fermenters
As the size of your fermenter increases the amount of power you have to put in
increased exponentially; also the mechanical stresses on cells increases.
Increasing the number of impellers helps but also has it limits.
For very large fermenters mixing is typically done with air bubbles; mixing with air
is also relatively gentle, reducing the mechanical stresses on cells. The formation
of dead zones where no mixing occurs is however more common in air-driven
fermenters.
Typical air-driven fermenters are shown in Figure 3.4:
Figure 3.4, from left to right: Airlift- concentric tubes, Airlift- external recycle,
Bubble column

3.3 Oxygen Transfer

Oxygen transfer from a gas bubble to a cell or clump of cells involves a large
number of steps:
i) Transfer of oxygen from bulk of bubble to gas-liquid interface
ii) Movement across gas –liquid interface
iii) Diffusion through stagnant liquid film surrounding the bubble
iv) Transport through bulk liquid
v) Diffusion through stagnant liquid film surrounding cell or multicellular

particle
vi) Movement across interface of a cell or a multicellular particle
vii) Movement into bulk of the cell or that of the interior of the multicellular
particle.
In practice diffusion through the stagnant film surrounding the bubble is usually the
major resistance to oxygen transfer. Oxygen transfer to single cells is usually not
limited; however, when cells form a multicellular particle oxygen transfer resistance
through the liquid film at the particle surface and from the surface to the particle
interior can be significant.
The rate of mass transfer of oxygen across a gas-liquid interface is described by:
∗
𝑁𝑁𝐴𝐴 = 𝑘𝑘𝐿𝐿 𝑎𝑎(𝐶𝐶𝐴𝐴𝐴𝐴 − 𝐶𝐶𝐴𝐴𝐴𝐴 )
In which NA is the volumetric rate of oxygen transfer and a the interfacial area per
∗
unit volume. 𝐶𝐶𝐴𝐴𝐴𝐴 is the solubility of oxygen in the aqueous phase, and CAL the actual
concentration in the liquid bulk.
The “L” indicates that the mass transfer rate is based on (and predominantly
determined by) mass transfer resistance in the liquid phase, rather than the gas
phase. This caused by the very low solubility of oxygen in water, which at room
temperature is only about 8 ppm.
The rate of oxygen transfer has to be (at least) the same as the oxygen uptake
rate. If the volumetric rate of oxygen transfer is NA and the volumetric rate of oxygen
uptake QO then at steady state:
NA = QO
The oxygen uptake rate is dependent on the cell concentration x. The specific
oxygen uptake rate is qO is defined as:
qO = QO / x

Hence at steady state:

𝑞𝑞𝑂𝑂 𝑥𝑥
𝑘𝑘𝐿𝐿 𝑎𝑎 = ∗
𝐶𝐶𝐴𝐴𝐿𝐿 − 𝐶𝐶𝐴𝐴𝐴𝐴
The value of kLa is strongly dependent on the bubble size. It is also strongly
dependent on the mobility of the gas-liquid interface and the internal circulation in
the gas phase. The value of the kLa is strongly influenced by factors such as the
presence of salts, sugars, surface active agents such as antifoams, cells,
temperature etc., and in practice to get a good estimate of the kLa it needs to be
measured experimentally.
Bubbles can coalesce (= combine to former larger bubbles) once released;

lowering the rate of oxygen transfer. Whether this occurs depends on many factors
such as the presence of surfactants. Bubbles can also be broken into smaller
bubbles if the amount of shear in the bioreactor is high, giving a higher oxygen
transfer rate. If a large amount of oxygen is needed a small (Rushton) turbine at
high speed is normally used in stirred reactors. The presence of large amounts of
bubbles in stirred tanks can affect the effectiveness of the stirrer by causing an
airpocket around the stirrer (flooding).
In stirred fermenters the value of kLa for non-coalescing, non-viscous media can be
predicted using:
𝑘𝑘𝐿𝐿 𝑎𝑎 = 2.0 × 10−3 (𝑃𝑃/𝑉𝑉)0.7 𝑢𝑢𝐺𝐺0.2
For coalescing systems:
𝑘𝑘𝐿𝐿 𝑎𝑎 = 2.6 × 10−2 (𝑃𝑃/𝑉𝑉)0.4 𝑢𝑢𝐺𝐺0.5
In which P is the power input, V the volume, and uG the superficial gas velocity (=
gas volumetric flow rate divided by the cross-sectional area of the fermenter)
In bubble columns and airlifts the kLa is very difficult to predict, and depends
strongly on the bubble size. The bubble size in turn depends highly on whether the
bubbles coalesce or not. When fine bubbles (diameter 0.5-1 mm) are formed in a
bubble column with a coalescing medium the bubbles will quickly merge as they
rise, giving coarse bubbles (diameter 4-6 mm); if coarse bubbles are formed at the
start then of course fine bubbles will never be obtained.
In all bubble columns the oxygen concentration in the gas bubbles will decrease
as they rise through the column. If the oxygen concentration in the liquid bulk phase
is about zero then the oxygen concentration in the gas phase in coarse bubble
systems will decline by roughly 0.55 % for each meter the bubble rises. In fine
bubble systems it can be up to 6 times higher.

