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Kinetics of Cellular Growth
Kinetics of Cellular Growth
Objectives:
At the end of this lecture and supplemental readings, the students will be able to
Discuss various phases of cell growth in batch culture and the model that describe them.
Lecture Outline
2. Batch Reactors
2.1.Batch Reactor Applications
2.2.Cell Growth in Batch Culture: Growth Curve
3. Biomass Yield
4. Growth as Function of Substrate Concentration: The Monod Equation
5. Maintenance Energy
6. Product Formation
7. Oxygen Demand
8. Oxygen Transfer
9. Effect of Temperature
10. Material Balance in Batch Reactor
APRollon ChE 233 BRD Lecture 2.7 How Cells Grow in Batch Culture Page 1 of 28
1. Cellular Metabolism as Redistribution of Energy
Cell functioning
CO2, H2O, heat
& maintenance
Substrate
The energy used for above (growth and maintenance) is derived from the energy provided by ATP.
When ATP ADP + Pi (phosphate): energy is released and allows work-requiring process to
happen.
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ATP used for the above growth and maintenance requirement are ATP formed in substrate-level
phosphorylation or electron transport chain.
In ethanol fermentation, the fungus grows while it coverts sugar in the feed to ethanol. In biological
wastewater treatment, microorganisms grow as they consume organic contaminants in the
wastewater and convert them to oxidized form and product, which is still of higher energy level.
As in any chemical reaction, reactants react and products are formed at definite proportions. In a
balanced chemical reaction, these proportions are indicated by the respective stoichiometric
coefficients of the reactants and products. Examples of balanced biochemical reactions are shown
in previous lectures. These balanced biochemical reactions show the amount of cells
(microorganisms or biomass) formed and the amount of products produced per unit amount of
substrate consumed. These proportions are known as yield of biomass on substrate (YSX) and yield
of product on substrate (YSP).
YSX ≡ amount of biomass produced per unit amount of substrate consumed.
YSP ≡ amount of product produced per unit amount of substrate consumed.
The relations for several types of bioprocesses are discussed below.
Aerobic growth on a single carbon source and energy source, a C-free nitrogen source,
no formation of products.
1
rS rX mS X Eqn. 1.1
YSX
where
rS ≡ substrate utilization rate (mol substrate.m-3.d-1)
rX ≡ biomass production rate (mol biomass.m-3.d-1)
mS ≡ maintenance coefficient (mol substrate.mol-1 biomass.d-1)
X ≡ biomass concentration (mol biomass.m-3)
The rate of biomass production (rX) is a function of the specific growth rate () and the biomass
concentration (X).
rX X Eqn. 1.2
where
≡ specific growth rate, i.e., amount of biomass formed per unit amount of biomass
present (d-1)
When there is product formation:
1 1
rS rX rP mS X Eqn. 1.3
YSX YSP
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where
rP ≡ product formation rate (mol product.m-3.d-1)
The rate of product formation (rX) can be expressed as function of the specific product formation
rate (qP) and the biomass concentration (X).
rP q P X Eqn. 1.4
where
qP ≡ specific product formation rate, i.e., amount of product formed per unit amount of
biomass present (mol product.mol-1 biomass.d-1)
Yield parameters are relatively independent of pH, medium composition and growth rate of the
cells. Thus, yield values determined under certain conditions of pH, etc., can be applied to other
conditions. For example, yield values for suspended cells can be taken as equal to the yield for
immobilized cells, as in biofilms. Theoretical yield values are derived from biochemical
pathways and stoichiometry.
Overall yield
The overall yield for biomass when there is no product formation:
rX X
YSXOV Eqn. 1.5
rS
X mS X
YSX
Multiplying by YSX/:
YSX
YSXOV Eqn. 1.6
Y
1 m S SX
The influence of several parameters can be illustrated by performing simulations with a set of
data. For example:
= 0.01 h-1
YSX = 0.55 kg.kg-1
mS = 0.025 kg.kg-1.h-1
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When the growth becomes too small ( << mS), too much substrate is used for maintenance and
the overall yield starts to decrease. If maintenance requirement can be neglected, the overall
yield YOVSX = YSX.
The maintenance and yield can be varied only in a limited range. The influence of these two
parameters is therefore limited.
The influence of several parameters can be illustrated by performing simulations with a set of
data. For example:
= 0.01 h-1
YSX = 0.55 kg.kg-1
YSP = 0.55 kg.kg-1
qP = 0.010 kg.kg-1.h-1
mS = 0.025 kg.kg-1.h-1
Homework 1. Draw a plot of the overall yield at different values of the pertinent parameters.
