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Bio-Process Kinetics

Process Biotechnology

Anondho WIJANARKO
University of I ndonesia





Simplified Lecture Notes



To Imanti
and
Adam, Musa, Isa and Muhammad
CONTENTS
Microbial Growth
Microbial Cell Growth
Classical & Empirical Growth Kinetics
Environmental Alteration Studies
Light I llumination Effect
Temperature Effect
Microbial Kinetic Studies
Non Elementer Reaction
Microbial Growth Reaction Kinetics
Microbial Growth
Microbial Cell Growth
Mode of Growth
Selective assimilation of nutrients and
convert into and also include Chemical
rearrangement of protoplasmic material
characteristic of the particular organism
Production of an increased amount of
nuclear substance and cell division
Growth Phase
Growth Phase
Induction Phase (Lag Phase)
Transient Phase (Acceleration Phase)
Exponential Phase
Stationary Phase (Declining Phase)
Death Phase
Question Sheet
Why microbial growth have an lag phase?
Why death phase could be occurred in
microbial growth?
What is essential nutrient for growth of
organism especially prokaryotes?
What is important factor for cell division?

Answer Sheet
Keywords
Environmental Adaptation
Saturated microbial population and rare of
nutrient
Generally : Organic materials/CO
2
,
Phosphate, Nitrate/NH
3
,
Sulfate, Mg
2+
, K
+
Mg
2+
Growth Approximation

Growth Constants
Exponential Stationary Growth Phase
Total Biomass Production(G)

Incident growth rate, Incident mean division rate (|)



Specific growth rate, Beginning mean division rate ()



Doubling time of population (t
D
); exponential growth
phase

dt
dX
X t t
X
X
~
(
(

=
1
1 2
ln
1
2
|
dt
dX
X
t
=

1
lim
0

) (
i m
X X G =

t
2 ln
=
D
Problem solving
Microbial population growth
A new microorganism has been discovered which at each cell
division yields three daughters. From the growth rate data below
calculate the mean time between successive cell divisions?

t [h] X[g/dm
3
]
0.0 0.10
0.5 0.15
1.0 0.23
1.5 0.34
2.0 0.51
Answer Sheet
| |
1
815 . 0
815 . 0
100 . 0
3

=
=
h
e X
dm
g t
|
|
4 ln
~
=
D
t
h t
D
70 . 1
815 . 0
4 ln
~
= =
Classical Growth Kinetics
Empirical Approximation
Monod Growth Kinetic


Tessier Growth Kinetic

Moser Growth Kinetic

Contois Growth
Kinetic
s
s
s
+
=
( )
1
1

+ =

| s K
s
( )
s
K s
e
/
1

= |
s X B
s
+
= |
Empirical Growth Kinetics
Medium constituent Inhibition
Andrews Growth
Kinetic



Aiba Growth Kinetic
i
2
K
s
+ +
=
s K
s
s
|
( ) p K s K
K s
p s
p
+ +

= |
Growth Kinetics
Multiple essential nutrient


Bailey Growth Kinetic
...
3 3
3
2 2
2
1 1
1

+
=
s K
s
s K
s
s K
s
s s s
|
Home Work
Which kinetic approximation do you choose in case
of microbial growth of Hepatotoxin produced
Oscilatoria Agardhii NIVA CYA 97 in low
temperature?
Which empirical equation that you choose of
inoculation of microorganism in case of multiple
content limitation of nutrients, such as Mg
2+
,
phosphate, Nitrate and organic compound?
Which kinetic approximation do you choose of
cultivation photosynthetic microorganism that did
not grew up in pH above 7.8?
Answer Sheet
Keywords
Aiba Equation
Hepatoxin was produced by all photosynthetic
heterocystis microbes inoculation in case of cold
environmental condition
Bailey Equation
Multiple limitation of nutrient contents
Andrew Equation
Generally, Increasing pH was increase culture [HCO
3
-
]
that was known as essential nutrient for growth
mechanism in cytoplasm and pH around 7.8 tend
optimum [HCO
3
-
] in case of inoculation of common
photosynthetic microbe.
Simple Bio-Production Kinetic
Cellular growth rate
Monod approximation

