2-Bacterial Growth Kinetics_F12

1
BACTERIAL GROWTH KINETICS

Growth Kinetics of Pure Bacterial Culture
in a Batch System (VH, p.444)
- Growth Phases of a Simple Batch
Culture

1) Lag phase
2) Accelerating growth phase
3) Log (exponential growth) phase
4) Declining growth phase
5) Stationary phase
6) Death or declining death phase
7) Log death phase


1. Lag Phase

a. Adaptation (acclimation) period
- the lag phase is the initial phase which represents the period (time) required for bacteria to
adapt to their new environment.
b. Constant number of cells
- during this phase, the individual bacterial cells increase in size, but the number of cells
remains unchanged.
c. Physiologically active
- they are very active physiologically and are synthesizing new enzymes and activating
factors.

2. Accelerating Growth Phase

a. Transition period from the lag phase to the log phase.
b. Cell is beginning to grow (increase in numbers) noticeably as enzyme systems are gearing up.

3. Log (Exponential Growth) Phase

a. Exponential growth
- during this phase, the bacterial cells divide regularly at a constant rate.
b. Straight line on semilog scale
- The logarithms of the number of cells plotted against time results in a straight line.
c. Maximum Rate of Substrate utilization
- A maximum growth rate occurs under optimal conditions, and substrate is removed from
the medium at the maximum rate.
- The growth rate is limited only by the bacteria's ability to process the substrate.
- food is in excess (not limiting) so that the rate of growth is only limited by the ability to
process the food
- Sometimes called "0-order growth" and growth rate is constant and maximum.
Endogenous Phase
5 6
4
3
7
1

2 S
Time
S
u
b
s
t
r
a
t
e

-

-

-

-
_
_
_
_

L
o
g

(
N
o
.

o
f

V
i
a
b
l
e

C
e
l
l
s
)
2

4. Declining Growth Phase

a. Transition period from the log phase to the stationary phase.
b. Decreasing growth rate
c. Exhaustion of essential nutrients
d. Accumulation of toxic metabolic products

- the growth rate can be limited either by the exhaustion of essential nutrients or by the
accumulation of toxic metabolic products.
- food becomes limiting factor and therefore growth rate and mass of bacteria are dependent
on the amount of food present.

5. Stationary Phase

a. The number of cells remains constant perhaps as a results of complete cessation of division or
the balancing of reproduction rate by an equivalent death rate.

- growth of new cells is balanced by the death of old cells.
- no increase in cell mass
- population is "stable"
- net growth rate, r
g
= 0

6. Death or declining Phase

a. The number of viable cells decreases slowly due to the fact that the death rate exceeds the
production rate of new cells (while the total mass may remain constant).
b. Depletion of essential nutrients
c. Accumulation of inhibitory products.

- Death occurs primarily as a result of depletion of essential nutrients and/or the accumulation
of inhibitory products.

7. Log Death Phase

a. Exponential death - "wholesale die-off"
- system is dead
- even if you add food, you will get no growth.







2-Bacterial Growth Kinetics_F12
3

Endogenous Phase

a. Near-starvation condition
- That portion of the bacterial growth curve encompassing parts of the stationary and
declining phases in which microorganisms are in near-starvation is more frequently called
the endogenous phase.
b. Most activated sludge treatment systems are operated in this phase since the microorganisms
flocculate rapidly and settle out of solution by gravity.
- food is scarce and the bacteria are forced to metabolize storage products and dead, lysed cells
- growth does not cease, but death exceeds it, net growth rate is negative.


REMARKS:

a. Growth curve is for a batch system (fed once) and pure culture.
- real biological treatment systems are typically continuously fed (and wasted).

b. Growth of bacteria is dependent upon many other factors which will be discussed in more
detail later.

c. Activated sludge and other biological treatment systems contain many types of organisms.
- i.e., biological treatment systems are ecosystems and as such the growth characteristics of
one organism may be affected by other organisms.

d. In general, cells settle much better when they are in endogenous phases.

e. In real biological treatment systems, you must operate at a positive growth rate or you will
eventually lose your bacteria.
4


Relationship between: (A) biological growth phase, (B) biological flocculation. Also shown are
the accumulation of the exocellular polymer (ECP) to microorganisms (Tenny and Verhoff)
Biomass vs. time
Turbidity vs. time
Exocellular polymer vs.
time
ECP/Biomass vs. time
2-Bacterial Growth Kinetics_F12
5
Filamentous organisms observed in activated sludge



Common characteristics of filamentous organisms observed in activated sludge; 1000 x phase
contrast(Water Pollution Control Federation)
6


Common characteristics of filamentous organisms observed in activated sludge; 1000 x phase
contrast(Water Pollution Control Federation)

