Lecture: Non-elementary Reaction Mechanisms & Bioreactors

David J. Keffer
Department of Chemical and Biomolecular Engineering
The University of Tennessee, Knoxville
dkeffer@utk.edu
last updated: November 16, 2011

This lecture is coordinated with Chapter 7 of Fogler.

Active Intermediates and Nonelementary Rate Laws

Some examples of nonelementary rate laws

rate laws with non-integer powers

CH3CHO  CH4 + CO

rate = k[CH3CHO]3/2

rate laws with a quotient

H2 + Br2  2HBr

3/ 2
k1CH 2 CBr
rate  2

CHBr  k 2CBr2

Rate laws of this form usually involve an active intermediate.

An active intermediate is a high-energy molecule that reacts virtually as fast as it is formed. As a

result, it is present in very small concentrations. Frequently, active intermediates are formed
during a high energy collision in which translational kinetic energy is converted into kinetic
energy associated with an internal degree of freedom, such as a vibrational mode.

A + M  A* + M

Pseudo-Steady-State Hypothesis

The active intermediate reacts as quickly as it is formed.

nr
rA*   ri , A*  0
i 1

1
example: decomposition of Azomethane (AZO)

(CH3)2N2  C2H6 + N2

mechanism

creation of an active intermediate

reaction 1: (CH3)2N2 + (CH3)2N2  (CH3)2N2 + (CH3)2N2*

annihilation of an active intermediate (reverse of reaction 1)

reaction 2: (CH3)2N2* + (CH3)2N2  (CH3)2N2 + (CH3)2N2

decomposition of an active intermediate

reaction 3: (CH3)2N2*  C2H6 + N2

Each step in the mechanism is an elementary reaction.

r1  k1C AZO
2

r2  k2C AZOC AZO*

r3  k3C AZO*

Write a mole balance on the active intermediate.

accumulation = in – out + generation

There are no in or out terms. The PSSH dictates that the accumulation is zero, so we have

0  r1  r2  r3  k1C AZO  k 2C AZOC AZO*  k3C AZO*

2

Solve for the concentration of the active intermediate.

2
k1C AZO
C AZO* 
k 2C AZO  k3

The rate of production of ethane is thus

2
k3k1C AZO
r3  k3C AZO* 
k 2C AZO  k3

2
At very low concentrations of AZO, k 2C AZO  k3 , so the observed rate is

r3  k1C AZO
2

At high concentrations of AZO, k 2C AZO  k3 , so the observed rate is

k3 k1
r3  C AZO
k2

In general

mechanism

creation of an active intermediate (M is any molecule)

reaction 1: A + M  M + A*

annihilation of an active intermediate (reverse of reaction 1)

reaction 2: A* + M  A + M

decomposition of an active intermediate (P is product(s))

reaction 3: A*  P

Each step in the mechanism is an elementary reaction.

r1  k1C ACM

r2  k2CM C A*

r3  k3C A*

active intermediate mole balance

0  r1  r2  r3  k1C ACM  k2CM C A*  k3C A*

Solve for the concentration of the active intermediate.

k1C ACM
C A* 
k 2CM  k3

3
Because the concentration of all molecules (M) inert or otherwise is constant, we observe the rate
of production as

k3k1C ACM
r3  k3C A*   kC A
k 2 CM  k3

This appears to be first order but it is not an elementary reaction.

Chain Reactions

Chain reactions has a sequence of steps

1. initiation: formation of the active intermediate
2. propagation or chain transfer: interaction of an active intermediate with reactant or
product to form another active intermediate
3. termination: deactivation of the active intermediate to form products

example: thermal cracking of ethane

step 1: initiation
k1
C 2 H 6  2CH 3 r1  k1CC2 H 6

step 2: propagation:
k2
C2 H 6  CH 3  CH 4  C2 H 5 r2  k 2CC2 H 6 CCH 
3

k3
C2 H 5  C2 H 4  H  r3  k3CC H 
2 5

k4
C2 H 6  H   C2 H 5  H 2 r4  k 4CC2 H 6 C H 

step 3: termination:
k5
2C2 H 5  C4 H 10 r5  k5CC H 
2
2 5

mole balances on the active intermediates

nr
0   i , ri
i 1

4
 nr
H●: 0   i ,H  ri  0  r1  0  r2  1  r3  1  r4  0  r5  r3  r4
i 1

nr
CH3●: 0   i ,CH  ri  1  r1  1  r2  0  r3  0  r4  0  r5  2r1  r2
3
i 1

nr
C2H5●: 0    ri  0  r1  1  r2  1  r3  1  r4  2  r5  r2  r3  r4  r5
i ,C2 H 5
i 1

