CBE 320b BIOCHEMICAL

ENGINEERING III COURSE NOTES

Instructor: Dr. A. Margaritis, Ph.D., P.Eng., F.C.I.C.
Professor of Biochemical Engineering

http://www.eng.uwo.ca/people/amargaritis/

DEPARTMENT OF CHEMICAL AND BIOCHEMICAL
ENGINEERING

The University of Western Ontario
Faculty of Engineering
A. Margaritis 2006-2007


TABLE OF CONTENTS
1. Introduction
Bioprocess Design
Novel Bioreactor Types
Design Criteria for Bioreactors
2. Aeration and Oxygen Mass
Transfer in Bioreactor
Systems
Oxygen Requirements by Microorganisms
The volumetric Mass Transfer Coefficient K
L
a
and Methods of Measurements
Empirical Correlations of K
L
a


3. Agitation of Bioreactor
Systems
4. Scale-up of Bioreactor
Systems
Scale-up Criteria
Example of Geometric Scale-up
5. Sterilization of Liquid Media
Kinetics of Thermal Death of Microorganisms
Batch Sterilization of Liquid Media
Continuous Sterilization of Liquid Media
Examples of Design for Continuous Liquid
Medium Sterilization in a Tubular Sterilizer

6. Air Sterilization by Fibrous
Bed Filters

Mechanisms of Air Filtration and Design of
Fibrous Packed Beds
Example of Design of Fibrous Packed Bed for
Air Sterilization
1. Introduction
GENERALIZED VIEW OF
BIOPROCESS

RAW MATERIALS
UPSTREAM PROCESSES
Inoculum
Preparation
Equipment
Sterilization
Media Formulation
and
Sterilization
BIOREACTOR - FERMENTER
Reaction Kinetics
and Bioactivity
Transport Phenomena
and Fluid Properties
Instrumentation
and Control
DOWNSTREAM PROCESSES
Separation
Recovery and
Purification
Waste Recovery,
Reuse and Treatment
THE BOTTOM LINE
REGULATION ECONOMICS HEALTH AND SAFETY
TYPICAL BIOPROCESS FLOW SHEET
RAW MATERIAS
Nutrients and Reactants
in Aqueous Solution
(may contain insoluble
organic and/or inorganic
materials)
Air
CELL SEPARATION
1). CELL DISTRUPTION
2). PRODUCT EXTRACTION
PRODUCT
CONCENTRATION
PROCESS
FINAL PRODUCT
DRYING
PURIFICATION
PRODUCT
SEPARATION
PREPARATION
OF BIOMASS
Innoculum Stages
FOAM CONTROL
Antifoam Addition
pH CONTROL
Acid-Alkali Addition
Extracellular
product
Intracellular
product
STERILIZATION
BIOREACTOR
Free Cells,
Immoblized Cells
or
Enzyme Bioreactor
PRODUCT RECOVERY
TABLE 1. Basic Bioreactor 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
______________________________________________________________________
TABLE 2. Summary of Bioreactor Systems
__________________________________________________________
Bioreactor Cell Systems Products
Design used
__________________________________________________________
Air-Lift Bioreactor Bacteria, Yeast and SCP, Enzymes, Secondary
other fungi metabolites, Surfactants

Fluidized-Bed Immobilized bacteria, Ethanol, Secondary
Bioreactor yeast and other fungi, metabolites, Wastewater
Activated sludge treatment

Microcarrier Immobilized (anchored) Interferons, Growth factors,
Bioreactor mammalian cells on Blood factors, Monoclonal
solid particles antibodies, Vaccines, Proteases,
Hormones

Surface Tissue mammalian, tissue Interferons, Growth factors,
Propagator growth on solid surface, Blood factors,
tissue engineering Monoclonal antibodies,
Vaccines, Proteases, Hormones

__________________________________________________________

TABLE 2. Summary of Bioreactor Systems
(Contd)
____________________________________________________________________________________________________
Bioreactor Cell Systems used Products
Design
________________________________________________________________________________________

Membrane Bioreactors, Bacteria, Yeasts, Ethanol, Monoclonal anti-
Hollow fibers and Mammalian cells, Plant bodies, Interferons, Growth
membranes used, cells factors, Medicinal products
Rotorfermentor

Modified Stirred Immobilized Bacteria, Ethanol, Monoclonal anti-
Tank Bioreactor Yeast, Plant cells bodies, Interferons, Growth
factors

Modified Packed- Immobilized Bacteria, Ethanol, Enzymes, Medicinal
Bed Bioreactor Yeasts and other fungi products

Tower and Loop Bacteria, Yeasts Single Cell Protein (SCP)
Bioreactors

________________________________________________________________________________________
TABLE 2. Summary of Bioreactor Systems
(Contd)
_______________________________________
_____
Bioreactor Cell System used Products
design
__________________________________________________________________________________________________________
___________
Vacuum Bioreactors Bacteria, Yeasts, Fungi Ethanol, Volatile
products