Tutorial Questions
1. The Kolmogorov scale gives the size of the smallest eddy in a turbulent flow. If the
1 2
Kolmogorov scale is smaller than 2 to 3 of the cell diameter then damage to a cell
in a stirred cell suspension is likely. It can be used to determine the maximum value
of the stirrer speed N that can be used without damaging the cells.
CHO cells with a diameter of 15 µm are grown in a fermenter with a working volume
of 2 litre. The fermenter is stirred with a Rushton turbine with a diameter D of 20
cm. The power input P into the fermenter can be described by:
P = 5 ρN 3 D 5
where ρ is the fluid density.
The Kolmogorov scale can be described by:
1/ 4
ν 3 
λ =  
ε 
where λ is the size of the smallest eddies, ε the local energy dissipation per unit
mass of liquid, and ν the kinematic viscosity of the fluid. The local energy
dissipation per unit mass can be calculated on the basis of the fluid mass in the
impeller zone, ρD 3 :
P = ερD 3
Calculate the maximum stirrer speed (in rotations per minute) that can be used
without damaging the cells. Given: ν = 1.3×10-6 m2 s-1 ρ = 1000 kg
-3
m
2. The gas holdup ε is the volume of gas / total volume.
If bubbles are formed with a uniform size d, show that the kLa can be estimated by:
6ε
𝑘𝑘𝑙𝑙 𝑎𝑎 = 𝑘𝑘
𝑑𝑑 𝑙𝑙

3.4 Oxygen Consumption in Bioreactors

Although some microorganisms can grow without oxygen, and in fact oxygen is
poisonous for strict anaerobes, in many cultures oxygen is needed for growth. The
rate of oxygen consumption by cells is described by the specific oxygen uptake
rate QO. Its value depends on the growth conditions, including the carbon source
used to grow the cells. Similar to the specific growth rate, a specific oxygen uptake
rate can be defined:
Qo=qox
Table 2 shows typical (specific) oxygen uptake rates for various cell types. It can
be seen that there is a large variation in the oxygen uptake rates, with animal and
plant cells having a much lower oxygen uptake rate than microorganisms.
Table 2. Typical oxygen uptake rates.
Cell culture Carbon Qo qo qo

source (mmol O2l-1hr-1) (mmol O2 mmol O2 g dry
cell-1 hr-1) weight-1 hr-1)
Escherichia coli Peptone 5 3.2×10-11
(bacteria)
Streptomyces Meat 16 4.1

griseus extract
(Bacteria)
Penicillium Lactose 30 1.2

chrysogenum
(mould)
Saccharomyces Ethanol 40 10
cerevisiae (yeast)
Nicotiana Sucrose 0.9 0.9

tabacum plant
cells
Chinese hamster Glucose/ 0.6 2.9×10-10

ovary animal cells glutamine

Figure 3.5 shows how the specific oxygen uptake rate depends on the dissolved
oxygen concentration. It can be seen that the oxygen uptake remains constant
unless it drops below a critical oxygen concentration. The oxygen uptake rate then
drops fast. Clearly it is important to keep the dissolved oxygen concentration in the
reactor above this level.
qo
Critical oxygen concentration
Dissolved oxygen concentration CA
Figure 3.5, Plot of oxygen consumption in bioreactor.
3.4.1 Oxygen Transfer in Bioreactors
The solubility of oxygen in water is very low. As a result it can be difficult to supply
cell cultures with sufficient oxygen, especially at larger scales; only at small scales
and when growing cells that don’t have a high oxygen demand can one rely on
diffusion to provide sufficient oxygen. Small scale animal cell culture, for example,
can be done by growing the cells on the surface of a dish with a thin layer of (static)
medium on top: only oxygen diffuses in to provide the cells with oxygen. In most
cases, however, more oxygen is needed, and this has to be done by bubbling air
through the medium. In a typical culture a gas flow of 1 volume of air per unit
volume of liquid per minute is used.
The air is sterilised over a filter and the dispersed in the reactor with a sparger -
often simply a metal plate with small holes or a porous frit. The small bubbles
formed may merge and form larger bubbles. A medium that promotes this effect is
called a coalescent medium. Pure water is an example of a coalescent medium.
Other media may prevent coalescence. A medium in which little coalescence
occurs is called a non-coalescent medium. The addition of salts and detergents to
the medium makes a medium more non-coalescent.
If an impeller is present in the reactor then the impeller will redistribute the bubbles
in the reactor. A distinction can be made between a flooded bioreactor in which the
impeller is surrounded by a lot of air and where little dispersion of bubbles occurs
(low stirrer speed/high gas flow rate) and a reactor in which the bubbles are fully
dispersed (see Figure 3.6).

When the impeller is flooded oxygen transfer is poor. Increasing the impeller speed
not only distributes bubbles better over the reactor but also breaks up bubbles into
smaller ones. This increases the air holdup (volume of air in reactor/total volume
of air and liquid and also increases the area of the bubbles available for oxygen
transfer.
Flooded
Flooded Partly dispersed Fully dispersed
Figure 3.6, Gas dispersion in a stirred reactor.
When the air finally emerges from the reactor the air is very humid. Prolonged
sparging of air can lead to the evaporation of large amounts of water so in many
fermenters a condenser is installed to prevent too much water from escaping.
If the oxygen consumption by the cells is too high then there are a number of things
you can do:
• Increase the gas flow (increasing risk of flooding)

• Increase the stirrer speed (however this also increases power
consumption)
• Increase the oxygen concentration in air (pure oxygen contains 5 times
more oxygen than air); however, pure oxygen is expensive.
3.4.2 Oxygen Transfer Rate and the “kla”
The path that oxygen has to take from a bubble to a cells is long and includes
transfer from the bulk of the bubble to the liquid-air interface, transfer across this
interface; transfer across the liquid film around the bubbles; transfer through the
liquid medium to the cells; transfer across the liquid film around cells; and transfer
across the cell-liquid interface. The limiting step is in most cases the transfer across
the liquid film surrounding a bubble. This is due to the low solubility of oxygen in
water.

