KMU 413

BIOTECHNOLOGY II

LECTURE NOTES 3

Asst. Prof. Hande Günan Yücel

12.10.2021
 PART 1

A Brief Summary on Microbial

Growth Curve

Yield Definitions
 Growth Kinetics in Batch Culture
After inoculation, the microorganisms selectively take up the substrates from the medium.

1) The Lag Phase:

• It is the adaptation period.
Typical growth curve for a bacterial • Cell mass may increase; but cell number does
population: not increase.
• Synthesis and/or repression of some enzymes
take place. (This mechanism depends on the
nutrients found in the growth media)
• If the lag phase is short, productivity is high.

Duration of lag phase depends on;

- inoculum concentration (it should be high,
minimum 5%-10% by volume)
- inoculum age (inoculum should consist of cells
at exponential phase, which are young and
active)
- Nutrients found in the medium
- Other growth factors (aeration, agitation,
etc.)

Diauxic growth: multiple lag phases

It can be observed if the medium contains two or
more carbon sources.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Growth Kinetics in Batch Culture
2) The exponential phase (logarithmic phase):

• Cells are now adapted to the new

environment.
• So cell mass and cell number increase rapidly.
• This is a balanced growth period. This
means all of the cell components grow at the
same rate.
• So, net specific growth rate calculated
from cell number and cell mass will be
same.
=µnetX, (X=Xo at t=0) (beginning of the
logarithmic phase)
ln=µnett X=X

(X and Xo are the cell concentrations at t and t=0)

ln
Doubling time: Time necessary for doubling microbial mass
τd= = Slope: µnet

Specific growth rate (according to cell mass concentration)

(τd )’= t

Specific growth rate (according to cell number concentration)

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Growth Kinetics in Batch Culture

3) The deceleration phase:

• At this phase, amount of one (or more)

of the essential nutrients is reduced or
toxic by-products are accumulated.
• This situation causes stress and
metabolism shifts to «survival» mode.

Unbalanced growth– τd is not equal to (τd )’

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Growth Kinetics in Batch Culture
4) The stationary phase:

• Cell growth rate=Cell death rate (Net

growth rate is zero)
• However, the cells are still metabolically active
and produce «secondary products».

The possible cases at this phase:

• Viable cell number may decrease; but cell
concentration may stay constant.
• Cells may burst and viable cell number
decrease. But, a second growth phase may
occur on lysis products (cryptic growth).
• Cells may not grow but produce secondary
products.

Conversion of cell mass into maintenance During this phase, the essential nutrients are
energy: consumed and cell catabolizes its own
=-kdX reserves for new building blocks and for energy
producing monomers--ENDOGENOUS
X=X
METABOLISM.

First order rate constant for Energy is always required for:

Cell mass endogeneous metabolism 1) An active membrane
concentration at 2) Essential metabolic activities (e.g. Motility)
the beginning of 3) Repairing the damages
the stationary MAINTENANCE ENERGY
phase
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Growth Kinetics in Batch Culture
5) The death phase (decline phase):
This phase begins because of nutrient depletion
and/or toxic product formation.

Conversion of cell mass into maintenance

energy:

=-(kd )’N
N=N
First order death rate constant

Cell concentration at the

end of the stationary
phase

It is possible to reestablish the growth by

transferring fresh inoculum.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Stoichiometric Coefficients Associated with Growth
Apparent Growth Yield

Y X/S = - Based on a substrate (for example glucose, which is

used as carbon and energy source)
(g dry cells/g substrate consumed)

YX/S is generally between 0.4 and 0.6 for most yeast and bacteria.

