Kinetic of Substrate utilization, product formation and

biomass production in cell culture

 Introduction
• When a small quantity of living cell is added to a liquid solution of
essential nutrients at a suitable temperature and pH, the cell will grow.
• Cell growth process got two different manifestation according to the
morphology of cell involved
• For unicellular organism which divide as they grow, increase in biomass (mass of
living matters) are accompanied by increases in the number of cells present.
• Increase the growth of mold size

• Associated with cell growth are two other processes:

• Uptake of some material from the cell’s environment and
• Release of metabolic end product into the surrounding.
Figure: Summary of some of the important parameters, phenomena and interactions
which determine cell population

Environment (Medium) Cell Population

Nutrients
Substrates
• Multicomponent • Multicomponent
• Reactions in solution Products • Cell to cell heterogeneity
• Acid-base equilibria • Multireaction
• Variable pH, T, ….. • Internal controls
Heat
• Changing rheological properties • Adaptability
(visocity,…..) • Stochastic
• Multiphase (G-L, L-L, G-L-L) • Genetic drift
• Spatial nonuniformity Mechanical interactions
Different perspective for cell population kinetic representation

Unstructured Structured

Most idealized case Multicomponent

Unsegregated

Balance growth average

Cell population cell
Treated as one description
(approximation)
Component
solute

“average cell” “average cell”

approx imation approx imation
Segregated

Multicomponent
Single component Balance growth
average
Heterogeneous cell
Individual cells (approximation)
description

Actual case
Other models used to describe cellular growth
 Basic Purposes of Reactors
- Mixing of substrates, contacting catalyst
- Mass transfer (G/L, L/L, G/S, L/S)
- Heat transfer
- Control of environment
- Containment (protection from/of environment)
Types
- Batch Reactor (BR)
- Continuously Stirred Tank Reactor (CSTR)
- Plug Flow Reactor (PFR)
- Packed Bed Reactor (PBR)
 Batch Reactor
Batch Reactors are defined as reactors in which no flow of mass across the
reactor boundaries, once the reactants have been charged.

Schematic Representation of Batch Reactors:

Stirrer

Liquid Surface
Tank V
 Growth curve of a batch culture

Figure : Growth curve of a batch culture . (a) acceleration phase (b) retardation phase and © declining phase

The figure shows an increase of cell at the start of the cultivation. This is due to the presence of enough nutrient for the
cell to grow. At same time the amount of nutrient decreases as it being consumed by the cell. Other side products such
as carbon dioxide or ethanol is also formed simultaneous.
• In batch cultures, the cell properties such as;
- size of cells
- internal nutrients
- metabolic function
vary considerably during the above growth phases.
• No apparent increase of the amount of cell at the start of cultivation, this is termed as the lag phase.
After this period, the number of cells increases exponentially thus, this stage is called exponential
growth phase;
- the cell properties tend to be constant
- last for a short period of time
• The next stage is the stationary phase, where the population of cell achieved it maximum number .
This is because
- all nutrient in the closed system has been used up by the cell
- lack of nutrient will eventually stop the cell from multiplying
• The final stage of cell cultivation is the death phase. the decrease of the number of cell occurs
exponentially which happens when the cell breaks open (lysed) . The rate of death normally follows
the first-order kinetics
Batch Reactor
Contd….
Material Balance in Batch Reactor

• A material balance on moles of component i shows that the rate of

accumulation of component i (given by the time derivative of total amount
of concentration i in the reactor) must be equal to the net rate of formation
of component i due to chemical reaction in the vessel.

