MICROBIAL GROWTH

AND
PRODUCT FORMATION
1.1 Phases of the Batch Growth Cycle
Lag phase
Exponential growth phase
Declining growth phase
Stationary phase
Death phase



Cell division occurs in the exponential phase.
The rate of increase of cell number (N) is proportional to the
no.of cells
Instead of cell no., we can use dry cell weight per volume X
(kgm-3) as a measure of cell concentration
During the exponential phase in a batch reactor we can write




) 1 ( X
dt
dX
=
where
is the specific growth rate of the cells
Eqn. (1) can be integrated from the end of the lag phase
( , ) to any point in the exponential phase (X, t) to
give
0
X X =
lag
t t =
0
X X =
) (
lag
t t
e

or
|
|
.
|

\
|
0
ln
X
X
=
) (
lag
t t
(2)
The time required for the cell numbers or dry weight to double,
ie the doubling time, is related to the specific growth rate by:




At low nutrient concentrations, depends on nutrient
concentration.
At high nutrient concentrations, reaches a maximum value.
The end of the exponential phase arises when some essential
nutrient is depleted, or when some toxic metabolite accumulates
to a sufficiently high level.
Following the exponential phase, the rate of exponential growth
decreases (declining growth phase) and is followed by the
stationary phase.
Following this is the death phase, when cell lysis occurs and the
population decreases.



d
t
d
t = ) 2 ( ln

2 ln
=
d
t
or
(3)

Monod assumes that only one substrate (the growth limiting

substrate, S) is important in determining the rate of cell growth.
For batch growth at constant volume:



where






At high substrate concentration, >> , eqn. (4) reduces to
zeroth order dependence on substrate concentration.



1.2 Relationship between growth rate and substrate concentration
) (
. .
max
S K
X S
dt
dX
s
+
=

(4)
max

is the maximum specific growth rate

s
K is the value of the limiting substrate
concentration which results in a growth
rate of half the maximum value
S
is the concentration of the limiting substrate
S
s
K
ie at S
s
K
max

>> ; =

(5)
At low substrate concentration, << and a first order dependence
results.





S
s
K
S
s
K
S
K
s
.
max
|
|
.
|

\
|
=

ie at <<
; from eqn (1) and eqn (4) we get

(6)
Value of varies with the type of microorganism.
Value of depends on the nature of substrate.





Values of are generally quite small, implying that is near
for much of the period of batch growth. This apparent zeroth order
dependence on justifies the term exponential growth.




s
K

max

S
The Arrhenius relationship generally holds for the temperature
dependence of ,




1.3 Temperature Effects

hence k = A. exp
) (
RT
E
a

becomes

) (
RT
E
a

= A. exp

(7)
where A is Arrhenius constant
Ea is activation Energy
R is Universal Gas Constant
T is temperature
The temperature dependence of growth rate comes from the fact
that growth employs enzymes. The processes catalysed by enzymes
often have optima associated with the rise in k due to rise in
temperature, and the denaturation of enzymes at elevate
temperatures.

The effect of pH on microbial growth parallels that observed for

enzymes.
The effect of pH on enzymes is associated with the ionization of
-amino and carboxyl terminal groups in proteins, in addition to
many other ionizable groups on constituent amino acids, which
could give rise to complex kinetics when pH is varied.



1.4 pH Effects

Oxygen is consumed together with substrate and converted to
biomass and products. Varying the amount of dissolved oxygen
concentration has the same effect as varying the concentration of
the limiting substrate.

o
1.5 The Effect of Dissolved Oxygen Concentration
A microbial cell consumes nutrients and generates products in
order to reproduce, through a set of reactions called cellular
metabolism.
Microorganisms are classified on the basis of their nutritional
and energy requirements for cellular metabolism.


1.6 Product Formation

1.6.1 Cellular Metabolism
Energy Source
Light
Chemical
(breaking of bonds)
Phototrophs
Chemotrophs
Organotrophs
Litotrophs
(oxidation of organic
material)
(oxidation of
inorganic material)

Carbon Source
Autotroph
Heterotroph
CO2 as source of carbon
organic sources of carbon
Most organisms we deal with are chemoheterotrophs, ie requiring
organic carbon source and a chemical source of energy.





1.6.2 Growth-Associated Product Formation
End products of energy and carbon metabolisms are called primary
metabolites

These products are referred to as growth-associated products, as their
rate of production parallels the growth of the cell population.

Examples: - Ethanol produced by anaerobic fermentation of glucose by
yeast
- Production of gluconic acid from glucose by Gluconobacter







1.6.3 Non Growth-Associated Product Formation
Most organisms we deal with are chemoheterotrophs, ie requiring
organic carbon source and a chemical source of energy.