Cyclone Bioreactors Bacteria, Yeasts, Fungi Commodity products,
SCP

Photochemical Photosynthetic bacteria, SCP, Algae, Medicinal
Bioreactors Algae, Cyano bacteria, plant products,
Plant Cell culture, r-DNA Monoclonal antibodies,
plant cells Vaccines, Interferons


________________________________________________________________________________________
Fig. 1.1. Schematic diagram of a tower bioreactor system with
perforated plates and co-current air liquid flow.
Medium
inlet
Air filter
Orifice
Compressed
air
Flow
meter
Peristaltic
pump
Medium
reservior
Constant temp.
water bath
Air exhoust
Pump
Jacket
Perforated
plate
Sparger
Broth
outlet
Sampling
nozzles
Fig. 1.2. Schematic diagram of a tower bioreactor system
with multiple impellers and liquid down comer and
counter-current air liquid flow
Perforated
plate
Downcomer
Baffle
Impeller
Feed Air
Product
Air
Fig. 1.3. ICI Deep Shaft Unit
AIR
PROCESS
AIR
OUTLET
RISER
DOWN-
COMER
SHAFT
LINING
INLET
SLUDGE
RECYCLE
START
-UP AIR
FIG. 1.4. EMLICHHEIM FLOWSHEET
AIR
COMPRESSOR
DEEP
SHAFT
B
FLOATATION
LAGOON
B
SAND
WASH
WATER
CLARIFIER
RECYCLE SLUDGE
RECYCLED
WATER
SETTLEMENT
TANT
CONDENSATE,
MAE-UP WATER, AND
FLOCCULATING AGENT
DECANTER
CENTRIFUGE
SOIL AND
SLUDGE
FIG. 1.5. Internal circulation patterns of fluidized Ca-alginate beads
containing immobilized cells of Z. mobilis. All dimensions in cm.
0.1
0.953
6.895
21.30
28.40
2.876
26.43
1.176 2.620 4.530
Outer draft tube
Inner draft tube
4 Jets
FIG. 1.6. Vacuum Fermenter
Dry ice
bath
Metering
pump
Receiving
tank
(bleed)
Filter
Filter
Fermenter
Vacuum
control
Receiving
tank
(product)
Condenser
Level
control
Heating
water
Medium
reservoir
Rheostat
Vacuum
pump
Air or O2
Chilled
water
2. Aeration and Oxygen Mass
Transfer in Bioreactor
Systems
Living Cells:
Bacteria,
Yeasts,
Plant cells,
Fungi,
Mammalian Cells
Require Molecular Oxygen O
2
as
final Electron Acceptor in Bioxidation
of Substrates (Sugars, Fats, Proteins,
etc.)
Substrate O2
Electrons
H2O
Products of
Oxidation
CO2
Products
Cell mass
FIG. 2.1. Bio-oxidation of Substrate with Molecular
Oxygen as the Final Electron Acceptor
OXIDATION-REDUCTION REACTION

Glucose is oxidized to make CO
2

Oxygen is reduced to make H
2
O

Fig. 2.1. Shows the biochemical pathway for
aerobic oxidation of carbohydrates, fatty
acids, and amino acids (AA) via the Tri-
carboxylic acid cycle (T.A.C.) and electron
Transport System.

Molecular oxygen O
2
accepts all the
electrons released from the substrates during
aerobic metabolism.



FIG. 2.2. Aerobic oxidation of carbohydrates, fatty acids, and amino acids via the TCA
cycle and the Electron Transport System (ETS) through which electrons are transported
and accepted by molecular oxygen (O
2
).

ATP is produced from the phosphorylation of ADP. The ETS is
composed of the following: FP
1
= NADH; FP
2
= succinate
dehydrogenase; Q = Co-enzyme Q; Cytochrome b, c, a, and a3.
The final electron acceptor O
2
is reduced to water. Oxygen comes
from the liquid phase and diffuses through the cell.
Pyruvate
Acetyl CoA
alpha-
Ketoglutarate
Marate
Isocitrate
Fumarate
Succinate
2H
2H
2H
2H
2H
2H
Citrate
CO2
CO2
NAD
FPi
FPi
ADP+Pi
Q b
ADP+Pi
ATP
ATP
c a a3
O2
H2O
ADP+Pi
CO2
Oxaloacetate
Amino acids
Fatty acids
Respiratory chain phosphorylation
--Electron transport along the respiratory chain--
OXIDATION-REDUCTION REACTION
(CONTD)
Question: How do we ensure that we
provide enough O
2
so that the cell
growth in a bioreactor is not limiting?

Answer: Must ensure that O
2
is
transferred fast enough from the air
bubbles (gas phase) to the liquid phase
(usually water) where all cells are
present and growing.

