How cells Grow?

Cells Growth

Cell growth is a Result of Replication and Change in Cell Size

Growth is the most essential response to physiochemical environment

Microbes grow under different physical, chemical and nutritional conditions

In suitable medium, organisms extract nutrients and convert them into

biological compounds

Part nutrients used for energy and part for biosynthesis and product formation;
Thus,
Substrates + Cells →Extracellular products + more cells
ΣS +X → SP + nX

Microbial growth is a autocatalytic reaction

Growth rate is directly related to cell concentration and cellular reproduction

is the normal outcome of reaction
 Cells Growth
Cell growth is the primary response of viable cells to substrates and nutrients.

Product formation is a secondary response.

The net specific growth rate defined as

1 dX
μnet 
X dt
μ net = μ g − k d
Where, X is the cell mass concentration (g/l)
t is time (h)
μnet is net specific growth rate (h-1)
μnet is difference between a gross specific growth rate, μg, and cell mass loss
rate,
kd, due to cell death or endogenous metabolism
 Cells Growth

Microbial growth also described in terms of cell number concentration

1 dN
μR 
N dt
Batch growth
Cell number concentration
a) hemocytometer (Petroff-Hausser slide)
b) viable cell counts (petri dish)
c) Particle counter
d) Light scattering techniques
 Cells Growth

Counting Cells: Hemocytometer

• Counting Cells: Hemocytometer.

• Advantage: accurate, typically low noise in measurement.
• Disadvantage: time consuming, carcinogenic, mutagenic stains.
 Cells Growth

Counting Cells: Plate Counts

• petri dish or dilution plate counts: count colonies
(CFUs = colony forming units) formed by
individual cells (dilute sample).
• Advantages: counts viable cells, fairly accurate.
• Disadvantages: Takes days.
 Cells Growth

Particle counter
 Cells Growth

Cell mass concentration

a) direct methods
→ dry weight (filtration or centrifugation)
→ packed cell volume (centrifugation)
→ optical density (light scattering, 600-700 nm), flow cytometry

b) indirect methods
→ measure biomolecule concentration and correlate to dry cell mass concentration.
(DNA, protein, ATP, NADH, product formation)

Example 1. NH4+ utilization during growth releases H+, amount of OH- added is
proportional to growth.
 Cells Growth

Organisms selectively take up dissolved nutrients from liquid medium converting

them into biomass
Five phases of cell growth

1. Lag
2. Logarithmic/exponential growth
3. Deceleration
4. Stationary phase
5. Death phase
Cells Growth
 Cells Growth

Lag phase

Occurs immediately after inoculation

Period of adaptation and re-organization when transferred to new medium

New enzymes may be produced and some may be repressed

Cell mass may increase slightly without increase in cell number density

Pseudo lag phase sometimes occurs due to small inoculum size or poor
conditions of inoculum
Low concentration of some nutrients and growth factors

E.g, low concentration of Mg2+ in the nutrient medium of Enterobacter

aerogenes increases the lag phase
 Cells Growth

Age of inoculum culture has a strong effect on the length of the lag phase
Age refers to the time maintained in a batch culture
Usually the lag period increases with the inoculum age
Sometimes an optimum inoculum age exists

Many commercial fermentation plants rely on batch culture

For high productivity the lag phase must be as short as possible

Minimize duration of lag phase cells

Should be adapted to growth medium and conditions
Should be young (or exponential phase cells) and active

Nutrient medium and conditions also need to be optimized

certain growth factors may be included
 Cells Growth
Multiple lag phases may be observed
Medium contains more than one carbon source (diauxic growth)
After one carbon source is exhausted the cells again adapt their metabolic
activity to utilize the second source
First carbon source is more readily utilizable than the 2nd
The more readily available carbon source represses enzymes required for
the 2nd
 Cells Growth
Logarithm or Exponential Growth Phase
After adaptation cells multiply rapidly and the cell mass and cell number
density increases exponentially with time

Period of balanced growth

All components of the cell grow at the same rate
Average composition of the cell remains similar
Thus, the net specific growth rate determined from cell number or cell mass
would be the same
Due to large nutrient concentration the growth rate is independent of nutrient
concentration