Substitute in the values for the rates

H●: 0  k 3C C H   k 4 C C 2 H 6 C H 
2 5

CH3●: 0  2k1CC2 H 6  k 2CC2 H 6 CCH 

3

C2H5●: 0  k 2CC2 H 6 CCH   k3CC H   k 4CC2 H 6 C H   k5CC H 

2
3 2 5 2 5

Solve:

k1
CH3●: CCH   2
3
k2

k3 CC2 H 5
H●: CH  
k 4 CC2 H 6

Substitute in for CH3●: and H● in the balance for C2H5●:

C2H5●: 0  2k1CC2 H 6  k3CC H   k3CC H   k5CC H 

2
2 5 2 5 2 5

Solve

k1
C2H5●: CC H   2 CC H
2 5
k5 2 6

k3 k1 1
H●: CH   2
k4 k5 CC2 H 6

5
The rate of disappearance of ethane is

r1  r2  r4  k1CC2 H 6  k 2CC2 H 6 CCH   k 4CC2 H 6 C H 

3

k1 CC2 H 6
r1  r2  r4  k1CC2 H 6  2k1CC2 H 6  k3 2
k 5 CC2 H 6

k1
r1  r2  r4  3k1CC2 H 6  k3 2 C C2 H 6
k5

The rate of production of C2H4 is

k1
r3  k3CC H   k3 2 CC H
2 5
k5 2 6

The rate of production of C4H10 is

r5  k5CC H   2k1CC2 H 6
2
2 5

Reaction Pathways

See Figures 7-2, 7-3 and 7-4 on pp. 391-393

Numerical Example

Provided below are two files that look at the same basic reaction using an active intermediate.
The first version of the file does not invoke the PSSH. The second version of the file does
invoke the PSSH. The purpose of including the files is several fold.

1. The first file, without using PSSH, shows that the same approach we have been using can be
used to model systems of reactions that result in non-elementary rate laws for the composite
reaction.

2. How one changes the calculations (or equivalently the code) to account for the PSSH.

3. The goodness of the PSSH is a function of the rate parameters, k1, k2 and k3. For the values
given, the results between the two versions of the code are similar. As one narrows the gap
between the slow reaction (k1) and the fast reactions (k2 and k3), one can see the goodness of
the PSSH approximation deteriorate.

6
Active Intermediate Example Input File without PSSH

function dydt = sysodeinput(x,y,nvec);

%
% active intermediates
% A + M --> A* + M
% A* + M --> A + M
% A* --> P
%
% A = A
% B = M
% C = A*
% D = P
%
% sample usage:
% [y,x]=sysode(2,2000,0,400,[10,40,0,0]);
%
CA = y(1); % mol/liter
CB = y(2);
CC = y(3);
CD = y(4);
%
% stoichiometry
%
nuA1 = -1;
nuB1 = 0;
nuC1 = 1;
nuD1 = 0;
%
nuA2 = 1;
nuB2 = 0;
nuC2 = -1;
nuD2 = 0;
%
nuA3 = 0;
nuB3 = 0;
nuC3 = -1;
nuD3 = 1;

%
% rate laws
%
k1 = 1.0e-3; % liters/mole/sec
r1 = k1*CA*CB; % mole/liter/sec
%
k2 = 1.0e-1; % liters/mole/sec
r2 = k2*CB*CC; % mole/liter/sec
%
k3 = 1.0e-0; % liters/mole/sec
r3 = k3*CC; % mole/liter/sec
%
% mole balances
%
dydt(1) = nuA1*r1 + nuA2*r2 + nuA3*r3;
dydt(2) = nuB1*r1 + nuB2*r2 + nuB3*r3;
dydt(3) = nuC1*r1 + nuC2*r2 + nuC3*r3;
dydt(4) = nuD1*r1 + nuD2*r2 + nuD3*r3;

