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Microbial Growth Kinetics


Motivation for studying cell growth kinetics
Cells are the biocatalyst. Different than enzymes, the
catalyst amount (cell) is changing during the catalytic
process.
Growth is a result of replication and changes in cell size.
Cells extract nutrients from the environment and convert
them into biological products.
Most products of interest will be associated with the growth
and activity of cells
Design of systems (e.g. system size and duration) requires
knowledge of
Accumulation rate of cells
Depletion rate of nutrients
Accumulation rate of desired product
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3
Methods used to measure microbial
growth
Direct Method
Cell number
Microscopic counts
Particle counter
Plate count
Cell mass
Direct method
Turbidity
Dry weight
Indirect method
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Viable counts (Fig. 6.21, 6.22)
Each colony on plate or filter arises from single live
cell
Only counting live cells
Need to carefully select growth medium
3
5
Microscopic counts (Fig. 6.23)
Need a microscope, special slides, high power
objective lens
Staining can be used to distinguish dead and live
cells
Suits for bacteria and yeasts
Cell pass through the
orifice and generate a
electric pulse
Weight of pulse
corresponding to cell
size
Suitable for discrete
cells in a particle free
medium
Particle counter
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7
Cell mass: Turbidity
Measuring cell mass
Cells act like large
particles that scatter visible
light
A spectrophotometer
sends a beam of visible
light through a culture and
measures how much light
is scattered
Measures both live and
dead cells
Blanking against medium
Cell dry weight method
Need to have solids free medium
Sample centrifuged or filtered, washed
with buffer
Dry at 80C for 24 hours, then measure
the cell dry weight
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Indirect Method
Quantifying cell concentration:
- indirect: direct method is inapplicable. (mold solid state
fermentation)
by measuring intracellular components such as
protein, DNA etc.
by measuring products or substrate of metabolism
by measuring physical properties of the broth such as
viscosity
Microbial Cell Growth Kinetics
- Introduction
- Growth patterns and kinetics in batch
culture
- growth phases
- effect of factors: oxygen supply
- heat generation
- Growth kinetics (Monod Equation)
- Growth in continuous culture (ideal
chemostat)
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Growth Kinetics
Introduction
- Autocatalytic reaction: The rate of growth is directly related
to cell concentration
substrates + cells extracellular products + more cells
S + X P + nX
S: substrate concentration (g/L); X: cell mass concentration (g/L);
P: product concentration (g/L); n: increased number of biomass.
Net specific growth rate (1/time):
t: the time
dt
dX
X
1
net

Growth Kinetics
Introduction
Net specific growth rate (1/time):
d
k
g
=
net
:
g

Gross specific growth rate (1/time)


:
d
k
The rate of loss of cell mass due to cell death
or endogenous metabolism
Endogenous metabolism: during the stationary phase,
the cell catabolizes cellular reserves for new building blocks and
for energy-producing monomers.
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Growth Kinetics
Introduction
Net specific replication rate (1/time):
dt
dN
N
1
R

: '
R

Gross specific replication rate (1/time)


:
d
k
The rate of cell death (1/time)
R

d
k = '
R
R

N : Cell number concentration (cell number /L)
Growth Kinetics
- Growth patterns and kinetics in batch culture
- growth phases
In batch culture:
- lag phase
- logrithmic or exponential growth phase
- deceleration phase
- stationary phase
- death phase
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Typical growth curve for a bacterial population
Lag phase
A period of adaptation for the cells to their
new environment
New enzymes are synthesized.
A slight increase in cell mass and volume, but no
increase in cell number
Prolonged by low inoculum volume, poor
inoculum condition (high % of dead cells), age of
inoculum, nutrient-poor medium
Multiple lag phases: (diauxic growth) medium
contains more than one carbon source
Batch Growth Kinetics
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Batch Growth Kinetics
Exponential growth phase
In this phase, the cells have adjusted to their
new environment and multiply rapidly
(exponentially)
Balanced growth all components of a cell grow
at the same rate.
Growth rate is independent of nutrient
concentration, as nutrients are in excess
Cell growth rate is highest in this phase
(1/time) rate growth specific maximum the is
R
m