The cellular metabolic control system is set to achieve maximum reproduction rates
 Cells Growth

1. Nutrient and substrate concentrations are large

2. Growth rate is independent of nutrient and substrate conc.
3. Cell number and mass concentrations increase exponentially

Exponential growth rate is first order and is characterized by a straight line on a semi-
log plot of ln(X) vs time

X- cell conc. at t= t
X0 – cell conc. At t = 0
 Cells Growth

Deceleration phase
Deceleration occurs due to
1. depletion of one or more nutrients or
2. accumulation of toxic by products of growth

Typically, changes occur in a short period

1. Unbalanced growth results
2. Cell composition and size will change; td ≠ t’d

In this phase stresses induced cause restructuring of cell to increase survival

The molecular mechanisms of repression and induction give rise to these observable
changes

Changes is cell physiology under conditions of nutrient limitation is more easily

studied in continuous culture
 Cells Growth
Stationary Phase
Though the net growth rate is zero (growth rate = death rate)
1. Cells are metabolic active and produce secondary metabolites (non
growth related products)
2. Certain metabolite production is enhanced due to metabolite
deregulation
E.g, antibiotics, hormones
During this phase the following may take place:
1. Total cell mass concentration may stay constant, but the viable cell
numbers may decrease
2. Cell lysis may occur and viable cell mass may drop; 2nd growth phase
may occur and cells may grow on lysis products of lysed cells (cryptic
growth)
3. Cells may not grow but may be active for secondary metabolite
production; cellular regulation changes when concentrations of certain
metabolites are low
 Cells Growth

Removal of inhibitory compounds will result in further growth if additional substrate is

provided
During stationary phase catabolism occurs to produce new building blocks and
energy-producing monomers
Endogeneous metabolism

Cell always expends energy to

1. Maintain an energized membrane (proton-motive force)
2. Transport nutrients, and for
3. Essential metabolic functions such are motility and repair of damage to
cellular structure
4. Energy expenditure called maintenance energy
Equation describing conversion or loss of cell mass into maintainance energy
is given by a first order differential equation
 Cells Growth

Termination of growith
Factors include:
1. Nutrient exhaustion
2. Toxic product accumulation

Inhibitory product if present will also slow growth once accumulated

Growth will slow down and stop
e.g., ethanol production by yeast causes inhibition to growth

Adverse effects of toxin can be overcome by

1. Dilution of toxified medium
2. Addition of unmetabolized chemical compound complexing with toxin
3. Simultaneous toxin removal
 Cells Growth

Death Phase
This follows the stationary phase and some cell death may start in stationary
phase

No clear demarcation exists between the last two phases

Dead cell lyse and intracellular nutrients are used by the living organisms during
stationary phase

Rate of death usually follows 1st order kinetics

X
 Cells Growth

During death phase cells may or may not lyse

Re-establishment of culture may be possible in the early stages

During death and stationary phase a distribution of properties among individuals

may exist in a population

For narrow distribution, cell death may occur nearly simultaneously

For broad distribution, a fraction will survive for an extended period

This fraction would dominate the re-establishment of a culture from inoculum

derived from the last two phases

Using an old inoculum may select variants of the original strain having altered
metabolic capabilities
 Cells Growth

Yield coefficient defined as the consumption of another material

e.g., growth yield in a fermentation is

X
YX = −
S S
An apparent (or observed) yield is defined at the end of batch growth

It is not a true constant since substrate utilization can vary

True (a constant) and apparent growth yield will be better understood while
discussing continuous culture

Yield coefficients based on other substrates or product formation may be defined

X P
YX = − YP = −
O2 O2 S S
 Cells Growth

Maintenance coefficient
Describes Cellular Maintenance

Specific rate of substrate uptake for cellular maintenance given as

dS dt m
m−
X
During stationary phase where, little external substrate is available endogeneous
metabolism of biomass components are used for maintenance

Microbial products: Classified in 3 major categories

1. Growth associated products
2. Non-Growth associated products
3. Mixed-growth associated products
 Cells Growth

1. Growth Associated Products:

Produced simultaneously with Microbial Growth

Specific rate of product formation is proportional to specific rate of growth, μg