Yield factor


Substrate Utilization

Product Formation
(Beginning of Stationary Phase)




s
s
s
+
=
( )
( )
( )
( )
dt
ds
dt
dX
s X
s
X
Y =
A
A
=
/
X
dt
X d
= |
( )
( )
( )
( )
dt
dX
dt
dP
X P
X
P
Y =
A
A
=
/
s K
X s

dt
X d
s
+

=
s K
X s
Y

dt
s d
s s X
+

=
/
s K
X s
Y
dt
P d
s
X P
+

=
/
Environmental
Alteration Studies
Microbial Growth Kinetic
Enviromental Condition
Direct Effects
Light Illumination (Energy Source)
Temperature
Essential nutrients content
Indirect Effects
Gas inlet volumetric rate
Gas inlet content
Liquid circulation rate
Non essential nutrients content

Light Illumination Effect
Oscillatoria agardhi Gomont
(Post AF, R de Witt, LC Mur, J. Plank. Res., 7 (1985) 487-495)
Temperature effect
Modified Arhenius Model
( ) ( )
R
d
H
R
d
S
a
T
RT E
e
T
e A
A A

+
=
1
/

Temperature Effect
Classification of
Microorganism

Question Sheet
What is happen if microorganism is at 90
o
C? Why?
In case of decreasing of temperature about 20
o
C
from optimum temperature, what is happen in case
of microbial growth rate?
In case of ethanol production that was S. sake have
ethanol tolerance around 10%, what do you do to
make an whisky industry?
Why a shade microbe does not grew well in high
light illumination and commonly have not high
temperature resistance?
Answer Sheet
Death, thermophile did not survived at temperature 85
o
C up.
Refer to Arhenius approximation, microbial growth was
decreased to quarter.
Ethanol production was set below 7-8% and purified to 40%
Photo-bleaching (chlorolysis), shade microbe commonly
psychrophile, that have a limitedness to growth at both of
high temperature and light illumination.
Problem solving
Temperature variation of Growth
Johnson, Eyring and Polisaar represent growth of E. coli between
18
o
C and 46
o
C by the following equation for the specific growth
rate :




Plot this function as log versus 1/T
Show that this equation can be represented as the product of two function
whose form is suggested by the plot in above part, and what explanation
rationalizes these two individual functions and the value of above
parameters?
In this interpretation of as function of T, what implicit assumption are
made with regard to irreversible deactivation?
|
.
|

\
|
+
|
.
|

\
|

=
T
T
T
75200
239 exp 1
7520
exp 10 96 . 9
9

Answer Sheet
|
.
|

\
|
=
T
7770
exp 10 84 . 6
12

0.0034364 17.494 18.000


0.0034247 19.178 19.000
0.0034130 21.011 20.000
0.0034014 23.005 21.000
0.0033898 25.174 22.000
0.0033784 27.530 23.000
0.0033670 30.089 24.000
0.0033557 32.867 25.000
0.0033445 35.880 26.000
0.0033333 39.147 27.000
0.0033223 42.687 28.000
0.0033113 46.520 29.000
0.0033003 50.667 30.000
0.0032895 55.151 31.000
0.0032787 59.990 32.000
0.0032680 65.199 33.000
0.0032573 70.773 34.000
0.0032468 76.667 35.000
0.0032362 82.737 36.000
0.0032258 88.620 37.000
0.0032154 93.488 38.000
0.0032051 95.692 39.000
0.0031949 92.579 40.000
0.0031847 81.570 41.000
0.0031746 63.206 42.000
0.0031646 42.683 43.000
0.0031546 25.748 44.000
0.0031447 14.433 45.000
0.0031348 7.7740 46.000
|
.
|