7
Active Intermediate Example Input File with PSSH

function dydt = sysodeinput(x,y,nvec);

%
% active intermediates
% A + M --> A* + M
% A* + M --> A + M
% A* --> P
%
% A = A
% B = M
% C = P
% A* is determined from PSSH
%
% sample usage:
% [y,x]=sysode(2,2000,0,400,[10,40,0]);
%
CA = y(1); % mol/liter
CB = y(2);
CC = y(3);
%
% stoichiometry
%
nuA1 = -1;
nuB1 = 0;
nuC1 = 0;
%
nuA2 = 1;
nuB2 = 0;
nuC2 = 0;
%
nuA3 = 0;
nuB3 = 0;
nuC3 = 1;
%
% rate constants
%
k1 = 1.0e-3; % liters/mole/sec
k2 = 1.0e-1; % liters/mole/sec
k3 = 1.0e-0; % liters/mole/sec
%
% PSSH expression for concentration of active intermediate
%
CAstar = k1*CA*CB/(k2*CB+k3);
%
% rate laws
%
r1 = k1*CA*CB; % mole/liter/sec
r2 = k2*CB*CAstar; % mole/liter/sec
r3 = k3*CAstar; % mole/liter/sec
%
% mole balances
%
dydt(1) = nuA1*r1 + nuA2*r2 + nuA3*r3;
dydt(2) = nuB1*r1 + nuB2*r2 + nuB3*r3;
dydt(3) = nuC1*r1 + nuC2*r2 + nuC3*r3;

8
Enzymatic Reaction Fundamentals

overall reaction: S = substrate, P = product

SP

reaction pathway E = enzyme

ES  ES  EP

enzymes are specific. Typically, one enzyme catalyze only one type of reaction.
Therefore, unwanted by-products of side-reactions are usually avoided in enzymatic reactions.

Enzyme-Substrate Complex

enzyme have a specific active site which binds the substrate to form the enzyme-substrate
complex.

If the enzyme is exposed to extreme temperatures or extreme pH, it may unfold, losing its active
sites, becoming denatured.

two popular models

lock-and-key (older but less preferred today)
induced fit
See Figure on page 396.

Six and only six classes of enzymes

1. oxidoreductases AH2 + B + E  A + BH2 + E reducing A

2. transferases AB + C + E  AC + B + E replacing B with C on A
3. hydrolases AB + H2O + E  AH + BOH + E splitting water in H and OH
4. isomerases A + E  isoA + E forming an isomer
5. lyases AB + E  A + B + E splitting a molecule
6. ligases A + B + E  AB + E joining two molecules

Enzymatic Reaction Mechanisms

example
substrate: S = urea
enzyme: E = urease
product : P = ammonia and carbon dioxide

reaction 1: formation of the enzymatic complex

k1
S  E  S  E* r1  k1C S C E

9
reaction 2: reverse of reaction 1
k2
S  E*  S  E r2  k 2C S E*

reaction 3: product formation

k3
S  E *  H 2O  2 NH 3  CO2  E r3  k3C S E* C H 2O

The challenge is that we can only measure the total E concentration.

Et  E  S  E *

A mole balance on the enzymatic species yields

nr
S  E* : 0   i ,S E* ri  1  r1  1  r2  1  r3  r1  r2  r3
i 1

S  E* : 0  k1C S C E  k 2C S E*  k3CS E* C H 2O

Solve for the concentration of the enzymatic complex

k1CS C E
S  E* : C S E* 
k 2  k3C H 2O

the rate of disappearance of substrate is given by a mole balance on S

nr
dCS
S:   i ,S ri  1  r1  1  r2  r1  r2
dt i 1

dCS
S:  k1CS C E  k 2C S E*
dt

dCS k1C S C E  k2 
S:  k1C S C E  k 2  k1   1CS C E
dt k 2  k 3 C H 2O  k 2  k 3C H O 
 2 

