Cell Growth

Cell Growth
Cells and Substrate Cells and Extracellular Product
+P
+ S +S +P
Autocatalytic reaction
Rate of microbial growth is characterized by the
specific growth rate ( (h
-1
))
X - cell mass (g cells/L)
t - time (h)
dt
dX
X
1
=
Determination of Cell Growth
Direct counting -
hemacytometer
Counting by diluting and
plating the cells on solid
medium
Particle counters
Dry cell weight
Cell volume by
centrifugation
Turbidity or optical
density - 560 - 600 nm
Measure amount of
product formed or
amount of substrate
consumed
Measure amount of
cellular protein or DNA
Mycelial growth or
extracellular
polysaccharide
formation - viscosity
Follow pH
Typical Batch Cell Growth
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Time (hr)
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)
Lag Phase
Exponential
Growth
Deceleration
Stationary
Phase
Death Phase
Lag Phase
Immediately after inoculation - period of adaptation
Reorganize their molecular constituents
Depending on the nutrients may have to synthesize new
enzymes or repress some enzymes
Cell mass may increase a little, without an increase in
cell number
Age of inoculum effects lag and amount
Rule of thumb to minimize lag phase - 5% by volume,
young cells
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Time (hr)
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Lag Phase
Exponential Growth
Phase
Logarithmic growth
Cells are adjusted to their environment
Cell mass & number increase exponentially with time.
Balanced growth - when all components of a cell grow
with the same rate. The average composition of the cell
remains constant with time
First order growth
t
X
X
X
dt
dX

=
|
|
.
|

\
|
=
0
ln
t
e X X
t X X

0
0
0 @
=
= =
or
0
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Time (hr)
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)
Exponential
Growth
Deceleration
Growth decelerates due to depletion of one or
more essential nutrients.
Accumulation of toxic by-products
Phase is short, changes occur over a short period
of time - unbalanced growth
Cell changes to increase the prospects of cellular
survival
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Time (hr)
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)
Deceleration
Phase
Stationary Phase
Net growth is zero, no cell division or death rate =
growth rate
Cells still produce secondary metabolites (non-
growth related products)
Production of certain metabolites is enhanced
during stationary phase (antibiotics) due to
metabolic deregulation
Cell catabolizes cellular reserves for new building
blocks and for energy-producing monomers.
Endogenous metabolism
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Time (hr)
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Stationary
Phase
Stationary Phase - cont.
The following can occur
Cell mass concentration may stay constant but number of
viable cells may decrease
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)
Cells may not be growing but may have active metabolism to
produce secondary metabolites. Cellular regulation changes
when concentrations of certain metabolites (C,N,P) are low.
Cell must use energy to maintain an energized
membrane and transport nutrients for essential
functions like motility and repair of cellular structures.
Energy expenditure is maintenance energy
Death Phase
Decline phase
Clear demarcation between stationary and death may be
difficult
Cells usually lyse
X
S
= Cell concentration at
end of stationary phase
k
d
= first order death constant
Transfer cells to fresh medium could have cells grow
Distribution of cells in medium
Using old inoculum may elect for variants of the strain
with altered metabolic capabilities.
t k
S
d
d
e X X
X k
dt
dX

=
=
0
0.1
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0.7
0 5 10 15 20 25
Time (hr)
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)
Death Phase
Yield Coefficients
Defined based on the amount of consumption of
another material
Growth yield
AS = AS
assimulation into biomass
+ AS
simulated into extracellular
product
+ AS
growth energy
+ AS
maintenance energy
Other yield coefficients may be defined such as
) (
) (
/
gsubstrate
gcells
S
X
Y
S X

A
A
=
2
/
2
O
X
Y
O X
A
A
=
S
P
Y
S P
A
A
=
/
or
Yield Coefficients - cont
Aerobic growth
Y
X/S
is usually 0.4 to 0.6 g cells/g substrate for
yeast and bacteria - Glucose
Y
X/O2
= 0.9 to 1.4 g/g - Oxygen
Anaerobic bacterial growth is less efficient
Y
X/S
= 0.2 to 0.4 g/g - glucose
Y
X/S
=0.6 to 1 g/g - methane
Maintenance Coefficient
Rate of uptake of a substrate for cellular maintenance
Stationary Phase - endogenous metabolism of biomass
components
Energy expenditure to
repair damaged cellular components
transfer nutrients and products in and out of cells
motility
adjust osmolarity of cells interior volume
( )
X
dt dS
m
m
/
=
Product Formation
Growth-associated products -
produced simultaneously to
growth (exponential, decline)
Constitutive Expressed Enzyme
Substrate Utilization enzymes
Non growth associated - takes
place during stationary phase
q
p
= constant
Antibiotics
RDX degradation
g X P p
Y
dt
dP
X
q
/
1
= =
0
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0 5 10 15 20 25
Time (hr)
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e
l
l