Note that:
The specific product formation rate, qP, is the main important variable that influences the
overall yield.
When qp decreases, the effect of the maintenance and growth increases, reducing the overall
yield of product on substrate.
When the specific growth rate increases, the overall yield decreases.
The parameters mS and YSX can only be varied in a limited range. They do not have so much
effect on overall yield for product.
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Specific growth rate
The specific growth of the microorganism is dependent on the substrate concentration (S) as well
as kinetic parameters (kS and m) that are characteristic of the microorganism.
S
max Eqn. 1.9
Ks S
where
m = maximum specific growth rate (d-1)
kS = Monod half-saturation constant (mol.m-3). This is the value of S at half the
maximum specific growth rate.
S = substrate concentration (mol.m-3)
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2. Batch Reactor (BR)
closed, well-mixed
(i.e. not continuously fed &
no withdrawal of reactor contents)
At start:
Nutrients are added and mixed in suspension.
Inoculation: Viable microorganisms are added.
Feeding: Input of substrate (that is, organic pollutants present in wastewater)
Biological process proceeds in time. Microorganisms grow. They feed on substrate. In
wastewater treatment, a consortium of microorganisms grows. The product of one group may
be the substrate for another group. Hence, substrate (i.e., organic pollutants) concentration
decreases while cell (biomass) concentration increases. Products are produced. Gaseous
products leave the liquid or aqueous system while dissolved products accumulate in the liquid
or aqueous system. There is no withdrawal of effluent during the batch process.
When, needed, BR may be provided with pH and temperature control.
When the desired biological process requires air, the BR is aerated.
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2.2.Batch Growth Curves
A. Lag phase
- cells adapt their-enzymatic equipment to new medium
A cell does not produce at all times large quantities of all enzymes it can produce on the basis of
its genetic material. It produces only those that it actually has to use in a given situation.
assimilatory enzyme - always required and always produced irrespective of the
composition of medium
inducible enzyme - produced only when needed - to be produced requires inducer,
e.g. a substrate of the enzyme or a compound structurally similar to its substrate
Production of enzymes also requires ATP + building blocks for protein synthesis.
Synthesis of enzymes which are not required is blocked by Depressor which acts on DNA. The
inducer counteracts repressor, hence, permit the concerned DNA to "order" induced synthesis of
that enzyme.
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Lag phase
After inoculation, microorganisms need to adjust to the new environment, i.e., to
prepare the enzymatic machinery for the specific cellular functions involve in
metabolism (process of growth anabolism).
Depending on the composition of nutrients, new enzymes are synthesized, the
synthesis of some other enzymes is repressed, and the internal machinery of cells is
adapted to the new environmental conditions.
Virtually no growth although metabolic activity is high. There can be increase in cell
mass but no increase in cell number density.
When the inoculum is small and has a low fraction of cells that are viable, there may
be a pseudo-lag phase, which is a result, not of adaptation, but of small inoculums
size or poor condition of the inoculum
1. Low concentration of some nutrients and growth factors may also cause a long lag phase.
Examples:
o The lag phase of Enterobacter aerogenes (formerly Aerobacter aerogenes) grown
in glucose and phosphate buffer medium increases as the concentration of Mg2+
(which is an activator of the enzyme phosphatase), is decreased.
o Even heterotrophic cells require CO2 fixation (to supplement intermediates
removed from key energy-producing metabolic cycles during rapid biosynthesis),
and excessive sparging can remove metabolically generated CO2 too rapidly for
cellular restructuring to be accomplished efficiently, particularly with a small
inoculum.
2. The age of the inoculum culture has a strong effect on the length of lag phase.
Age refers to how long a culture has been maintained in a batch culture.
Usually, the lag period increases with the age of the inoculum. In some cases, there is an
optimal inoculums age resulting in minimum lag period.
To minimize the duration of the lag phase, cells should be adapted to the growth medium and
conditions before inoculation, cells should be young (or exponential phase cells) and active,
and the inoculum size should be large (5% to 10% by volume).
The nutrient medium may need to be optimized, and certain growth factors are included to
minimize the lag phase.
Multiple lag phases may be observed when the medium contains more than one carbon source.
