1

Agitation and Aeration

in Submerged Culture
Agitation in Fermentation
Agitation provides
Improved mixing
Heat transfer
Mass transfer
Adverse effects of agitation
Rupture of cell walls
Change of morphology of filamentous MOs
Variation in the efficient of growth & rate of growth
Variation in the rate of product formation
Power consumption
Power consumption in agitation is a function of
physical properties, operating condition and vessel and
impeller geometry
The power consumption P ( in mechanical agitation)
can be calculated by
P = 2 r N F
Power number (N
P
) is the ration of drag force on the
impeller to inertial force is given by dimensional
analysis:
N
P
=
N - agitation speed
r - distance from the axis to the
point of the force measurement
Shear Sensitive Mixing
Mechanically agitated fermenter is effective in
mixing of fermenter contents
suspension of cells
breakup of air bubbles for enhanced oxygenation
prevention of forming large cell aggregates
Shear generated by the agitator can disrupt the cell
membrane and eventually kill some microorganisms,
animal and plant cells
Shear is responsible for the deactivation of enzymes
For optimum operation of an agitated fermentation
system, understanding of hydrodynamics involved in
shear sensitive mixing is important
Aeration
Supply of molecular O
2
to the surface of each microbial
cell is of primary importance in aerobic fermentation
MOs utilize O
2
for
growth
product formation
oxidation of substances
provision of energy through respiration
Oxygen is poorly soluble in water
The solubility of oxygen at 4
o
C in pure water is only 8
mg l
-1
Glucose is 6000 times more soluble in water than O
2
The solubility of oxygen decreases with increasing
temperature and increasing concentration of solutes in
the solution
O
2
requirement of cells (depends on type of MO) is on
the order of 1 g/L h.
Dissolved Oxygen (DO) in a fully saturated fermentation
medium is sufficient only for a less than a minute
Hence continuous supply of O
2
is required
O
2
supply to culture is generally by sterile air (20.8%O
2
)
In certain cases, O
2
, is mixed with air to increase the
solubility
Need appropriately designed gas mixing device
Mixing is required to disperse air bubbles and to enhance
O
2
transfer in the fermentation broth
2
Oxygen Demand
Depend on,
Type of culture
Age of culture
eg. Log phase cultures demand more O
2
than lag or
stationary phase cultures
Nature of end product
eg. High O
2
level is required for gluconic acid and
citric acid fermentation
Step 1 - Diffusion through the bubble to the gas-liquid interface
Step 2 - Diffusion across the gas-liquid interface
Step 3 - Diffusion through the bubble boundary layer
Step 4 - Movement through the bulk liquid by forced convection and diffusion
Step 5-9: Movement through the floc
Step 2 - Diffusion across the gas-liquid interface
Movement of oxygen from the gas phase to the
liquid phase
Magnitudes of individual resistances depend on bubble and liquid
phase hydrodynamics, composition and rheological properties,
density and activity cells and gas liquid interfacial phenomena
Major goals in aerobic fermenter design is to reduce O
2
transfer
resistances
Sum of all resistances (gas film, liquid film and
interfacial resistances) = overall resistance to transfer of O
2
into the cell
Resistances to oxygen transfer Oxygen Transfer Rate
Assuming O
2
transfer from gas to liquid phase is controlled
by the liquid film resistance around air bubbles oxygen
transfer rate (OTR) is expressed as
dC
L
/dt = OTR = k
L
a ( C
*
- C
L
)
k
L
= oxygen transfer coefficient
a = gas - liquid interfacial area per unit liquid volume
C
*
& C
L
= saturation and actual DO conc. in the liquid
OTR depends on
Oxygen transfer coefficient (k
L
)
Interfacial area (a)
Concentration driving force (C
*
-C
L
)
Oxygen Transfer Coefficient - k
L
The mass transfer coefficient (k
L
) represents the rate at
which oxygen molecules move through the boundary
layer to the bulk liquid
k
L
increases with increasing turbulence
k
L
can be increased by
