KMU 413
BIOTECHNOLOGY II
LECTURE NOTES-4
19.10.2021
How Cells Grow in
Continuous Culture?
Continuous Culture
Fresh nutrient medium is continuously supplied to a well-stirred culture.
Products and cells are simultaneously withdrawn.
Growth and product formation can be maintained for prolonged periods in continuous
culture.
After a certain period of time, the system usually reaches a steady state (cell, product,
and substrate concentrations remain constant).
Continuous culture provides constant environmental conditions for growth and product
formation and supplies uniform-quality product.
Continuous culture is an important tool to determine the response of microorganisms to
their environment and to produce the desired products under optimal environmental
conditions.
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
Some Specific Devices for Continuous Culture
The primary types of continuous cultivation devices:
Chemostat
Turbidostat
Plug flow reactors (PFR)
1) Chemostat:
Cellular growth is usually limited by one essential
nutrient.
Other nutrients are in excess.
When a chemostat is at steady state, the nutrient,
product, and cell concentrations are constant.
So, the name chemostat refers to constant
chemical environment.
A typical «chemostat»
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
Some Specific Devices for Continuous Culture
2) Turbidostat:
Cell concentration in the vessel is
maintained constant.
Optical density is monitored and feed flow
rate is controlled due to it.
When the turbidity of the medium exceeds
the set point, a pump is activated and fresh
medium is added.
The culture volume is kept constant by
removing an equal amount of culture fluid.
The turbidostat is less used than the
A typical «turbidostat» chemostat because it is more complicated
than a chemostat and because the
environment is dynamic.
Turbidostats can be very useful in selecting
subpopulations able to withstand a desired Engineering
environmental stress (for example, high ethanol Point of View
concentrations), because the cell concentration is
maintained constant. The selection of variants or mutants
with desirable properties is very important.
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
Some Specific Devices for Continuous Culture
3) A Plug Flow Reactor (PFR)
It can also be used for continuous cultivation purposes.
There is no backmixing in an ideal PFR.
Liquid recycle is required.
In a PFR, substrate and cell concentrations vary with axial position in the vessel.
In waste treatment, some units approach PFR behavior, and multistage chemostats tend to
approach PFR dynamics if the number of stages is large (five or more).
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
Chemostat
The most common tool.
It can be used for;
Investigate the effects of environmental
conditions on cell behaviours.
Determine the system parameters.
Select one cell type.
Engineering
Point of View
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
MATERIAL BALANCE ON THE CELL
CONCENTRATION IN AN IDEAL
CHEMSOTAT
The Ideal Chemostat
Mixing An ideal chemostat is the same as a perfectly mixed
continuous-flow, stirred-tank reactor (CFSTR).
Most chemostats require some control elements, such as pH
and dissolved oxygen control units.
Feed Output
Fresh sterile medium is fed to the completely mixed and
aerated (if required) reactor, and cell suspension is removed
at the same rate. Liquid volume in the reactor is kept
constant.
Aeration
A material balance on the cell concentration
𝑑𝑋
𝑑𝑡
Mass flow Mass flow Mass flow Mass flow Mass flow
rate of cells rate of cells rate of cell rate of cell = rate of cell
- + -
into the - out of the growth loss accumulation
reactor reactor
F is the volumetric flow rate of nutrient solution (l/h),
VR is the culture volume (l) (assumed constant),
X is the cell concentration (g/l),
µg and kd are growth and endogenous (or death) rate constants, respectively (h -1 ).
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
If «kd» is used, this means that the primary mechanism for cell mass decrease is
endogenous metabolism.
If «k’d» is used, this means that the primary mechanism for cell mass decrease is cell
death and lysis metabolism.
BUT; if this equation is written in terms of cell number, kd could only be a cell
death rate.
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
DILUTION RATE
(Nutrient Exchange Rate)
Equation on cell balance in
continuous flow bioreactor.
D: Dilution rate
Equation becomes:
Assumptions:
1. Feed medium is sterile, Xo=0
2. kd<<µg, kd=0 µg=D
3. Chemostats are generally operated as st-st, dX/dt=0
In a chemostat:
Cells are removed at a rate equal to their growth rate.
Growth rate of cells is equal to the dilution rate.
This makes the chemostat a powerful experimental tool that can be easily
controlled and operated.
Monod equation can be used in a chemostat since growth rate is limited by at least one
substrate:
A plot of 1/µg versus 1/S can be used to estimate values for µm and Ks
BİOREACTOR WASHOUT: If D is set at a value greater than µm, the
culture cannot reproduce quickly enough and is washed out.
For D < µm, the equation is rearranged:
You can predict the substrate concentration.
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
MATERIAL BALANCE ON THE
SUBSTRATE CONCENTRATION IN AN
IDEAL CHEMSOTAT
Material balance on the limiting substrate in the absence of endogenous
metabolism
S0 : feed substrate concentration (g/l)
S : outlet substrate concentration (g/l)
qP : the specific rate of extracellular product formation
YM X/S : Maximum cell yield coefficient (g cells / g substrate)
YP/S : Product yield coefficient (g product / g substrate)
Assumptions:
1. Extracellular product formation is negligible, qP=0
2. No endogenous metabolism, kd=0
3. Reactor is operated as st-st, dX/dt=0
Due to the assumptions 2 and 3, it was expressed that µg=D.
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
SO;
This equation shows «how
does X change with D?»
In the Presence of Endogenous Metabolism
If you consider the effect the inclusion of endogenous metabolism, the equation becomes:
Substitute this
equation into
Here, YMX/S is constant.
It is the maximum yield at
the conditions with no Divide by X
endogenous metabolism
or maintenance energy.
Apparent Yield: It varies with growth conditions if
kd > 0.
1/ When YX/S is written, it should be interpreted as
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
Y AP
Divide by D
Maintenance
coefficient
based on
substrate «S»
𝐾 𝑠 𝜇𝑔
𝑆=
𝜇𝑚 − 𝜇𝑔
In the presence of endogenous metabolism
=D+
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
MATERIAL BALANCE ON THE
PRODUCT CONCENTRATION IN AN
IDEAL CHEMSOTAT
Productivity in Chemostat
The balance on product formation gives:
For nongrowth-associated product formation qP is a constant (β),
For growth-associated products it is a function of µg.
If the «product formation term «qP» is not neglected in the substrate balance
equation:
This equ. was
obtained at
previous page.
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
The productivity of a chemostat for product (Prp) can be found from DP.
The productivity of a chemostat for biomass (Prx) can be found from DX.
(Setting the derivative equal to zero).
The optimal value of D (Dopt ) will depend on whether endogenous
metabolism and/or product formation are considered.
When kd = 0 and qP = 0, Dopt for biomass production (DX) becomes:
S0 is usually much greater than Ks.
So, Dopt will approach D = µm or the washout point.
Stable chemostat operation with D=µm is very difficult. A value of D slightly less than Dopt
may be a good choice.
Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002
Example 6.4 from your textbook
Example 6.5 from your textbook