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o
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(
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/
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)
0
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0 5 10 15 20 25
Time (hr)
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)
Product Formation - cont
Mixed-growth-associated
Luedeking-Piret Equation
o = 0 non-growth associated
| = 0 only growth associated
o = Y
P/X
Examples
Lactic acid fermentation
Xanthan gum production
| o + =
g p
q
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Time (hr)
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)
Temperature Effects
Optimum temperature for cell growth
psychrophiles (T<20)
mesophiles (20<T<50)
thermophiles (T>50)
Above optimum temperature the growth rate decreases
and thermal death results
Both and k
d
vary according to the Arrhenius Equation
= Ae
-E
a
/RT
, k
d
= A'e
-E
d
/RT
Activation energy for cell growth 10-20 kcal/mol
Activation energy for thermal death 60-80 kcal/mol
Thermal death is more sensitive than growth.
X k
dt
dX
d
) ( =
Temperature Effects - cont.
Temperature affects product formation,
Optimum for growth not necessarily optimum for product
formation.
Temperature sensitive promoter product
Yield coefficient effected.
Maintenance coefficient increases with increasing temperature
Decreasing the yield coefficient.
Reaction rate may be greater than the diffusion rate at
high temperatures.
Usually assume things are reaction limited
Molecular diffusion activation energy = 6 kcal/mol
Bioreactions = 10 kcal/mol.
Increase rate lowering activation energy diffusion limited.
pH Effects
pH affects
H
+
concentration enzyme activity growth rate
Optimal for growth not necessarily optimum for
product formation.
Ranges:
3 to 8 - bacteria
3 to 6 - yeast
3 to 7 - molds
5 to 6 - plants
6.5 to 7.4- animal cells
pH Effects - cont.
Most cells can control intracellular pH in
the presence of fluctuations in external pH
changes.
Examples
Nitrogen source - ammonia - decrease in pH
add base
Nitrogen source - Nitrate - reduce to ammonia
increase in pH add acid
Production of either acids or bases change pH
adapt to higher or lower pH.
Dissolved Oxygen
Limiting in aerobic
cultivation since it is
sparingly soluble in water
Saturation 25 C 1 atm 7 ppm
Salts alter solubility
High temperatures decreases
oxygen solubility
Sparge oxygen throughout
fermentation.
O
2
Oxygen Transfer Rate
Oxygen transfer is limited by dissolving oxygen
in liquid cell medium, then can diffuse to cells
Oxygen transfer from gas to liquid
N
O2
= k
l
a (C* - C
L
) = OTR
k
l
oxygen transfer coefficient (cm/h)
a gas-liquid interfacial are (cm
2
/cm
3
)
k
l
a volumetric oxygen transfer coefficient (h
-1
)
C* saturated DO concentration (mg/l)
C
l
actual DO concentration in the broth (mg/l)
N
O2
rate of oxygen transfer (mgO
2
/lh)
Oxygen Uptake Rate (OUR)
Q
O2
- specific rate of oxygen consumption (mg O
2
/l h)
Y
X/O2
- oxygen yield coefficient (g dw cells / g O
2
)
X cell concentration (g dw cells/l)
If oxygen transport is limiting then rate of oxygen cons
= rate of oxygen transfer or OUR = OTR
or
2
2
/ O X
O
Y
X
Q OUR

= =
( )
( )
L L O X
L L
O X
O
C C a k Y
dt
dX
OTR C C a k
Y
X
Q OUR
=
= = = =
*
/
*
/
2
2
2

Oxygen Limitation
Growth rate varies linearly with dissolved
oxygen concentration under oxygen
limitation conditions.
Overcome oxygen limitation
sparge reactor continuously
oxygen enriched air,
H
2
O
2
work under higher pressure - 1 to 2 atm.
Redox Potential
Influences the rate of oxidative-reductive reactions
Function of DO pH, ion concentrations
Electrochemical potential of a fermentation, E
h
E
0
- constant
F - Faraday constant
R - gas constant
P
O2
- partial pressure of oxygen (atm)
measured in millivolts
Reduce Redox potential by
purging with nitrogen
addition of reducing agents like cysteine HCl or Na
2
S
+
+ = H
F
RT
P
F
RT
E
O h
log
3 . 2
log
4
3 . 2
2
Dissolved CO
2
Performance of cells
High concentration may be toxic
Some DCO
2
required for metabolic
functions of cells,
Controlled by changing amount in air
supply or changing the agitation speed.
Ionic Strength
Effects transport of nutrients in and out of cells, metabolic
functions of cells, and the solubility of certain nutrients

C
i
- ion concentration
Z
i
- ion charge
Examples
Glucose - above 200 g/l ethanol fermentations - reduction in
water activity
NaCl - above 40 g/l high osmotic pressure
Inhibition can be overcome by intermittent feeding
of substrate - Fed batch reactor
2
2
1
i i
Z C I

=
Heat Generation during
Microbial Growth Q
GR
V
L
- reactor volume
X cell concentration (g/l)
1/Y
H
- metabolic heat evolved per gram of cell mass
produced (kJ/g cells)

net
- net growth rate
Aerobic fermentation - rate of heat evolution can be
roughly correlated to the rate of oxygen uptake
Q
GR
= 0.12 Q
O2
H
net L GR
Y
X V Q
1
=
40 to 50% of energy is
converted to ATP rest
is released as heat
Y
H
Determination - Thermo
AH
s
- heat of combustion of
substrate (kJ/g substrate) -
combusting substrate
Y
X/S
- substrate yield coefficient
(g cell/g substrate)
AH
c
- heat of combustion of
cells (kJ/g cells) - combusting
cells ( 20 to 25 kJ/g cells)
1/Y
H
- metabolic heat evolved
per gram of cell mass produced
(kJ/g cells)
H
C
S X
S
Y
H
Y
H 1
/
+ A =
A
Substrate + O
2
CO
2
+ H
2
O
Total Combustion
AH
S
CO
2
+ H
2
O + Microbial Cells
Microbial
Growth &
Respiration
1/Y
H
Combustion
of Microbial
Cells AH
C
Blue- Easily measured
Rearrange Heat Equation to Solve for Y
H
Example Values for Y
H
values for
glucose 0.42 g/kcal
acetate 0.3 g/kcal
ethanol 0.18 g/kcal
methane 0.061 g/kcal
Y
H
Determination - Thermo
C S X S
S X
H
H Y H
Y
Y
A A
=
/
/
Degree of oxidation
of the substrate
influences the amount
of heat released.