Lecture Outline
CHNG 3804
Biological Systems
Kinetics and Modelling
Background
A catalyst is a substance that affect the rate of reaction
without altering the reaction equilibrium.
Enzymes, enzyme complexes, cell organelles and whole
cells are catalysts in bioprocesses.
The bio-catalyst are either viable or non-viable, growing
or non-growing.
In Most bioprocesses the reaction is homogenous (i.e.
there is not temperature and concentration gradients in
the reactor.
Reaction Yield
The extent to which reactants are converted to products
is expressed as the yield of reaction.
The theoretical yield that can be obtained from
equilibrium reaction can be different from actual yield.
Yield =
Mass or mole of product present
Total Mass or mole of reactant consumed
As Engineers we need to be able to
Measure the rate at which
Cells grow
Products are formed
By-products are formed
Predict the rates
Cells grow
Products are formed
By-products are formed
Reaction Thermodynamics
A + bB yY + zZ
C y Cz
K = Y bZ
C A CB
As the temperature increases, K
increases,
therefore
the
equilibrium concentration of
product increases. However,
other factors that should be
considered is the activity of the
enzyme.
0
Grxn
RT
0
0
Hrxn
Srxn
ln K =
+
RT
R
LnK =
Reaction Rate
Volumetric rate. The total mass of
reactant converted in a system depends
on the size of the reactor. It is more
common to specify the rate of reaction as
the rate per unit volume (rA). The
volumetric rate of reaction is calculated in
a constant volume.
aA + bB yY + zZ
dMA
=Mi Mo R A
dt
dMA
RA =
dt
R A 1 dMA
rA =
=
V
V dt
dC A
rA =
dt
Reaction Rate in Biological System
Mass of particular enzyme or cell added
to the system is usually unknown. To
overcome this issue, the enzyme quantity
is expressed as unit of activity measured
at a specified condition. It is generally
defined as the amount of catalyst that
used to convert 1 mol substrate per
minute at the optimal pH, temperature
and
substrate
concentration.
The
specific rate of reaction (rA) can be
reported as kg per unit enzyme per
second.
The comparison between two different
strains of an enzyme can be made by
comparing the specific rate not a
volumetric rate.
dMA
dt
1 1 dMA
rA = ( or )
X E
dt
1 1 dC A
rA = ( or )
x e dt
RA =
X: cell concentration,
e: enzyme concentration
Kinetics of a Reaction
rA = kCaA CBb
k = Ae
E
RT
ln k = ln A
The kinetics of many biological reactions are either zeroorder, first order or a combination of these called MichealisMenten kinetics.
Bacterial Growth
Growth of population of bacteria
Factor affecting growth
Nutritional requirement
E
RT
Temperature
Major factors affecting growth rate of bacteria
Upper limit determined by
Protein termolability
lysis
Collapse of cell membrane
Lower limit determined by
Membrane gelling
Freezing point of cellular water
Growth temperature limit differ widely for different
bacteria
Growth Rate and Temperature
GR = b( T Tm )
Effect of T on growth rate of bacteria does not
follow chemical reaction kinetics
Linear relationship between square root of growth
rate and T
Tm: minimum growth T (K)
T: growth temperature (K)
b: constant
GR: growth rate of bacterial culture
Bacteria Grouped According to Ability to
Grow at Different Temperatures
Growth Temperature ( C)
Group
Min.
Optimum
Max.
Thermophiles
40-50
55-75
60-80
Mesophiles
10-15
30-45
35-47
Psychrotrophs
obligate
Facultative
-5-5
15-18
19-22
-5-5
23-30
30-35
Nutrient Concentration
Growth rate depends on the type and amount of
nutrients supplied.
Growth of E-Coli in minimal medium + glucose is less
than that obtained by growth in complete medium
(e.g. nutrient broth) containing amino acids and
growth factors.
Growth rate is proportional to concentration of
limiting nutrient
Growth inhibited in excessively high nutrient
concentrations.