This expression is no good in a practical sense, since we can only measure the total enzyme
concentration.

k1CS CE
CEt  C E  CS E*  C E 
k 2  k3C H 2O

10
 1 k 2  k3C H 2O
C E  C Et  C Et
1
k1CS k1CS  k 2  k3C H 2O
k 2  k 3 C H 2O

Substituting this into the balance for the substrate yields

dCS  k2  k 2  k 3 C H 2O
 k1   1 CC
dt  k 2  k 3C H O  k1CS  k 2  k3C H O S Et
 2  2

k1k3
 CS C Et C H 2O
k1CS  k 2  k3C H 2O

Michaelis-Menton Equation

The reaction is carried out in aqueous solution, so water is in excess and can be considered
constant.

kcat  k3C H 2O

kcat  k 2
KM 
k1

Putting these new definitions into the balance for the substrate yields

dCS kcat
 CS C Et
dt CS  K M

The turnover number is given by k cat . This is the number of substrates converted to product per
unit time. For example, for the enzyme catalase with the substrate H2O2, the turnover number is
40x106 s-1. Forty million hydrogen peroxide molecules are decomposed by this enzyme every
second.

The constant KM is the Michaelis constant. It is a measure of the attraction of the enzyme for its
substrate. It is also called the affinity constant. For the example above, the Michaelis constant is
1.1 M.

If we define another variable, Vmax, which represent the maximum rate of reaction for a given
total enzyme concentration,

Vmax  kcat C Et

We have

11
 dCS V C
  max S
dt CS  K M

This is called the Michaelis-Menten equation.

See Figure 7-6 on page 400.

At low concentrations of substrate, C S  K M ,

dCS V C
  max S
dt KM

The reaction is apparently first order in the substrate.

At high concentrations of substrate, C S  K M ,

dCS V C
  max S  Vmax
dt CS

The reaction is apparently zeroth order in the substrate.

dCS V
Furthermore, K M is the concentration that yields   max .
dt 2

Michaelis-Menten plot is –rate vs concentration of substrate. (See Figure 7.6)

dCS V C
  max S
dt CS  K M

Lineweaver-Burk plot is -1/rate vs 1/CS. Invert equation above. (See Figure E7-3.1) Straight
line.

1 C  KM 1 K 1
  S   M
dC S Vmax C S Vmax Vmax C S
dt

So you can get Vmax from the intercept and Km from the slope.

12
If the generation of product is reversible:

reaction 1: formation of the enzymatic complex

k1
S  E  S  E* r1  k1C S C E

reaction 2: reverse of reaction 1

k2
S  E*  S  E r2  k 2C S E*

reaction 3: product formation

k3
S  E *  H 2O  P  E r3  k3C S E* C H 2O

reaction 4: product returns to enzyme

k4
P  E  S  E *  H 2O r4  k 4C P CE

The challenge is that we can only measure the total E concentration.

Et  E  S  E *

A mole balance on the enzymatic species yields

nr
S  E* : 0   i ,S E* ri  1  r1  1  r2  1  r3  1  r4  r1  r2  r3  r4
i 1

S  E* : 0  k1CS C E  k 2C S E*  k3C S E* C H 2O  k 4C P C E

Solve for the concentration of the enzymatic complex

k1CS C E  k 4C P C E
S  E* : CS E* 
k 2  k3C H 2O

the rate of disappearance of substrate is given by a mole balance on S

nr
dCS
S:   i ,S ri  1  r1  1  r2  r1  r2
dt i 1

dCS
S:  k1CS C E  k 2C S E*
dt

13
 dCS k C C  k 4C P C E   k1k3C H 2O CS  k 2 k 4C P 
S:  k1CS C E  k 2 1 S E  C
dt k 2  k 3 C H 2O  k 2  k 3 C H 2O  E
 

This expression is no good in a practical sense, since we can only measure the total enzyme
concentration.

k1CS C E  k 4C P CE
C Et  C E  CS E*  C E 
k 2  k3C H 2O

1 k 2  k3CH 2O
CE  C Et  C Et
k1CS  k 4C P k 2  k3C H 2O  k1CS  k 4CP
1
k 2  k3C H 2O