net
= =
Batch Growth Kinetics
Exponential growth phase
Doubling time of cell mass: the time required to double
the microbial mass:
net net net
693 . 0 2 ln
0
/ ln

t = = =
X X
d
10
Batch Growth Kinetics
Deceleration growth phase
Very short phase, during which growth decelerates
due to either:
Depletion of one or more essential nutrients
The accumulation of toxic by-products of growth
(e.g. Ethanol in yeast fermentations)
Period of unbalanced growth: Cells undergo
internal restructuring to increase their chances
of survival
Batch Growth Kinetics
Stationary Phase:
With the exhaustion of nutrients (S0) and build-up
of waste and secondary metabolic products
- The growth rate equals the death rate.
- There is no net growth in the organism population.
- Cells may have active metabolism to produce secondary
metabolites.
Primary metabolites are growth-related: ethanol by S.
cerevisae.
Secondary metabolites are non-growth-related:
antibiotics, pigments.
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Batch Growth Kinetics
Stationary phase
- 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)
- Endogenous metabolism occurs by catabolizing cellular
reserves for new building blocks and energy-producing
monomer (maintenance energy).
The rate describing the conversion of cell mass into
maintenance energy or the loss of cell mass due to cell
lysis:
. metabolism endogenous for constant rate the is
d
d
k
X k
dt
dX
=
Batch Growth Kinetics
Death Phase:
The living organism population decreases with time,
due to a lack of nutrients and toxic metabolic by-
products.
The rate of death usually follows:
constant. rate death order - first the is
'
'
d
d
k
N k
dt
dN
=
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Kinetic Pattern of Growth and
Product Formation
Growth-associated Non growth-associated Mixed-growth-associated
exercise:
A simple batch fermentation of an aerobic bacterium growing
on substrate, S, gave the results listed below
How would you find maximal net growth rate and Y
X/S
from the data
set?
How would you determine the minimum substrate concentration
needed to produce 50 kg cells in 1000 L of medium (assume the
inoculums contribution is negligible)?
time(h) cell(g/L) glucose(g/L)
0 1.25 100
9 2.45 92
16 5.1 90.4
23 10.5 76.9
30 22 48.1
34 33 20.6
36 37.5 9.38
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14
Maintenance coefficient: describe the specific rate
of substrate uptake for cellular maintenance
X
dt
dS
m
m
] [
=
energy
e maintenanc energy growth
product
lar extracellu
an into
on assimilati
biomass into
on assimilati
S S S S S A + A + A + A = A
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Segregated model
All cells are treated as
individuals in a population
Need to be able to
measure individual traits
for this type of model to
be useful
Non-segregated model
All cells are behave in
the same way in some
average sense
Mathematically much
simper
Include a detailed
description of
intracellular events
Structured model Unstructured model
No details in terms of
intracellular reactions,
only biomass is
considered
Mathematical models
Mathematical models can be used to assist in predicting cell growth,
substrate utilization and product formation. Models provide valuable
tools for process design and control.
Types of model for cell growth include:
Quantifying Growth Kinetics
Unstructured model: assuming fixed cell composition.
Applicable to balanced-growth condition:
- exponential growth phase in batch culture
- single-stage, steady state continuous culture
- cell response is fast compared to external changes
- the magnitude of the external changes is not too large
(e.g. 10%-20% variation from initial conditions).
Nonsegregation model: assuming all cells are the same
in the culture.
Monod equation: unstructured and nonsegregation model
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Quantifying Growth Kinetics
Monod equation: unstructured and
nonsegregation model
Assumption:
- a single enzyme system with Michaelis-Menten kinetics
is responsible for uptake of substrate S, and the amount
of that enzyme or its activity is sufficient low to be
growth-rate limiting.
- the relationship of specific growth rate to substrate
concentration assumes the form of saturation kinetics.
- a single chemical species is growth-rate limiting while
changes in other nutrient concentrations have no effect.
Monod type saturation growth kinetics
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Quantifying Growth Kinetics
Monod equation: when applied to cellular
systems, the gross specific growth rate
(1/time)
S
S
K
S
m
g
+
=

is the maximum specific growth rate (1/time).