S = S + Sassimilatied
+ into S
an
+
growth energy
S
assimilation into maintenance
extracellular energy
biomass
product

Y X/O2 = - Yield coefficient based on other nutrient

Y P/S = - Yield coefficient based on product formation

Maintenance coefficient: Substrate uptake rate for cellular maintenance.

m=
 Product Yield
1) Growth-associated products: They are formed simultaneously with cell growth.

qP =
qP =Y P/X µg «Specific product formation rate» α «specific growth rate»
Example: Enzyme
If endogeneous metabolism is not zero, µg µnet production

2) Nongrowth-associated products: They are formed at the stationary growth

phase.
«Specific product formation rate» is constant. Example: Antibiotic
production such as penicilin
qP =β=constant

3) Mixed-growth-associated products: They are formed at the slow growth and

stationary growth phase.
Example: Lactic acid
qP =α µg β fermentation
 Growth-associated Mixed-growth-associated Nongrowth-associated

qP =
qP =α µg +β qP =β=constant
qP =Y P/X µg

Example: Enzyme Example: Lactic acid Example: Antibiotic

production fermentation production such as penicilin

“Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002 Let’s take this equation:
 qP =α µg + β (Luedeking–Piret equation).

If α=0, qP = β Product is only non-growth associated.

If β =0, qP = α µg Product is only growth associated and α

would be equal to Y P/X
EXAMPLE 6.1 FROM YOUR TEXTBOOK
 We should
Given Given
calculate
Time (h) Cell Concentration (g/L) ln (Cell Concentration) Glucose concentration (g/L)
0 1.25 0.2231 100
9 2.45 0.8961 97
16 5.1 1.6292 90.4
23 10.5 2.3514 76.9
30 22 3.0910 48.1
34 33 3.4965 20.6
36 37.5 3.6243 9.38
40 41 3.7136 0.63

Exponential phase Exponential phase

has started. has finished.
In exponential phase;
ln=µnett
 PART 2

Effects of Environmental Conditions on Growth Kinetics

1. Temperature
2. pH
3. Nitrogen source
4. Dissolved oxygen (DO)
5. Redox potential
6. Dissolved carbon dioxide (DCO2)
7. Ionic strength
8. Substrate concentrations
 1. Temperature
As temperature is increased toward its optimum value, growth rate doubles
for every 10oC increase in temperature.
Above the optimum temperature, growth rate decreases and thermal death
may occur.
The net specific replication rate when the
temperature is above the optimum value.
T effect on
At high temperatures, thermal death rate is greater than the growth rate. cell growth
So, viable cell concentration decreases.
Both parameters vary with temperature
according to the Arrhenius equation.
Activation Activation
energy for energy for
growth thermal death

Optimum temperature for cell growth and product formation may be different.
T effect on
When temperature is increased above the optimum value, maintenance product
requirements also increase. This results in a decrease in the yield formation
coefficient.
At high temperatures, rate of bioreaction may be higher than the diffusion T effect on
rate. So, diffusion becomes rate-limiting step. rate limiting
step
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Typical Variation of Cell Growth Rate with Temperature

Typical growth rate change of E. coli with temperature

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 2. pH (Hydrogen Ion Concentration)
pH directly affects the enzyme activities.
Generally, acceptable pH range varies about the optimum by
When pH differs from the optimum value, maintenance energy requirements increase.

Bacteria: 3-8
Yeasts: 3-6
Optimum pH ranges for different
Molds: 3-7
cells.
Plant cells: 5-6
Animal cells: 6.5-7.5

Differences in the optimum pH value can be Engineering

used to select one organism over another Point of View
one.

It is possible to adapt cultures to

a wider range of pH values.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
  pH can change because of the production of organic acids, the utilization of acids
(particularly amino acids), or the production of bases.
 The evolution or supply of CO2 can alter pH greatly in some systems (e.g., seawater or
animal cell culture).

pH control by means of a buffer or an active pH control system is

very important.

3. Nitrogen Source

If is the nitrogen source is If nitrate is the nitrogen

ammonium, hydrogen ions source, hydrogen ions are
are released into the removed from the medium,
medium resulting in a resulting in an increase in
decrease in pH. pH.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 4. Dissolved Oxygen (DO)
Oxygen is sparingly soluble in water. So, it may be a limiting substrate.
If oxygen is the rate-limiting factor, specific growth rate varies with DO concentration
according to saturation kinetics. Below a critical concentration, growth approaches a
first-order rate dependence on the dissolved-oxygen concentration.
Above a critical oxygen concentration, the growth rate becomes independent of the
dissolved-oxygen concentration.