Oxygen and Carbon Dioxide
Ability to grow in presence of O2 is variable.
Obligate Aerobic use O2 as a terminal electron
acceptor for respiratory to produce H2O.
2O2 + 2e + 2H+
Medium Populations
For each species of microorganisms, growth is
most rapid at certain degree of alkalinity or
acidity.
Bacteria usually grow in the pH range of 4-8.
Yeasts prefer a pH range of 3-6.
Molds grow well in a pH range of 3-7.
Higher eukaryotic cells (human and animal)
grow best in a range of 6.5-7.5.
H2O
Facultative anaerobic: Tolerant to presence or
absence of O2. Grow better in presence of O2.
Water Availability
Not all water in and around cell is available for use in
cellular reactions.
Water bound by molecules is unavailable. The higher the
solute concentration, the less available the water in
solution.
In order to use water bound up by solutes, energy must
be expended by the cell.
Reduction of water availability by addition of sugars, salt,
drying is basis of food preservation.
Most bacteria grow only in aw range of 0.85-1.
aw = mole of pure water/(mole of pure water + moles of
solute)
H2O2 + O2
pH
Most bacteria can tolerate range of 1.5 to 2 pH unit either side of their
optimum.
Bacteria are well buffered internally to cope with small changes in pH.
Larger change in pH require energy to pump H+ ions into or out of the cell.
pH affects:
Type of acid are important
Absorption and solubility of ions
Dissociation of molecules
Transport mechanisms
Enzyme activity
Organic acid act as ionophores and increase permeability of membrane which
lead to disruption of transport mechanism, energy generation, etc.
Growth of Populations
Given a supply of nutrients, bacteria will grow at
a logarithmic rate
N = N02kt
K: growth rate (generation per hour)
Mean generation time (MGT) is a time taken for
a population to double (1/K)
Phases of Growth
Lag phase: time required for the cell to repair
sub lethal damage and adapt to new
environment
Exponential phase: Grow at a max. rate for that
environment.
Stationary phase: cell division balanced by cell
death
Death phase: death rate greater than growth
rate
Microbial Growth
Typical Simplified Batch growth given in
microbiology text books.
Stationary
Death
Log Cell
Number
Exponential
Lag
Time
Modeling/Data Analysis
Noise
Exacerbated the need to take derivatives
Need to consider experimental errors and
the uncertainty of parameters.
Mathematical Models
A mathematical model is a set of relationships
between inputs (manipulated variables) and outputs
(the fermenter states).
In the case of fermentations, the inputs are typically
the conditions in the fermenter, such as temperature,
media composition and substrate concentration.
The outputs are typically biomass, products and byproducts.
In more complex models intracellular compounds
may also be included.
Uses of Models
The motivations for modelling fermentations are varied,
reasons including;
improving understanding,
process control,
fermenter simulation,
optimisation and
estimation of conditions within the fermenter.
These different motivations for modelling make different
types of models appropriate.
The intended use of the model dictates its type and
complexity.
As a general rule, the model should be as simple as
possible but be able to represent the broad range of the
fermentations behaviour.
Simulations are not Experiments
Simulation allows data to be collected at
any frequency, and the data is free from
experimental noise.
However data from a fermentation model
should be considered to be subject to the
same uncertainty as the fermentation data
to which it was fitted.
Models are Approximations
Types of Models
To make a complete and all encompassing model, it
would be necessary to describe the workings of cells at
the atomic level.
The interactions within cells are extremely complex.
E.coli contains approximately 2000 different proteins,
making an exact description of microbial metabolism
beyond the capability of any model.
Hence, any model will need to be an approximation - the
level of detail used in the approximation will be
dependent on the data available and the purpose of the
model.