Ks is the saturation constant or half-velocity constant (g/l)
when the substrate concentration S>> Ks
(exponential growth phase),
m g
=
When endogenous metabolism is unimportant,
net g
=
Monod vs. Michaelis-Menten:
recap of differences
Monod
Growth
Empirical
K
s
, 1/t
Michaelis Menten
No growth; constant E
Derived from theory
K
m
v, mg/L-t
Simlarities are shape of curves, form of function, parameter
estimation techniques.
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Determination of Monod Parameters
Batch: X, S, t lnX ~ t , get
m
(slope) from data
in exponential phase.
t
m
t
net

X
X
~ =
0
ln
Woolf) - (Hanes
Burk) - Lineweaver (
1 1 1
0 ,
m m
S
g
m m
S
g
d
S
m
g
S K S
S
K
K
S K
S
Xdt
dX

+ =
+ =
~
+
= =
Classical Growth Kinetics
Empirical Approximation
Monod Growth Kinetic
Tessier Growth Kinetic
Moser Growth Kinetic
Contois Growth Kinetic
S
S
s
+
=
m

g
( )
1
1

+ =
n
s m g
s K
( )
s
K S
m g
e

= 1
S X K
S
sx
m g
+
=
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Substrate Inhibition
Non-competitive inhibition
I s
m
g
s I
s
I
m
g
K S S K
S
K K if
S
K
K
S
/
_
) 1 )( 1 (
2
+ +
=
>>
+ +
=

Competitive inhibition
S
K
S
K
S
I
S
m
g
+ +
=
) 1 (

Product Inhibition
Non-competitive inhibition
S K
K
P
S
s
I
m
g
+
+
=
) 1 /(

Competitive inhibition
S
K
P
K
S
I
S
m
g
+ +
=
) 1 (

20
Presence of Toxic compounds
Non-competitive inhibition
) )( 1 ( S K
K
I
S
s
I
m
g
+ +
=

Competitive inhibition
S
K
I
K
S
I
S
m
g
+ +
=
) 1 (

S
K
I
Ks
K
I
S
I
I
m
g
+ +
+
=
) 1 /(
) 1 /(

Un-competitive inhibition
Lead to cell death
'
d g
k
+
=
S
S
s
m

Batch Growth Kinetics
Effect of factors: aerobic growth is more efficient.
- Dissolved oxygen (DO)
- aerobic fermentation requires oxygen
- oxygen gas is sparingly soluble in water
- specific growth rate may be limited by DO if DO is
below a critical oxygen concentration.
Critical oxygen concentration: the growth rate becomes
independent of DO concentration.
bacteria and yeast: 5%-10% of the saturated DO
mold: 10%-50% of the saturated DO
The saturated DO in aqueous solution is 7 ppm at 25
o
C
and 1 atm.
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Batch Growth Kinetics
Effect of factors:
- Dissolved oxygen (DO)
The rate of oxygen transfer (OTR) from the gas to liquid phase is
given by:
OTR = No2 = KLa (C*-CL)
K
L
is the oxygen transfer coefficient (cm/h),
a is the gas-liquid interfacial area (cm
2
/cm
3
)
K
L
a is the volumetric oxygen transfer coefficient (h
-1
)
C* is saturated DO concentration (mg/l);
C
L
is the actual DO concentration (mg/l);
No
2
is the rate of oxygen transfer (OTR) (mgO
2
/l.h)
Batch Growth Kinetics
Effect of factors:
- Dissolved oxygen (DO)
Oxygen Uptake Rate (OUR) is oxygen consumption rate by
microbes. If the maintenance requirement of O2 is negligible
compared to growth, then
) * (
2
/
C C a K
Y
X
L
O X
g
=