LIQUID PHASE
O2
O2
O2
O2
Dissolved O2
in liquid phase,
nutrients
(medium mostly
water)
AIR BUBBLE
LIQUID FILM
CELL
O2
INTERNAL
CELL
RESISTANCE
LIQUID FILM
CELL-LIQUD
INTERFACE
Electron
Transport
System +
TCA cycle
enzymes
GAS FILM
GAS-LIQUD
INTERFACE
FIG. 2.3. 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
LIQUID PHASE (CONTD)
At Steady-state with no O
2

accumulation in the liquid phase:



What are the O
2
requirements of
microorganisms?
Rate of O2 Transfer (OTR) = Rate of O
2
Uptake (OUR)
(Air bubbles Liquid) by Growing Cells
2.1 OXYGEN REQUIREMENTS OF
MICROORGANISMS
We define: Q
O2
= Respiration rate coefficient for
a given microorganism.
Units of QO
2
:
(mass of O
2
consumed) (unit wt. of dry biomass) .
(time)
Biomass means the mass of cells in a
bioreactor vessel.
Some units of Q
O2
:
mM O
2
/(g dry wt. of biomass) (hr.)
gO
2
/(g dry wt.) (hr.)
LO
2
/(mg dry wt.) (hr.)
CONVERSION FACTORS:
1 M O
2
= 32 x 10
-6
g O
2

1 L = 1 x 10
-6
L at S.T.P.
1 mole O
2
= 22.4 L O
2
at S.T.P.

In general:
Q
O2
= f(microbial species and type of cell, age of
cell, nutrient conc. in liquid medium, dissolved O
2

conc., temperature, pH, etc.)
For a given: 1) type of species of cell
2) age of cell
3) nutrient concentration
4) temperature
5) pH

and if O
2
concentration, C
L
, is the limiting factor in cell
growth, then Q
O2
is a strong function of dissolved O
2

concentration C
L
(= mg O
2
/L). The relationship between Q
O2

and C
L
is of the Monod type.
OxygenCONC. (C
L
)
Q
O
2
0
2
4
6
8
10
12
0 2 4 6 8 10 12 14 16 18 20
Q
O
2
max
K
O
2
Q
O
2
max
/2
Q
O
2
C
LCRIT.
FIG. 2.4. Respiration coefficient Q
O2
as a function of the dissolved oxygen concentration
C
L
.
where: K
O2
= O
2
conc. at Q
O2 max
/2
C
L CRIT.
= Critical O
2
conc. beyond which O
2
is
not limiting
Q
O2
= Q
O2max
= constant
At C
LCRIT.
respiration enzymes of Electron Transport System are saturated
with O
2
.

When O
2
conc. is the limiting substrate then






analogous to the Monod equation:

max
.S
= ________ (S = substrate conc. (g/L)
K
S
+ S

= 1 dX (h
-1
) [K
s
= S (g/L), at
max
/2]
X dt

( ) 1 . 2
.
2
2
2
L
L MAX
C
C Q
Q
+ K
=
O
O
O
Table 1 shows typical values of Q
O2
measured by
Warburg respirometer.
Table 2 shows typical data for critical oxygen
concentration C
L,CRIT
. (mmol O
2
/L).
FIG. 2 shows the variation of Q
O2
with
fermentation time for the microorganism
Bacillus subtilis, where Q
O2
reaches a maximum
value during the exponential growth phase.
FIG.3 shows the effect of agitation rate (revolutions
per minute) on the value of Q
O2
for the bacterium
Nocardia erythropolis, growing on hexadecane to
produce biosurfactants.
________________________________________________________________________
Microbial Species Temp. Culture Resp. Rate Coeff.
(
o
C) age (hr.) Q
O2
(L O
2
)/
(mg dry wt.) (hr.)
_____________________________________________________________
B. aerogenes 36; 30 17; 48 47; 50
Azotobacter choococcum 22 36 2,000-10,000
A. subtilis (cells) 37 6-8 170
C. subtilis (spores) 32 98-147 10
Corynebacteria species 30 48-96 67
E. coli 40; 32 20 200; 272
L. bulgaricus 45; 37 8 55; 34
Micrococcus luteus 35 30-34 15
Microbacterium avium 37 84 1
Mycobacterium tuberculosis 38 252 4
Pseudomonas fluorescens 26 30 58
________________________________________________________________________
TABLE 1. Cell suspensions in glucose. Oxygen uptake determined in
constant volume Warburg respirometer
________________________________________________________________________
Microorganism Temp. (
o
C) C
L CRIT
.
(mmol O
2
)/L
_____________________________________________________________