Thus,

d
culture volume molar concentration of component i 
dt
 moles i formed by reaction 
 culture volume . 
 unit culture volume  unit time 
Contd….

or, d
VR  ci   VR  rfi
dt
where
VR  culture volume
moles i
ci 
unit culture volume

moles of i formed by reaction

rfi 
unit culture volume  unit time
• If no liquid is added or removed from the reactor and if gas stripping of
culture liquid is negligible, then VR is constant. Thus we can write
dci
 rfi
dt

A similar balance may be formed in terms of mass or number of density of a

component.
if the component i is contain in a gas stream entering or leaving the reactor,
the corresponding terms giving the net rate of component i addition to the
reactor by gas flow must by added to the above material balances.
 Characteristics properties of batch reactor
- Each batch is a closed system.
- The total mass of each batch is fixed.
- The volume or density of each batch may vary as reaction proceeds.
- The energy of each batch may vary as reaction proceeds; heat exchanger may be
provided to control temperature.
- The reaction (residence) time for elements of the reacting fluid is the same.
- The operation of the reactor is inherently unsteady-state; batch composition
changes with respect to time.
- At any time, the batch is uniform in composition, temperature because of the
efficient and vigorous stirring
 Continuous Stirred Tank Reactor (CSTR)
Continuous Stirred Tank Reactors (CSTR) are defined to be flow reactors
characterized by intense mixing so that the properties anywhere inside the
reactor are exactly the same as that of the exist stream.
Schematic Representation of CSTR:
Stirrer

Liquid Surface
Input Rate V

Output Rate
 CSTR
• Batch and continuous culture system differ in that, in a continuous culture
system, nutrients are supplied to the cell at a constant rate and in order to
maintain a constant volume of biomass in the reactor, an equal volume of cell
culture is removed.
• This will allow the cell population to reach a steady - state condition.
• Similar to the batch cultivation, the air is pumped into the culture vessel
through a sterile filter. Bubbling of air provides
• - supplying air for the growth of aerobic culture
• - it also ciculate and agitate the culture
• - pressurise the head space of the culture vessel such that to provide a force
during the removal of the media ( and cells) from the vesselfor analysis (OD,
call viability etc)
 Characteristics of CSTR
- The flow through the vessel(s), both input and out streams, is continuous but not
necessary at a constant rate.
- The system mass inside each vessel is not necessary fixed.
- The volume or density of each batch may vary as reaction proceeds.
- The energy of each batch may vary as reaction proceeds; heat exchanger may be
provided to control temperature.
- The reaction (residence) time for elements of the reacting fluid is the same.
- The operation of the reactor may be steady state or unsteady-state.
- The fluid properties are uniform in composition, temperature anywhere in the
vessel because of the efficient and vigorous stirring
CSTR

Schematic of CSTR
Contd……..
CSTRs
Chemostat:
A completely mixed continuous stirred-tank reactor for the cultivation of cells are called
chemostats.
It has the following configurations
• Mixing is supplied by means of an impeller, rising gas bubbles or both
• The mixing is so vigorous that each phase of the vessel contents is uniform composition
• The liquid effluent has the same composition as the reactor contents
• Control flow rate and concentration of growth-limiting nutrient of liquid medium entering
and exiting a growth chamber (bioreactor)
• Control
• pH
• Temperature
• Concentration of terminal electron acceptor
• Concentration of toxic by-products of metabolism
 Contd……..
• Because of complete mixing, the dissolved-oxygen is the same throughout the
bulk liquid phase. This is the crucial importance in considering aerated
CSTRs
• The aeration system maintains dissolved oxygen in the CSTR above the
limiting concentration
• As the vessel is well stirred, has adequate heat removal capacity, and is
equipped with a satisfactory temperature controller, we can assume it a
isothermal at the desired temperature and proceed with investigation of
microbial reaction process
Contd……..
• In the steady state, where all the concentrations within the vessel are
independent of time, we can write
Rate of addition  rate of removal rate of formation
      0
 to reactor   from reactor   within reactor 

• Let VR donate the total volume of culture within the reactor, the steady-state
CSTR material balance can be write

F cif  ci   VR rfi  0
Where F= volumetric flow rate of feed and effluent liquid stream
cif = component i molar concentration in the feed stream
ci = component i concentration in the reaction mixture and in the
effluent stream.
Contd……..
• Rearranging the equation we can write
rfi  ci  cif   Dci  cif 
F
VR
The parameter D is called dilution rate and defined as

F
D
VR
The rate of formation could be easily evaluated based upon measurements of
the (steady state) inlet and exit stream.

