Support Bioprocess Ahmet

Chapter 3: Enzymes
David Shonnard
Department of Chemical Engineering
Michigan Technological University
1
David R. Shonnard Michigan Technological University
Presentation Outline:
Lectures 4 and 5
l Introduction to Enzymes
l Kinetics of Enzyme-Catalyzed Reactions
l Effects of Environmental Conditions on Kinetics
l Inhibition of Enzyme Catalyzed Reactions
l Immobilized Enzyme Systems
l Industrial Uses of Enzymes
2
1
Introduction to Enzymes (3.1 and 3.2)
Constituents of Enzymes
Protein
molecule(s)
Co-factors
Co-enzymes
“Bioprocess Engineering: Basic Concepts

Shuler and Kargi, Prentice Hall, 2002
3
Introduction to Enzymes (3.1)
Naming of Enzymes
• adding suffix -ase to
→ substrate converted (e,g, urease)
→ reaction catalyzed (e,g, dehydrogenase)
Enzymes Facts
• over 2,000 known enzymes

• more efficient than chemical catalyses
• high molecular weight (15,000<MW<106 Daltons)
4
2
Major Classes of Enzymes (Table 3.1)
Oxidoreductases
• oxidation – reduction reactions
Transferases
• transfer of whole functional groups (e.g. NH2 group)
Hydrolases
• Hydrolysis reactions involving various functional groups
Lyases
• Additions to double bonds
Isomerases
• oxidation – reduction reactions
Ligases
• formation of bonds with ATP cleavage
5
How Enzymes Work (3.2)
Lower the activation energy of enzyme-substrate complex

 ∆G oA2  1 2
−
 RT 
 Enzyme-
rate2 e
=  ∆ Go  substrate
rate1 −
A1

complex
e
RT 
= 108
for ∆GoA1 = 18 kcal / mole
∆GoA2 = 7 kcal / mole
“Bioprocess Engineering: Basic Concepts, Shuler and Kargi, Prentice Hall, 2002
Lock and Key Model Enzyme-

substrate
Active Site complex
6
3
Enzyme Kinetics (3.3)
Michaelis-Menten Kinetics
E + S ←
k -1

k1
→ ES 
k2
→ E + P
Substrate Enzyme- Product

Substrate
Complex
Relate product reaction rate to measurable quantities
7
Enzyme Kinetics (3.3)
Mass Balance Equations

d[P]
Product Rate υ = = k 2 [ES]
dt
d[ES]
ES Rate = k1 [E][S] - k-1[ES] - k 2 [ES]
dt
Enzyme Balance [E] = [Eo ] - [ES]
8
4
Michaelis-Menten Kinetics (eqn. 3.8)
Rapid Equilibrium Assumption (formation of ES is rapid)

k -1 [E][S]
Equilibrium Constant K'm = =
(relates [ES] to [E] , [S] k1 [ES]
Combine K’m equation with E balance equation

[Eo ][S]
[ES] =
K'm + [S]
maximum
Product Rate Eqn. - Michaelis-Menten Equation
rate
k2 [E o ][S] Vm [S]
υ = k 2 [ES] = ' =
K m + [S] K'm + [S]
9
Michaelis-Menten Kinetics (cont.)
Saturation Kinetics
Vm=k2[Eo]
d[P]
υ=
dt K’m = [S] when υ = 1/2 Vm
|
Michaelis Menten Constant
[S]
• at high [S] → υ=Vm (constant) - why? → all active sites on E filled with S
• if K’m is small → S has high affinity for E
10
5
Experimental Determination of Vm and K’m
Saturation Kinetics
So
P
[Eo] [So]
[P]
or
[S]
Batch Reactor d[P]
υ = t=0
measure dt
t [S] [P] . t
→ determine rate (slope) from measured data
11
Plotting Experimental Data
Double-Reciprocal Plot (Lineweaver-Burk Plot, eqn. 3.13)
Vm [S] 1 1 K' 1
rearrange υ = = + m
K'm + [S] υ Vm Vm [S]
Limitation:
K’m not
determined 1/υ
accurately. '
-1/Vm '
Data at low [S] ' K'm
Influence -1/K’m ' slope =
Regression Vm
too much.
1/[S]
12
6
Other Types of Plots
K’m
Eadie-Hofstee Plot (eqn. 3.14) υ
'
Vm '
υ '
υ = Vm + K'm
[S]
υ /[S]
Hanes-Woolf Plot (eqn. 3.15)

1/Vm
[S] /υ
[S] K'm 1 K’m / Vm
= + [S] '
υ Vm Vm '
'
[S]
13
Complex Enzyme Kinetics: Inhibition
A) Competitive Inhibition (eqn. 3.20 - 3.23)
Vm [S] E + S ←k
 k
→ ES 
-1 k
→ E + P
1 2
υ =
 [I]  +
K'm 1 +  + [S]
I → Inhibitor binds to active site on enzyme
 KI 
b KI
EI → Enzyme / Inhibitor complex
• K'm, app → net effect of I is to increase K’m
• overcome inhibition by increasing [S]
14
7
Complex Enzyme Kinetics: Inhibition
B) Noncompetitive Inhibition (eqn. 3.24 - 3.27)
Vm [S] E + S ←
k -1

k1
→ ES 
k2
→ E + P
υ =
 [I]  ' + +
1+ K  (Km +[S]) I I
 I
b KI b KI
EI + S ←
→ ESI
Vm
Vm, app =
 [I]  → net effect of I is to reduce Vm
1 + 
 KI 
• overcome inhibition by blocking inhibitor
15
More Inhibition Kinetics (eqn. 3.28 - 3.38)
C) Uncompetitive Inhibition • have similar mechanisms

D) Substrate Inhibition by inhibiting on ES
16
8
Summary of Inhibition Kinetics
17
Temperature Effects on Enzyme Kinetics
The rate of enzyme conversion of substrate will increase with

temperature up to an optimum. Above this temperature,
enzyme activity will decrease as enzyme denatures (Tertiary
structure lost). Figure 3.15 shows a typical response.
Temperature -k d t
[E] = [Eo ] e
activation -E d / RT
kd = Ad e
Vm = k2 [Eo], T<Topt
E d = deactivation energy
= k2 [E], T>Topt Topt (40 -130 kcal / mole)
[E] is concentration
of active enzyme
Temperature
k2 = A e-E a / RT deactivation
E a = activation energy
(4 - 20 kcal / mole)
18
9
pH Effects on Enzyme Kinetics (eqn. 3.40 - 3.44)
Enzymes are active only over a small pH range
Reasons
1. Tertiary structure is pH-dependent
2. Active site functional group charges are pH-dependent
E + H+ “Bioprocess Engineering: Basic Concepts,