Azotobacter vinelandii 30 0.018-0.049
E. coli 37.8 0.0082
E. coli 15 0.0031
Serratia marcescens 31 ~ 0.015
Pseudomonas denitrificans 30 ~ 0.009
Yeast 34.8 0.0046
Yeast 20 0.0037
Penicillium chrysogenum 24 ~ 0.022
Penicillium chrysogenum 30 ~ 0.009
Aspergillus oryzae 30 ~ 0.020
________________________________________________________________________
-
Adopted from R. K. Finn, P.81 in: N. Blakebrough (ed),
Biochemical Engineering Science. Vol. 1, Academic Press, Inc., New
York, 1967

TABLE 2. Typical values of C
L CRIT
in the Presence of Substrate
-
FIG. 2. 5a: Oxygen uptake rate, Q
O2
X () and broth viscosity ()during batch aerobic fermentation of Bacillus subtilis. b: Respiration
rate coefficient, Q
O2
() and volumetric mass transfer coefficient, K
L
a ().
Taken from A.Richard and A. Margaritis, Rheology, Oxygen Transfer, and Molecular Weight Characteristics of Poly(glutamic acid)
Fermentation by B. subtilis, Biotechnology and Bioengineering, Vol. 82 No. 3, p. 299-305, (2003)
FIG. 2.6. Effect of agitation on the respiration coefficient (Q
O2
) in a 20 L batch
fermentation of Nocardia erythropolis. () 250 r.p.m, () 375 r.p.m, () 500 r.p.m.
(Adopted from Kennedy et al. In Dev. Ind. Microbiol., 20 (1978) 623-630)
2.2 THE VOLUMETRIC MASS
TRANSFER COEFFICIENT
k
L
a AND METHODS OF
MEASUREMENT
Mass Balance of Oxygen in Unit Liquid
Volume
AIR BUBBLE
LIQUID FILM
GAS FILM
GAS-LIQUD
INTERFACE
L
k
a
C
L
*
UNIT LIQUID
VOLUME
CELLS
(CONC. X)
O2 C
L
OXYGEN
(CONC. C )
L BULK
LIQUID
PHASE
O2 TRANSFER
FIG. 2.7 Schematic diagram of the mass balance of oxygen transfer in unit liquid volume
Mass Balance of Oxygen in Unit Liquid
Volume (Contd)
Rate of = net rate of O
2

Accumulation supply from air
of O
2
bubbles rate of
O
2
consumption by
cells


dC
L
dt
= k
L
a(C
*
L
- C
L
) - Q
O2
X......(2.2)
Mass Balance of Oxygen in Unit Liquid
Volume (Contd)
where: dC
L
/dt in (mmol O
2
/L.h)
k
L
a in (h
-1
)
C
*
L
, C
L
in (mmol O
2
/L)
Q
O2
in (mmol O
2
/(g dry
wt. cell)(h)
X in (g dry wt. Cell/L)
Mass Balance of Oxygen in Unit Liquid
Volume (Contd)
At steady state:







dC
L
dt
k
L
a(C
*
L
- C
L
) = Q
O2
X.........(2.3)
= 0
At all times C
L
= constant
Mass Balance of Oxygen in Unit Liquid
Volume (Contd)
Oxygen transfer rate from air
bubbles to liquid = OTR

OTR = k
L
a (C
*
L
C
L
)



OTR
k
L
a =
(C
*
L
- C
L
)
......(2.4)
Mass Balance of Oxygen in Unit Liquid
Volume (Contd)
For a given OTR and C
L
*
(= Py
O2
/H), please note that as
k
L
a increases, then C
L
also increases.

Where:
C
L
*
= saturated oxygen conc. (mole O
2
/Lit)
P = total pressure inside air bubble (atm)
y
O2
= mole fraction of oxygen in air (0.21)
H = Henrys constant (atm.Lit/mole O
2
)

This is an important way of controlling the dissolved
oxygen concentration C
L
which also affects the metabolic
activity of aerobic cells their rate of growth and the rate
of production of different metabolic products.

For pure oxygen, y
O2
= 1.00
Methods of Measurement of K
L
a
in a Bioreactor
Two basic methods for Measuring
K
L
a

Chemical methods (no cells present)

Physical Methods (with/without
cells)
Chemical Methods of K
L
a
Measurement
The Sulphite Batch Oxidation Method.
SO
3
2-
F,
Water out
Water in
rpm
Motor
Influent
Air flow, rate
Air outlet
FIG. 2.8. Schematic diagram of a stirred tank batch reactor
Chemical Methods of K
L
a
Measurement (Contd)
Liquid Solution = 0.5 M Na
2
SO
3
(Sodium
sulphite), with Cu
++
as catalyst.

Sparge air through the bioreactor vessel at a
given volumetric flow rate Q and impeller
speed (R.P.M.)