Reducing stagnant film thickness (reduce the thickness of
the boundary layer)
increasing the rate at which molecules travel through the
boundary layer
increase rate of surface renewal
Mass transfer coefficient is also affected by interfacial
phenomena
k
L
is a function of physical properties and the vessel
geometry
Hydro-dynamics in multiphase mixing (fermentation
broth) is complex
Hence difficult/impossible to derive a theoretical
correlation for k
L
So empirical correlations are obtained by fitting
experiment data
These correlations are expressed by dimensionless
groups
3
Interfacial area - a
The interfacial area is determined by the
aeration rate
bubble diameter
The smaller the bubble size the larger the interfacial
area
Small bubbles rise slowly through the liquid and so
spend longer time in contact with the liquid - enhances
mass transfer
However slow rise bubbles create less turbulence which
in turn reduce k
L
and mass transfer
Variables that influence interfacial area are sparger
design, fermenter volume, airflow rate and bubble size
Concentration driving force ( C* - C
L
)
C* is the concentration of oxygen in gas liquid interface which is
equal to the saturation concentration of oxygen in the reactor
The value of C* is determined by the partial pressure of oxygen
in the gas phase (Po
2
), on Henry's Law and on the medium's
Henry's constant for oxygen (Ho)
Relationship between gas partial pressure (P
O2
) and liquid-phase
oxygen conc. (C
*
) obeys Henry's law,
P
O2
= Ho C
*
H - Henry's law constant specific to a given gas component and liquid phase and
temperature
C
*
- is a function P
O2
which also depends on the fraction of O
2
in the gas phase
and its total pressure
Therefore, OTR increases with pressure
The value of a medium's Henry's constant for oxygen is
dependent on the concentration of salts and sugars and on
temperature
Eg: Henry's constant for water at 25
o
C and 35
o
C is:
Henry's constant increases with temperature and thus the
solubility of oxygen decreases with temperature
Temperature
o
C Henry's constant (atm.mg
-1
.l)
25 0.0258
35 0.0299
Why k
L
a
It is difficult to measure both k
L
and a in a
fermentation
The two terms are combined in the term k
L
a
and termed volumetric transfer coefficient
(VTC)
Higher the value of k
L
a higher the aeration rate
k
L
a = f ( , D, , etc.)
Factors affecting k
L
a in fermentations
Air flow rate
Degree of agitation
Medium Composition
Effect of biomass
Presence of anti-foam agents
Rheological properties of the broth
Air flow rate
Air flow rate : 0.5 1.5 vvm
If high air flow rates are used OTR falls rapidly due to
impellers becoming flooded
4
Degree of agitation
Agitation assists oxygen transfer in different ways
Disperse air bubbles/break in to small bubbles
Delays the escape of air bubbles from the liquid
Prevents coalescence of air bubbles
Decreases the size of the boundary layer
Degree of agitation may be measured by the amount of
power consumption
k
L
a and Power Consumption
k
L
a Gassed power consumption for unit volume is
affected by large number of variables
Impeller speed
No of impellers
Impeller size
Culture rheology
Superficial air velocity
Gassed power consumption per unit volume
k
L
a (P
g
/V)
k
L
a and Power Consumption
Gassed power consumption per unit volume
k
L
a = k (P
g
/V)
x
V
s
y
P
g
power absorption in an aerated system
V liquid volume
V
s
Superficial air velocity
k, x, y empirical factors specific to the system under
investigation. x depends on the size/scale of the
fermentation (laboratory, pilot or production)
eg: k
L
a = k (P
g
/V)
0.95
V
s
0.67
cooper et. al. (1944)
One impeller
Sulphite oxidation technique
Influence of medium composition
Inorganic Electrolytes
Inorganic salts as solutes increase the surface tension of pure
water
These solutes make the whole solution, interior and surface,
very much more cohesive or strongly bound together
Increase K
L
a
Alcohol
Addition alcohol to water reduces surface tension.
Cause increase in surface area and a corresponding decrease in
average bubble diameter.