2-Bacterial Growth Kinetics_F12
7
Bacterial Growth Kinetics

a. For generalization of results, quantitative descriptions of bacterial growth

- To generalize results, quantitative descriptions of bacterial growth in a culture
are essential.

b. For evaluation and interpretation of experimental results

- Mathematical models have been successfully used in evaluation and interpretation of
experimental results.

c. The experimental data may be rendered more meaningful and concise if they are analyzed in
terms of the various growth kinetic parameters.

d. The basic kinetic constants include:
1) specific growth rate,
2) maximum specific growth rate,
m
3) maximum rate of substrate utilization per unit mass of organisms, k
4) half-saturation constant or Monod constant, Ks
5) yield coefficient, Y
6) endogenous decay coefficient (death rate constant), k
d




Batch Experiment

It has been observed that if one of the essential requirements for growth is present in only limited
amounts, the limitimg factor affects on the rate of growth. For example, if substrate is the growth
limiting factor, the varing growth of the bacteria may be observed by running a batch experiment
using different concentrations of the substrate.



S
1
< S
2
< S
3
< S
4
< S
5





8
2)
S
5
S
5
S
4
S
4
No. of Log
5
S
3
cells S
3
No. of
Cells
S
2
S
2

S
1
S
1

1
Time Time
















The Monod Model

a. The effect of substrate concentration on the specific growth rate is shown in Figure 1.

(a) The Monod Plot

3

S
1
S
2
S
3
S
4
S
5
S

2
2-Bacterial Growth Kinetics_F12
9

S

K
S

m
/2

Fig. 1. Effect of substrate concentration on the specific growth rate.
(3
rd
: DC 350; 4
th
DC 459 Fig. 6-18) Monod growth rate constant as a function of limiting food
concentration


b. It has been observed that if one of the essential requirements (e.g, substrate) for growth is
present in only limited amounts, the effect of a limitimg factor on the rate of growth can be
defined by the equation proposed by Monod (1942):


m
S
= ----------- (1)
Ks + S

where = specific growth rate, T
-1
(day
-1
)

m
= maximum specific growth rate, T
-1
(day
-1
)
S = growth rate limiting substrate concentration, ML
-3
(mg/L)
Ks = half-saturation coefficient; substrate concentration at one-half the maximum growth
rate, ML
-3
(mg/L)
= half-velocity constant (by Metcalf & Eddy)
= Monod constant


c. The Monod expression provides a continuous transition between first and zero order kinetics
based on growth limitimg substrate concentration.

d. If Ks >> S,
'
m
s
S
k S
K

= =
where k =
m
/ Ks

the model would be reduced to a first order expression

10
e. If Ks << S,
m
m
S
S

= =
the model would become a zero order expression

Note: The half-saturation constant is characteristic of a specific organism, substrate, and culture
conditions.


The lineweaver-Burk Equation

1 Ks + S Ks S
--- = ------------ = ---------- + ----------

m
S
m
S
m
S

1 Ks 1 1
--- = ------- ----- + -------

m
S
m






a. The lineweaver-Burk plot is obtained by plotting 1/ versus 1/S.
b. This line represents a straight line with a slope of Ks/u
m
and an intercept of 1/
m

on the 1/ axis.

Lineweaver-Burk plot

1/ Slope = Ks/
m
Intercept = =1/
m
1/S
2-Bacterial Growth Kinetics_F12
11
The Eadie Plot
Eadie plot

m
S
= ------------
Ks + S

(Ks + S) =
m
S
Ks + S =
m
S
Ks = (
m
- ) S


m
-
m

-- = ------------ = ------- -----
S K
s
K
s
K
m


1
m

-- = - ---- + ------
S K
s
K
s





Rate of Bacterial Growth, ,
g
g
dX
r
dt
| |
|
\ .


a. In the batch culture, the specific growth rate () is defined in the exponentical growth phase.
b. Since bacteria increase their number at a constant rate in this phase, the rate of growth is
defined by the relationship:

m
g
g
S dX
r X X
dt Ks S

| | | |
= = =
| |
+
\ . \ .


where r
g
= rate of bacterial growth, M L
-3
T
-1
(mg/L d)
X = concentration of viable bacteria (in VSS) at time t, ML
-3
(mg/L)
= specific growth rate, T
-1
(d
-1
)

m
s
S
K S

=
+


The parameter represent the rate of growth per unit concentration of bacteria and has the
dimension of reciprocal time:


1
g
dX
X dt

| |
=
|
\ .

m
/Ks
/S
Slope = 1/K
s

12
Rate of Substrate Utilization, ,
su
u
dS
r
dt
| |
|
\ .

,
su
u
dS
r
dt
| |
|
\ .
= rate of substrate utilization, ML
-3
T
-1
(mg/L-d)

Cell Yield, Y

a. Through bacterial growth, a portion of substrate is converted to new bacterial cells and a
portion is oxidized to end product releasing usable energy and heat energy.


g u
X S
t t
A A | | | |

| |
A A
\ . \ .


b. The number of bacterial cells or the amount
of biomas formed per unit amount of
substrate utilized has been reported to be
nearly constant for a given organism and
substrate.