The critical oxygen concentration is about 5% to

10% of the saturated DO concentration for
bacteria and yeast and about 10% to 50% of the
saturated DO concentration for mold cultures
Aerobic

Saturated DO concentration in water at 25oC

and 1 atm pressure is about 7 ppm.

Facultative

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Oxygen is usually introduced to fermentation broth by sparging air through the broth.
Oxygen transfer from gas bubbles to cells is usually limited by oxygen transfer through the
liquid film surrounding the gas bubbles.

Oxygen transfer rate from the

gas to liquid phase
kL is the oxygen transfer coefficient (cm/h),
a is the gas–liquid interfacial area (cm2 /cm3 ),
kLa is the volumetric oxygen transfer coefficient (h-1 ),
C* is saturated DO concentration (mg/l),
CL is the actual DO concentration in the broth (mg/l),
NO2 is the rate of oxygen transfer (mg O2/l◊h).

Oxygen uptake rate

qO2 is the specific rate of oxygen consumption (mg O2/g dw cells◊h),

YX/O2 is the yield coefficient on oxygen (g dw cells/g O2), X is cell
concentration (g dw cells/l)

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 When oxygen transfer is the rate-limiting step, the rate of oxygen consumption is equal to
the rate of oxygen transfer:

Under oxygen-transfer limited conditions, growth rate varies nearly linearly with the
oxygen transfer rate.

DO limitations should be overcome. For this Engineering

purpose, Point of View
 oxygen-enriched air or pure oxygen can be used.
 high pressure can be preferred.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 5. Redox Potential

It affects the rate and extent of many oxidative–reductive reactions.

In a fermentation medium, the redox potential is a complex function of;
 DO
 pH
 other ion concentrations (reducing and oxidizing agents)

Electrochemical
potential of a
fermentation medium
electrochemical potential is measured in millivolts by a
pH/voltmeter and PO2 is in atmospheres.

Redox potential can be reduced ;

by passing nitrogen gas
by the addition of reducing agents

Redox potential can be increased ;

by passing oxygen gas
by the addition of oxidizing agents

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 6. Dissolved Carbon Dioxide (DCO2)

Very high DCO2 concentrations may be toxic.

On the other hand, cells require a certain DCO2 level for proper metabolic
functions.
Engineering
The dissolved carbon dioxide
concentration can be controlled by Point of View
changing the CO2 content of the
air supply and the agitation
speed.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 7. Ionic Strength

It affects;
 transport of certain nutrients in and out of cells
 metabolic functions of cells
 solubility of certain nutrients, (e.g. dissolved oxygen)

where C is the concentration of an ion, Zi is its charge, and I is the ionic

strength of the medium.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 7. High Substrate Concentrations

High substrate concentrations cause inhibitory effects.

 What is the inhibitory level of the substrate? It changes depending on the type of cells
and substrate. Glucose may be inhibitory at concentrations above 200 g/l due to a
reduction in water activity.
 Some salts such as NaCl may be inhibitory at concentrations above 40 g/l due to high
osmotic pressure.
 Some refractory compounds, such as phenol, toluene, and methanol, are inhibitory at
much lower concentrations (e.g., 1 g/l).

Engineering
How can the substrate inhibition be overcome? Point of View
It is possible by intermittent addition of the
substrate to the medium.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 PART 3

Quantifying Gowth
Kinetics
Unstructured and
Nonsegregated Models
1) Substrate-limited growth
2) Models with growth inhibitors
3) Logistic Equation
 Quantifying Gowth Kinetics

It is possible to think of the growth dynamics in terms of kinetic descriptions.

Structured Models:
 Cell mass is divided into Unstructured Models:
components such as These models present a prediction for
element or molecule balanced growth.
according to this type of
models. So, they can be used for batch
growth at exponential phase and
 These models present a continuous growth at steady state.
prediction for the change in
these components with cell These models are simpler and more
growth. common.