Make sure that [SO
3
-2
] is in excess (i.e. 0.5 M
Na
2
SO
3
)
Chemical Methods of K
L
a Measurement
(Contd)
Oxygen oxidizes the sulphite ion to
sulphate.
SO
3
-2
+
1
2
O
2
Cu
++
SO
4
-2
.......(2.5)
(SULPHITE) (SULPHATE)
The rate of chemical reaction is extremely
fast.
The controlling step is diffusion of O
2
molecules through the liquid film
surrounding the air bubbles.
Chemical Methods of K
L
a Measurement
(Contd)
Rate of reaction = R = k
2
[O
2
][SO
3
-2
]
~ k
1
[O
2
] =
= -

i.e. k
1
~ k
2
[SO
3
-2
]
= constant
2
d[SO
3
-2
]
1
dt
Chemical Methods of K
L
a Measurement
(Contd)

i.e. R is zero order to sulphite concentration

[SO
3
-2
] because it is in excess.
? From stoichiometry shown in Eq. (2.5)
dt
1
d[SO
3
-2
]
2
R = (- )
= (K
L
a)(C
L
*
- C
L
)...(2.6)
Chemical Methods of K
L
a Measurement
(Contd)
The reaction with [SO
3
-2
] is extremely
fast.
As a result, the O
2
gas molecules are
consumed as soon as they diffuse into
the liquid phase.
Therefore, the D.O. concentration in
the liquid phase, C
L
~ 0.
Chemical Methods of K
L
a Measurement
(Contd)
Equation (2.6) becomes:

R = (K
L
a)(C
L
*
) = (K
L
a)( )
Py
O2
H
= (-
1
2
)
d[SO
3
-2
]
dt
......(2.7)


Assuming a perfeftly mixed vessel,
Chemical Methods of K
L
a Measurement
(Contd)
Use iodometric titration to measure
[SO
3
-2
] as a function of time, t, as the
air bubbles pass through the
bioreactor vessel at a given R.P.M.
Chemical Methods of K
L
a Measurement
(Contd)
SLOPE = - ~ -
d[SO
3
-2
]
dt t
[SO3
-2
]
TIME, t, (min)
[
S
O
3
-
2
]
0
10
20
30
40
50
60
70
80
0 2 4 6 8
SLOPE = - ~ -
d[ SO
3
- 2
]
dt t
[ SO
3
- 2
]
FIG. 2.9. Concentration of SO
3
-2
as a function of oxidation time
Chemical Methods of K
L
a Measurement
(Contd)
For a given:
Aeration rate Q
Agitation Speed R.P.M.
Total air pressure P
Volumetric mass transfer coefficient
K
L
a can be calculated from Equation
(2.7) as:


K
L
a =
)(H)
(- )(
2
t
[SO
3
-2
]
1
Py
O2
......(2.8)
-



In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
Consider a Stirred Tank Bioreactor System,
Where Cell Growth takes Place at a Given
Set of Conditions:
Aeration
Agitation
pH
Temperature
Medium Composition
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
The Bioreactor Vessel is Equipped with:
The D.O. Probe, Connected to a D.O. Analyzer.
Chart Recorder:
To Measure Signal from D.O. Probe and
Measure On-line the D.O. Concentration in the
liquid phase of the Bioreactor.
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
The D.O. Probe Measures the
Py
O2
Partial Pressure (Py
O2
) of
dissolved O
2
in the liquid
phase, which means that it
measures H
O2
C
L
.
Where:
H
O2
= Henrys Constant for O
2
in
Water
C
L
= D.O. Concentration In the
Liquid
Phase (Mass of O
2
/L)
In Situ Measurement of K
L
a, Q
O2
,
and C
L
*
During Cell Growth in a
Bioreactor (Contd)
Fig. 2.10 Set up of a Stirred tank Bioreactor with Dissolved Oxygen Probe, pH probe and
accessories.
Acid
DO2
1
4 9
pH
7
8
12
11
2
10
6
14
rpm
Alkali
13
15
15
16
5
3
1. Feed
2. Flow meter
3. Ring sparger
4. Impeller
5. Motor
6. Shaft
7. pH probe
8. D.O. probe
9. Baffle
10. To Condenser
11. D.O. meter
12. pH meter
13. Speed controller
14. Water Jacket
15. Thermometer
16. Chart recorder
Water out
30 deg.
water in
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a
Bioreactor (Contd)
Turning air ON and OFF while Maintaining the
same R.P.M. we can:
Record the D.O. Probe Output in the Chart
Recorder.
From these Data, we can get
K
L
a,
Q
O2
,
C
L
*

at given in-situ Bioreactor Conditions.

In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
The ON-OFF Operation takes 5 min, during which time:
Cell Concentration X (g /L) ~ Constant.
We make sure that the D.O. Concentration C
L

never falls below the critical oxygen concentration
C
CRT
,which means that the respiration rate
coefficient Q
O2
= Q
O2Max
= Constant.