Shuler and Kargi, Prentice Hall, 2002 pHopt
b K2 Between
pK1 and
EH + S ←K'
m
→ EHS 
k2
→ EH + P
pK2
b K1 Vm [S]
pHopt
υ =
EH+2  K [H+ ]
K'm 1 + +2 +  +[S]
 [H ] K1 
K 'm,app → ∞ at high and low [H+ ] 19

Immobilized Enzyme Systems

Immobilization - Definition
The containment of enzyme solution within a confined space for
the purpose of retaining and re-using enzyme in processing
equipment. There are many advantages that accompany
immobilized enzymes and many methods for immobilization.
20
10
Immobilized Enzyme Systems
Advantages
1. Reduce costs of operation compared to free enzyme systems
where additional separation and purification steps are needed.
2. Some immobilization methods can increase enzyme activity.
3. A model system to study enzyme action in membrane-bound
enzymes that occur in the cell.
Disadvantages
1. Many immobilized enzymes exhibit lower activity compared to
free enzymes.
2. More expensive to prepare than free enzymes.
3. Mass transfer limitations due to immobilization methods.
21
Methods of Enzyme Immobilization
22
11
Matrix Entrapment of Enzymes
Matrix Entrapment
The enzyme solution is mixed with a polymeric fluid that solidifies
into various forms, depending on application (usually small
beads). The polymeric material is semi-permeable. Large
molecular weight enzymes can not diffuse out, but smaller
substrate and product molecules can.
Matrices for Entrapment

• Ca-alginate
• Agar
• Polyacrylamide
• Collagen
23
Membrane Entrapment
Membrane Materials
Enzymes solution may be confined between thin semi-permeable
membranes. Membrane materials include;
• Nylon • Cellulose
• Polysulfone • Polyacrylate
Membrane Configurations
Hollow fiber configuration is a popular arrangement for separating
enzyme from substrate and product solution.
Hollow fibers containing Mobile fluid outside
a stationary enzyme fiber tubes containing
solution substrate and products
24
12
Membrane Entrapment:
Diffusion Processes
Solution Substrate
E
E ∗ E E
E E
Hollow fiber E E
Solution
Product
25
Surface Immobilization: Adsorption
Adsorption: Attachment of enzymes to stationary solids by

weak physical forces (van der Waals or dispersion forces).
Active site is normally unaffected and nearly full activity is
observed. Desorption of enzymes is a common problem.
Solid Support Materials:

• Alumina • Silica
• Porous Glass • Ceramics
• Diatomaceous Earth • Clay
• Cellulose Materials • Activated Carbon
• Ion Exchange Resin • Starch
26
13
Surface Immobilization: Covalent Bonding
Covalent Bonding: The retention of enzyme on support

surfaces by covalent bonding between functional groups on the
enzyme and those on the support surface.
Functional Groups on Enzymes:
• Amino (protein-NH2) • Carboxyl (protein-COOH)

• Hydroxyl (protein-OH) • Sulfhydryl (Protein-SH)
Active site of enzyme must not participate in covalent

bonding. Enzyme inhibitors are added to enzyme solution
during covalent bonding treatment.
27
Surface Immobilization: Support Bonding
28
14
Surface Immobilization: Support Bonding
“Bioprocess Engineering: Basic Concepts, Shuler and Kargi, Prentice Hall, 2002 29
Diffusional Limitations:
Immobilized Enzyme Systems (section 3.4.2)
Diffusional limitations are observed to various degrees in all

immobilized enzyme systems. This occurs because substrate
must diffuse from the bulk solution up to the surface of the
immobilized enzyme prior to reaction. The rate of diffusion
relative to enzyme reaction rate determines whether limitations
on intrinsic enzyme kinetics is observed or not.
Damkohler Number
maximum rate of reaction Vm'
Da = =
maximum rate of diffusion k L [Sb ]
If Da>>1, diffusion rate is limiting the observed rate
If Da<<1, reaction rate is limiting.
30
15
Diffusional Effects on Surface-Bound Enzymes
on Non-porous Supports
EL, Enzyme Loading (mg enzyme/cm2)
Vm
Vm '= • EL /s•cm2 )
(mole
[Eo ]
Vm [Ss]
Js = k L ([S
b]-[S
s])=
K m + [Ss]
Eqn. 3.53
31
Diffusional Effects on Surface-Bound Enzymes

on Non-porous Supports (cont.)
Graphical Solution to Eqn. 3.53
(A) is reaction kinetics
(B) is mass transfer rate
Intersection is solution for [Ss ]
Figure 3.18 is useful for

observing effects of
• stirring rate (kL)
• changes in [Sb]
• changes in enzyme loading
32
16
Diffusional Effects in Enzymes Immobilized
in a Porous Matrix
Enzymes within a porous matrix

Substrate Mass Balance Equation
 d2 [S] 2 d[S] Vm' '[S]

De  2 +  =
 dr r dr  K m + [S]
Effective diffusivity mole/(s•cm3 support)
= Sb ; negligible film resistance
Boundary Conditions
at r = R, [S] = [Ss ]
d[S]
ar r = 0, =0
dr
33
Diffusional Effects in Enzymes Immobilized

in a Porous Matrix
Dimensional Substrate Mass Balance Equation
[S] r K
S= , r= , β= m
[Ss ] R [Ss ]
 d2 S 2 dS S
 = φ
2
 2+
 dr r dr  1+S/ β
Boundary Conditions
at r = 1, S = 1
dS Vm'' / K m
ar r = 0, =0 φ = R = Thiele Modulus
dr De
34
17
Effectiveness of Immobilized Enzymes
Rate of reaction within matrix (rs) is equal to
the rate of diffusion through matrix surface (Ns)
d[S]
rs = Ns = - 4πR2 De
dr r = R
Vm'' [Ss ]
rs = η
Km + [Ss ]
η = 1, no diffusion limitations
η < 1, diffusion limits reaction rate
3  1 1
η =  -  = effectiveness factor (for β → ∞ and φ → ∞)
φ  tanh φ φ
35
Effectiveness Factor and

Thiele Modulus/Michaelis Constant
36
18
Effectiveness Factor and
Particle Radius/Enzyme Loading
Vm''
37
Overview of Industrial and Medicinal Enzymes

Table 3.6
Major
Industrial
Enzymes
Table 3.6
38
19
Production Statistics of Industrial Enzymes,
(1990)
World Market
Enzyme Type Market ($) Share (%)
Proteases alkaline (detergents) 100 MM 25.0
other alkaline 24 MM 6.0
neutral 48 MM 12.0
animal rennet 26 MM 6.5
microbial rennet 14 MM 3.5
trypsins 12 MM 3.0
other acid proteases 12 MM 3.0
39
Production Statistics of Industrial Enzymes