• The dilution rate is equal to the number of tank liquid volumes which pass
through the vessel per unit time.
• D is reciprocal of the mean holding time or mean residence time or space time
 Advantages and Disadvantages
Types of Advantages Disadvantages
operation
Versatile: can be used for different reaction everyday High labor cost: skilled labour is required
Safe: can be properly sterilized. Much idle time: sterilization growth of inoculum,
Batch cleaning after fermentation
Little risk of infection or strain mutation.
Safety problem: when filling , emptying , cleaning
Complete conversion of substrate is possible
Works all the time: low labour cost, good utilization of Often disappointing: promised continuous
reactor production for months fails due to (a) infection eg a
short interruption of the continuous feed sterilization.
Often efficient: due to the autocatalytic nature of microbial
reactions, the productivity can be high. (b) spontaneous mutation of microorganism to non-
Continuous,
producing strain
steady state Automation may be very appealing.
Very inflexible: can rarely be used for other
(Chemostat) Constant product quality
productions without substantial retrofitting
Downstream: all the downstream process equipment
must be designed for low volumetric rate, continuous
operation
Combines the advantages of batch and continuous Some of the advantages of both batch and continuous
operation. operation but the advantages far outweigh the
Semi-batch
disadvantages, and fed-batch is used to produce both
(Fed-batch) Excellent for control and optimization of a given production
biomass (baker’s yeast) and important secondary
criterion
metabolites (e.g. penicillin)
 Kinetics of Balance Growth
• The net rate of cell mass growth rx is given by the equation
Where,
rx   x
x = cell mass per unit culture volume
µ = the specific growth rate of the cell

• Using this representation in the steady-state CSTR material balance for cell
mass gives
D x f  D   x
• The feed stream is normally sterile medium. Therefore xf = 0 and Dxf=0.
Contd….

• A cell population > 0 can be maintained if the specific

growth rate µ is balanced by the dilution rate
• In this case, nonzero cell population can be maintained
if D = µ
• i.e., when the culture has adjusted so that its specific
growth rate is equal to the dilution rate.
• Experiment with Bacillus linens culture confirmed the
indeterminate nature of population level. After a
steady continuous operation at 6 h, two subsequent
interruption of the culture was observed.
• In this case, a portion of the reactor contents
consisting of the cell plus medium was removed and
replaced by medium alone
• Following each interruption, the system achieved a
new steady population of different size
 Monod Growth Kinetics
Types of media
• Synthetic medium is one in which chemical composition is well defined.
e.g., minerals based with necessary carbon, nitrogen and energy as well as
vitamins
• Complex media contain material of undefined composition. e.g., mixed
with unknown extract chemicals. Complex media including beef broth,
blood infusion broth, corn-steep liquor, sewage.
• The general goal in making a medium is to support good growth and/or high rate
of product synthesis
• Should not supplied too much nutrient. Excessive nutrient can inhibit or even
poison cell growth
• If the cells grow too extensively, their accumulated metabolic end will often
disrupt the normal biochemical processes of the cells
• Therefore, the growth process are limited by limiting the amount of nutrient in
the medium
 Monod Growth Kinetics
• If the concentration of one essential medium constituent is varied while the
concentrations of all other medium components are kept constant, the growth
rate changes in a hyperbolic way
 Monod Growth Kinetics
• A functional relationship between the specific growth rate m and essential
compound’s concentration was proposed by Monod in 1942.
max s
•  -----------------7.10
Ks  s
Here µmax is the maximum growth rate achieved and Ks is a saturation constant,
when s>>Ks and the concentrations of all other essential nutrients are
unchanged.
Ks is that value of the limiting nutrient concentration at which the specific
growth rate is half its maximum value. It is the division between the lower
concentration range, where  is strongly dependent on s. and the higher range
where  becomes independent of s
 Contd…..