1 dP
qp = = YP  g
X dt X
μg is not the same as μnet since endogeneous metabolism is non-zero

E.g. Production of a constitutive enzyme is an example of a growth associated bio-

product
 Cells Growth

2. Non-growth product formation:

Takes place during Stationary Phase
Growth rate is zero
Specific rate of product formation is constant

qp = β = constant
E.g. Many secondary metabolites, such as antibiotics (e.g., penicillin) are non-growth
associated products
 Cells Growth

Mixed-growth Products:
Formation takes place during Slow Growth and Stationary Phase

The specific rate of product formation is given by the Luedeking-Piret equation:

qp = αμg + β

If α = 0 the product is only non-growth associated

If β = 0 the product would be only growth associated and α = YP/X

E.g. Lactic acid fermentation, xanthan gum and some secondary

metabolites are examples
 Cells Growth

Kinetic pattern

a. Growth associated products

b. Mixed-Growth associated products
c. Non-growth associated products
 Cells Growth

Effect of environmental conditions : Temp, pH, and dissolved O2

Temperature
1. Psychrophiles - <200C
2. Mesophiles 20 to 500c
3. Thermophiles >500C

μmax doubles for every 100C increase in temperature towards optimal temperature

 dX  =  − k X
dN
= (  ' R − k 'd ) N  dt  ( d )
dt
μ’ = A e-Ea /RT

k’d = A’e-Ed / RT
Ea – activation energy for growth (10 to 20 kcal/mol)
Ed- activation energy for death (60 to 80 kcal/mol)
 Cells Growth

Temperature may affect the rate limiting step

Maintenance coefficient may increase when the T increases over optimal temp.

Arrhenius plot of growth rate (E. coli)

Rich complex medium

Glucose-mineral salt medium

 Cells Growth

Effect of pH
Optimal pH for growth may be different from product formation
pH range varies by organism
bacteria (most) pH = 3 to 8
yeast pH = 3 to 6
plants pH = 5 to 6
animals pH = 6.5 to 7.5

Microorganism have the ability to control pH inside the cell, but this requires
maintainance energy

pH can change due to

utilization of substrates; NH4+ releases H+,
NO3 - consumes H+
Can change production of organic acids, amino acids, CO2, bases
 Cells Growth

Effect of dissolved oxygen

O2 may be a limiting substrate for aerobic fermentation, since O2 is sparingly

soluble in water
High cell concentration – O2 rate limiting step

Above critical oxygen concentration - independent

5 to 10% of saturated DO (~7 mg O2/L) for bacteria/yeast

Glucose – rate limiting

 Cells Growth

How to supply oxygen?

- specific rate of oxygen consumption (mg O2/g dw of cells.h)

- yield of oxygen transfer (mg O2/l.h

X – cell concentration (g dw cells/l)
 Cells Growth

When the oxygen transfer is the rate-limiting step, the rate of oxygen
consumption is equal to the rate of oxygen transfer. Moreover, if the maintenance
requirement of oxygen is negligible compared to growth, then

How to overcome DO limitations?

Use oxygen enriched air or pure oxygen

High atmospheric pressure

 Cells Growth

Other factors
1. Redox potential: Complex function of DO, pH, and ion concentration

2. Dissolved CO2 (DCO2); to high or low DCO2 is toxic

3. Ionic strength - affects transport of nutrients in and out of cells, and solubility of
O2

4. Substrate concentration - inhibition

glucose (100 g/L), ethanol (10 g/L), NH4+ (5 g/L)
 Cells Growth

Heat generation by growth

Only 40 to 50% of the energy stored in a carbon substrate is converted
to biological energy (ATP) during aerobic metabolism. The
remainder is released as heat upon conversion to CO2 and H2O

Heat generated can be calculated using the heat of combustion of substrate

and of cellular material.
 Cells Growth

Heat of combustion = Metabolic heat + Heat of combustion of cellular material

On a per gram of substrate basis

- Heat of combustion of substrate (kJ/g substrate)

YX/S – substrate yield coefficient (g cell/g substrate)

- Heat of combustion of cells (kJ/g cells)