X
2
X
1

-------- = Y = yield coefficient
S
1
S
2


X
2
X
1

---------- = Y
S
2
S
1


where Y = bacterial cell yield = yield coefficient = maximum yield coefficient, MM
-1
(mg
VSS/mg BOD)
= the ratio of the mass of cells formed to the mass of substrate consumed
X
2
= concentration of viable bacteria at time t = 2, M L
-3

X
1
= concentration of viable bacteria at time t = 1, M L
-3

S
2
= concentration of growth limiting substrate at time t = 2, M L
-3

S
1
= concentration of growth limiting substrate at time t = 1, M L
-3



X
2
X
1
S
2
S
1

---------- = -Y -----------
t
2
- t
1
t
2
t
1


dX dS
Y
dt dt
| | | |
=
| |
\ . \ .

So X


Xo S
1
Time, t
X
,

S
2
2-Bacterial Growth Kinetics_F12
13
g u
dX dS
Y
dt dt
| | | |
=
| |
\ . \ .


where
su
u
dS
r
dt
| |
=
|
\ .
= rate of substrate utilization, M L
-3
T
-1


Note: since utilization has meaning of negative, the sign is changed.

( )
( )

g
u
dX
dt
Y
dS
dt
=


Cell yield depends on (ME, p 371)
1) the oxidation state of the carbon source and nutrient elements
2) the degree of polymerization of the substrate
3) pathways of metabolism
4) growth rate
5) various physical parameters of cultivation


The Relationship between the Rate of Bacterial Growth and the Rate of Substrate
Utilization

c. The relationship between the rate of growth and rate of substrate utilization can be expressed
as:

g u
dX dS
Y
dt dt
| | | |
=
| |
\ . \ .
or r
g
= Y r
su



1 1
m
su
u g s s
dS dX SX kSX
r
dt Y dt Y K S K S
| | | |
= = = =
| |
+ +
\ . \ .


where k = maximum rate of substrate utilization per unit mass of organisms, T
-1
(d
-1
)


m
k
Y

= or
m
kY =
m
g
g
dX S kYS
r X X X
dt Ks S Ks S

| | | | | |
= = = =
| | |
+ +
\ . \ . \ .




14

Note: In a continuous flow reactor, CSTR,

su
u
dS So S
r
dt u
| |
= =
|
\ .


u
dS
So S dt
U
X X u
| |
|

\ .
= =



Other Rate Expressions

The substrate utilization rate, r
su
r
kSX
Ks S
su
=
+


1) When S >> Ks, r
kSX
Ks S
kX
su
=
+
= .. 1
st
order expression

- If the system is in endogenous phase and excess microbes,
r
su
= - k .. zero order expression (8-15), ME, p 373


2) When S << Ks, r
kSX
Ks S
k SX
su
=
+
= ' where k = k/Ks . 2
nd
order expression

- The system is not operated with excess microbes, thus the number of bacteria is a rate limiting
factor.
- If the system is in endogenous phase and excess microbes

rsu = - kS

3) Empirical expression r k X
S
S
su
o
= ' (8-18)



Exponential growth phase

Mathematical expression of main principles in the bacterial growth kinetics (in the exponential growth phase) are:

r
dX
dt
X
g
g
=
|
\

|
.
| =
(1)

2-Bacterial Growth Kinetics_F12
15

=
+
m
s
S
K S
(2)

dX
dt
Y
dS
dt
g u
|
\

|
.
| =
|
\

|
.
|
(3)

m
o
o o
o
t P
X
X
Q
YS X X
YS
=
|
\

|
.
|
|
\

|
.
| ( ) ln ( ) ln
(4)

where

P
K Y S Y X
YS X
s o o
o o
=
+ +
+


Q
K Y
YS X
s
o o
=
+


Equations (1), (2), and (3) are the basic expression of bacterial growth kinetics. Using these basic kinetic expressions, develop the bacterial
growth model [ Equation (4)] that describes the temporal bacterial growth.

Show clearly your work step by step. Define all the terms used in the equation, and give all the assumptions used.