\
|
=

T
53000
exp 10 96 . 5
72

Answer Sheet
Deactivation line
This decreasing line presented a rapid decrease in growth rate as the temperature
approaches the upper limit for survival of the microorganism tend that the most
thermally sensitive essential protein denatures and this hypothesis also has been
confirmed in several instance by genetic studies in which mutation of a single gene
has caused a large change in the maximum tolerable temperature for
microorganism
One physical mechanism for this phenomenon is obvious as the temperature
increases, the atoms in the enzyme molecule have greater energies and a greater
tendency to move. Eventually, they acquire sufficient energy to overcome the
weak interactions holding the globular protein structure together, and deactivation
folows.
Activation line
This increasing line clarified at low temperature, apparently, the metabolic activity
of cell increase with increasing temperature as the activities of its enzyme rises.
Notice in this activation line, that was also commonly called to Arrhenius plot,
that classical Arrhenius behavior appears at low temperature, exactly, below of
the maximum tolerable temperature for microorganism.
The implicit assumption are made regard to irreversible deactivation :
irreversibility alteration of whole active forms of enzyme to inactive forms in
case of a large change in the maximum tolerable temperature for each
microorganism

Temperature Effect
Cellular Consideration
Psychrophile
Obligate
Protococcus Agardh SS 100-3
Oscillatoria redekei Van Goor
Oscillatoria sp. SS 100-5
Facultative
Anabena cylindrica Lemmerman
Oscillatoria Agardhi Gomont
Nostoc commune Antartica
Mesophile
Synechococcus leopoliensis
Anabaena variabilis IAM M3
Microcystis Aeruginosa IAM M228
Thermophile
Mastigocladus laminosus HTF
Synechococcus lividus OH75S
Synechcocus elongatus It 7S

Why optimum specific growth rate values of
psychrophile factually, lower than
thermophile?
Why GC content of microbial DNA is
important for classification of organism in
terms of growth rate dependence on
temperature?
What is DBI?
Answer Sheet
Keywords
Arhenius Limitation
Guanin Cytosin of DNA have a very strong of 3 pairs
hydrogen bound, that was responsible in high temperature
tolerance characteristic of microbe.
DBI was value that defined content of double bond of
cellular membrane fatty acid in the each strains plasma-
membrane.
Problem solving

Please solve 3 numbers for mark of 90
or 4 numbers for mark of 100
Number b and e must be done
a. Calculate | of each strain
at S=0.2 g/dm
3
?
b. Calculate A of each strain!!!
Calculate at 285 K and 310 K
of each strain!
Think deeply and carefully before
answer these question!!!
c. Define DBI and GC Content!
d. Write DBI equation!
e. For each type of strain
Calculate the DBI?
Calculate GC contens (GC
DNA
)?
What type? Why?


O. agardhi A. nidulans S. lividus
Fatty acids contents
14:0 0 0.011 0
14:1 0 0.012 0
16:0 0.292 0.477 0.54
16:1 0.217 0.385 0.1
16:2 0.033 0 0
18:0 0.004 0.037 0.22
18:1 0.073 0.074 0.14
18:2 0.146 0 0
18:3 0.235 0 0
Growth Characteristic
T
m
355 K 361 K 371K
T 295 K 301 K 328 K
K
i
0.001 g/dm
3
10 g/dm
3
1 g/dm
3
K
m
10 g/dm
3
10 g/dm
3
100 g/dm
3
0.012 h
1
0.014 h
1
0.048 h
1

E
a
46.5 Kj/mole 128 Kj/mole 240 Kj/mole

DNA
GC C
m
T
o
+ = 0 . 41 3 . 69 ) (
K mole
J
R

= 314 . 8
RT
E
a
e A

=
i
m
K
s
s
K
+ +
=
1
1
|
Answer Sheet
| | [h
-1
] A [h
-1
]
Oscillatoria agardhi Gomont 0.003984 0.0000478 2.06*10
6
Anacystis nidulans 0.01960 0.0010584 2.29*10
20
Synechococcus lividus 0.001995 0.0000958 8.00*10
36
[h
-1
]
(285K) (310K) DBI GC
DNA