1/YH – Metabolic heat evolved per gram of cell mass produced (kJ/g cells)
 Cells Growth

Typical ΔHC = 20 to 25 kJ/g dcw

The degree of oxidation of S has a large effect on 1/ YH

 Cells Growth

The total rate of heat evolution in a batch fermentation is

Removal of heat?
Metabolic heat released during fermentation can be removed by circulating
cooling water through an external jacket around the reactor vessel or through a
coiled tube within the reactor.
 Cells Growth
Modeling of cell growth
The complete description of the growth kinetics of a culture would involve
recognition of the structured nature of each cell and the segregation of culture
into individual cells
Structured Models:
Model divides cell mass into components (by molecule or by element) and predicts
how these components change as a result of growth. These models are very
complex and not used very often.

Unstructured Models:
Model assumes balanced growth where cell components (fixed cell mass) do not
change with time. Much less complex and much more commonly used. Valid for
batch growth during exponential growth stage and also for continuous culture during
steady-state operation. Look at cell macroscopically: skip internal kinetics and
regulation

Valid in single-stage, steady-state continuous culture & log phase of batch culture

Fails during any transient condition

 Cells Growth

Unstructured model – Monod model

Substrate-limited growth : Relationship of specific growth rate to substrate

concentration
Assumptions
•Single chemical species, S uptake is growth-rate limiting step
•Other nutrients does not have effect
•Single enzyme is evolved in the uptake of S
SIMILAR TO MICHAELIS-MENTEN KINETICS MODEL

Monod equation
S>>Ks μ = μmax
S<<KS μ = μmax S/KS
Cells Growth
 Cells Growth

This monod equation describes substrate-limited growth only when growth is slow
and population density is low.

The basis of the Monod equation does not really hold true, however, the equation is
used frequently for many bacterial cultures.

At high population levels the build up of toxic metabolic by-products is more

 Cells Growth
Models with growth inhibitors
Growth can be inhibited by product/substrate or inhibitory substances present
In the medium

Substrate inhibition – competitive or non competitive

use a controller for feeding substrate to avoid excess substrate present

max [S]
=
Competitive
 [S] 
KS 1 + + [S]
 K I 

max
Non-competitive =
 [S]  KS 
1+ 1+
 KI   [S]
 Cells Growth

Product inhibition: How to avoid?

Example: Ethanol fermentation from glucose. ETOH is inhibitor at concentrations

above 5%.
 Cells Growth

Inhibition by toxic compounds

 Cells Growth

Sigmoidal shape: Batch growth curve

We relate changes in S to changes in X through YX/S

Relationship between microbial yield (Y) and substrate
consumption is:

Integrate over X and S to get

 Cells Growth
By integrating above equation:

This expression provides a sigmoidal form of the rate expression

Notice that the expression does not contain any kD' so the reduction in cell growth
must be due to loss of substrate and not due to the presence of toxic by-products.
 Cells Growth

This equation requires a predetermined knowledge of the maximum cell mass in

a particular environment.

Logistic equations are a set of equations that characterize GROWTH in terms of

CARRYING CAPACITY (CC). Specific growth is related to amount of unused CC.

Carrying capacity: The maximum population of a given species that can

survive indefinitely in a given environment (given the food, habitat, water
and other necessities available in the environment).

Boundary condition
X(0) = X0
 Cells Growth in continuous culture

Design of Chemostat
CO2 and air out
Substrate Cells
Substrate
Products
Chemostat or
continuous
flow stirred
tank reactor
(CSTR)

Air or
oxygen
 Cells Growth in continuous culture

Chemostat

1. Growth limited by one nutrient

2. At steady state nutrient, cell, and product
concn same
3. Control of pH, temp. agitation, dissolved
oxygen
4. Sterilization required
 Cells Growth

Turbidostat

Cell concentration is maintained by monitoring the optical density of the culture

 Cells Growth (Modeling)

The ideal Chemostat

• evaluate KS, μmax, YX/S and other system parameters
• study changes in environment and effects on cell physiology
• select for cells with desired metabolic capabilities (e.g. selection for cells
capable of degrading a toxic compound)

Why derive mass balance equation?