(cont., 1990)
World Market
Enzyme Market ($) Share (%)
α-amylases 20 MM 5.0
β-amylases 52 MM 13.0
Glucose isomerase (soft drinks) 24 MM 6.0
Pectinase (Juice/Wine Making) 12 MM 3.0
Lipase (Soaps/detergents, cheese..) 12 MM 3.0
All Others 44 MM 11.0
40
20
“Typical” Production of Industrial Enzymes
41
Medicinal Uses of Enzymes
Used for Diagnosis and Therapy
Trypsin and Streptokinase - as antiinflammatory agents

Lysozyme - as an antibiotic for gram-positive cells
Urokinase - as an agent to dissolve blood clots
Asparaginase - an anticancer drug (cancer cells need asparagine)
Glucose oxidase - blood levels; glucose → gluconic acid + H2O2
Tissue Plasminogen Activator (TPA) - dissolves blood clots
42
21
Enzymes and Biosensors
Membrane-bound redox enzymes constitute a large and important class of

enzymes. The cell membrane provides the scaffolding upon which these
enzymes arrange into systems for multi-step catalytic processes. The
reconstruction of portions of this redox catalytic machinery, interfaced to an
electrical circuit, leads to novel sensing devices.
Copyright ©Monbouquette Laboratory, UCLA
43
Enzymes and Biosensors
Copyright © 1995, 1996, 1997, 1998, 1999, 2000 The Nitrate Elimination Co., Inc.; All Rights Reserved
These enzymes are immobilized on "beads" with an electron-carrying

dye. In this formulation, the reduction of nitrate to environmentally safe
nitrogen gas is driven by a low voltage direct current.
44
22
Enzyme Engineering
Bioluminescence a Biomarker for Toxicity of
HPV Chemicals and in Drug Development
Eileen Kim, Ph.D. student, and Cambrex Corporation
62 kDa molecular weight oxygenase

Yellow green light emission at 560 nm
Quantum yield: 88 photon/cycle
Light output proportional to [ATP]
Firefly Luciferase
45
Inhibition of Luciferase
Strong inhibition of Luciferase

120
by chloroform, a High Production
100
Volume (HPV) chemical. This inhibition
limits the assay applications. It is
80 desired to engineer a Luciferase
that is not inhibited by HPV chemicals
% Activity
60
Wild Type Lucif erase
CHCl3MutLuc2
40
20
0
0 0.5 1 1.5 2
Chloroform Conc. (%)
Figure 2. Inhibition of Luciferase Activity by increasing the concentration of Chloroform
Kim et al., AIChE Annual Meeting Presentation Record, November 16, 2003, San Francisco, CA
46
23
Recombinant Luciferase
Source
(firefly) DNA Cloning vector
Luc gene
Fragmentation Enzymatically
Target
linearize Produce protein
DNA (luc)
from cloned gene
Join target DNA
And cloning vector
Protein encoded
Introduce DNA into host cell By cloned gene
DNA
construct Isolate cells with cloned gene
Host cell
47
Inhibition of Luciferase
Inhibition by chloroform is much reduced

120
in the mutant Luciferase compared to the
100
wild type.
80
% Activity
60
Wild Type Lucif erase
CHCl3MutLuc2
40
20
0
0 0.5 1 1.5 2
Chloroform Conc. (%)
Figure 2. Inhibition of Luciferase Activity by increasing the concentration of Chloroform
Kim et al., AIChE Annual Meeting Presentation Record, November 16, 2003, San Francisco, CA
48
24
Chapter 9: Operating Bioreactors
David Shonnard
1
Presentation Outline:
l Choosing Cultivation Methods
l Modifying Batch and Continuous Reactors
l Immobilized Cell Systems
2
1
Choosing the Cultivation Method
The Choice of Bioreactor Affects Many Aspects of

Bioprocessing.
1. Product concentration and purity
2. Degree of substrate conversion
3. Yields of cells and products
4. Capitol cost in a process (>50% total capital expenses)
Further Considerations in Choosing a Bioreactor.

1. Biocatalyst. (immobilized or suspended)
2. Separations and purification processes
3
Batch or Continuous Culture?
These choices represent extremes in bioreactor choices
Productivity →for cell mass or growth-associated products

Batch Culture: assume kd = 0 and qp = 0
rb = rate of cell mass production in 1 batch cycle

Xm - X YM S
rb = = X/S o Exponential growth time
tc tc
1 Xm Lag time
t c = batch cycle time = ln + tl Harvest &
µmax Xo Preparation
4
2
Batch or Continuous Culture? (cont.)
Continuous Culture: assume kd = 0 and qp = 0

rc = rate of cell mass production in continuous culture
rc = Dopt Xopt
dDX KS
set = 0 ⇒ Dopt = µ max (1- )
dD KS + So
KS Dopt
Xopt = YXM/ S (So - ) = YXM/ S (So + KS - (KS (So + KS ) )
µmax - Dopt
KS
DoptXopt = YXM/ S µmax (1- ) (So + KS - (KS (So + KS ) )
KS + So
≈ YXM/S µmax So when KS << So
5
Comparing Rates in Batch and Continuous Culture
rc YXM/S µmax So X
= = ln m + t lµ max
rb  1 X  Xo
YXM/S So /  ln m + t l 
 µmax Xo 
A commercial fermentation with
Xm
= 20, t l = 5 hr, and µ max = 1.0 hr
-1
Xo
rc
= 8 ⇒ Continuous culture method is ~ 10 times
rb more productive for primary products
(biomass & growth associated products
6
3
Why is it that most commercial bioprocess are Batch??
1. Secondary Product Productivity → is > in batch culture

(SPs require very low concentrations of S, S << Sopt)
2. Genetic Instability → makes continuous culture less productive

(revertants are formed and can out-compete highly selected and
and productive strains in continuous culture.)
3. Operability and Reliability

(sterility and equipment reliability > for batch culture)
4. Market Economics
(Batch is flexible → can product many products per year)
7
Most Bioprocesses are Based on Batch Culture

(In terms of number, mostly for secondary, high value products)
High Volume Bioprocesses are Based on Continuous Culture