Using the D.O. probe output and a recorder we
measure directly the D.O. concentration as a
function of time, t.
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
While we maintain the same R.P.M. of the bioreactor impeller, we
turn the AIR-OFF. During the AIR-OFF period the following
conditions apply:
Rate of Supply of O
2
= 0
No Air Present in the Bioreactor
K
L
a = 0 because a = 0, no air bubbles present
Using Eq. 2.2 for O
2
Mass Balance, we have:


We know cell concentration X by measuring it.
Therefore, we calculate Q
O2
because we also measure
the slope Q
O2
X.
dC
L
dt
= 0 - Q
O2
X
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
Fig. 2.11 Shows D.O. concentration C
L
inside the
bioreactor = f(t) when Air is turned Off and On, always
keeping the R.P.M. of the impeller the same to provide
good mixing of the liquid phase.
After a period of about 5 min, a liquid sample is taken
from the bioreactor to measure the cell concentration X
(g dry wt./L).
The K
L
a, Q
O2,
and C
L
*
values correspond to that
specific fermentation time and given cell growth
conditions.
We can do many AIR-OFF and AIR-ON
measurements to get all three parameters K
L
a, Q
O2,

and C
L
*
as a function of total batch fermentation time.
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
TIME (MIN)
D
O
2

C
O
N
C
.

C
L

(
m
M

O
2
/
L
)
AIR-OFF
AIR-ON
C
L,CRIT
3 - 5
C
L
STEADY-STATE
FIG. 2.11. Transient Air-Off, Air-On Experiment in a Bioreactor System
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a
Bioreactor (Contd)
During the AIR-OFF period the D.O. concentration C
L
is plotted
as a function of time t from which we get the slope = - Q
O2
X, as
shown in Fig. 2.12.
Time, t (min)
C
L

(
m
M
O
2
/
L
)
0
1
2
3
4
0 1 2 3 4 5 6 7 8 9 10
SLOPE = - Q
O2
X
FIG. 2.12. D.O. concentration C
L
as function of time during AIR-OFF period.
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
AIR-ON Period
During this period the following oxygen mass balance
equation applies:


From the C
L
vs. time (t) data we can get


dC
L
dt
= K
L
a (C
L*
- C
L
) - Q
O2
X
dC
L
dt
~
t
C
L
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
Re-arranging Eq. 2.2 and solving for C
L
we get Eq. 2.9




By plotting C
L
vs. at a given fermentation time, t,

we can get the slope which is equal to
dC
L
dt
+ C
L
*.....(2.9)
CL =
K
L
a
1
-
Q
O2
X +
dC
L
dt
+
Q
O2
X
K
L
a
1
-
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
and therefore, the value of K
L
a is found, and the
intercept also gives the value of



During the Air-On Period:
C
L
*
= Constant
Q
O2
= Constant
K
L
a = Constant
C
L
, dC
L
/dt vary with time t
Py
O2
H
C
L
*
=
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a
Bioreactor (Contd)
[dC
L
/dt+Q
O2
X]
C
L

(
m
g
O
2
/
L
)
0.8
1.4
2.0
2.6
3.2
3.8
4.4
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
SLOPE = -1/k
L
a
Intercept = C
L
*
FIG. 2.13. D.O. concentration C
L
as function of [dC
L
/dt + Q
O2
X] during AIR-ON period.
In Situ Measurement of K
L
a, Q
O2
, and
C
L
*
During Cell Growth in a Bioreactor
(Contd)
Figures 2.8 and 2.9 show batch aerobic fermentation results in a
stirred tank bioreactor system for the production of the
biopolymer poly(glutamic acid) produced by Bacillus subtilis
obtained by A. Richard and A. Margaritis.
Reference: A. Richard and A. Margaritis (2003), 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 .
Please read chapter 8, Bioproducts and Economics pp. 609-685,
in Book Biochemical Engineering by H.W. Blanch and D.S.
Clark, Marcel Dekker, Inc., New York (1996). This material is
useful for the Plant Design Course, CBE 497 (4
th
year).
In Situ Measurement of K
L
a, Q
O2
,
and C
L
*
During Cell Growth in a
Bioreactor (Contd)
FIG. 2.14. Batch fermentation kinetics of Bacillus subtilis IFO 3335 during polyglutamic acid production. Biomass, X (); dissolved
oxygen concentration, C
L
(); Polyglutamic acid (PGA) concentration, P ().
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).
In Situ Measurement of K
L
a, Q
O2
,
and C
L
*
During Cell Growth in a
Bioreactor (Contd)
FIG. 2.15. Dynamic air-on/air-off data during Poly(glutamic acid (PGA) production by Bacillus subtilis IFO 3335
(fermentation time = 26 h). Dissolved oxygen concentration C
L
() 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).
2.3. EMPIRICAL CORRELATIONS
OF K
L
a
A large number of Empirical
Correlations Exist for K
L
and K
L
a for
Agitated and Aerated Bioreactor
Vessels.
General Background Reading:
Textbook by H.W. Blanch and D.S.
Clark Biochemical Engineering,
Chapter 5. Transport Processes,
pp. 343-415. Publisher: Marcel Dekker,
Inc., New York, 1996.
Consider a Stirred Tank Bioreactor
Vessel at a given:

P
g
V
L
D
T
L
H
AIR, Q
Q = Vol. air flow rate
@S.T.P.
D
T
= Tank diameter
H
L
= Liquid height (un-
gassed)
V
L
= Working Liquid
volume (un-gassed)
P
g
= Gassed power
P = Un-gassed power

Impeller Speed R.P.M.
Aeration Rate Q

Working Liquid Volume V
L

of the Vessel
FIG. 2.16. Typical stirred tank bioreactor vessel


Most Empirical Correlations for K
L
a have the
following form



Where:
K
L
a = Vol. mass transfer coefficient
P
g
= Gassed power supplied by
mechanical impeller for mixing of
bioreactor vessel.
V
L
= Liquid working volume of
bioreactor vessel




K
L
a = C
P
g
V
L
m
U
g
k
................(2.10)
EMPIRICAL CORRELATIONS
OF K
L
a


U
g
= Superficial air velocity


m, k = Exponents, constants
The values for C, m, and k depend greatly on the ionic strength of the
aqueous phase in the bioreactor.
Ionic strength, I, of the solution in the bioreactor is defined by Equation 2.11.

I = E(Z
i
2
C
i
)(2.11)
Where:
I = Ionic strength of solution, (g ions/L)
Z
i
= Electric charge of ionic species i, present in the solution
e.g.
SO
4
-2
= has Z
i
= -2
Na
+
has Z
i
= +1
Ag
+
has Z
i
= +1
C
i
= Concentration of ionic species in the solution = (g-ions/L)

Cross-sectional area of
bioreactor vessel
Vol. air flow rate @ S.T.P.
=
EMPIRICAL CORRELATIONS
OF K
L
a
Constants C, m, and k also depend on:
Temperature, T
pH
Physical properties of the solution
Presence of other nutrients
For Pure Water at pH = 7, T = 25
o
C, the following
empirical correlation applies:
K
L
a = (0.026)
P
g
V
L
0.4
U
g
0.5
....(2.12)
EMPIRICAL CORRELATIONS
OF K
L
a
Where:
K
L
a = Vol. mass transfer coefficient (s
-1
)
P
g
= Gassed power (W)
U
g
= Superficial air velocity (m s
-1
)
Note: The values of C = 0.026, exponents
0.4 and 0.5 in Eq. 2.12 can be used
only with the units of K
L
a, P
g
and
U
g
specified above.
A log-log plot of experimental data according to Equation
2.10 is shown in the following figure.











Taking the log on both sides of Eq. 2.10, we get

log (K
L
a) = log (C) + k log (U
g
) + m log (P
g
/V
L
).

log (P
g
/V
L
)
l
o
g

K
L
a
SLOPE = m
U
g
= CONSTANT
FIG. 2.17. A log-log plot of experimental data according to Equ. 2.10.
Definition of gas-holdup, H
o
, in an agitated and
aerated vessel
T
V
AIR
LIQUID PHASE,
VL
AIR BUBBLES,
Vg (DISPERSED
PHASE)
H
o
= gas hold-up =
Volume occupied by gas phase
Total volume
(V
T
) Total volume = Liquid Volume (V
L
)+Gas volume (V
g
)
H
o
=
V
g

V
g
+V
L
.........................(2.13)
FIG. 2.18. Typical agitated and aerated stirred tank bioreactor vessel
Assuming a monodispersed size distribution of air
bubbles each having the same diameter d
B
, then the
gas hold-up H
o
is related to the interfacial specific
gas-liquid area and d
B
according to Eq. 2.14.



Where:
H
o
= dimensionless
d
B
= bubble diameter, m
a = interfacial specific area, m
2
/m
3
= m
-1

Eq. 2.14 can be used as an approximation for a
rough estimate of specific interfacial area a (m
2
/m
3

of total volume)

.........................(2.14)
d
B

6H
o

a =
3. AGITATION OF BIOREACTOR
SYSTEMS
Fig. 3.1 shows the dimensions of what is called a
standard stirred tank bioreactor vessel with
Baffles.
FIG. 3.1. Standard Stirred Tank Bioreactor Geometry [Adopted from S. Aiba, A.E.
Humphrey and N.F. Millis. Bubble Aeration and Mechanical Agitation. In Biochemical
Engineering, 2
nd
Ed., Academic Press, Inc., New York (1973) 174].
Geometric Ratios for a Standard Bioreactor
Vessel
Impeller D
i
/D
t
H
L
/D
t
L
i
/D
i
W
i
/D
i
H
b
/D
i
W
b
/D
t
No. Baffles
Type