1. Describe dynamics of cell growth, substrate utilization, and product
formation.
2. Useful for control of bioreactors.
3. Evaluate kinetic and yield parameters.
4. Determine the optimum values for bioreactor operating
parameters.
Cells Growth (Modeling)

Schematic diagram of Chemostat

 Cells Growth (Modeling)

Mass balance of on cells

FXO – FX + μgX VR - kdX VR = dX/dt VR

F = in and out volumetric flow rate (L / hr)

X = bioreactor and outlet cell mass concentration (g /L)
Xo = inlet cell mass concentration (g /L)
μ = specific cell growth rate neglecting endogenous metabolism (hr-1)
kd = endogenous cell loss rate constant (hr-1)
VR = Volume of fluid in the reactor
 Cells Growth

d
dX
DX 0 + X ( − D − kd )− =
dt
Chemostats are normally operated at steady-state, dX/dt = 0.
Assume a sterile feed (Xo = 0), and kd is so small that is neglected, kd = 0.

=D
 Cells Growth

In a chemostat, cells are removed at a rate equal to their growth rate, and the growth
rate of cells is equal to the dilution rate.

Growth rate can be manipulated as an independent parameter and makes it powerful

experimental tool.

Setting the Dilution (D) rate sets the Growth Rate

Now substitute Monod equation:

S as a function of D

S= Steady state limiting

substrate concentration

There is an upper limit on D, or the cells will washed out of the bioreactor. i.e. D>>
μmax
 Cells Growth

How is X affected by D?

A similar mass balance equation for S in the absence of endogenous metabolism

 Cells Growth

When the extracellular product formation is negligible and the system is steady
State i.e qp = 0 and dS/dt = 0

=D

At steady state, μg = D
 Cells Growth

S= Steady state limiting

substrate concentration

The above equations are written on the assumption of endogeneous metabolism

Is absent or negligible.

Y(X/S) is assumed to be constant

 Cells Growth

Allowing for endogenous metabolism (kd > 0):

If we assume that endogeneous metabolism occurs, the yield coefficient changes

With limiting nutrient and growth rate.

FXO – FX + μgX VR - kdX VR = dX/dt VR

dX/dt = DX0 + (μg – kd – D)X

Steady-state substrate balance, and extracell P=0, then

 Cells Growth

D
X = YX / S (S0 − S)
D + kd

What does kd represent?

• Shuler uses kd to represent changes in cell mass due to
endogenous respiration and kd’ for changes in cell mass due to
cell death and lysis.
– Endogenous respiration: catabolism of cellular reserves for
continued maintenance and energy.
 Cells Growth

kd is small

D
X = YX / S (S0 − S)
D + kd kd is not negligible
 Cells Growth
Finding the True Yield Coefficient
YMX/S is constant (Maximum yield
coefficient without endogenous
metabolism or maintenance energy)

D  D + kd  This approach is not valid for direct

−  true  = 0 conversion of S into maintainance energy
YX / S  YX / S 
app

ms – Maintenance
Coefficient
YA/PX/S = Apparent
yield
Cells Growth
 Cells Growth

Product mass balance

Please do it as the home work

 Biomass production

Chemostat with recycle

α = recycle ratio
C = cell concin the
recycle/cell conc in effluent
X1 = cell concentration in
reactor effluent
X2 = cell concentration in
effluent from separator
F- nutrient flow rate
Xo – initial cell concn
 Biomass production

Mass balance

VR – culture volume

At steady state: Xo = 0 (sterile feed)

solving for μ

Since C > 1 and α (1-C) < 0, then μ < D

A chemostat can be operated at dilution rates higher than the specific
growth rate when cell recycle is used
 Biomass production

Substitute Monod Eqn. into above, solve for S

 Biomass production

Material balance for growth-limiting substrate

(11 + α) D = F/V

At steady state dS/dt = 0

(1 + α)

Solving for X

D/μ =
 Biomass production

Comparison of chemostat with and without cell recycle

R1 – biomass output rate without recyle

R2 - biomass output rate with recyle
 Biomass production

Multistage Chemostat Systems

Production of secondary metabolite production - the growth and product formation
Steps need to be separated because optimal conditions for each step are different