(mostly for large volume, lower value, growth associated products --
ethanol production, waste treatment, single-cell protein production)
8
4
Modified Bioreactors: Chemostat with Recycle
To keep the cell concentration higher than the normal steady-

state level, cells in the effluent can be recycled back to the
reactor.
Advantages of Cell Recycle
1. Increase productivity for biomass production
2. Increase stability by dampening perturbations of input stream

properties
9
Chemostat with Recycle: Schematic Diagram

Figure 9.1
Mass balance envelope
α = recycle ratio
C = cell concentration ratio
X1 = cell concentration in
reactor effluent
X2 = cell concentration in
effluent from separator
• Centrifuge
“Bioprocess Engineering: • Microfilter
Basic Concepts”
Shuler and Kargi,
Recycle Stream • Settling Tank
Prentice Hall, 2002
10
5
Chemostat with Recycle: Biomass Balance
dX1
FX o + α FCX1 - (1 + α )FX1 + VR µX1 = VR
dt
dX1
at steady - state (= 0) and sterile feed (Xo = 0)
dt
αFCX1 - (1+ α )FX1 + VRµX1 = 0
and solving for µ
µ = [1 + α (1- C)]D
Since C > 1 and α (1- C) < 0, then µ < D
A chemostat can be operated at dilution rates higher than the

specific growth rate when cell recycle is used
11
Chemostat with Recycle: Biomass Balance
µ = [1 + α (1- C)]D
µmax S
Monod Equation, µ =
KS + S
Substitute Monod Eqn. into above, solve for S
KSD(1 + α (1- C))

S =
µmax - D(1 + α (1- C))
12
6
Chemostat with Recycle: Substrate Balance
µX 1 dS
FSo + α FS - (1
+ α )FS - VR M = VR
Y X /S dt
dS
at steady
-state (= 0)
dt
µX 1
FSo + α FS - (1
+ α )FS - VR =0
Y XM/S
and solving 1for
1 X
D M D 1
X1 = YX /S (So -S); But=
µ µ [1+ α (1-C)]
M
Y (So -S)
M
Y X /S  K SD(1+ α (1-C)) 
 µmax - D(1+ α (1-C))
X /S
X1 = = So -
[1+ α (1-C)] [1+ α(1-C)]
13
Chemostat with Recycle: Comparison

Figure 9.2
“Bioprocess Engineering:
Basic Concepts”
Shuler and Kargi,
X1(recycle) Prentice Hall, 2002
=DX2
=DX1
α=0.5, C=2.0, µmax=1.0 hr-1, KS=0.01 g/L, YMX/S=0.5

X1 = cell concentration in reactor effluent with no recycle
X1(recycle) = cell concentration in effluent with recycle
14
7
Multiple Chemostat Systems
Applicable to fermentations in which growth and product

formation need to be separated into stages: .
P1 P2
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
Growth stage Product formation stage
15
Multiple Chemostat Systems (cont.)
1. Genetically Engineered Cells:
Recombinant
DNA
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
Translate to protein product
16
8
Features of Genetically Engineered Cells:
→ have inserted recombinant DNA (plasmids) which allow for the

production of a desired protein product.
→ GE cells grow more slowly than original non-modified strain

(due to the extra metabolic burden of producing product).
→ Genetic Instability causes the GE culture to (slowly) lose ability

to produce product. The non-plasmid carrying cells or the cells
with mutation in the plasmid (revertants) grow faster.
17
Genetically Engineered Cells (cont.):
In the first stage, only cell growth occurs and no inducer is

added for product formation. The GE cells grow at the
maximum rate and are not out-competed in the first chemostat
by revertant cells. When cell concentrations are high, an
inducer is added in the latter (or last) chemostat to produce
product at a very high rate.
18
9
2-Stage Chemostat System Analysis
Stage 1 - cell growth conditions, kd=0, qp=0, steady-state
µmax S1
µ1 = = D1 from biomass balance
KS + S1
KS D1 F
rearranging, S1 = where D1 =
µmax - D1 V1
X1 = YXM/ S (So - S1 ) from substrate balance
19
‘
Stage 2 - product formation conditions, kd=0, F =0, steady-state
dX2
FX1 - FX2 + V2 µ2 X2 = V2 = 0 biomass balance
dt
µ max S2 X F
µ2 = = D2 (1- 1 ) where D2 =
KS + S2 X2 V2
µ 2X2 q PX 2 dS
FS1 - FS2 - V2 M
- V2 = V2 2 = 0 substrate balance
Y X /S YP /S dt
dP2
FP1 - FP2 + V2q PX2 = V2 = 0 product balance
dt
20
10
‘
Stage 2 - product formation conditions, kd=0, F =0, steady-state
µ max S2 X1
µ2 = = D2 (1- ) biomass balance
KS + S2 X2
 µ X q X 
S2 = S1 -  2 M2 + P 2  substrate balance
 D2 YX /S D2 YP/ S 
2 equations, 2 unknowns (S2 ,X2 )
dP2
FP1 - FP2 + V2q PX2 = V2 = 0 product balance
dt
use X2 in product balance to solve for P2
21
Examples of Batch / Continuous Bioreactors:

Ethanol Production from Corn
• 3.5 billion gallons EtOH / yr in the US

• Small distributed plants; , <100 million gallons EtOH/yr
Glacial Lakes Energy, LLC; Watertown, SD
Badger State Ethanol, VeraSun Energy, LLC;

Monroe, WI Aurora, SD
22
11
Examples of Batch / Continuous Bioreactors:
Ethanol Production from Corn
23
Fed-Batch Operation
Useful in Antibiotic Fermentation
→ reactor is fed continuously (or intermittently)

reactor is emptied periodically
→ purpose is to maintain low substrate concentration, S
→ useful in overcoming substrate inhibition or catabolic

repression, so that product formation increases.
24
12
Basic Concepts”
Fed-Batch Operation (cont.) Shuler and Kargi,
Prentice Hall, 2002
Before t = 0, almost all of the substrate, t=0

So, in the initial volume, Vo, is converted
to biomass, Xm, with little product form- t
ation (X=Xm≈YX/SSo) and P≈ 0. i
At t=0, feed is started at a low flow rate n
c
such that substrate is utilized as fast as
r
It enters the reactor. Therefore, S remains e
very low in the reactor and X continues to a
s
maintain at ≈YX/SSo over time. The volume i
increases with time in the reactor and n
g
Product formation continues.
tw cycle time
25
Fed-Batch Operation (cont.)
Behavior of X, S, P, V, and µ over time
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
26
13
Analysis of Fed-Batch Operation
dV
Volume: = F ⇒ V = Vo + Ft
dt
0
0 d(XV) dX dV
Biomass: FX o + VµX = = V +X
dt dt dt
dV 1 dV F
VµX = X ⇒ µ = = = D
dt V dt V
F F Do
µ = = =
V Vo + Ft 1 + Do t
27
Analysis of Fed-Batch Operation (cont.)
Total Biomass: X t (g cells) vs time

 dX t   dV
d t 
X
V − Xt
dX  V  dt   dt 
= 0 or = 2
= 0
dt dt V
dX t X t dV
rearranging = = X m F = YX / SSoF
dt V dt
integrating X t = Xto + YX /SSo Ft = (Vo + Ft)X m
28
14
Analysis of Fed-Batch Operation (cont.)