 These models are very

complicated.
In general, «segregation» is not a critical component so «nonsegregated» models will be
satisfactory for this lesson.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Using Unstructured Nonsegregated Models to Predict Specific Growth Rate

1. Substrate-limited growth
 This figure is an example of
saturation kinetics.
 This means, a single chemical
species (S) limits the growth rate.
 So, increase in S influences
growth rate, while changes in
other nutrient concentrations
have no effect.
Change in the specific growth rate of E. coli with
nutrient concentration

1) Langmuir–Hinshelwood (or Hougen–Watson)

kinetics in traditional chemical kinetics
2) Michaelis–Menten kinetics for enzyme
reactions.
3) Monod equation for growth kinetics:

The most common kinetic model

for cell growth

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 µ,max

½ µ,max

µg = specific cell growth rate (hr-1) (If endogeneous metabolism is unimportant, µnet
= µg).
µm = maximum specific cell growth rate (hr-1) (S >> Ks )
S = substrate concentration (g/L)
KS = Saturation constant (g/L)
KS=S for µ = 1/2 µm
In general, µg = µm for S >> Ks

µg = (µm /Ks)S for S < < Ks.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Monod equation is valid for substrate-limited growth.

It is possible to relate the environmental conditions only to «S» if the growth is slow and
population density is low.

At high population levels, the buildup of toxic metabolic by-products becomes more
important.

The rate expressions for

high cell population

S0 is the initial concentration of the substrate

Ks0 is dimensionless

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 2. Models with growth inhibitors

At high concentrations of
1) substrate
2) product
3) inhibitory substances
growth becomes inhibited and growth rate depends on inhibitor concentration.

The inhibition pattern of microbial growth is analogous to enzyme inhibition

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 1. Substrate inhibition:
 At high substrate concentrations, microbial growth rate is inhibited by the substrate.
 Substrate inhibition of growth may be competitive or noncompetitive (as in enzyme
kinetics).

if KI >> Ks

Competitive substrate
inhibition

Noncompetitive
substrate inhibition

KI values are different.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 2. Product inhibition:
 At high product concentrations, microbial growth rate is inhibited by the substrate.
 Product inhibition of growth may be competitive or noncompetitive and in some cases
underlying mechanism is not known.

Competitive product Noncompetitive

inhibition product inhibition

For example ethanol fermentation from glucose.

Ethanol is the inhibitor at concentrations above about 5%

Other rate expressions used for ethanol inhibition

(Pm is the product concentration at which growth
stops and Kp is the product inhibition constant).

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 3. İnhibition by toxic compounds:
 This type of inhibition may be competitive, noncompetitive or uncompetitive.

Competitive inhibition Noncompetitive inhibition Uncompetitive inhibition

In some cases, toxic compounds cause cells death. The net specific rate expression
becomes:

(k’d is the death-rate constant (h-1 ))

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 3. Logistic Equation

Batch growth curve assumes a sigmoidal shape

This shape can be predicted by combining the

Monod equation with the growth equation and an
equation for the yield of cell mass based on
substrate consumption.

(Assumption: No endogenous metabolism)

Logistic Equation

Integrated Form of
Logistic Equation
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Logistic Equation
This equation requires a predetermined knowledge of
the maximum cell mass in a particular environment.
Logistic equations are a set of This maximum cell mass is denoted as ; carrying
equations that characterize growth capacity.
in terms of carrying capacity.

A usual approach:
Specific growth rate is related to the amount of
unused carrying capacity

In your textbook, it is written as «X». Please be aware that

this is the average value of X!
Integration with boundary condition t=0, X=Xo

Logistic curve

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Example 6.2 in Your Textbook

PLEASE TRY TO PREPARE THIS TABLE AND THEN CHECK THE RESULTS.
Solution
Divide by X

You can take X(infinite) as

10.8 g/L.

A value of k = 0.24 h-1

would describe most of
the data.

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
 Another approach would be to take the log of the above equation:

and to fit the data to this equation and estimate k from the intercept. In this case k would be
about 0.25 h-1 .

Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002