Mass transfer across films can be described by the film theory. The film theory
leads to the expression:
NA = kla (CA*-CA)
NA is the oxygen transfer rate.

kl is the liquid phase mass transfer coefficient.
a is the area of interface available for mass transfer per volume
CA is the oxygen concentration in the water bulk phase.
CA* is the equilibrium oxygen concentration in the liquid phase, i.e. the
concentration of oxygen in the water phase if there was an equilibrium between the
oxygen concentration in the gas phase and the water phase.
The oxygen transfer rate must be sufficient to provide the cells with the required
oxygen and keep the oxygen concentration in the liquid bulk medium above its
critical value.
3.4.3 Measurement of Oxygen Concentrations
The measurement of the oxygen concentration during fermentation is important. In

most aerobic fermentation it is essential that the oxygen concentration does not fall
below a certain level. Oxygen concentration can be measured in the gas phase or
the liquid phase.
The measurement of the oxygen concentration in the gas phase is usually done
with a paramagnetic oxygen analyser or (preferably) a mass spectrometer.
Changes in the oxygen concentration in the gas phase in small reactors can be
quite small and therefore hard to measure. Carbon dioxide can also be measured
in the gas phase - this is usually done by infrared analysis.
The alternative to the measurement of the oxygen concentration in the gas phase
is to measure the oxygen concentration in the liquid phase. Figure 3.7 shows a
sketch of a dissolved oxygen electrode. The oxygen diffuses across a membrane
to be reduced at a cathode; the oxygen concentration is determined by either the
voltage across the assembly or the current produced by the reduction of the
oxygen. If air is used as the gas flow then calibration of an oxygen sensor is done
by first sparging air into cell-free medium (oxygen tension = 100%) and then N2
(oxygen tension = 0%).
Cathod
Electrolyte
solution
Oxygen transfer
Membrane
Bulk medium
Figure 3.7. An oxygen probe

3.4.4 Measurement of the Oxygen Transfer Rate (kla)
Static method
The static method involves measurement of the oxygen concentration in the inflow
gas stream and the outflow gas stream. The difference in the mass of oxygen
between them is the oxygen consumed in the fermenter- which is the same as the
amount of oxygen transferred. A steady state balance over the gases coming in
and out of the reactor gives
VLNA = FginCAgin - FgoutCAgout
Dynamic method
The dynamic method for measuring the kla involves the following steps:
• Sparge air into a fermenter with a well-calibrated dissolved oxygen sensor
until you reach steady state. At that stage the oxygen transfer into the
medium equals the consumption of oxygen by the cells, and the dissolved
oxygen concentration remains constant at C AL .
• Turn the air supply off, wait a while, and then turn it back on again. The
trace of the oxygen concentration as a function of time should look like
that in Figure 3.8, and slowly get back to its steady state value C AL
C AL
concentration
Air off C AL
Oxygen
Air on
t0 t1 t2 Time
Figure 3.8, Variation of the dissolved oxygen concentration during the dynamic
measurement of kla.
The recovery of the oxygen concentration CA can be described by:
dC AL
= kla (C AL
*
− C AL ) − Qo
dt

Assuming the rate of oxygen consumption Qo is constant throughout and has the
same value as the value at steady state:
dC AL
= kla (C AL
*
− C AL ) − Qo = kla (C AL
*
− C AL ) − kla (C AL
*
− C AL ) = kla (C AL − C AL )
dt
dC AL
= kladt
(C AL − C AL )
CAL 2 t2
dC AL
∫
CAL1 (C AL − C AL )
= ∫ kladt
t1
C AL − C AL1
ln = kla (t 2 − t1 )
(C AL − C AL 2 )
C AL − C AL1
A plot of ln versus t2-t1 gives a line with slope kla.
(C AL − C AL 2 )
C AL − C AL1
ln
(C AL − C AL 2 )
Slope = kla
t2-t1

Tutorial Questions
1. When air is sparged in water at atmospheric pressure the maximum

solubility of oxygen in water is 8 ppm. Air contains about 20% oxygen.
Calculate the oxygen concentration in water (in kg O2/m3) when the sensor
is calibrated with air and the output of a calibrated sensor is 60%. Calculate
the expected concentration if the water is saturated with pure oxygen.
2. We use the static method to determine the kla in a 20 L fermenter.

We sparge air into the fermenter with a pressure of 1 atm and a temperature
of 22 oC. The flowrate of air is 0.23 L s-1 and it contains 21% oxygen. The
exit gas contains 20.1 % oxygen and has a pressure of 1.48 atm and a
temperature of 30oC. The exit flow rate is 0.15 l s-1. R = 0.082057 atm K-1
gmol-1.
a) Calculate Qo
b) The oxygen concentration in the liquid is found to be 82% (when
compared with medium saturated with air at 1 atm pressure). The
solubility of oxygen in water when air is sparged into the medium at
atmospheric pressure is 8 ppm. Estimate kla. MW O2= 32.
3. We use the dynamic method to determine kla. We find:
Time O2 tension
5 sec 50%
20 sec 60%
The steady state concentration of O2 is 78 %. Calculate the kla.