Product Formation: total product, Pt = PV
For many secondary products, the specific rate of
product formation is a constant = q P
dPt
= q P Xt = q P (Vo + Ft) X m
dt
Ft
integrating, Pt = Pto + q PX m (Vo + )t
2
Po Vo V Dt
or P = + q PX m ( o + )t
V V 2
Po Vo Vo Ft
or P = + q PX m ( + )t
(Vo + Ft) (Vo + Ft) 2(Vo + Ft)
29
Immobilized Cell Systems; 9.4
Restriction of cell mobility within a confined space
Potential Advantages:
1. Provides high cell concentrations per unit of reactor volume.
2. Eliminates the need for costly cell recovery and recycle.
3. May allow very high volumetric productivities.
4. May provide higher product yields, genetic stability, and shear damage
protection.
5. May provide favorable microenvironments such as cell-cell contact,

nutrient-product gradients, and pH gradients resulting in higher yields.
30
15
Immobilized Cell Systems; 9.4
Potential Disadvantages/Problems:
1. If cells are growing (as opposed to being in stationary phase) and/or

evolve gas (CO2), physical disruption of immobilization matrix could
result.
2. Products must be excreted from the cell to be recovered easily.
3. Mass transfer limitations may occur as in immobilized enzyme

systems.
31
Methods of Immobilization
Active Immobilization:
1. Entrapment in a Porous Matrix:
Inert/solid core
cells
Polymeric Beads:
Polymers:
agar, alginate
κ-carrageenan
porous
polyacrylamide
polymer
gelatin, collagen
matrix
32
16
Methods of Immobilization (cont.)
Encapsulation:
hollow spherical particle
liquid core with cells
“less severe mass transfer limitations”
Membrane:
nylon, collodion, semipermeable membrane
polystyrene,
polylysine-alginate hydrogel
Cellulose acetate-ethyl acetate
33
Hollow Fiber Membrane Reactor:

liquid in shell side
shell
nutrients products
nutrients products
tube
semi-permeable membrane
cells
34
17
2. Cell Binding to Inert Supports:

micropores
Micro-porous Supports: dP>4 dC
• cells in
micropores
“mass transfer limitations occur”
porous glass, porous silica, alumina

ceramics, gelatin, activated carbon
Wood chips, poly propylene ion-exchange resins
(DEAE-Sephadex, CMC-), Sepharose
35
Binding Forces:
+
- + ion exchange
Electrostatic Attraction -- --
- - - + support
+
cell
Hydrogen Bonding C-O-
support
|| HO
O
36
18
Adhesion of bacteria to Sand Particles:
Eliminating Electrostatic Repulsion
37
Binding Forces:
Covalent Bonding: (review enzyme covalent bonding)
Support materials: CMC-carbodiimide

support functional groups
-OH, -NH2, -COOH
Binding to proteins on cell surface
38
19
Overview of Active Cell Immobilization Methods:
Adsorption Adsorption
Support Capacity Strength
Porous silica low weak
Wood chips high weak
Ion-exchange resins high moderate
CMC high high
39
Passive Immobilization:
support liquid phase
• wastewater treatment
• mold fermentations Biofilm
• fouling of processing equipment (biopolymer +
polysaccharides)
40
20
Analysis of Biofilm Mass Transfer
Figures 9.1, 9.12
Substrate/product
O2 diffusion in biofilms
diffusion in biofilms
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
41
Analysis of Biofilm Mass Transfer (cont.)
Differential Substrate Balance: Material volume

in biofilm,
Rate of diffusion ∆V = ∆x ∆y ∆z
out through the
∆x Rate of diffusion
area ∆x ∆z
in through the
dS dS area ∆x ∆z
-De ∆x ∆z -De ∆x ∆z
dy dy
y +∆y
y+∆y y ∆z y
∆y
Rate of substrate 1 µmax S
X ∆x ∆y ∆z
consumption in the M
YX / S KS + S
volume ∆V = ∆x ∆y ∆z
(due to cell growth, or 1 qp S
X ∆x ∆y ∆z
product formation) YP /S KS + S
42
21
Differential Substrate Balance at Steady-State:
Rate of diffusion Rate of diffusion Rate of substrate

in through the
area ∆x ∆z
- out through the
area ∆x ∆z
- consumption in the
volume ∆V = ∆x ∆y ∆z
=0
dS  dS  1 µmax S
-De ∆x ∆z -  -De ∆x ∆z - M X ∆x ∆y ∆z = 0
dy y  dy y+ ∆y  YX / S KS + S
Divide through by ∆x ∆y ∆z and switch order of first 2 terms

dS dS
De - De
dy y +∆y
dy y 1 µ max S
- X = 0
∆y YXM/S KS + S
43
Differential Substrate Balance at Steady-State:
1 µmax S
2
dS
De = X eqn 9.49
dy 2 YXM/ S KS + S
Boundary Conditions
S = Soi at y = 0 (at the biofilm / liquid interface)
dS
= 0, at y = L (at the biofilm / support interface)
dy
44
22
Dimensionless Substrate Balance at Steady-State:

2
d S φ S 2
A numerical
= eqn 9.51
dy 2 1+ β S solution is
S y So required
where S= , y= , β= ,
So L KS
µ maxX Analytical
and φ = L M "Thiele Modulus"
solution is
Y De KS X/S
possible for
Boundary Conditions 0 order and
S = 1 at y = 0 (at the biofilm / liquid interface) 1st order
kinetics
dS
= 0, at y = 1 (at the biofilm / support interface)
dy
45
Zero Order Substrate Consumption Kinetics:
d 2S φ2 S
= , for β >> 1, and φ < 1
dy 2
1+ β S
d 2S φ2
= zero - order substrate consumption kinetics
dy 2 β
d dS φ2  dS φ2
dy dy
=
β
⇒ ∫ d dy  = ∫ β
dy
dS φ2
= y + C1
dy β
46
23

dS
Boundary condition # 2, at y = 1, = 0
dy
φ2 φ2
0= (1) + C1 ⇒ C1 = -
β β
dS φ2 φ2  φ2 φ2 
dy
=
β
y -
β
integrate again, ∫ dS = ∫  β y -
β 
dy
φ2 2 φ2
S= y - y + C2
2β β
47
Boundary condition #1, at y = 0, S = 1