Flat-Blade 0.33 1.0 0.25 0.2 1.0 0.1 4
Turbine

Paddle 0. 3 3 1.0 - 0.25 1.0 0.1 4
impeller

Marine 0.33 1.0 pitch = D
i
1.0 0.1 4
Propeller

Where:
D
t
= tank diameter,
H
L
= liquid height
D
i
= impeller diameter
H
b
= impeller distance from bottom of vessel
W
b
= baffle width
L
i
= impeller blade length
W
i
= impeller blade height
FIG. 3.2 A. Different Impeller Types. (a) Marine-type propellers; (b) Flat-blade
turbine, W
i
= D
i
/5. Disk flat-blade turbine, W
i
= D
i
/5, D
i
= 2D
t
/3, L
i
= D
i
/4; (d)
Curved-blade turbine, Wi = D
i
/3; (e) Pitched-blade turbine, W
i
= D
i
/8; and (f)
Shrouded turbine, W
i
= D
i
/8.
FIG. 3.2 B. 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.
3.1 Mixing and Power Requirements for
Newtonian Fluids in a Stirred Tank
FIG. 3.3 N
P
vs. N
Re
; the power characteristics are shown by the power number, N
P
, and the
modified Reynolds number, N
Re
, 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].
Fig. 3.3 shows relationship between N
P
and
N
Re
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 Equ.
3.1

N
P
= Pg
c
/n
3
D
i
5
(3.1)

The impeller Reynolds number is given
by Equ. 3.2

N
Re
= nD
i
2
/..................(3.2)

Where:
N
Re
= dimensionless Reynolds number
N
P
= dimensionless Power number




P = Un-gassed power for liquid (no air), W
g
c
= 1, for SI units system
n = Impeller rotational speed, revolutions per
sec., (s
-1
)
D
i
= Impeller diameter, m
= Density of liquid, kg/m
3
= Viscosity of liquid, (N.m)/(s)

For six-bladed flat-blade turbine impeller (cf.
Fig. 3.3), the mixing becomes fully turbulent at
an impeller Reynolds number N
Re
= 3,000.

Power number N
P
= 6 (constant) at N
Re
> 3,000









Different Types of impellers have
different power characteristics Fig. 3.3.
For six-bladed flat turbine and for
turbulent conditions:
N
P
= 6 = Pg
c
/n
3
D
i
5

or P = (6)(n
3
D
i
5
)/(g
c
)..(3.3)
At N
Re
= 3,000 the corresponding
impeller speed is:
n = (3,000)()/(D
i
2
)()(3.4)

Eq. 3.4 is an estimate of the minimum impeller
speed, n, of a 6-flat blade turbine impeller for the
on-set of turbulent flow within the stirred tank
bioreactor vessel.
Eq. 3.3 shows that for a fluid of a given density,
:
P n
3
D
i
5


This is an important consideration for bioreactor
vessel scale-up.

Eq. 3.1 is used to find the un-gassed power, P, at
a given:
impeller diameter, D
i
and
impeller speed, n.

For aerobic fermentation (aerated) bioreactors:

P
g
(gassed) < P (un-gassed) power
since
eff
(effective density) <
P
g
/P < 1

The aeration number, Na, is defined by Equ. 3.5 and is
used to quantify the power ratio Pg/P as a function of
aeration rate Q
g
, as shown in Fig. 3.4.
For water:
N
a
= Q
g
/nD
i
3
(3.5)
Where:
N
a
= aeration number (dimensionless)
Q
g
= Volumetric flow rate of air (m
3
at STP/s)
n = impeller rotational speed, revolutions per
second (s
-1
).
D
i
= impeller diameter (m).

FIG. 3.4 Power requirements for agitation in a gassed system. The ordinate and abscissa are
degree of power decrease, Pg/P, and the aeration number, Na. Parameters are the types of
impellers, whose representative geometrical ratios in agitated vessels are also shown in the
figure. [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) 176].
Fig. 3.4 shows the relationship between
Pg/P ratio and Aeration Number, Na,
for three types of mechanical impellers:
Flat-blade turbine (A)
Vaned disk impeller with
different vanes (n
p
= 4, 6, 8, 16)
curves, B, C, D, E
Paddle impeller

Calculation of the Required Volumetric
Mass Transfer Coefficient, K
L
a, During
Fermentation, and Gassed Power, P
g
.

At Steady-State Operation of an Aerobic
Fermentation:
OTR = OUR
K
L
a[C
L
*
- C
L
] = Q
O2
X.(3.6)
For a given Q
O2
, X, and (C
L
*
- C
L
), K
L
a can
be calculated using Eq. 3.6.

For a given V
L
and U
g
, P
g
can be calculated
using the empirical correlation for K
L
a given
by Eq. 3.7.

K
L
a = C [P
g
/V
L
]
m
[U
g
]
k
3.7

Figs. 3.3 and 3.4 are used in combination to find the
correct rotational impeller speed, n, to deliver the
required P
g
at a given U
g
, for the required value of
K
L
a.