Substituting this into the balance for the substrate yields

dCS   k1k3C H 2O CS  k 2 k 4C P    k1k3C H 2O CS  k 2 k 4C P  k 2  k 3 C H 2O

 CE    C
dt  k  k C   k  k C  k 2  k3C H O  k1CS  k 4C P Et
 2 3 H 2 O   2 3 H 2 O  2

 k1k3C H 2O CS  k 2 k 4C P
 CE
k 2  k3C H 2O  k1CS  k 4C P t

k cat  k 3C H 2 O

dCS  k1k cat CS  k 2 k 4C P

 CE
dt k 2  k cat  k1C S  k 4C P t

k cat  k 2
KM 
k1

k4
 kcat CS  k 2
CP
dCS k1
 C Et
dt k
K M  CS  4 C P
k1

Vmax  kcat C Et

14
 k4 V
 VmaxCS  k 2 C P max
dCS k1 kcat

dt k4
K M  CS  C P
k1

k4
KP 
k1 ratio of formation of enzymatic complex from substrate to that from product

Vmax
 Vmax CS  k 2 K P C P
dCS kcat

dt K M  CS  K P C P

kcat k k k k
KC   cat 1  3 1 C H 2O
k2 K P k2 k4 k2 k4 ratio of many rates…

Vmax  CS  P 
C

dCS
  K C

dt K M  CS  K P C P
(equation 7-29) of Fogler

This is the Briggs-Haldane Equation.

Section 7.2 Batch Reactors for Enzyme Reactions

In the simplest case, we have in a batch reactor a mole balance on the substrate

accumulation = generation

dCS
 Sr
dt

dCS V C
  max S
dt CS  K M

Reminder: This is the Michaelis-Menten equation. It can be analytically integrated.

CS  K M
dCS  Vmax dt
CS

15
  1 1  V
  dCS   max dt
 K M CS  KM

 1 1 
CS t
Vmax
  K M CS  S K M t dt

CS ,o 
  dC  
o

 
1
CS  CS ,o   ln CS    Vmax t  to 
KM  C S ,o  KM

This expression relates reactor time to substrate concentration. It can not be analytically solved
for the substrate concentration.

Effect of Temperature on Enyzmatic Reactions

Enzymatic reactions are very sensitive to temperature and have an optimum temperature.
See Figure 7-8. page 407.

The same can be said for pH.

16
Inhibition of Enzyme Reactions

Inhibitors are a molecule or species that renders the enzyme unable to catalyze a specific
reaction. In biological systems, some inhibitors have negative effects (e.g. cyanide acts an
inhibitor to a single enzyme in the aerobic oxidation process that will lead to death) and positive
effects (e.g. in the treatement of leukemia). Aspirin inhibits enzyme that catalyzes the synthesis
of a compound involved in the pain-producing process.

Three most common types of reversible inhibition are

competitive - inhibitor and substrate compete for the same active site
uncompetitive – inhibitor deactivates the enzyme-substrate complex
noncompetitive – enzyme has two types of sites and the presence of the inhibitor in one of
them deactivates the enzyme

Competitive Inhibition

reaction 1: formation of the enzymatic complex

k1
S  ES  E r1  k1C S C E

reaction 2: reverse of reaction 1

k2
S  ES  E r2  k 2C S E

reaction 3: product formation

k3
S  E W  P  E r3  k3C S E CW

reaction 4: formation of the inhibited enzymatic complex

k4
I  EI E r4  k 4C I C E

reaction 5: reverse of reaction 4

k5
I  EI  E r5  k5C I E

The rate of the formation of the product is still the rate of reaction 3.
We use the PSSH to determine the concentrations of both the substrate-enzyme and inhibitor-
enzyme complex.