φ2 2 φ2
1= (0 ) - (0) + C2 ⇒ C2 = 1
β β
φ2 2 φ2 φ2  y 2 
S= y - y + 1 or S=  - y + 1
2β β β 2 
φ2
for << 1
β
48
24
Biofilm Effectiveness
The effectiveness factor is calculated by dividing the rate of substrate

diffusion into the biofilm by the maximum substrate consumption rate.
Solve for the Effectiveness Factor, η
dS  µ S X 
NS AS = - ASDe = η  M max o  (A L)
dy y =0
 YX / S (KS + So )  S
Rate of substrate Volumetric rate of

diffusion into biofilm substrate consumption
through an area AS at within the biofilm in a
the surface at y = 0 volume (ASL)
49
Effectiveness Factor
Biofilm is most
effective for β >>1
η increases as φ
decreases for any
value of β
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
50
25
Spherical Particle of Immobilized Cells
Figure 9.14
“Bioprocess Engineering: VP is particle volume

Basic Concepts”
Shuler and Kargi,
AP is particle area
Prentice Hall, 2002
51
Analysis of Mass Transfer in Spherical Particle
Dimensionless Substrate Balance at Steady-State:

2
d S 2 dS φ S 2
+ = eqn 9.58
dr 2 r dr 1+ β S
S r So
where S= , r= , β= ,
So R KS
µ maxX
and φ = R "Thiele Modulus"
YXM/ SDe KS
Boundary Conditions
S = 1 at r = 1 (at the particle / liquid interface)
dS
= 0, at r = 0 (at the particle center)
dr
52
26
Particle Effectiveness
If all of the particle cells “see” substrate at a concentration So or high

enough to grow maximally, then the particle is said to have an
effectiveness of 1.
Rate of S consumption by a single particle
dS  µ S X 
NS AP = - APDe = η  M max o  VP
dr r=R  YX /S (KS + So ) 
Rate of substrate Volumetric rate of

diffusion into particle substrate consumption
through an area AP at within the particle in a
the surface at r = R volume (VP)
53
Particle Effectiveness (cont.)
Equation 9.58 can be solved analytically for limiting cases:
Case 1, for So<<KS (very dilute substrate)
1  1 1
η = −
φ  tanh 3φ 3φ 
VP µmax X
φ = M
"Thiele Modulus"
AP YX /SDeKS
54
27
Case 2, for So>>KS (very concentrated substrate)
 d2 S 2 dS  µ maxS µ X
De  2 +  = X = max
 dr r dr  Y M
X/S (K S + S) YXM/ S
Boundary Conditions
S = So at r = R (at the particle / liquid interface)
dS
= 0, at r = 0 (at the particle center)
dr
55
Case 2, for So>>KS

Use a variable transformation, S=S’/r
2
1 d S' µ maxX At a critical radius (rcr ), S = 0
2
= M
r dr YX /SDe µmax X 2 2
0 = So - M (R - rcr )
Solution for S is; 6 YX /SDe
µ maxX  rcr 
2
6 De So YXM/ S
S = So - (R2 - r 2 ) = 1 -
 R
M
6Y X/S De µmax X R 2
56
28
Case 2, for So>>KS
µmax X 4
π (R3 - rcr3 ) 3
YXM/ S 3 r 
η = µ maxX 4 = 1- cr
R 
M π R3
YX /S 3
or
3
 6 De So YXM/ S  2
η = 1- 1 - 
 µmax X R 2 
57
Bioreactors Using Immobilized Cells

Figure 9.15
The single particle analysis for η can be used in the analysis of

bioreactors having immobilized cells: “Bioprocess Engineering:
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
Consider a plug flow reactor filled with immobilized cell particles
58
29
Bioreactors Using Immobilized Cells (cont.)
A differential balance on a thin slice of particles within the reactor:
So differential
volume element F, So-dSo
z+dz
H
z
z
F, So
0 FSo z - FSo z +dz = N S a A dz

Rate of mass
Rate of Rate of
Soi transfer into
substrate flow - substrate flow =
particles within
into element out of element
element
59
Using the definition of η:
 µ S X 
NS AP = η  M max o  VP
 YX /S (KS + So ) 
dSo  µ S X   VP 
-F = η  M max o   a A
dz  YX / S (KS + So )   A P 
2 3
where a = surface area of particle per unit volume of bed (cm / cm bed)
A = cross - sectional area of the bed (cm2 )
60
30
At z = 0, So = Soi: integrating assuming η is constant
S   µ V X a A
KS ln oi  + (Soi - So ) = η  maxM P H
 So   YX /S F A P 
for low substrate concentration (Soi << KS )
S   µ VP X a A 
ln o  = - η  max H
 Soi   YXM/S F A P KS 
V 
note x = x  P  a (average cell mass conc. in the bed)
 AP 
61
31
Chapter 10:
Sterilization and Bioreactor Operation
David Shonnard
1
Sterilization Methods and Kinetics: 10.4
Sterility: the absence of detectable levels of viable organisms in a

culture medium or in a gas
Reasons for Sterilization

1. Economic penalty is high for loss of sterility
2. Many fermentations must be absolutely devoid of foreign
organisms
3. Vaccines must have only killed viruses
4. Recombinant DNA fermentations - exit streams must be
sterilized
2
1
Sterilization Agents
1. Thermal - preferred for economical large-scale sterilizations of

liquids and equipment.
2. Chemical - preferred for heat-sensitive equipment
→ ethylene oxide (gas) for equipment
→ 70% ethanol-water (pH=2) for equipment/surfaces
→ 3% sodium hypochlorite for equipment
3. Radiation - uv for surfaces, x-rays for liquids (costly/safety)
4. Filtration
→ membrane filters having uniform micropores
→ depth filters of glass wool
3
Kinetics of Thermal Sterilization (Death)
Practical considerations:
1. Not all organisms have identical death kinetics.
→ (increasing difficulty; vegetative cells < spores < virus)
2. Individuals within a population of the same organism may
respond differently
From Probability Theory:
p(t) = the probability that an individual is still viable at time t.
-kdt
p(t)= e (simplest form assuming 1st ord
4
2
Kinetics of Thermal Sterilization (cont.)
E[N(t)]= expected value (E) of the numb

of individual organis
at time t after sterilization starts.
-k t
= No p(t)= No e d
where o is Nthe initial number of individuals
N(t) -k t N(t)
= ed or = -kd t survival
ln " cur
No No
5
Temperature Effects on the

Kinetics of Thermal Sterilization
increasing
Arrhenius Equation
-kd
-E od / RT
kd = α e N(t
ln
α = constant (time ) -1 No
R = gas constant
T = absolute temperature
E od = activation energy for death t
(50 -150 kcal / g - mole) for spores
(2 - 20 kcal / g - mole) for vitamins / growth factors
6
3
Population Effects on the
Kinetics of Thermal Sterilization
Most Thermal Sterilizations are at 121˚C
Organism kd (min-1)
Vegetative cells >1010
Spores 0.5 to 5.0
Spores are the primary concern during thermal sterilization
7
System Variables for Thermal Sterilization
Primary System Variables in Thermal Sterilization