3.5 Scaling Up and Scaling Down

We will first look at general approaches for scaling processes up (or down), and
then look at how we could scale up typical fermenters.
General approaches used for scaling up and down
As getting the scale-up of a process wrong can be an expensive mistake a large

variety of methods have been developed to help with scaling up. Methods for
scaling up and down can be divided into:
• Duplication
• “Real” scale up :
o By use of a microscale model of the process at different scales as

a guide(e.g. by modelling the fluid dynamics in a at different scales
using finite element modelling, starting with the Navier-Stokes
equations)
o By use of a simplified model of the process (e.g. by representing
the dispersion of chemicals in a reactor with a tanks-in-series
model) as a guide in scale-up
o By scaling up by keeping dimensional numbers constant. This
helps to prevent regime changes.
o By the use of rules of thumb
o By trial and error.
Critical when scaling up is an understanding which phenomena occur in a process.

Not all phenomena change at different scales. Many phenomena related to
thermodynamics such as phase equilibria and reaction kinetics are scale
independent, whilst phenomena related with transport processes (momentum, heat
& mass transfer) are often highly dependent on the scale of the process
Dimensional analysis is a particularly useful technique. Dimensional analysis gives

you dimensionless numbers. Dimensionless numbers are useful because they tell
you what phenomena dominate in a system. If we take the Reynolds number as an
ρ𝑢𝑢𝑢𝑢
example, then from fluid dynamics we know that 𝑅𝑅𝑅𝑅 = . Re can be seen as a
µ
ratio of forces, i.e. Re is the ratio of the inertial and the viscous forces in a fluid. If
Re is small the viscous forces dominate, and we have a viscous or laminar flow. If
Re is large inertial forces dominate, and the flow is turbulent. If we have a system
in which Re is important and we calculate Re to be 100,000, then if we scale up
the system and keep Re 100,000 then we can be pretty sure that the flow in the
scaled up system is also turbulent. Thus, by keeping the dimensionless number
constant during scale up we have ensured that no regime change from turbulent
flow to laminar flow has occurred.
A variety of methods have been developed for finding dimensionless numbers.

Some Information about some of the methods can be found in the appendix.

3.5.1 Hierarchy of Scaling
It usually best to do scaling up in the following order:

Geometric similarity:
This involves scaling up the size of all parts of the system geometrically. For
example, if you double the height of the reactor you also double the width. Similarly,
if you double the width of the vessel you also double the diameter of your stirrer.
Mechanical similarity:
Mechanical similarity can be divided into two parts:
• Static similarity
• Kinematic similarity
• Dynamic similarity
Static similarity means that the tension is the same at different scales and therefore
deformation.
Kinematic similarity means both length and time scales are similar. In practice that
means the path / flow pattern is the same.
Dynamic similarity means that you keep the forces in the system the same. This
includes important dimensionless number such as Re.
Thermal similarity
This means that for a geometrically similar system (same shape, proportion etc)
the thermal profiles are the same.
Chemical similarity
This means that for a geometrically similar system the concentration profiles are
the same.
The reasons for having this order should be clear. For example, if we have to scale
up a plane then to have the same flow pattern around the small and large plane
they will have to have the same shape. If we want to have the same profile of
reaction rates in a catalyst particle with a different size then we have to first ensure
that the temperature profile is the same as the reaction rate is dependent on the
temperature. Only then should we worry about concentration profiles.

3.5.2 Scaling Up of Bioreactors
Scaling up and scaling down is often particularly difficult in bioprocessing because

of the non-linear nature of biological materials and the heterogeneous nature of the
materials. Also, because of the very large changes that can occur in biological
systems in length scales (e.g. subcellular materials nm, cells microns, cell
aggregates mm, fermenter meters) and time scales (e.g. mixing seconds, cell
division hours), regime changes occur regularly.
Scaling up bioreactors ensuring geometric similarity
Scaling up ensuring geometric similarity is relatively straightforward- see also the

previous section on bioreactors which tells you the relative sizes of a “standard”
bioreactor.
Scaling up bioreactors ensuring mechanical similarity
We will first assume the reactor is ungassed. Parameters that are relevant to the
mechanical forces in the system include:
• Power input P (units kg/(m2s3))

• Density ρ (units kg/m3)
• Stirrer diameter Di (units m)
• Stirrer speed Ni (units 1/s)
• Dynamic viscosity µ (units kg/(ms))
• Gravitational constant g (units m/s2)
Dimensional analysis of the mechanical forces in the reactor results in the following
expression:
𝛼𝛼
𝑃𝑃 𝜌𝜌𝑁𝑁𝑖𝑖 𝐷𝐷𝑖𝑖2 𝑁𝑁𝑖𝑖 𝐷𝐷𝑖𝑖2 𝛽𝛽
= 𝑐𝑐 � � ( )
𝜌𝜌𝑁𝑁𝑖𝑖3 𝐷𝐷𝑖𝑖5 𝜇𝜇 𝑔𝑔
The first term is the power number Np:

𝑃𝑃
𝑁𝑁𝑝𝑝 =
𝜌𝜌𝑁𝑁𝑖𝑖3 𝐷𝐷𝑖𝑖5
We have seen the second term before as it is the Reynold number.