Actually, the concentrations of the substrate-enzyme complex is unaffected by the inhibitor.

nr
SE: 0   i ,S E ri  1  r1  1  r2  1  r3  r1  r2  r3
i 1

SE: 0  k1C S C E  k 2CS E  k3C S E C H 2O

17
Solve for the concentration of the enzymatic complex

k1CS C E k 2  k3C H 2O
SE: C S E  or CE  CS E
k 2  k3C H 2O k1CS
The concentrations of the inhibitor-enzyme complex is unaffected by the substrate.

nr
I E: 0   i ,I E ri  1  r4  1  r5  r4  r5
i 1

I E: 0  k 4 C I C E  k 5C I  E

Solve for the concentration of the enzymatic complex

k 4C I C E
I E: C I E 
k5

The rate will be expressed in terms of the total enzyme concentration

Et  ES  E  I  E

k 4C I C E  kC 
C Et  C E C S E   C S E  1  4 I C E
k5  k5 
 k C  k 2  k 3 C H 2O   kC  k 2  k 3 C H 2O 
 C S E  1  4 I  C S E  1  1  4 I  C S  E
 k5  k1C S   k5  k1CS 

The rate of production therefore becomes

1
r3  k3C S E C H 2O  k3C H 2O C Et
 k C  k 2  k 3 C H 2O
1  1  4 I 
 k5  k1C S

kcat  k3C H 2O

1
r3  k cat C Et
 kC  k 2  k cat
1  1  4 I 
 k5  k1C S

18
 kcat  k 2
KM  Vmax  kcat C Et
k1 and

1
r3  Vmax
 kC  KM
1  1  4 I 
 k5  CS

k5
KI 
k4

Vmax C S
r3 
 C 
C S  1  I  K M
 KI 

an effective Michaelis constant is observed

 C 
K M'  1  I  K M
 KI 

Uncompetitive Inhibition

reaction 1: formation of the enzymatic complex

k1
S  ES  E r1  k1C S C E

reaction 2: reverse of reaction 1

k2
S  ES  E r2  k 2C S E

reaction 3: product formation

k3
S EP  E r3  k3C S E

reaction 4: formation of the inhibited substrate-enzymatic complex

k4
I  S EI E S r4  k 4C I C S E

reaction 5: reverse of reaction 4

k5
I E S I  S E r5  k5C I E S

The rate of the formation of the product is still the rate of reaction 3.

19
We use the PSSH to determine the concentrations of both the substrate-enzyme and inhibitor-
substrate-enzyme complex.

nr
SE: 0   i ,S E ri  r1  r2  r3  r4  r5
i 1

SE: 0  k1C S C E  k 2C S E  k3C S E  k 4C I C S E  k5C I E S

nr
I ES: 0   i ,I ES ri  r4  r5  k 4C I CS E  k5C I ES
i 1

Solve for the concentration of the inhibited enzymatic complex

k4
I ES: C I  E S  C I CS E
k5

Substitute into balance for substrate complex

k4
SE: 0  k1CS C E  k 2CS E  k3CS E  k 4C I CS E  k5 C I CS E
k5
Simplify

k 2  k3
SE: CE  CS E
k1CS

The rate will be expressed in terms of the total enzyme concentration

C Et  C E C S E C I E S

k 2  k3 k  k  k3 k 4 
C Et  C S E C S E  4 C I C S E  1  2  C I C S E
k1C S k5  k1C S k5 

The rate of production therefore becomes

1
r3  k3CS E  k3 C Et
k 2  k3 k 4
1  CI
k1CS k5

20
 k3  k 2 k5
KM  Vmax  k3C Et KI 
k1 and and k4

Vmax Vmax C S
r3  
K
1 M  I
C  C 
K M  1  I C S
CS K I  KI 

Noncompetitive Inhibition

reaction 1: formation of the enzymatic complex

k1
S  ES  E r1  k1C S C E

reaction 2: reverse of reaction 1

k2
S  ES  E r2  k 2C S E

reaction 3: product formation

k3
S EP  E r3  k3C S E

reaction 4: formation of the inhibited enzymatic complex

k4
I  EI E r4  k 4C I C E

reaction 5: reverse of reaction 4

k5
I  EI  E r5  k5C I E

reaction 6: formation of the inhibited substrate-enzymatic complex

k6
I  S EI E S r6  k 6C I C S E

reaction 7: reverse of reaction 6

k7
I E S I  S E r7  k 7 C I E S

reaction 8: formation of the inhibited substrate-enzymatic complex

k8
S  I EI E S r8  k8C S C I E

reaction 9: reverse of reaction 8

k9
I  E  S S  I  E r9  k9C I E S

The rate of the formation of the product is still the rate of reaction 3.
21
We use the PSSH to determine the concentrations of both the substrate-enzyme and inhibitor-
enzyme and inhibitor-substrate enzyme complexes.