1. Initial concentration of organisms
2. Temperature, T
3. Time (t) of exposure at temperature T.
[1
-Po(t)
Probability of an Unsuccessful Ferme
No
[1-Po(t)]= 1-[1-p(t)]
-kdt N o
= 1-[1-e ] (for a homogeneous popul
8
4
Sterilization Chart
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
9
System Variables for Thermal Sterilization
Use of Sterilization Charts:
1. Specify 1-Po(t) which is acceptable (e.g. 10-3)
2. Determine No in the system.
3. Read kdt from the chart.
4. Knowing kd for the spores (or cells), obtain the required time, t.
10
5
Scale-up of Sterilization
→ in batch sterilization, scale-up of small-scale sterilization data

to a much larger scale will result in unsuccessful sterilization
4
1-Liter Vessel -L Vessel 10
no = 10 spores
/L4 15 4
= 10 spores
/L
n 15
o
4
N o = (1 L)(n
o) o = (10 L)(n
o) N
-kd t no -kd t 104 no
[1-Po(t)]= 1-[1-e ] = 1-[1-e
-Po(t)][1 ]
= .003 = 1-5x10-14
≈ 1
11
Batch vs. Continuous Sterilization
Batch
1. Longer heat-up/cool down time
2. Incomplete mixing
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
12
6
Batch vs. Continuous Sterilization
Continuous 1. Shorter time

2. Higher temperature
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
13
Sterilization of Gases
→ aerobic fermentations require 0.1 to 1.0 (L air / (L liquid • min))

→ 50,000 L fermenter requires 7x106 to 7x107 L air/day
→ microorganism concentrations in air are about 1-10 / L air
Methods for Air Sterilization at Inlet Exit gas must

1. Adiabatic compression, 220˚C for 30 seconds be filtered
2. Continuous Filtration: → pathogenic

→ depth filters (glass wool filters) → recombinant
→ surface filters (membrane cartridges) DNA cells
3. Economics ≈ 25% of production costs for air system
14
7
Design and Operation of Bioreactors
Types of Bioreactors
1. Reactors with Mechanical Agitation see Fig. 10.1A
a) disperse gas bubbles throughout tank
b) increase residence time of bubbles
c) shear large bubbles to smaller bubbles
d) disk type or turbine type (dI ≈ 0.3 dT) see Fig. 10.3
e) provide high kLa values
f) baffles (4) augment mixing (≈ 0.1 dT)
2. Bubble Column see Fig. 10.1B

a) disperse gas bubbles throughout tank
b) perforated plates enhance gas dispersion and mixing
15
Figure 10.1A
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
16
8
Figure 10.3
(1st Edition)
Rushton
Impeller
Axial flow (Disk-type)
hydrofoil
Impeller
• lower energy demand
• comparable gas transfer
• superior axial mixing
• lower shear stress
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
17
Design and Operation of Bioreactors (cont.)
Types of Bioreactors
3. Loop Reactors see Fig. 10.1 C, D, E
a) bubble rising in draft tube causes mixing
b) mixing enhanced by an impeller or a jet pump
Materials of Construction:
Glass Vessels: Volume < 500 Liters
Stainless Steel Vessels: All Volumes
316 ss for vessel
314 ss for covers & jackets
18
9
Figure 10.1B
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
19
Reactor Geometry and Layout
Figure 10.2:
a) height to diameter ratio of 2 to 3

b) sterile air inlet and sparger
c) baffle plates & impellers
d) cooling coils
e) foam breaker
f) working volume (liquid capacity) ≈ 0.75 vessel volume
20
10
Figure 10.2
Height to Diameter
Ratio of 2 - 3
VL ≈ 0.75 VR
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
21
Reactor Types in Industry
Nonstirred/Nonaerated Vessels:
a) most fermentations in terms of total volume
b) food fermentations (beer, wine, diary products)
Stirred / and (or) Aerated Vessels:

a) most fermentations in terms of numbers of units
22
11
Aeration and Heat Transfer
Aeration and heat transfer requirements often limit the

design of commercial reactors
A multiplication sign
Aeration Design Equation: (OUR = OTR)
Oxygen Uptake Rate: OUR = X qO

2
X = cell concentration (g cells / L) - ranges from 1 to 5

q O 2 = specific O 2 uptake rate (Yield) [mmol O 2 / (g cells hr)]
(2 to 90; bacteria, yeast, molds)
23
Table 10.1
Basic Concepts”
Shuler and Kargi,
Prentice Hall, 2002
24
12
Oxygen Transfer Rate
Oxygen Transfer Rate: OTR = k L a (C * -C)

k La = volumetric mass transfer coefficient (hr-1 )
C * = O2 concentration in water at the bubble / water interface
PO partial pressure of O2 in air (Pa)
≅ 2
=
HO Henry's Law Constant for O2 (Pa / (mole O 2 / L))
2
C L = O2 concentration in the bulk water (mole O2 / L)
→ temperature, pressure, & salt concentration affect C*

→ vessel geometry, operation, and fluid properties affect kLa
25
kLa for Stirred Tanks
Oxygen Transfer Rate: OTR = k L a (C * -C)

0.4
 Pg 
k La = k   (vS )0.5 (N) 0.5 see equation 10.2a
 VR 
k = empirical constant (fluid and reactor - specific)
Pg = power requirement for an aerated bioreactor
Units
VR = bioreactor volume depend
vS = superficial gas exit speed = (Fa / A) upon
correlation
Fa = volumetric flow rate of air
data
A = bioreactor cross - sectional area
N = impeller rotation speed
26
13
Pg Correlation
0.45
 P2 N D3  Pg  Q 
Pg = K  u 0.56 i  or = f a3
 Qa  Pu  N Di 
 Q 
N A = aeration number =  a 3 
 N Di 
K = emperical constant (reactor geometry - specific)
Units
Pu = power requirement for an ungassed bioreactor depend
Di = impeller diameter upon
correlation
Qa = aeration rate = (Fa / VR ) data
27
“Biochemical Engineering”
Blanch and Clark,
Pu Correlation Marcel Dekker, 1997
(Figure 5.20 of Blanch and Clark)
28
14
“Biochemical Engineering”
Blanch and Clark,
NA Correlation Marcel Dekker, 1997
(Figure 5.22 of Blanch and Clark)
29
Example Problem
A 10, 000 liter (of liquid) bioreactor contains 5 g / L of growing cells

q O2 = 20 mmole O 2 / (g cells hr)
D T = 2 m, D i = 1 m, (6 - blade turbine agitator) x 3 blades
For 1 liquid volume per minute aeration rate (air), can the
OTR = OUR for N = 100 rpm?
30
15
Example Problem Solution
Pu : power requirement for ungassed reactor