𝜌𝜌𝑁𝑁𝑖𝑖 𝐷𝐷𝑖𝑖2
𝑅𝑅𝑅𝑅 =
𝜇𝜇
The Reynolds number tells you whether the flow is laminar or turbulent.
The third is the Froude number Fr, and this gives you of the ratio between inertial
and gravitational forces.
𝐹𝐹𝐹𝐹 =
𝑔𝑔
If mixing is relatively strong then the dependency on Fr is small and the power
number mainly depends on Re.

Figure 3.9 shows the plot of Np versus Re:
100
10
Rushton
Np turbine
1 Pitched blade
turbine
Marine
propeller
0.1
1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06
Re
Figure 3.9, Plot of power number Np vs. Reynolds number Re.
At low Re we get laminar flow around the stirrer and the value of Np depends on
Re. At high Re, however, the flow around the stirrer is turbulent, and Np is
independent of Re.
If we increase the reactor size geometrically then the impeller diameter Di

increases geometrically with it. If we are already in the turbulent region (which we
want to be as it improves mixing) then increasing Re by increasing Di has no effect
on Np.- Np is a constant.
𝑃𝑃
If Np is a constant then 𝑁𝑁𝑝𝑝 = 3 𝐷𝐷5 is a constant. Di is determined by the size of the
𝜌𝜌𝑁𝑁𝑖𝑖 𝑖𝑖
reactor so what about Ni and P? To investigate the effect of changing Ni, we will
look at the mixing time.
To determine what determines the mixing time in a stirred reactor we can make the
following analysis:
The pumping capacity φp of a stirrer, i.e. the amount of fluid that is moved by the
stirrer whilst it rotates through the fluid, is proportional to the tip speed (which is
proportional to NiDi) multiplied with the area that the stirrer covers whilst moving
through the fluid (which is proportional to Di2 ).
φp ∝ NiDi3
The time tc it takes to distribute this amount of fluid over the tank is proportional to
the volume of the tank and inversely proportional to the pumping capacity:
tc ∝ V/φp = Di3/NiDi3 = 1/Ni
Just distributing it once is not enough. – going around 4 times may be, so tm = 4tc.
Either way, we find that tm ∝ 1/Ni In other words, to keep the mixing time constant
at different scales we need to keep Ni constant. However, if the power number Np

is constant and the stirrer speed Ni is constant and Di increases then we have a
problem because the power P we have to supply is given by:
𝑃𝑃 = 𝑁𝑁𝑝𝑝 𝜌𝜌𝑁𝑁𝑖𝑖3 𝐷𝐷𝑖𝑖5
The power we need to supply increases by 𝐷𝐷𝑖𝑖5 – even a moderate scale-up leads
to a large increase in power use.
Scaling up bioreactors with regard to mechanical forces - the compromise
We would like to scale up a bioreactor by keeping the dimensionless numbers

constant as this would allow us to prevent regime changes whilst scaling up.
However, we when we tried to do this we saw that scaling up on the basis of the
dimensionless Reynolds number Re /dimensionless Power number Np or the
dimensionless mixing time was not possible as the power needed was too high.
Clearly, we have a problem with power. This is simply because power is dependent
on Di5, and this makes power consumption rise very fast when Di is increased. A
compromise is needed.
Commonly used compromises are:
• Scale up with constant P/V

• Scale up with constant Vtip (= constant NDi as vtip = πNDi)
𝑃𝑃 𝑁𝑁p ρ𝑁𝑁i3 𝐷𝐷i5 𝑁𝑁p ρ𝑁𝑁i3 𝐷𝐷i2

P/V = constant means = 𝑘𝑘𝐷𝐷i3
= = constant as Vtank ∝ Di3
𝑉𝑉 𝑘𝑘
A change in Di has much less effect on P/V than on P.
Scaling up by keeping P/V constant is typically done where mixing is an issue.

Scaling up by keeping Vtip constant is done on the other hand when shear stress is
important. This is because the highest shear rates (and therefore shear stresses)
in a stirred system occur at the tip of the impeller, and this highest shear rate is
proportional to the tip speed. The importance of shear depends on the cell type you
are looking at. Table 1 shows that in general:
Table 3, cell types and shear sensitivity.
Cell type Size Wall Shear sensitivity

Microbial cells such as 1-10 micron Yes Little
yeasts and bacteria
Filamentous microbes ~100 Yes Moderate
micron
Microbial pellets 1-5 mm Yes Moderate
Plant cells ~50 micron Yes Moderate to high
Animal cells 10-20 no High
micron
Scaling up on the basis of P/V constant is therefore a good choice for single celled
microbes, whilst for animal cells it would be vtip = constant.