nr
SE: 0   i ,S E ri  r1  r2  r3  r6  r7
i 1

SE: 0  k1C S C E  k 2C S E  k3C S E  k6C I C S E  k 7 C I E S

nr
I E: 0   i ,I E ri  r4  r5  r8  r9  k 4C I C E  k5C I E  k8CS C I E  k9C I ES
i 1

nr
I ES: 0   i ,I ES ri  r6  r7  r8  r9  k6C I CS E  k7 C I ES  k8CS C I E  k9C I ES
i 1

Solve for the concentrations of I  E and I  E  S first.

k 4 C I C E  k9 C I  E S
I E: CI E 
k 5  k8 C S

k 4 C I C E  k9 C I  E S
I ES: 0  k 6 C I C S  E  k 7 C I  E  S  k8C S  k9 C I  E S
k 5  k8C S

k 4 k8C S C I C E
k6C I C S E 
k 5  k8 C S
C I  E S 
kkC
k 7  9 8 S  k9
k 5  k8C S

Substitute into balance for substrate complex

k 4 k8C S C I C E
k6C I C S E 
k 5  k8 C S
SE: 0  k1C S C E  k 2CS E  k3CS E  k6C I CS E  k 7
kkC
k7  9 8 S  k9
k 5  k8C S

Simplify

   k 7 k 4 k 8C S C I 
   
k  k  k C  k 7 k6C I C   k C  k 5  k8C S C
SE:
 2 3 6 I
k 9 k 8C S  S E  1 S k 9 k8C S  E
 k   k 9   k   k 9 
k 5  k8C S k 5  k8C S
7 7
   

22
  
 
k k k C  k 7 k6C I 
 2 3 6 I
kkC 
 k 7  9 8 S  k9 
k 5  k8C S
SE: CE   C S E
 k 7 k 4 k 8C S C I 
 k 5  k 8C S 
 k1CS  
 kkC 
 k 7  9 8 S  k9 
 k 5  k 8C S 

Simplify

 k  k9 k5  k8CS   k9 k8CS k 2  k3  k 6C I   k5  k8C S k 7 k 6C I 

C E   7 C S E
 k 7  k9 k5  k8CS   k9 k8CS k1CS  k5  k8CS k 7 k 4 k8C S C I 

The rate will be expressed in terms of the total enzyme concentration

C Et  C E C S E C I E C I ES

Substitute in for E, I  E and I  E  S , and solve for C S E in terms of C Et . Substitute this

expression in the rate of production. (I skipped all these algebraic steps, as did Fogler.)

The rate of production therefore becomes

r3  k3C S E

Vmax C S
r3 
  End Result from Fogler. Noncompetitive
K M  CS 1  C I 
 KI 

Compare with:

Vmax C S
r3 
 C  Competitive
C S  1  I  K M
 KI 

Vmax C S
r3 
 C  Uncompetitive
K M  1  I CS
 KI 

23
Substrate Inhibition: In any of these cases, the inhibitor could be the substrate. Substitute S for I
in these results for rate law.

Enzyme co-factors: In many enzymatic reactions, you need two substrates. A rate law could be
derived following these same procedures.

24
Bioreactors

A bioreactor is a reactor that sustains and supports life for cells and tissue cultures.

reaction

cells + substrate  more cells + product

Monod equation for cell growth

rg  CC

where rg , is the rate of growth in grams/liter/second

 , is specific growth rate in 1/second
CC , is the cell concentration in grams/liter

CS
   max
K S  CS

where C S , is the substrate concentration in grams/liter

K S , is the Monod constant in grams/liter

Monod equation for bacterial cell growth rate

CS
rg   maxCC
K S  CS

Compare to the Michaelis-Menten equation for enzymatic reaction rates without inhibition.

dC S V C
  max S
dt K M  CS

In the case of many substrates, the concentration of the limiting substrate is used.