Bacterial Death Rate, Endogenous Death (Decay) Rate, ,
d
d
dX
r
dt
| |
|
\ .

d d
d
dX
r k X
dt
| |
= =
|
\ .


where
r
d
= endogenous decay rate, , ML
-3
T
-1
(mg L
-1
d
-1
)
k
d
= death rate constant = engogenous decay coefficient, T
-1
(d
-1
)


Endogenous Phase - near starvation phase

dX
Net rate of bacterial growth, r
g
, (-----)
g

net

dt

Net Growth Rate = Growth Rate - Death Rate

'
net
g
g g d
dX dX dX
r
dt dt dt
| | | | | |
= =
| | |
\ . \ . \ .

Where ,
d
g d
dX dX
X k X
dt dt

| | | |
= =
| |
\ . \ .



16
'
( )
net
g d net
g
dX
r k X X
dt

| |
= = =
|
\ .


in which

m
net d d
s s
S Y k S
k k
K S K S

= =
+ +



where k =
m
/ Y
= maximum rate of substrate utilization per unit mass of organisms, T
-1
(d
-1
)


'
net
g net d
g s
dX Y k S
r X k X
dt K S

| |
| |
= = =
| |
+
\ .
\ .



dX
Endogenous Phase Net rate of bacterial growth, r
g
, (-----)
g

net

dt

dX dX dX
r
g
= (----)
g

net
= (----)
g
(-----)
d
= X k
d
X = ( k
d
) X =
net
X =

X
dt dt dt

d
u
dS
Y k X
dt
| |
=
|
\ .


dX
r
g
= (-----)
g

net
= net rate of bacterial growth, ML
-3
T
-1

dt

where =
net
= net specific growth rate, T
-1


dX
m
S X dS
since (----)
g
= ----------- = Y (----)
u

dt Ks + S dt

dX
m
S X dS
r
g
= (----)
g

net
= ----------- - k
d
X = Y (----)
u
- k
d
X = Y r
su
- k
d
X
dt Ks + S dt


2-Bacterial Growth Kinetics_F12
17


dX
m
S kY S
r
g
= (----)
g

net
= (---------- k
d
) X =
net
X = (------------ k
d
) X
dt Ks + S Ks + S

where
m
= k Y



Note:

m
S Y (
m
/Y) S
=
net
= ----------- k
d
= ---------------- k
d

Ks + S Ks + S

Y k S
= ----------- k
d
since
m
/Y = k
m
= Y k
Ks + S

The above expression will be used in developing mass balance on X (see CSTR model).



Observed Growth Yield, Y
obs


(dX/dt)
g

net
Y
obs
= --------------
(dS/dt)
u




Substrate Utilization Rate, r
su


1 1
m
su
u g s
s
S dS dX
r X
dt Y dt Y K S
kSX
K S
| |
| | | |
= = =
| | |
+
\ . \ .
\ .
=
+


Specific Substrate Utilization Rate, U

u
dS
dt
U
X
| |
|
\ .
= (1)

18
where (dS/dt)
u
has positive value

u
dS kSX
dt Ks S
| |
=
|
+
\ .
(2)

Dividing (2) by X yields

u
dS
kS dt
U
X Ks S
| |
|
\ .
= =
+


kS
U
Ks S
=
+
(3)

Rearranging (3) for S as a function of U

U(Ks +S) = kS
UKs + US = kS
kS US = U Ks
S(k-U) = UKs

U Ks
S
k U
=

This is an expression for S as a function of U.
- as U increases, S decreases


In a CSTR,

( )
u
dS
So S So So S dt
U
X X X So
QSo So S F
E
VX So M
u u
| |
|

\ .
= = =
| || | | |
= =
| | |
\ .\ . \ .


where E = (So - S)/ So = substrate removal efficiency
= V/Q = hydraulic retention time
= aeration time, hr
F/M = Food to microorganisms ratio

----------------------------------------
Find S as a function of U:

Since
kS
U
Ks S
=
+

2-Bacterial Growth Kinetics_F12
19
U (Ks+S) = kS

UKs US = kS

kS US = U Ks

S (k U) = U Ks


UKs
S
k U
=

As U increases, S decreases


Evaluation of k and Ks

Linearlize Equation (3), Specific Substrate Utilization Rate eqn.

kS
U
Ks S
=
+
(1)

1 Ks S Ks S
U kS kS kS
+
= = +

1 1 1 Ks
U k S k
= +

Or, dividing Eq. (1) by S gives

U k
S Ks S
=
+


Inverse the eqn

1 S Ks S Ks
S
U k k k
+
= = +




1/U Slope = Ks/k
Intercept = =1/k
1/S

S/U Slope = 1/k
Intercept = =Ks/k
S