This phenomenon, known as diauxic growth, is caused by a shift in metabolic pathways in the
middle of a growth cycle. After one carbon source is exhausted, the cells adapt their metabolic
activities to utilize the second carbon source. The first carbon source is more readily utilizable than
the second, and the presence of more readily available carbon source represses the synthesis of the
enzymes required for the metabolism of the second substrate.
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B. Exponential growth phase (a.k.a. logarithmic growth phase or maximum growth phase)
The increase in number of cells (or mass) is proportional to the number of cells.
Integral form: ln X = t + C where C = ln X(t=O) (Eqn. 2.2)
X = e(t + C)
The slope of the straight part (logarithmic phase) of the growth curve (X vs, t) represents .
Relation between and doubling time (td):
at ti , have X1 ln X1 = t, + C
at t2, have X = 2Xl ln 2X1 = t2 + C
---------------------
ln 2 = (t2-ti) = td
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C. Deceleration growth phase
(Eqn. 2.4)
(Eqn. 2.5)
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D. Stationary phase
End of deceleration growth phase: Growth becomes equal to rate of death no net
growth
Even though the net growth rate is zero during the stationary phase, cells are still
metabolically active and produce secondary metabolites. Primary metabolites are growth-
related products and secondary metabolites are non-growth related. In fact, the
production of certain metabolites is enhanced during the stationary phase (e.g. antibiotics,
some hormones) due to metabolite deregulation.
During the course of the stationary phase, one or more of the following phenomena may take
place:
a. Total cell mass concentration may stay constant, but the number of viable cells may
decrease.
b. Cell lysis may occur, and viable cell mass may drop. A second growth phase may
occur, and cells may grow on lysis products of lysed cells (cryptic growth).
c. Cells may not be growing but may have active metabolism to produce secondary
metabolites. Cellular regulation changes when concentrations of certain metabolites
(carbon, nitrogen, phosphate) are low. Secondary metabolites are produced as a result
of metabolite deregulation.
During the stationary phase, the cell catabolizes cellular reserves for new building blocks and
for energy-producing monomers. This is called endogenous metabolism.
The cell must always expend energy to maintain an energized membrane (i.e. proton-motive
force) and transport of nutrients and for essential metabolic functions such as motility and
repair of damage to cellular structures. This energy expenditure is called maintenance energy.
As such, maintenance energy and endogenous metabolism are not limited to the stationary
phase but become dominant in the stationary phase. The maintenance or endogenous
expenditure is just a small fraction of the total cell needs during maximum growth.
When the primary metabolism diminishes as in the stationary phase, the endogenous
metabolism becomes dominant. Often, dead cells lyse, and intracellular nutrients released into
the medium are used by the living organisms during stationary phase.
The reason for termination of growth may be either exhaustion of an essential nutrient or
accumulation of toxic products. If an inhibitory product is produced and accumulates in the
medium, the growth rate will slow down, depending on inhibitor production, and at a certain
level of inhibitor concentration, growth will stop.
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E. Phase of logarithmic death
Death phase
Cell decay rate > growth rate there is net decay!
Cell lysis prevails, i.e. breaking open of the cell membrane and the subsequent release
of cell contents into the medium.
Cell lysis reduces cell number until no living cells are left.
The death rate can be thought of as a first-order reaction. Because S is zero, µG is zero
starting from the stationary phase:
rX = dX/dt = kdX (Eqn. 2.6)
Or lnX = kdt + C Chick’s law (Eqn. 2.7)
During the death phase, cells may or may not lyse, and the reestablishment of the culture may
be possible in the early death phase if cells are transferred into a nutrient-rich medium.
In both the death and stationary phases, there is a distribution of properties among individuals
in a population. With a narrow distribution, cell death will occur nearly simultaneously. With
a broad distribution, a fraction of the population may survive for an extended period. It is this
fraction that would dominate the reestablishment of a culture from inoculum derived from
stationary- or death-phase cultures.
Thus, using an old inoculum may select for variants of the original strain having altered
metabolic capabilities.
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3. Biomass Yield
A growth yield accounts for the substrate consumption in the first two terms (right hand
side of above equation) would be more logical.
Maintenance needs, are required whether the cell is growing or simply staying functional
(or viable), although they can be growth rate dependent in some fashion.
Conversion to extracellular products may not be directly related to the cell biomass
growth, although it is at least related to maintenance needs.
For organisms growing aerobically on glucose, YFX/S is typically 0.4 - 0.6 g/g for most
yeast and bacteria, while YFX/O2 is 0.9 - 1.4 g/g.
Anaerobic growth is less efficient, and the yield factor is reduced substantially.