Scaling up bioreactors ensuring thermal similarity
As the temperatures at which bioprocess occur are moderate, and little heat is
produced, ensuring thermal similarity when scaling up is usually not a problem.
Scaling up bioreactors ensuring chemical similarity
The issues related to chemical similarity include:
• Mixing
• Oxygen transfer
• Diffusion (in immobilised cell systems)
We already discussed mixing when discussing keeping mechanical similarity. We

found that keeping the (dimensionless) mixing time constant was not doable as the
required power was too high – the compromise solution was to keep P/V constant.
Keeping similarity in oxygen transfer can be looked at from different angles. The
first issue that needs to be looked at is the dispersion of bubbles. When scaling up
an aerated bioreactor it is important to ensure that the gas flow is fully dispersed.
Dimensional analysis of gas flows in reactors tells you that the following
dimensionless numbers are important for bubble dispersion:
• The gas flow number FLg

• The Froude number Fr
𝐹𝐹𝑔𝑔
𝐹𝐹𝐹𝐹𝑔𝑔 =
and
𝑁𝑁𝑖𝑖2 𝐷𝐷𝑖𝑖
𝐹𝐹𝐹𝐹 =
𝑔𝑔
Froude we have seen before. The gas flow number tells you the ratio of the gas
flow and the flow coming from the stirrer.
When the stirrer doesn’t disperse the gas it is said to be flooded. When it does it is
loaded.
When using a Rushton turbine in a standard reactor the transition between flooding
𝐷𝐷 3.5
and loading occurs when. 𝐹𝐹𝐹𝐹𝑔𝑔 = 30 � 𝑖𝑖 � 𝐹𝐹𝐹𝐹
𝐷𝐷𝑇𝑇
Any value of Flg larger than this gives flooding.
The fact that the gas is being dispersed by the stirrer doesn’t mean it is fully
dispersed. When using a Rushton turbine in standard reactor, full dispersion occurs
when:
𝐷𝐷𝑖𝑖 0.5
𝐹𝐹𝐹𝐹𝑔𝑔 ≤ 0.2 � � 𝐹𝐹𝐹𝐹 0.5
𝐷𝐷𝑇𝑇
A value of Flg higher than this leads to gas not being fully dispersed.

Having ensured that the bubbles are fully dispersed in the reactor, and therefore
oxygen transfer occurs all across the reactor, we can address the issue of actually
getting the oxygen to the cells. One option is to scale up ensuring the oxygen
concentration in the medium stays constant- this is relatively easy to implement,
for example by measuring the oxygen concentration in the medium and using this
measurement to control the gas flow rate. Another option is to ensure the kla stays
constant during scale-up.
A useful correlation for keeping kla constant is:
𝑃𝑃𝑇𝑇 𝛽𝛽
𝑘𝑘𝑘𝑘𝑘𝑘 = 𝐴𝐴( )𝛼𝛼 𝑢𝑢𝑔𝑔
𝑉𝑉𝐿𝐿
PT is the total power input- by the stirrer as well as the gas. The power input by the
stirred can be estimated using Np if the gas flow is low; that by gas from Pgas =
FgρgHL
HL is the liquid height. VL is the liquid volume.
The kla is dependent on both the bubble size and the number of bubbles/gas
holdup. Bubble size is determined by a large number of factors, including medium
properties (e.g. whether coalescence occurs) and the energy input by the stirrer.
Medium properties we can’t do much about- and energy input by the stirrer, as we
have seen before, is best determined by P/V or vtip. Constant gas holdup would
therefore be a good criterion for scale up. In practice instead of gas holdup the
volumetric gas flow rate is kept constant, i.e. Fg/VT. A typical value of Fg/VT is 1 vvm
– 1 volume of gas per unit volume of tank per minute.
Summary
Scale up of bioreactors is complex, but as rules of thumb one could use:
• Scale up geometrically first
• Scale up power on basis of P/V or vtip, the former particularly when mixing
is important and cells are reasonably shear resistant, the latter when cells
are shear sensitive.
• Where oxygen transfer is important try to keep the oxygen concentration

in the medium/kla constant.
• Keeping Fg/V constant can help with this.

Appendix 1. Determination of Dimensionless Numbers

A variety of methods have been developed for finding dimensionless numbers.
They include the use of ODEs, the Gauss- Jordan reduction methods, and the
methods of Rayleigh and Gukhman. I’ll discuss two:
Method 1. From an ODE
The determination which dimensionless numbers describe a process is relatively

easy if you already have a model of the process. For example, we have a process
in which a chemical A is converted by a first order reaction.
A mass balance over the reactor gives:
Accumulation = In – Out + Production - Consumption

𝑑𝑑(𝑉𝑉𝑉𝑉)
= 𝐹𝐹𝐶𝐶𝑖𝑖𝑖𝑖 − 𝐹𝐹𝐹𝐹 + 0 − 𝑉𝑉𝑉𝑉𝑉𝑉
𝑑𝑑𝑑𝑑
Rearranging gives:
𝐶𝐶
𝑉𝑉 𝑑𝑑(𝐶𝐶𝑖𝑖𝑖𝑖 ) 𝑉𝑉𝑉𝑉 𝐶𝐶
= 1 − 𝐶𝐶/𝐶𝐶𝑖𝑖𝑖𝑖 −
𝐹𝐹 𝑑𝑑𝑑𝑑 𝐹𝐹 𝐶𝐶𝑖𝑖𝑖𝑖
C/Cin is the dimensionless concentration