• The Ks values for E. coli strains growing in glucose- and tryptophan-limiting

media are 0.22 x 10-4 M and 1.1 ng/ml, respectively
 Contd…..

• The value of Ks is rather small. Thus s>>Ks and the term s/(Ks + s) may be
regarded simply as an adequate description for calculating the derivation of µ
and µmax as the concentration of s become smaller
• The relation also suggests that the specific growth rate is finite (µ ≠ 0) for any
finite concentration of the rate limiting component

• When the population growth is related to limiting nutrient as proposed by

Monod, a definite connections emerge among reactor
• operating conditons
• microbial kinetics
• and stoichiometric parameters
 Contd…..
• To show this we can write a mass balance on limiting substrate which couples
to the cell mass balance since µ depends on s
• In the substrate balance we can write the yield factor

• The steady-state mass balance on substrate is then

mass of cells formed

• YX / S  ---------7.11
mass of substrate consumed

Ds f  s  
• 1
x  0
• YX / S
Contd…..
• Putting the value of µ
• The corresponding cell mass balance is

max sx
Ds f  s   0
YX / S s  K s 
• -----------------7.12

  max s 
 •
 D  x  Dx f  0

• ---------------------7.13
 K s s 

• These two equation are called Monod chemostat model equation

 Contd…..
• For the common use of sterile feed (xf = 0), thus the can be solved for x and
s to yield

 DK s 
x sterile feed  Y X / S  s f  
  max  D  ----------7.14

and DK s
x sterile feed  ------------------7.15
 max  D
Above two equation contain the explicit steady state dependence of x and s on flow rate
(D = F/V). For very slow flows at given volume, D 0 ; thus s tends to zero.
• As D increases continuously, s increases first linearly with D and then
still more rapidly as D → µmax . The cell-mass concentration x declines
with the same functional behavior : first linearly in D, then diminishing
more rapidly as D → µmax.
• At this point as D approaches µmax , x become zero. The dilution rate D
has just surpassed the maximum possible growth rate, and the only
steady-state solutions is x = 0 .
• This condition of loss of all cells at steady state, termed wash out , occurs
for D greater than Dmax , where x = 0 then ,

Dmax = µmax sf / Ks + sf --------------7.16

Figure 7.6. Dependence of effluent substrate concentration s, cell concentration x,
and cell production rate xD on continuous culture dilution rate D as computed from
Monod chemostat model. ( µmax = 1 h-1 , Ks = 0.2 g/L, Yx/s = 0.5 , sf = 10 g/L)
  DK s 
x sterile feed  Y X / S  s f  
  max  D 
D
- Equation 7.21 allows calculation
of the concentration of product in
the effluent.
- The rate of product output is
then given by pD, which is
maximized for constant Yp/x
- Then D has the value specified
in equation 7.18.
 Kinetics Implication of Endogenous and Maintenance
Metabolism
• The data presented in Figure 7.8 for A.
aerogenes show a marked decline in
cell concentration as dilution rate
becomes small.
• Similar behavior has also been
observed for the food yeast Torula
utilis.
• This trend, which is contrary to the
Monod chemostat model, can be
explained by including the possibility
of endogeneous metabolism in the
model.
• By endogenous metabolism we mean
that there are reactions in cells which -------------7.22
consume cell substances. Thus we
might write.
• The additional term in equation 7.22
can be interpreted formally as a cell
death rate.
• Such a modification of the Monod model is also consistent with other
available data. For example , if the rate of respiration of an aerobic
culture is proportional to the rate of substrate utilization.