Thus, increase in K
L
a
Antifoam Agent
All antifoams are surfactants and they decrease the
surface tension of pure liquid by adsorbing strongly at
relatively low bulk concentration
Antifoam agents which accelerate the thinning promote
bubble coalescence
Addition of the antifoam agent causes the bubble size
to grow and the interfacial area is reduced
Many antifoams which are necessary to control foaming
in fermentation reduce OTR by as much as 50%
Therefore antifoam additions must be kept to an
absolute minimum
Effect of the addition of antifoam on bubble size in a
bubble column
With no antifoam added With antifoam added
5
Rheological properties of the broth
Fermentation broth consists of
Liquid medium
Microbial mass
Product produced
Air bubbles
Rheology of the broth is affected by
Composition of the medium
Concentration of biomass
Morphology of biomass
Concentration of the product
Rheology of the broth has a great influence on k
L
a and
agitation
Problems with O
2
transfer from gas to liquid
Coalescence of air bubble
Mycelia may form agglomerates
Mixing problems
Medium
containing starch
Viscosity
increases
Degrade starch
Viscosity
decreases
Development of mycelia
MO growth
Depending on rheological
properties of the broth
agitation can increase or
decrease viscosity and then
k
L
a
k
L
a 1/viscosity
Buckland et. al. (1988)
k
L
a decreases proportionately
with the square root of broth
viscosity
Viscosity of the Newtonian broth does not vary with
agitation but Non-Newtonian does
Microbial biomass
Biomass concentration and its morphology has a
profound effect on fermentation
Most bacterial and yeast fermentations give rise
to non- viscous Newtonian broths turbulent
flow can be achieved
Fungal fermentations become highly viscous and
non Newtonian broths
Method to increase OTR in fermenter
Increase total pressure
Enrich air with pure O
2
Use pure O
2
Only low flow rates is required to meet demand
Will not strip CO
2
and other toxic gasses
Unable to maintain positive pressure
Oxygen free radical formation- toxic to
essential enzymes/MOs
High Cost of production
Important
Excess aeration cause problems such as excessive foam
and high N
2
N
2
has sweeping effect in moving CO
2
N content of air for aeration is enhanced removal of
gases and inhibitory metabolic products, esp. CO
2
from
culture
Addition of antifoaming agents may be necessary
6
Oxygen as a growth limiting nutrient
DO is an important substrate in aerobic fermentations
and may be a limiting substrate
Oxygen transfer problem arises at high cell densities -
growth is limited by O
2
availability in liquid phase
DOC can affect the specific growth rate
Therefore, oxygen concentration should be maintained
at a value above C
crit
Effect of Dissolved Oxygen (DO) on specific oxygen
uptake rate (QO
2
) follows Michaelis-Menten type
relationship
QO
2
increases with increasing DO with a first order
relationship up to C
crit
No further increase in oxygen uptake rate occurs above
C
crit
DO must be maintained > C
crit
in order to obtain
maximum growth/biomass production
Above this value is independent of DO
Even temporary depletion of dissolved oxygen below C
crit
may cause irreversible cell damage
O
2
only slightly soluble - needs to be supplied continuously
to culture to ensure that oxygen absorption rate oxygen
consumption rate
In cultures with limiting substrate, Monod equation can be
used to expressed the specific growth rate ( ) of
microorganisms;
=
max
(C
S
/ K
S
+ C
S
)
- specific growth rate
K
S
- saturation constant
C
S
- substrate concentration
So for oxygen limitation
=
max
C
L
/(K
0
+C
L
)
For bacteria, yeast and fungi, C
crit
ranges from 0.0003 to 0.05 mMol/L
depending on the form of growth and on the extent of pellet or
aggregate formation
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12
CL
Q
Q
max
C
crit
Q
max/2
The dissolved oxygen concentration in a reactor is thus
determined by the balance between the oxygen transfer
rate (OTR) and oxygen uptake rate (OUR)
dC
L
/dt = OTR - OUR
x 2 L
*
L
L
.C QO - ) C a(C k
dt
dC
x
L o
L m
x/o
L
*
L
L
C .