ρL N D 2i
Re = Reynold's Number =
µL
ρ L = 1,000 kg / m3 µL = 10-3 Newton s / m2
 1,000 kg   100 s-1 (12 m2 )1 Newton 
 
 m3   60   (kg m / s2 ) 
Re =
−3 Newton s
10 2
m
= 1.67x106
31
Example Problem Solution (cont.)
From Figure 5.20 of Blanch and Clark

Pu
Power number = 4 =
ρ L N3 D5i
Pu = 4 ( ρ L N3 D5i ) for 1 impeller
 kg   100 -1 5 5  2 2
4 kg m / s
= 4 1,000 3  s  (1 m ) = 1.852x10 (Watts)
 m  60  s
 2
4 kg m / s
2
 4
Pu (3 impellers) = 31.852x10 (Watts) = 5.62x10 Watts
 s 
= 74.5 HP
32
16
Qa
Pg : NA (aeration no.) = 3
N Di
 liters   −3 m3 

10,000  10 
min   liter 
NA = = 0.10
(100 min -1 )(1 m)3
From Figure 5.22
Pg
= 0.42 ⇒ Pg = (.42)(5.56x10 4 Watts)
Pu
= 2.335x104 Watts
= 31.3 HP
33
0.4
 Pg  HP   0.5 0.5
k La (mmole O2 / (l hr atm) = 0.60 

(v S ) (N(rpm))
 3
 VR 10 liters 
Pg 31.3 HP HP
= = 3.13 3
VR (10)(103 liters) 10 liters
 cm3 
10 4 liters / min  10 3 
 liter  cm
vS = = 318.3
π 2 2 cm 2 min
(2 m) (10 )
4 m
k La = 0.60(3.13)0.4 (318.3)0.5 (200)0.5 = 169 (mmole O2 / (l hr atm)
34
17
 g cells   mmoles O2 
 liter   
OUR = X q O = 5 20
2 g cells hr 
mmoles O2
= 100
liter hr
OTR = k La(PO - P*)

2
mg O 2
P * for CL = 1 = H O CL
liter 2
 
 0.21 atm   mg O 2 
= 1 = 0.0263 atm
 mg O2   liter 
8 
 liter 
35
OTR = k La(PO - P*)

2
mmoles O 2
= 169 (0.21 − 0.0263) atm
liter hr atm
mmoles O 2
= 31.05
liter hr
Since OUR > OTR, we must modify the bioreactor operation

in order to bring them into balance
• increase N
• use pure O2 rather than air.
36
18
Basic Concepts”
Measurement of OTR Shuler and Kargi,
Prentice Hall, 2002
N2 O2
∞ ∞
CL
N2 O2
t=0
dC L
= kLa(C*-CL )
dt
t = 0,CL = 0
ln(C
* -CL ) = -(kL a)
37
Heat Generation Rate: Aerobic Growth
Q GR ≈ 0.12 (OUR
 kca   mmol O2 
 L •hr  L •hr 
38
19
Heat Generation Rate: Agitation
Pg (power input aerated

Q agit =
VR (working volume of r
 1 hp
≈ 
100 ga

39
Heat Balance
HRR (Heat Removal Rate) = U A ∆TLM

U = overall heat transfer coefficient
A = surface area of heat transfer surface
∆TLM = log mean temperature difference between
the bioreactor fluid and cooling fluid
(T - t1 ) − (T - t 2 )
=
ln[(T - t1 ) − (T - t 2 )]
T = bioreactor fluid temperature
t1 = cooling water inlet temperature
t 2 = cooling water outlet temperature
40
20

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Chapter 3: Enzymes

l Kinetics of Enzyme-Catalyzed Reactions

l Effects of Environmental Conditions on Kinetics

l Inhibition of Enzyme Catalyzed Reactions

l Immobilized Enzyme Systems

l Industrial Uses of Enzymes

“Bioprocess Engineering: Basic Concepts

Introduction to Enzymes (3.1)

• over 2,000 known enzymes

How Enzymes Work (3.2)

Lower the activation energy of enzyme-substrate complex

Lock and Key Model Enzyme-

Substrate Enzyme- Product

Relate product reaction rate to measurable quantities

Enzyme Kinetics (3.3)

Mass Balance Equations

Rapid Equilibrium Assumption (formation of ES is rapid)

Combine K’m equation with E balance equation

Michaelis-Menten Kinetics (cont.)

→ determine rate (slope) from measured data

Plotting Experimental Data

Double-Reciprocal Plot (Lineweaver-Burk Plot, eqn. 3.13)

Hanes-Woolf Plot (eqn. 3.15)

Complex Enzyme Kinetics: Inhibition

A) Competitive Inhibition (eqn. 3.20 - 3.23)

• K'm, app → net effect of I is to increase K’m

• overcome inhibition by increasing [S]

B) Noncompetitive Inhibition (eqn. 3.24 - 3.27)

More Inhibition Kinetics (eqn. 3.28 - 3.38)

C) Uncompetitive Inhibition • have similar mechanisms

Temperature Effects on Enzyme Kinetics

The rate of enzyme conversion of substrate will increase with

E + H+ “Bioprocess Engineering: Basic Concepts,

K 'm,app → ∞ at high and low [H+ ] 19

Immobilized Enzyme Systems

Methods of Enzyme Immobilization

Matrices for Entrapment

Surface Immobilization: Adsorption

Adsorption: Attachment of enzymes to stationary solids by

Solid Support Materials:

Covalent Bonding: The retention of enzyme on support

Functional Groups on Enzymes:

• Amino (protein-NH2) • Carboxyl (protein-COOH)

Active site of enzyme must not participate in covalent

Surface Immobilization: Support Bonding

Diffusional limitations are observed to various degrees in all

EL, Enzyme Loading (mg enzyme/cm2)

Diffusional Effects on Surface-Bound Enzymes

Graphical Solution to Eqn. 3.53

(A) is reaction kinetics

(B) is mass transfer rate

Intersection is solution for [Ss ]

Figure 3.18 is useful for

Enzymes within a porous matrix

 d2 [S] 2 d[S] Vm' '[S]

Diffusional Effects in Enzymes Immobilized

Dimensional Substrate Mass Balance Equation

Effectiveness Factor and

Overview of Industrial and Medicinal Enzymes

Production Statistics of Industrial Enzymes

Medicinal Uses of Enzymes

Used for Diagnosis and Therapy

Trypsin and Streptokinase - as antiinflammatory agents

Membrane-bound redox enzymes constitute a large and important class of