Oscillatoria agardhi Gomont 0.00618 0.0 1.353 0.310 psychrophile

Anacystis nidulans 0.00306 0.0618 0.471 0.456 mesophile

Synechococcus lividus 0.0 0.0479 0.240 0.700 thermophile


DBI was value that defined content of double bond of cellular membrane fatty acid in the each strains
plasma-membrane.
Percentage of Guanin & Cytosin in whole of DNA
( ) { }
{ } FA
n N UFA n
DBI
n


=
:
0
Microbial Kinetic Studies
Non Elementer Reaction
Common reaction rate



N = integer Elementer
N = non integer Non Elementer
Non Elementer Example :
n
s k
dt
ds
=
HBr Br H 2
2 2
+
| | | | | |
| |
2 2
2
2
3
2 1 2
' ] [
'
Br k HBr
H Br k
dt
Br d
+

=
CO CH CHO CH +
4 3
| |
| |
2
3
3
3
CHO CH k
dt
CHO CH d
=
Reaction Mechanism
2 2 2 2
1
2 O H N H NO
k
+ +

| |
0
2 2
=
dt
O H d
2 2
O H
O H N H NO
2 2 2
2 2 2 + +
O H H O H
k
2 2 2 2
2
2

+
| | | | | | | | 0
2 2 2 2 2 1
= H O H k H NO k | | | |
2
2
1
2 2
NO
k
k
O H =
| |
| | | | | | | | | | | |
2
2
1 2
2
2
1
2 2 2 2 2
2
H NO k H NO
k
k
k H O H k
dt
O H d
=
|
|
.
|

\
|
= =
Reaction Mechanism
2 2
1
1
2 O N NO
k
k

2 2 2 2
/ O H O N
| |
0
2 2
=
dt
O N d
O H O H H
k
2 2 2 2
2
3

+
| |
0
2 2
=
dt
O H d
O H N H NO
2 2 2
2 2 2 + +
2 2 2 2 2 2
2
O H N H O N
k
+ +

| | | | | | | | 0
2 2 2 3 2 2 2 2
= H O H k H O N k
| | | |
2 2
3
2
2 2
O N
k
k
O H =
| |
| | | |
| |
| |
| | | |
| |
| |
2
2
2
2 1
2 1
2
2
2
2 1
1
2 2 2 2 2
2
1 1 H
H
NO
k k
k k
H
H
NO
k k
k
k H O N k
dt
N d
+

+

=
|
|
.
|

\
|
+ +
= =

| | | | | | | | 0
2 2 2 2 2 2 1
2
1
=

H O N k O N k NO k | |
| |
| |
2
2
2 1
1
2 2
1 H
NO
k k
k
O N
+

+
=

Question Sheet
What is mechanism path?
What definition of intermediate species?
What was become determining factor of
reaction rate?

Answer Sheet
Mechanism path is microscopic description of a
chemical reaction that was composed in term of
elementer reactions
Intermediate species is an imaginary reactant that
was proposed in mechanism path and was predicted
have a share in deciding of reaction rate
Slowest elementer reaction of the proposed
mechanism path

Microbial Growth
Enzymatic Reaction/Kinetic consideration
Michaelis-Menten Kinetics
Reaction mechanism


Kinetic equation


Substrate Actvation and Inhibition
Reaction mechanism


Kinetic equation


Product Activation and Inhibition
Reaction mechanism


Kinetic equation








s K
s
m
+

=

|
ES S E
k
k

+
1
1
E P ES
k
+

2
ES S E
k
k

+
1
1
2
2
2
ES S ES
k
k

+
E P ES
k
+

3
i m
K s s K
s
/
2
+ +

=

|
ES S E
k
k

+
1
1
E P ES
k
+

2
ESP P ES
k
k

+
3
3
( ) p K s K
K s
p s
p
+ +

= |
Michaelis-Menten Kinetics
Reaction mechanism



Kinetic derivation

ES S E
k
k

+
1
1
E P ES
k
+

2
| | ES E E + =
0
| |
| | ( ) | |
| | | | ( ) ES E S
k k
k
E S
k k
k
ES
ES k k E S k ES
ES
dt
d
dt
d