Some products are produced in batch cultures at the end of the
exponential phase.

They are formed from secondary metabolism, ie they are
secondary metabolites, and are non growth-associated products, and
their kinetics do not depend on rate of growth of culture

Examples :- Production of the antibiotic penicillin by P. chrysogenum

1.6.4 The Intermediate Class of Products

An intermediate class of products, where product formation kinetics lie
between the two classes above, are called partially growth-associated
products.

Examples :- amino acids, lactic acid, citric acid, extracellular
polysaccharides (eg xantan), solvents (eg acetone).



1.7 Fermentation Profiles
An intermediate class of products, where product formation kinetics lie
between the two classes above, are called partially growth-associated
products.

Once a potentially useful fermentation process is identified, its time
course, or fermentation profile is plotted for use as a basis for
comparison in later scale-up and optimization of process parameters.




1.8 Growth Yield
Growth yield is defined as





= Y
dS
dX
If defined as
Y
is constant; then
dX YdS =
If the boundary conditions are:
at
at
0 0
, , 0 S S X X t = = =
S S X X t t = = = , ,
then
X X S S Y =
0 0
) (
thus ) (
0 0
S S Y X X + =
(8)
Now take edn (4)
) (
. .
max
S K
X S
dt
dX
s
+
=

(4)
Since
) 1 ( X
dt
dX
=
Thus
) (
max
S K
S
s
+
=
(9)
Now take eqn. (8)
) (
0 0
S S Y X X + = (8)
From eqn. (8) we get:
X X S S Y =
0 0
) (
or
0 0
YS X X YS + =
and
Y
X X
S
Y
YS X X
S
) ( ) (
0
0
0 0

=
+
=
Put this expression for S into eqn.(9) we get:
|
.
|

\
|

+
|
.
|

\
|

=
Y
X X
S K
Y
X X
S
s
0
0
0
0
max

But from eqn.(1) we have:
) 1 ( X
dt
dX
=
ie
=
dT
dX
X
1
so
|
.
|

\
|

+
|
|
.
|

\
|
|
.
|

\
|

=
Y
X X
S K
Y
X X
S
dT
dX
X
s
0
0
0
0
max
1

(10)
Integrating eqn.(10), we get:
|
|
.
|

\
|
+
|
|
|
|
.
|

\
|
|
.
|

\
|
+

|
.
|

\
|
|
|
|
|
.
|

\
|
|
.
|

\
|
+
|
.
|

\
|
+ +
=
0
0 0
0
0
0
0
0
0
0
max
.
.
ln ln .
S Y
X X S Y
Y
X
S
K
X
X
Y
X
S
Y
X
K S
t
s
s

(11)
1.9 Productivity and Production Rate
If we assume the Monod model holds, and if we define the
conversion from the limiting substrate S to microorganism biomass as
the yield , and if we assume that there is no lag phase, and
that there is no cell death, then what is the productivity of a constant
volume fermentation system?
dS
dX
Y =
Biomass productivity is defined as how much biomass we can get per
unit volume of fermenter, per unit time (e.g. kg/m/hr).

For a batch system, time is needed between runs, to clean the
fermenter, load the medium, and sterilize it. This time is called the

Down Time .



d
t
A plot of biomass concentration vs time will look as follows:
X
Maximum productivity here
Point m where
dT
dX
is maximum
0
X
Maximum production rate is at point m.

Maximum productivity may be later since maximum
productivity is not calculated from time zero.

Productivity is overall production rate, ie involving down time
d
t
Now recall Monods eqn (9) ie
(9)
) (
max
S K
S
s
+
=
The general plot of versus is as follows

S
The value of varies between substrates. If for a particular
substrate the value of is very small, then from eqn. (9) will
have a value close to max for most of the fermentation, untill
substrate S is almost exhausted, and until that point is reached, the
process has zero order kinetics.
s
K
s
K
So a process with a small is less sensitive to substrate
concentration
And a process with a big is more sensitive to substrate
concentration
s
K
s
K
In a process with a small the approximation to zero order
kinetics occur from high S right down to very low values of S
The exponential phase will extend further before the microb will
begin to be affected by the depletion in S
s
K
Each student please pick a fermentation process for the
production of a different product, either from the list given
below, or from outside the list, and find and copy its
fermentation profiles on a piece of squared paper, make a
photocopy, keep the original, and submit the photocopy
during the next lecture:

Penicillin
Amino acid
Citric acid
Xantan
Acetone
Ethanol
Biopolymer
Erythromycin
Yeast
ASSIGNMENT 1
ASSIGNMENT 2
Use your original fermentation profiles
drawn on the squared paper and assuming
the down time of each fermentation is 4
hours, apply the method suggested in the
lecture to estimate the productivity of
biomass production for your fermentation.
d
t