Sufficient oxygen supply:


OTR OUR
When oxygen transfer is the rate-limiting step, at steady state,
the rate of oxygen consumption is equal to the rate of oxygen
transfer.
/h) O (mg
2
/
2
2
O X
g
o
Y
X
X q OUR

= =
h) - cells dw /g O (mg n consumptio O of rate specific the is
2 2
2
o
q
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Batch Growth Kinetics
Effect of factors:
- Dissolved oxygen (DO)
Question: Oxygen is to be supplied for yeast production. If
oxygen uptake rate (OUR) is 15g/l medium-h for a required
yeast growth, and the oxygen transfer rate (OTR) is 10 g/l
medium-h. Is such oxygen transfer rate sufficient to maintain
the required yeast growth? If the required growth has to be
maintained, how to improve the oxygen transfer rate?
Answers: OUR=15g/l medium-h > OTR=10 g/l medium-h
insufficient oxygen supply rate.
Oxygen transfer rate is limiting.
) * (
/
2
C C a
L
k
O X
Y
X
g
=

Increase k
L
a so that
Cells Growth in Continuous Culture
Continuous culture: fresh nutrient medium is
continually supplied to a well-stirred culture and
products and cells are simultaneously withdrawn.
At steady state, concentrations of cells, products
and substrates are constant.
In batch culture: the culture environment changes
continually.
growth, product formation and substrate
utilization terminate after a certain time interval.
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A continuous-culture laboratory setup
medium
Products,
cells
Ideal Chemostat
Same as perfectly mixed continuous-flow, stirred-tank
reactor (CFSTR).
- Control elements: pH, dissolved oxygen, temperature
- Fresh sterile medium is fed to the completely mixed
and aerated (if required) reactor.
- Suspension is removed at the same rate.
- Liquid volume in the reactor is kept constant.
24
25
g
g
g
net
net
M
M
M
26
27
28
Cell Growth in Ideal Chemostat
At steady state, X
0
=0, k
d
0, q
p
=0 , Monod equation applied,
Cell Productivity: DX
S
S
K
S
m
D
g
+
= =

D
m
D K
S
S

)
0
( )
0
(
/ / D
m
D
s
K
S Y S S Y X
M
S X
M
S X
= =

opt
D
dD
DX d
= 0
) (
) )
0
(
0
(
)
0
1 (
/
s
K S
s
K
s
K S Y
opt
X
S
s
K
s
K
m opt
D
M
S X
+ + =
+
=
Washed out: If D is set at a value greater than
m
(D >
m
),
the culture cannot reproduce quickly enough to maintain itself.
Cell Growth in Ideal Chemostat
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.05 0.1 0.15 0.2 0.25
D (1/hr)
S
,

X

(
g
/
L
)
0
0.05
0.1
0.15
0.2
0.25
0.3
D
X

(
g
/
L
-
h
r
)
X S
DX

m
= 0.2 hr
-1
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When the endogenous metabolism is not negligible,
and product formation is negligible, k
d
=0; q
p
=0
d m
d s
d g
k D
k D K
S
k D

+
=
=

) (
S K
S
k D
s
m
d g
+
= + =

maintenance
cell growth
d
M
s x
k D
D
S S Y X
+
= ) (
0 /
D
m
Y Y
D Y
k
Y Y
Y
k D
X
S S
D
Y
X
S S D
s
d
M
s x
d
M
s x
g
M
S X
AP
S X
M
S X
M
S X
AP
S X
+ =
+ =
=
+

=
/ /
/ / /
1 1
1 1
0
) (
/
0
/
0

M
S X
Y
k
m
d
s
/
=
substrate
Cell:
Re-organize cell equation
Divide by D
Maintenance:
Experimental S and X data
collected at varying D could be
used to find constants Y
X/S
and m
Cells Growth in Continuous Culture
Ideal Chemostat
from obtained be can k Then
1/D. against )
S
X
( 1/ plotting by
s experiment chemostat from obtained be can and
d
0
/ /
/
S
Y Y
m Y
AP
S X
AP
S X
s
M
S X