+
=
+
=
+ =
=

0
2 1
1
2 1
1
2 1 1
0
| | 0
0
= ES
| |
| |
( )
| |
m
dt
d
dt
d
K S
S P
k
k k
S
S E k
ES k P
k
k k
S
S E
ES
+

=
|
|
.
|

\
|
+
+

= =
+
+

=

max
1
2 1
0 2
2
1
2 1
0
Home Work
Please exhibit kinetic derivation of substrate
activation and inhibition?
Please exhibit kinetic derivation of product
activation and inhibition?
What do you think about reaction kinetic if
K
m
is high that was indicated in
bioremediation of toluene by C. nivalis?

Answer Sheet
Substrate Activation & Inhibition
Reaction mechanism


Kinetic equation



Equation constant



i m
K s s K
s
/
2
+ +

=

|
ES S E
k
k

+
1
1
2
2
2
ES S ES
k
k

+
E P ES
k
+

3
1
3 1
k
k k
K
m
+
=

2
2
k
k
K
i

=
0 3
E k =
Answer Sheet
Product Activation & Inhibition
Reaction mechanism


Kinetic equation



Equation constant


Reaction kinetic become First order


X C X
s
K
X
s K
s
X
dt
dX
m
m
=
+
=
+

= =
1

|
ES S E
k
k

+
1
1
E P ES
k
+

2
ESP P ES
k
k

+
3
3
( ) p K s K
K s
p s
p
+ +

= |
|
|
.
|

\
|

+
=

3
3
1
2 1
k
k
k
k k
K
s
3
3
k
k
K
p

=
0 2
E k =
Problems
Solve problems in Biochemical Engineering
Fundamental (JE Bailey and DF Ollis) of
page 446 to 447 number 7.2 to 7.3 !!!
Problem solving
Cellulose Hydrolysis Kinetics
The enzymes which degrade cellulose, producing the dimer cellobiose, a
simplified reaction network can be written :





Where G
1
, G
2
are insoluble cellulose and soluble ccllubiose and E
1
is
indicative of the enzyme involved in the slowest step leading to cellobiose.
Derive a reaction rate of this hydrolysis studies?
1 2 1 2
1 2 1 1
1 1 1 1
3
3
2
1
E G E G
E G E G
E G E G
k
k
k
k

+
+
+
Answer Sheet
Fast reaction

Slow reaction

Inhibition step

Intermediate species
1 2 1 2
1 2 1 1
1 1 1 1
3
3
2
1
E G E G
E G E G
E G E G
k
k
k
k

+
+
+
| | | |
1 1 1 2
& E G E G
| | | | | | | |
1 2 1 1 1 0
E G E G E E + + =
| |
| |
0
0
1 2
1 1
=
=
dt
E G d
dt
E G d
| | | | | |
| | | | | | 0
0
1 2 3 1 2 3
1 1 2 1 1 1
=
=

E G k E G k
E G k E G k
| | | | | |
| | | | | |
1 2
3
3
1 2
1 1
2
1
1 1
E G
k
k
E G
E G
k
k
E G
=
=

| | | | { }
2 1
0
1
3
3
2
1
1 G G
E
E
k
k
k
k

+ +
=
Rate of cellulose hydrolysis Reaction
| |
| | | | | |
| |
| | | | | | | |
| |
| | | | { } | |
| |
| | | | { }
| | | | { }
2 1
0
2 3 1 1
2
1 2 3 1 1
2
1 2 3 1 1
2
1
2
2
1 2 3 1 1 2
2
3
3
2
1
1 G G
E
G k G k
dt
G d
E G k G k
dt
G d
E G k E G
k
k
k
dt
G d
E G k E G k
dt
G d
k
k
k
k

+ +
=
=
=
=
| |
| | | | { }
| | | | { }
2 1
2 1
0 2
2
3
3
2
1
2
3
2
1
1 G G
G G
E k
dt
G d
k
k
k
k
k
k
k
k

+ +

=