In constructing a model it is necessary to
select its type and complexity. Models can
be classified as follows:
Structured
Unstructured
Segregated
Unsegregated
Commonly Made Assumptions for
Unstructured Models
Types of Models
Unstructured
Structured
Unsegregated Most idealized case
Assumes cells have a
Cell population all treated number of components,
as identical
but that an average cell
can describe the system.
Segregated
Single component
Heterogeneous individual
cells
Homogenous cell population
Cell composition invariant
Perfect mixing
Uniform gas concentration
Assumes cells have a
number of components.
Description of cell-to-cell
Heterogeneity is included
Actual case
Microbial Growth
Many but not all microorganisms grow
exponentially
Dependent on type of microorganism
Dependent on sugar/substrate
concentration
Concept of Yield Coefficient
Quantifies energy of conversion of
substance to cell mass/cell number
Cell yield coefficient, Yx/s, defines as
Yx/s = dx/dS
Cell yield coefficient is rate of change of
cell conc. with respect to substrate conc.
Commonly Used Yield Coefficients
Yx/s = growth yield on substrate
YP/s = Product yield on substrate
Yx/O2 = growth yield on oxygen
Yx/ATP = growth yield per mole ATP generated.
YH = gram cell generated per unit heat evolution
Growth Rate vs Nutrient Concentration
During most of batch cultivation, specific growth rate is
constant and independent of changing nutrient Conc.
Growth rate is chemical reaction and hence varies with
reactant Conc.
Reactants in cell cultivation are essential nutrients or
substrates.
Limiting substrate: is the compound that affects cell
growth in such a manner that increasing or decreasing
the Conc. of the substrate increase or decrease cell
growth proportionally.
Microbial Growth
Growth Rate
Typical Simplified Batch growth given in
microbiology text books.
For most microorganisms the growth rate
is dependent on sugar concentration
Stationary
Death
Log Cell
Number
Exponential
Lag
S
Time
Growth Equations
Biomass formation is most simply described by
assuming that the rate of growth is proportional to the
number or mass of cells present.
dX
= X
dt
This proportionality is termed the specific growth rate
and is generally symbolised by and has units of hr-1.
This proportionality is not however constant, and is
affected by many factors such as the type of substrate
and its concentration.
Mycelial fermentations generally dont fit this model well
Growth Equations
In the following equations, a number of parameters and
variables are common;
Specific growth rate
max
Maximum specific growth rate
S
Substrate concentration (typically glucose)
X
Biomass concentration
The models all contain additional parameters to express
the relationship between the specific growth rate and the
substrate and biomass concentrations.
Growth Equations - Monod
=
max S
km + S
Monod assumed that at low substrate
concentrations the growth rate is first-order with
respect to substrate concentration, whilst at
higher concentrations the growth rate was
independent of the glucose concentration.
The parameter km is the substrate concentration
at which the growth rate is half of the maximum.
Monod Kinetics
Typical value of Km quite small
Specific growth rate during lag phase is constant.
Saturation constant (affinity constant, Km) inversely
proportional to affinity of the organism for its substrate.
Substance
Glucose
Km
(mg/L)
1.0
Organism
Enterobacter Aerogenes
Glucose
2.0-4.0
Escherichia coli
Glucose
25.0
Saccharomyces cerevisiae
Ribose
3.0
Hansenula polymorphia
Ammonia
0.1
Eni aerogenes
Growth Equations - Tessier
= max (1 e
Ka
Tessiers model also predicts a decrease in the
specific growth rate with decreasing substrate
concentrations.
This model also predicts that the specific growth
rate reaches a maximum value which is
practically independent of substrate
concentration.
Like Monods model, Tessiers approaches this
maximum asymptotically.
Growth Equations - Monod
Monods equation is analogous to the MichaelisMenten enzyme kinetics, and some authors
have tried to give the equation physical
meaning, assuming that the cells growth could
be taken as limited by a single enzyme reaction.
However, Monod arrived at his equation
empirically and suggested that any sigmoidal
curve could be fitted to the experimental data
Limitation of Monod Equation
Monod equation should not be applied
when the growth conditions are changing
rapidly.