𝑉𝑉𝑉𝑉
is the Damköhler number.
𝐹𝐹
The Damköhler number is the ratio of the reaction time and the residence time. It
tells you whether the reaction has fully completed when the fluid emerges from the
outlet (large Damköhler number) or only partly (low Damköhler number).
Method 2. Method of Rayleigh
When using the Method of Rayleigh for finding dimensionless numbers is to write
down a list of the parameters that you think are important for describing the
process.
For example, when scaling up the flow in a pipe, and investigating what determines
the pressure drop in the pipe, we can assume that the following parameters are
important:
Parameter Unit
∆P Kg/(ms2)
ρ Kg/m3
µ Kg/(ms)
L m
D m
U m/s
We can write:
∆𝑃𝑃 = 𝜌𝜌𝛼𝛼 𝜇𝜇 𝛽𝛽 𝐿𝐿𝛾𝛾 𝐷𝐷 𝛿𝛿 𝑢𝑢𝜀𝜀
Units:
𝑘𝑘𝑘𝑘 𝑘𝑘𝑘𝑘 𝑘𝑘𝑘𝑘 𝑚𝑚
2
= ( 3 )𝛼𝛼 ( )𝛽𝛽 𝑚𝑚𝛾𝛾 𝑚𝑚𝛿𝛿 ( )𝜀𝜀
𝑚𝑚𝑠𝑠 𝑚𝑚 𝑚𝑚𝑚𝑚 𝑠𝑠

To keep the dimensions correct:

Mass: 1=α+β α = 1- β
Time: -2 = -β - ε ε=2-β
Length: -1 = -3α - β + γ + δ + ε
-1 = -3(1-β) - β + γ + δ + 2-β
0 = β+γ+δ
δ = -β - γ
µ β 𝐿𝐿 𝛾𝛾
∆𝑃𝑃 = ρ1−β µ𝛽𝛽 𝐿𝐿𝛾𝛾 𝐷𝐷 −β−γ 𝑢𝑢2−β = ρ𝑢𝑢2 � � ( )
ρ𝑢𝑢𝑢𝑢 𝐷𝐷
∆𝑃𝑃 µ β 𝐿𝐿 𝛾𝛾
( 2) = � � ( )
ρ𝑢𝑢 ρ𝑢𝑢𝑢𝑢 𝐷𝐷
So the dimensionless numbers are:

∆𝑃𝑃 µ 𝐿𝐿
: the Euler number =1/Re : the Reynolds number : the
𝜌𝜌𝑢𝑢2 ρ𝑢𝑢𝑢𝑢 𝐷𝐷
dimensionless length

List of dimensionless numbers
Standard
Name Definition Field of application
symbol
Archimedes fluid mechanics (motion of fluids due to

Ar
number density differences)
Arrhenius chemistry (ratio of activation energy to

number thermal energy)
fluid mechanics (onset of instabilities in

Atwood
A fluid mixtures due to density
number
differences)
Bejan number
fluid mechanics (dimensionless
(fluid Be
pressure drop along a channel)
mechanics)
Bejan number thermodynamics (ratio of heat transfer

(thermodyna- Be irreversibility to total irreversibility due to
mics) heat transfer and fluid friction)
Bingham fluid mechanics, rheology (ratio of yield

Bm
number stress to viscous stress)
heat transfer (surface vs. volume

Biot number Bi
conductivity of solids)
chemistry (residence-time distribution;

Bodenstein
Bo or Bd similar to the axial mass transfer Peclet
number
number)
geology, fluid mechanics, porous media

Bond number Bo (buoyant versus capillary forces, similar
to the Eötvös number)
heat transfer, fluid mechanics

Brinkman
Br (conduction from a wall to a viscous
number
fluid)

Standard
symbol
Capillary porous media, fluid mechanics (viscous

Ca
number forces versus surface tension)
Damkohler chemistry (reaction time scales vs.

Da
number residence time)
porous media (ratio of permeability to

Darcy number Da
cross-sectional area)
Dean number D turbulent flow (vortices in curved ducts)
Deborah
De rheology (viscoelastic fluids)
number
colloid science (ratio of electric surface

Dukhin
Du conductivity to the electric bulk
number
conductivity in heterogeneous systems)
convective heat transfer (characterizes

Eckert number Ec dissipation of energy; ratio of kinetic
energy to enthalpy)
Eötvös fluid mechanics (shape of bubbles or

Eo
number drops)
Ericksen fluid dynamics (liquid crystal flow

Er
number behaviour; viscous over elastic forces)
hydrodynamics (stream pressure

Euler number Eu
versus inertia forces)
Fourier heat transfer, mass transfer (ratio of

Fo
number diffusive rate versus storage rate)

Standard
symbol
fluid mechanics (wave and surface

Froude
Fr behaviour; ratio of a body's inertia to
number
gravitational forces)
Galileo fluid mechanics (gravitational over

Ga
number viscous forces)
heat transfer, fluid mechanics (laminar

Graetz
Gz flow through a conduit; also used in
number
mass transfer)
Grashof heat transfer, natural convection (ratio

Gr
number of the buoyancy to viscous force)
chemical engineering (adsorption

Hatta number Ha
enhancement due to chemical reaction)
heat transfer (ratio of the buoyancy to

Hagen number Hg
viscous force in forced convection)
chemistry (ratio of sensible to latent

Jakob number Ja energy absorbed during liquid-vapor
phase change)
gas dynamics (ratio of the molecular

Knudsen
Kn mean free path length to a
number
representative physical length scale)
fluid dynamics (free convection within

Laplace
La immiscible fluids; ratio of surface
number
tension to momentum-transport)
Lagrange ∆𝑃𝑃𝑃𝑃 Fluid dynamics (ratio of pressure and