----------------------------7.23

• Then from equation (7.8), (7.22) and (7.23) it follows that the specific
respiration rate is

----------------------------7.24
• Figure 7.9 displays experimental data which agree
nearly with eq 7.24
• Observed variation in the yield coefficient Y and
D also support a growth rate of the form given by
eq. 7.22. If the rate of substrate disappreance is

-----7.25

• Where Y'x/s is the true coefficient using equation (7.22)

and (7.25) and the defination of Yx/s ( remember Yx/s
is an overall stoichiometric coefficient equal to the total
mass of cells produced divided by the total mass of
substrate consumed , so that with sterile feed - rs =
Dx/Yx/s) gives

-----7.26
At larger dilution rate, continuous culture behavior can deviate significantly
from the Monod chemostat model as shown in Fig. 7.10.
 Other forms of growth kinetics
• Other related form of specific growth rate
dependence have been proposed which is
particular instances give better fits to
experimental data . -----7.29
• For example Teissier , Moser and Contois
suggest following model.
• The first two example render algebraic
solution of the growth equations much
more difficult than the Monod form -----7.30
• The equation of Contois contains an
apparent Michaelis constant which is
proportional to biomass concentration x .
• This last term will therefore diminish the
maximum growth rate as the population
density increases, eventually leading to µ -----7.31
∞ x-1
• The specific growth rate may be inhibited by medium constituents such as substrate or
product. An example due to Andrews propose that substrate inhibition be treated by the form.

--------7.32
• Alcohol fermentation provides an example of product inhibition ; the anerobic glucose
fermentation by yeast has been treated by Aiba, Shoda and Nagatani with specific growth
function of the type

--------7.33

• It is possible that two ( or more) substrate may simultaneously be growth limiting . While few
date are avaibale , a Monod dependence on each limiting nutrient may be proposed , so that

--------7.34

• In the absence of convincing data for this form , we may regard it simply as a useful indicator
that growth depends on several limiting nutrients.
 Other Environmental Effects on Growth Kinetics
• During balanced growth, only a single parameters µ [ or the
population doubling time td ( = ln 2/µ)] is required to characterize
population growth kinetics.
• For this reasons, the magnitude of the specific growth rate µ is
widely used to describe the influence of the cell environment on the
cells performance.
• Consider first the influence of temperature : the range of
temperature capable of supporting life as we know lies between
roughly -5 and 95oC
• Procaryotes can be classified according to the temperature interval
in which they grow, as shown in table 7.2
• The data in Fig. 7.11 for growth of E. coli dramatically illustrate the
strong influence of temperature
• Apparatenly at low temperature the metabolic activity of the cell increases
with increasing temperature as the activities of its enzyme rise.
• When the most thermally sensitive essential protein denatures, however ,
the cell dies.
• This hypothesis has been confirmed in several instances by genetic studies
in which mutation of a single gene has caused a large change in the
maximum tolerable temperature for a microorganism
• Since protein configuration and activity are pH dependent
• Hence, cellular transport processes, reactions, and hence growth rates to
depend on pH.
• Bacterial growth rates are ususally maximum in the range of pH from 6.5 to
7.5.
• This is exemplified by the data for E. coli shown in Fig. 7.12.
• There is exceptions, however acidophilic bacteria which grow at pH 2.0.
• Yeasts grow best in the pH range 4 to 5, while pH optimum for mold growth
are usually between 5 and 7. Most yeast and molds grow over a wide range
of pH from 3.0 to 8,5.
 Transient growth kinetics

• During certain intervals in batch cultivation or during start up or disturbances to

continuous flow reactors, cell populations grow in a transient state in which
more complicated kinetics behavior may be observed. Different types of kinetic
models may be required to describe different transient growth situations.
• Growth-cylce phases for batch cultivation in a typical batch process the
number of living cells varies with time, as shown in Fig. 7.13. After a lag
phase, where no increase in cell numbers is evident, a period of rapid
growth ensues, during which the cell numbers increase exponentially with
time.
• Normally in closed vessel the cells cannot multiply indefinetly, and a
stationary phase follows the period of exponential growth. At this point the
population achieves its maximum size. Eventually a decline in cell numbers
occurs duing the death phase. Here an exponentially decrease in the
number of living individuals is often observed.
Growth -Cylce Phases for Batch Cultivation
 contd……
Each phase is of potential importance in microbiological process
• General objective of a good design may be to
• minimize the length of the lag phase ,
• maximize the rate and length of the exponential phase,
• slowing the onset of the transition to stationary growth.
• To achieve such goals, we should understand the variables which influence the phase of batch
growth.
• The shock of rapid switch to a new environment has several effects on the living cell
• control and regulation of enzyme activity include an adaptive characteristics: when presented
with a new nutrient, its assimilation is achieved by cell production of new enzymes.
• thus transfer of glucose-bred culture in its exponential phase to a lactose medium will necessarily
result in a time interval of insignificant cell division rate while the enzymes and cofactors for the
lactose metabolic pathway are synthesized in the cell.
• Similarly, variation in the concentration of nutrients may cause a lag phase. If the new nutrient
medium is richer in a limiting nutrients, some time and nutrient will be expended in
nonmultiplicative growth while large concentration of metabolizing enzymes are created.
• A decreased nutrient level may result in no lag at all; the exponential rate may resume
immediately but at a slower pace.
 contd……