At extremely low substrate concentration
When the growth is inhibited by high substrate
or product concentrations.
Growth Equations - Moser
=
max
1 + K s S
Mosers equation, if =1, can be derived from Monods
simply by dividing both the numerator and the
denominator by the substrate concentration.
It has a numerical disadvantage, if the substrate
concentration goes to zero, as the value of the specific
growth rate becomes indeterminate rather than zero.
This problem can be overcome by multiplying the
numerator and the denominator by S.
Growth Equations - Contois
=
max S
Bx + S
The Contois equation is similar to that of Monod,
however it assumes that the specific growth rate
decreases with both decreasing substrate and increasing
biomass concentrations.
At low biomass and high substrate concentrations, the
specific growth rate asymptotically approaches its
maximum value.
It would be better to include the effect of biomass/lack of
nutrients separately as this could cause trouble with fed
batch fermentations.
Growth Equations - Logistic
= kx(1-x)
The logistic equation assumes that the
growth rate decreases as the biomass
concentration increases.
Hence, the effect of decreasing nutrient
concentration is included implicitly.
This can be a problem if extra nutrients
are fed!
Growth Equations
All of these equations predict that the growth
rate decreases as the substrate concentration
decreases.
A number of authors have looked at these
different types of models and found few
differences between them.
Some equations have the advantage that they
can be integrated analytically.
However as the growth equation is generally
part of a much larger model, this is of little use.
Also necessary to include by-product inhibition
terms.
Growth Equations - Blackman
= max
for Cs > maxB
= Cs/B
for Cs <= maxB
Blackmans equation is a piece-wise linear
approximation of Monods model.
Growth Equations - Haldane
=
max S
km + S + S 2 / K I
Haldanes model incorporates substrate
inhibition into Monods equation.
That is, the Haldane model can describe a
decrease in the specific growth rate due to an
excessive substrate concentration
This can occur during batch yeast fermentations
if high sugar concentrations are used (>150g/L)
Determination of max and km
Method 1
Starting from Monod Model if S>>km can
assume
dX
= max X
dt
Linearise/Determine max from log plot.
Reasonable approximation in the initial stages
of fermentations if:
Substrate concentration large compared to cell
concentration
Similarities between Enzyme
Kinetics and Microbial Growth
Determination of max and km
Method 2
Determine (dX/dt) from experimental data (noise is a
problem here)
Graph vs S
max is the maximum value of
Km is the value of S at which = max
Method 3
Construct model and use a minimisation routine to
determine parameters
Requires a lot of noise free data for complex models
Enzymes [E] react with substrates [S] to form
products.
Experimentally it was observed by Brown (1902)
that the action of Invertase on Sucrose to
produce Glucose and Fructose was
first order with respect to sucrose concentrations at
low concentrations
and zero order with respect to sucrose concentrations
at higher concentrations
An explanation was formalised by Henri (1903)
and more completely by Michaelis and Menten
(1913)
Enzyme kinetics
Michaelis Menten Kinetics
Ks
[E ][S ] = K
[ES ] S
-d[S]0/dt
k2
E + S ES E + P
zero order
kinetics
/2
dP
dt
v = k 2 [ES ]
v=
first order
kinetics
K
[E0 ] = [E ] + [ES ]
[E ] = [E0 ] [ES ]
[S]0
Michaelis Menten Kinetics
Same form of
equation as Monod
([E0 ] [ES ])[S ] = K
S
[ES ]
[ES ] = [E0 ][S ]
[S ] + K S
k 2 [E0 ][S ]
v=
[S ] + K S
v [S ]
v = max
[S ] + K M
Michaelis Menten
kinetics assumes that
the combination of the
Enzyme and the
substrate is rapid and
reversible.