=
M
S X
Y
k
m
d
s
/
=
Endogenous metabolism (X
0
=0, k
d
> 0, q
p
=0) is considered
D
m
Y Y
s
M
S X
AP
S X
+ =
/ /
1 1
30
If the endogenous metabolism and product formation are all
present, k
d
=0; q
p
=0
s p
M
s x
p d
M
s x
M
S X
d
p X
M
S X
net
d m
d s
d
s
m
Y
Y
q k D
D
S S Y X
X
Y
k
Y
q
Y
S S D
k D
k D K
S
k
S K
S
D
/
/
0 /
/ / /
0
) (
0 ) ( ) (
) (
+ +
=
= + +

+
=

+
=

Product formation
maintenance
cell growth
substrate
Cell:
m
Example CSTR problem
Calculate the substrate concentration in the feed (g/L) of a CSTR to
produce 5 g/L cell biomass. Assume the feed is sterile, there is no
product in the feed stream, cell death is negligible, product formation is
negligible, maintenance is negligible and the system is at steady
state. You are given the following data for the system and
microorganism:
Volume = 1000L
Feed rate = 50 L/hr

m
= 0.1 hr
-1
Ks = 1 (g/L)
Y
x/s
= 0.5
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Example: A new strain of yeast is being considered for biomass production. The
following data were obtained using a chemostat. An influent substrate concentration of
800 mg/L and an excess of oxygen were used at 1 ph of 5.5 and T=35C. Using the
following data, calculate u
max
, ks, Y
x/s
M
, and ms, assuming
net
=
max
S/(k
s
+S)-k
d
Dilution rate (hr
-1
) Substrate(mg/L) Cell Conc. (mg/L)
0.1 16.7 366
0.2 33.5 407
0.3 59.4 408
0.4 101 404
0.5 169 371
0.6 298 299
0.7 702 59
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63
Flow cytometer (FACS)
Florescent antibodies are
added to culture and cells in
small droplets are sent
through a detector single file
Computers count and
characterize cells as they
pass, and deflect cells with
desired characteristics
Can count and keep live cells
flow-cytometry.de/img/fcm.gif
Summary of Growth Kinetics
dt
dX
X
1
net

- Autocatalytic reaction: The rate of growth is directly related
to cell concentration
Net specific growth rate (1/time):
d
k
g
=
net
- Cell concentration determination
- Growth patterns and kinetics in batch culture
- lag phase
- logrithmic or exponential growth phase:
- deceleration phase
- stationary phase: endogenous metabolism
- death phase
d
,
0
ln t t
net

X
X
=
X
d
k
dt
dX
=
33
Summary of Growth Kinetics
- Effect of factors:
- Dissolved oxygen:
) * ( : rate nsfer oxygen tra
/
: rate n consumptio oxygen
2
C C a
L
k
O X
Y
X
g

- Temperature, pH, ionic strength, substrate


concentration.
- Heat evolution:
H S X
Y
c
H
Y
s
H 1
/
+ A =
A
Summary of Growth Kinetics
-Cell growth in continuous culture:
q
p
=0, k
d
>0, X
0
=0, Monod equation is applied:
d
k D
D
S S
M
S X
Y X
d
k D
m
d
k D Ks
S
d
k D
g
d
k
S Ks
S
m
d
k
g net
+
=

+
= + =

+
= =
)
0
(
/
) (
;


Productivities: DP, DX
D
s
m
Y Y
M
S X
AP
S X
+ =
/ /
1 1
34
Summary of Growth Kinetics
-Cell growth in continuous culture:
q
p
>0, k
d
>0, X
0
=0, Monod equation is applied:
s p
M
S X
p d
Y
Y
q k D
D
S S
M
S X
Y X
/
/
)
0
(
/
+ +
=
d
k D
g
+ =
d
k D
m
d
k D Ks
S

+
=

) (
X
p
q DP =
Productivities: DP, DX
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