• Many of the intracellular enzyme require activation by small molecules

(vitamins, cofactor) or ions ( activators) which may have appreciable
permeability through the cell membranes.
• Transfer of a small culture volume or inoculum to a large volume of medium
will cause outward diffusion of these requisites for catalysis into the bulk
medium if the new medium is lacking in these species or differs appreciably in
ionic strength.
• The rate of growth will fall, corresponding to the lower concentrations of such species
inside the cell, and again a lag will appear while new machinery to generate such
activators is assembled.
• If essential activators are diluted (vitamins and ions which the cell cannot produce
internally), the total level of cell activity must diminish irrevocably.
 contd……

• The size of the transferred inoculum is also an important variable

• young cell population shows no lag upon transfer into a medium rich
in metabolic intermediates such as amino acids, the same inoculum
transferred into ammonium sulfate medium loses these vital
intermediates into solution
• with cultures in exponential growth at the time of transfer, their
original medium may already contain a reasonable bulk
concentration of intermediates, and the dilution on transfer will have
a lesser effect.
• Transfer of an old culture (approaching or in stationary phase) into
ammonium sulfate medium results in a longer lag.
• Multiple lag phase may contd……

sometimes be observed
when the medium contains
multiple carbon sources
(Fig. 7.14)
• This phenomenon, known
as diauxic growth, is caused
by a shift in metabolic
patterns in the midst of
growth.
• After one carbon substrate
is exhausted, the cell must
divert its energies from
growth to "retool" for the
new carbon supply.
• A possible explanation for Figure 7.14. In a medium containing initially equal amount
this serial utilization of glucose and xylose , diauxic growth of E. coli is observed
phenomenon is catabolic in batch culture.
repression.
 contd……

• Design to minimize culture and process times normally includes minimization

of the lag times associated with each new batch culture. Which can be
achieved as:
• The inoculating culture should be as active as possible and the
inoculation carried out in the exponential-growth phase
• The culture medium used to grow the inoculum should be as close as
possible to the final full scale fermentation composition.
• Use of reasonably large inoculum ( order of 5 -10 % of the new
medium volume) is recommended to avoid undue loss by diffusion of
required intermediate or activators.
contd……
Growth Phase for Batch Cultivation: Toxin effect
 Figure 7.15 illustrate this behavior. In the
plot of n ( the number density of cells at
stationary phase) for the bacterium A.
aerogenes vs lactose concentration
,however , distinct various in equation 7. 41
are obvious.

Figure 7.15. Dependence of maximum

batch population size on the initial
concentration of growth limiting
nutrients. (a) Pseudomonas sp. in
fructose medium and A. aerogenes in
(b) lactose and (c) ammonium tartarate
media.
• Equation (7.42) indicates that growth ceases only when ct , reaches a
particular level ct = 1/b . Dilution of a given toxified medium or addition
of a non-nutritive substance which complexes with a given toxin should
allow further growth and a consequent increase in the maximal
stationary phase biomass concentration xs.
• If growth halts due to nutrients exhaustion, dilution with a nonnutritive
volume produces no changes in xs.
• These criteria may be roughly utilized to determine the cause of growth
decline and eventual halt.
Unstructured growth model