C K
.C
Y
1
- ) C a(C k
dt
dC
7
When oxygen transfer is the rate limiting step, assuming energy
requirement for cell maintenance is negligible
OUR = OTR
QO
2
= K
L
a (C
*
- C
L
) (1)
Y
x/O
= X/O = (dCx/dt)/(dO
2
/dt)
dO
2
/dt = (dCx/dt)/ Y
x/O
= . C
x
/Y
x/O
. C
x
/Y
x/O
= K
L
a (C
*
- C
L
)
dC
x
/dt = Y
x/O
. K
L
a (C
*
- C
L
)
Q
max
= maximum specific O
2
uptake rate (specific O
2
demand by microorganism)
The value is constant for growth of MO in a well
defined environment.
Value of Q
max
for some microorganisms
A. niger - 30 mMole/g cell/hr
P. chrysogenum - 3.9 mMoles/g cells/hr
E. coli - 10.8 mMoles/g cells/hr
O
2
demand for hydrocarbon is 2.5 higher than
carbohydrate
Simple sugar easy to metabolize High O
2
demand
Complex sugar difficult to metabolize low O
2
demand
Q
max
for Penicillium spp. grown on different sugars;
Glucose - 13.4 mMol/L.h
Sucrose - 6.7 mMol/L.h
Lactose - 4.9 mMol/L.h
Aerobic Fermenter Design
Aerobic fermenter must has adequate aeration and
agitation to meet the metabolic requirements of mo.
The mixing should not cause damage to MO and power
consumption should be as low as possible
Most aerobic fermenters employ spargers underneath an
impeller and mechanical agitation to reduce air bubble
size and to increase gas liquid contact area
Non-mechanical agitated fermenter (e.g. bubble column
and air lift fermenters) have advantage of lower
equipment and power cost. May not be efficient in
viscous fermentation broths such as fungal and
actinomycete fermentations
Mechanical agitation (STR) - enhances heat transfer,
reduces inhomogenity within the liquid and aids
suspension of solid particles
In STR, large diameter and low speed impeller with a
small number of blades is required for mixing while high
speed and small diameter impeller with many lades is
required to increase interfacial area
Non-agitated fermentations are normally carried out in
tall vessel where aeration is sufficient to produce high
turbulence but greater energy input in the production of
compressed air is required
Another consideration in determining choice - many
industries need to process different products in same
plant - flexible systems that can be modified is favoured
Measurement of k
L
a
Sodium Sulfite Oxidation Method
Dynamic Gassing-out methods
Static Gassing-out method
Dynamic Gassing-out method
Oxygen balancing method
8
Fermenter scale up
Scale up should be done with out a decrease in yield
Model and prototype should be similar to each other
Factors involved in scale up
Inoculum development
Sterilization
Environmental conditions
Nutrients
pH
Temperature
DO
Shear
Foam
Affected by aeration and
agitation
Conditions that should be satisfied
1. Geometric similarity of the physical boundaries:
The model and the prototype must be the same
shape
All linear dimensions of the model must be related
to the corresponding dimensions of the prototype by
a constant scale factor
Theoretically scale-up is done based on geometric and
dynamic similarity of the two systems
2. Dynamic similarity of the flow fields:
Ratio of flow velocities of corresponding fluid particles
is the same in model and prototype
Ratio of all forces acting on corresponding fluid
particles is the same in model and prototype
forces act on a fluid element in a fermenter during agitation are:
viscosity force F
V
drag force on impeller F
D
gravity force F
G
When dynamic similarity of two flow fields with
geometrically similar boundaries is achieved, the flow
fields exhibit geometrically similar flow patterns
According to Newtons equation of viscosity
Which can be assumed to be
Further and
So,
Dynamic similarity between model (m) and prototype (p)
is achieved when
or when
Further Reynolds number
and similarly
Therefore dynamic similarity when the values of non-
dimensional parameter are the same at geometrically
similar locations
However, it is difficult to achieve this similarity when
more than one dimensionless groups are involved and
hence scale up criteria should be applied
9
Scale-up Criteria
Power consumption per unit volume P
mo
/v is employed as
a criterion for scale-up
Since liquid volume is proportional to D
I
3
for geometrically
similar systems,
P
mo
/D
I
3
- Power per unit volume
For constant P
mo
/D
I
3
Impeller tip speed
Reynolds Number and
Model
(20L)
Prototype
(2500 L)
P
o
/v 1 1
D
I
1 5
N 1 0.34
N
Re
1 8.5