Lg 𝐿𝐿𝑔𝑔 =
number 𝑣𝑣μ viscous forces)
heat and mass transfer (ratio of thermal

Lewis number Le
to mass diffusivity)

Standard
symbol
gas dynamics (compressible flow;

Mach number M or Ma
dimensionless velocity)
fluid mechanics (Marangoni flow;

Marangoni
Mg thermal surface tension forces over
number
viscous forces)
Morton fluid dynamics (determination of

Mo
number bubble/drop shape)
Power number Np 𝑁𝑁𝑝𝑝 = 𝑃𝑃/(ρ𝑁𝑁 3 𝐷𝐷 5 ) Fluid dynamics (power)
Nusselt heat transfer (forced convection; ratio of

Nu
number convective to conductive heat transfer)
heat transfer (advection–diffusion

Péclet number Pe problems; total momentum transfer to
molecular heat transfer)
Prandtl heat transfer (ratio of viscous diffusion

Pr
number rate over thermal diffusion rate)
reaction engineering (ratio of heat

Prater number β evolution to heat conduction within a
catalyst pellet)
Rayleigh heat transfer (buoyancy versus viscous

Ra
number forces in free convection)
Reynolds fluid mechanics (ratio of fluid inertial and

Re
number viscous forces)
fluid dynamics (effect of buoyancy on

Richardson
Ri flow stability; ratio of potential over
number
kinetic energy)

Standard
symbol
Roshko fluid dynamics (oscillating flow, vortex

Ro
number shedding)
Schmidt mass transfer (viscous over molecular

Sc
number diffusion rate)
mass transfer (forced convection; ratio

Sherwood
Sh of convective to diffusive mass
number
transport)
Stanton heat transfer and fluid dynamics (forced

St
number convection)
phase change, thermodynamics (ratio

Stefan number Ste
of sensible heat to latent heat)
particles suspensions (ratio of

Stokes
Stk or Sk characteristic time of particle to time of
number
flow)
fluid dynamics (continuous and

Strouhal
St or Sr pulsating flow; nondimensional
number
frequency)
fluid dynamics (rotating fluid flows;

Taylor number Ta inertial forces due to rotation of a fluid
versus viscous forces)
multiphase flow (strongly curved

Weber number We surfaces; ratio of inertia to surface
tension)
Weissenberg viscoelastic flows (shear rate times the

Wi
number relaxation time)

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Topic 3

3.1 Bioreactors for Cell Culture 61

Appendix 1: Determination of Dimensionless Numbers 83

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

By the end of this topic, you should be able to:

• Suggest a suitable reactor system for a given biological agent.

• Explain the application of different bioreactors.

• Determine the oxygen transfer in bioreactors.

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

3.1 Bioreactors for Cell Culture

• They have to provide an environment in which cells are active; in most

A variety of other factors may also be important, e.g. no foaming.

Industrial scale cell culture is typically performed in suspension culture. Such

Some production processes use immobilised cells. Actual culture of immobilised

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

The impeller used in stirred tank reactors is typically a Rushton turbine or a

Mixing in fermenters involves three consecutive steps:

• Distribution over the vessel;

• Dispersion of the material by eddies formed by the impeller;

• Homogeneity at the smallest scale (smaller than the smallest eddy) is

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

Suspension of solids in stirred tanks: the Zwietering equation

The Zwietering equation is:

S ν L d 0.2 [ g∆ρ / ρ L ]0.45 X 0.13

Nmin is minimum stirrer speed

νL is the liquid kinetic viscosity

d is the particle diameter

g is the gravitational constant

∆ρ is the difference in the density of the particle and the fluid

ρ L is the density of the liquid

X is the weight percentage of particles

Table 1. Values of S for a selection of impellers.

Impeller Dimpeller/Dtank Himpeller/Dtank S

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

Air driven fermenters

Typical air-driven fermenters are shown in Figure 3.4:

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

3.3 Oxygen Transfer

i) Transfer of oxygen from bulk of bubble to gas-liquid interface

ii) Movement across gas –liquid interface

iii) Diffusion through stagnant liquid film surrounding the bubble

iv) Transport through bulk liquid

v) Diffusion through stagnant liquid film surrounding cell or multicellular

vi) Movement across interface of a cell or a multicellular particle

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

Hence at steady state:

Bubbles can coalesce (= combine to former larger bubbles) once released;

𝑘𝑘𝐿𝐿 𝑎𝑎 = 2.0 × 10−3 (𝑃𝑃/𝑉𝑉)0.7 𝑢𝑢𝐺𝐺0.2

For coalescing systems:

𝑘𝑘𝐿𝐿 𝑎𝑎 = 2.6 × 10−2 (𝑃𝑃/𝑉𝑉)0.4 𝑢𝑢𝐺𝐺0.5

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

where ρ is the fluid density.

The Kolmogorov scale can be described by:

2. The gas holdup ε is the volume of gas / total volume.

©HERIOT-W ATT UNIVERSITY B40DD Oct 2016 v1

3.4 Oxygen Consumption in Bioreactors

Table 2. Typical oxygen uptake rates.

Cell culture Carbon Qo qo qo

Streptomyces Meat 16 4.1

Penicillium Lactose 30 1.2

Nicotiana Sucrose 0.9 0.9

Chinese hamster Glucose/ 0.6 2.9×10-10