In the presence of inhibition, an empirical factor is added

CS
rg  k obs  max CC
K S  CS

25
 n
 C 
where kobs  1  *p  ,
 C 
 p 

C p is concentration of product
C *p is concentration of product where metabolism ceases
n is an empirical constant.

There are other empirical growth equations as well:

  C 
rg   maxCC 1  exp  S  Tessier equation
  k 

 1 
rg   max CC   
Moser equation
1  kCS 

Cell death rate

rd  k d  kt Ct CC

where k d is an environmental cell death rate

kt is a toxicity death rate
Ct is the concentration of the toxin

Growth and death are a strong function of temperature.

Stoichiometry

Cells + Substrate  more cells + Product

We don’t have clean stoichiometry.

Instead, we shall use yield coefficients,

mass of new cells formed CC

Yc / s  
mass of substrate consumed C S

1
Yc / s 
Ys / c

Product formation can take place during different phases of the cell growth cycle.

26
Product formation during the exponential growth phase

CS
rp  Y p / c rg  Yp / c CC  Y p / c  max CC
K S  CS

Product formation during the stationary phase where no cell growth occurs, we relate product
growth to substrate consumption

rp  Y p / s rs

mass of product formed C P

Yp / s  
mass of substrate consumed CS

Part of the substrate must be used to maintain a cell’s daily activities: maintenance utilization
term

mass of substrate consumed for maintenance

m
mass of cells  time

typical value m = 0.05 g S per g dry weight per hour

The rate of substrate consumption for maintenance

rsm  mCC

If it is possible to sort out the S consumed for cell growth and S consumed for P then

S  Yc/ s C + Y p / s P

mass of new cells formed CC

Yc / s  
mass of substrate consumed C S

mass of new cells formed

Yc/ s 
mass of substrate consumed to form new cells

mass of product formed C P

Yp / s  
mass of substrate consumed CS

mass of product formed

Y p / s 
mass of substrate consumed to form product

substrate utilization

27
balance

acculumation = in – out + generation

assume batch reactor – no in and no out

assume steady state – no accumulation

net generation = rate consumed by cells + rate consumed to form product + rate consumed for
maintenance

 rs  Ys/ c rg  Ys/ p rp  mCC

During the growth phase, you may not be able to separate purpose of substrate consumption

 rs  Ys / c rg  mCC

The product rate of formation is then

rp  Y p / c rg

During stationary phase, growth is zero and these equations don’t work

CSn
rp  k p CC (Monod equation)
K Sn  CSn

Sn = secondary nutrient

CSn
 rSn  YSn / p rp  mCC  YSn / p k p CC  mCC
K Sn  CSn

Determine yield coefficients experimentally.

These equations apply for two asymptotic cases. Production during exponential growth phase.
Production during the stationary phase. Some reactors don’t fall into either of these asymptotic
cases.

28
Mass Balances

chemostat = CSTR with microorganisms

acc = in – out + generation

Cell balance

 Fin CC ,in  Fout CC  V rg  rd 

dCC
V
dt

Cell balance has a growth and death term. Usually, CC ,in  0 .

substrate balance

dC S
V  Fin C S ,in  Fout C S  Vrs
dt

For a batch reactor, no in and out terms.

Cell Balance

 V rg  rd 
dCC
V
dt

Substrate balance

growth phase

dC S
V  Vrs  VYs / c rg  VmCC
dt

stationary phase

dC S
V  Vrs  VYs / p rp  VmCC
dt

product

dC P
V  Vrp  VY p / s rs (seems obviously incorrect since some substrate is used for
dt
maintenance, not production)

29
This seems better:

dC P
V  Vrp  VY p / s rs (seems obviously incorrect since )
dt

Other notes:

dilution rate

F 1
D 
V R

CSTRs at steady state

dCC
cells:  0  DCC ,in  DCC  rg  rd
dt

dC S
substrate:  0  DC S ,in  DC S  rs
dt

dC P
substrate:  0  DC P ,in  DC P  rp
dt

These are algebraic equations.

The dilution rate can be adjusted to optimize reactor performance.

30