With substrates that are more or less reduced than glucose, the value of the apparent yield
will change. For methane, YFX/S would assume values of 0.6 - 1.0 g/g, with the
corresponding YFX/O2 decreasing to about 0.2 g/g. In most cases, the yield of biomass on a
carbon-energy source is 1.0 - 0.4 g biomass per gram of carbon consumed.
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4. Growth Rate as Function of Substrate Concentration
where:
μ is the specific growth rate of the microorganisms
μmax is the maximum specific growth rate of the microorganisms
S is the concentration of the limiting substrate for growth
Ks is the "half-velocity constant"—the value of S when μ/μmax = 0.5
At low S such that S <<< Ks: First order (constant: µmax/Ks). At high S such that S >>> Ks: Zero
order (µ = µmax)
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5. Maintenance Energy
Maintenance coefficient – describes the specific rate of substrate uptake for cellular
maintenance, or
During the stationary phase, where little substrate is available, endogenous metabolism of
biomass components is used for maintenance energy.
Note:
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6. Product Formation
Examples: Lactic acid, xanthan gum, some secondary metabolites from cell culture.
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7. Oxygen Demand for Aerobic Microorganisms
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For example, with Azotobacter vinelandii at a DO = 0.05 mg/L, the growth rate is about
50% of maximum even if a large amount of glucose is present. However, the maximum
amount of cells formed is not determined by the DO, as oxygen is continually resupplied.
If glucose were totally consumed, growth would cease even if DO = 0.05 mg/L. Thus, the
extent of growth (mass of cells formed) would depend on glucose, while the growth rate
for most of the culture period would depend on the value of DO.
For aerobic microorganisms:
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,
depending on the pellet size of molds.
Saturated DO concentration in water at 25°C and 1 atm pressure is about 7 ppm. The presence of
dissolved salts and organics can alter the saturation value, while increasingly high temperatures
decrease the saturation value.
Oxygen is usually introduced to the fermentation broth by sparging air through the broth.
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8. Oxygen transfer
Oxygen transfer from gas bubbles to cells is usually limited by oxygen transfer through the
liquid film surrounding the gas bubbles. The rate of oxygen transfer from the gas to liquid phase
is given by:
When oxygen transfer is the rate-limiting step, the rate of oxygen consumption is equal to the
rate of oxygen transfer. If the maintenance requirement of O2 is negligible compared to growth,
then
In batch reactor with negligible medium volume loss (due to air sparging):
where C0* is the oxygen saturation concentration in pure water (Table 11.3),
C* is the oxygen saturation concentration in the medium,
Hj is the oxygen solubility interaction constants (Table 11.4),
Zj is the ionic charge of ionic species j, and
Cj is the concentration of species j in the fermentation medium.
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9. Effect of Temperature
Microorganisms have various ranges in temperature in which they thrive and where they have
their maximum growth. They can be classified according to these growth temperatures.
Optimum temperature is the temperature at which they grow at maximum specific growth rate.
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10. Effect of pH
Hydrogen ion concentration affects the activity of enzyme. Thus, it also affects microbial growth
rate.
Optimal pH for growth may be different from that for product formation. Acceptable pH range
varies from optimum pH by 1 – 2 pH units.
Many organisms can maintain intracellular pH at relatively constant level even when pH of its
environment fluctuates. maintenance energy increases.
pH may be affected by the N-source:
o if ammonium as sole N source: H+ are released into the medium (consume ammonia)
decrease in pH
o if nitrate: H+ is removed from the media (to reduce nitrate to ammonia) increase in pH
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11. Balance Equation for Batch Reactor (BR)
S X + P
substrate cells product
dC Sl 1 1
Substrate balance: V C SlV q P C XlV Eqn. 2.8
dt YSl YSP
dC Xl
Biomass Balance: V C XlV k d C XlV Eqn. 2.9
dt
dC Pl
Product Balance: V q P C XlV Eqn. 2.10
dt
mass
concentration substrate
product cell
time
Initial rate of substrate consumption is slow as cell growth is in the lag phase.
As substrate is consumed, product and cell mass are formed.
Net cell growth ceases when substrate is depleted.
Eventually cell mass will decrease as product mass is virtually constant.
In environmental process in BR where the starting medium is not sterile (i.e. other
organisms, provided other growth factors are sufficiently available)
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o Fed-Batch Reactor (FBR)
BR: One cycle (lag, exponential, stationary, and death) new cycle
considerable downtime (time in which reactor is not productive)
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