Lineweaver Burk Plot
K 1
1
1
=
+ m
v vmax vmax S
1
v
Km
vmax
Simple
But, tends to crowd
data at high substrate
concentrations
Where
vmax = k2 [E0 ]
and
KM = KS
1
Km
1
S
Eadie Hofstee Plot
vmax
v = vmax
Km
Hanes Plot
K mv
S
v max
Km
v
S
vmax
S
S
K
=
+ m
v vmax vmax
Km
vmax
Production Kinetics of Low Molecular
Weight Compound in Cell Culture
Class of metabolite
Examples of low molecular
weight fermentation
product
Products directly associated with
generation of energy in the cell
rp=(YPX+mp)x
Ethanol, acetic acid, acetone,
lactic acid, butanol from anaerobic
fermentation
Products indirectly associated with Amino acids, citric acid
energy generation
Products for which there is no
clear direct or indirect coupling to
energy generation.
S
v
Substrate Uptake
Biomass
Substrate for growth
CO2+H2O
Substrate for maintenance
CO2+H2O
Cell
Substrate for production
Penicillin, vitamins, streptomycin
A complex equation has been derived
for each specific system.
Substrate Uptake
Equation below typical for batch system
dS
1 dX
1 dP
=
mX
dt
YX / S dt
YP / S dt
Substrate (sugar) uptake is generally assumed to be
linearly related to
Cell numbers Maintenance Energy (m)
Cell growth Yield Coefficient (YX/S)
Product formation - Yield Coefficient (YP/S)
For simulations need to ensure that sugar
concentration is not < 0
CO2+H2O
Excreted product
Duran, Bioprocess Engineering principle
Kinetics of Cell Death
The kinetics of cell death is important
consideration in
Design of sterilisation process.
Analysis of fermentations where substantial viability
loss is expected.
Consider to be first order in most cases.
lnN = lnN0-kdt
It is a function of temperature
More complex form equation is used for spores
and as the above profile is not linear in such
systems.
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Parameter Determination
Product Formation
Can determine the yield coefficient by the
amount of biomass (or product) formed
divided by the amount of sugar consumed.
This can be a problem if biomass and
product are formed simultaneously.
Product formation is generally proportional
to cell numbers/mass
Can be complicated by factors such as
By Product Inhibition
By Product Inhibition
The production of By-products such as
Inhibitory By-Products
Substrate concentrations
Dissolved Oxygen concentrations
Typical Inhibition Model
Acetate (E.coli)
Ethanol (Yeast)
Inhibition term = 1
Have been found to reduce growth and
production rates
Effect on growth and production can be
different
Product Formation Rate
Inhibitory By-Product
by product tolerance
uninhibited
production rate
Pmax is the concentration at which growth/production
stops, n is typically 1-2.
Can cause problems numerically when running
simulation due to fractional indices of negative numbers.
Can also cause errors in product formation parameters
by exaggerating the effect of low by-product
concentrations
By Product Inhibition
Red Xs data
Blue Line Model n=1
Green line Model
n~2
Without extra data
hard to tell if n=1
over predicts both
P
Pmax
Running Models
Need to solve several ODEs
Biomass
Substrate
Product
By-product
Oxygen
Software
Matlab, Simulink
Excel (Eulers method, need small step size, simple
models)
11
Review
Kinetics of cell growth
Types of models that can be used
Some of the pitfalls when modelling
Types of Software that can be used to
solve model equations.
References
Pauline M. Doran, Bioprocess Engineering
Principles, Elsevier Academic Press, 2004.
M. L. Shuler and F. kargi, Bioprocess Engineering:
Basic concepts, Prentice-Hall (1992).
Bailey, J. E., Ollis, D. F., Biochemical Engineering
fundamentals, McGraw Hill (1986)
Wang, D I C, Cooney, C H, Demain, A L, Humphery A
E, Lilly M D, Fermentation and enzyme technology, J.
Wiley, 1979.
Aiba, S, Humphery A E and Millis, N F, Biochemical
engineering, Academic press 1973.
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