2-Bacterial Growth Kinetics_F11.

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BACTERIAL GROWTH KINETICS

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

Log (No. of Viable Cells)

Endogenous Phase

5
4

3
7
1
2

1. Lag Phase

Substrate - - - -

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

a. Adaptation (acclimation) period

Time
- 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.

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 = 0
6. Death or declining Phase
a. The number of viable cells decreases slowly while the total mass may remain constant due to
the fact that the death rate exceeds the production rate of new cells.
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.
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.

2-Bacterial Growth Kinetics_F11.doc

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 BWT system 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.
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 substrate utilization rate, k
4) half-saturation constant or Monod constant, Ks
5) yield coefficient, Y
6) endogenous decay coefficient (death rate constant), kd

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.

S1

<

S2

<

S3

<

S4

<

S5

2)
S5
S4
No. of
cells

S3

S5
Log
No. of
Cells

S4
S3

S2

S2
S1

S1
1
Time

Time

3
2

S1

S2

S3

S4
S

S5

2-Bacterial Growth Kinetics_F11.doc

The Monod Model

a. The effect of substrate concentration on the specific growth rate is shown in Figure 1.
(a) The Monod Plot
m

m/2

KS
S

Fig. 1. Effect of substrate concentration on the specific growth rate.

(3rd: DC 350; 4th 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
= ----------Ks + S

(1)

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
Ks

= k 'S

where k =m / Ks
the model would be reduced to a first order expression

e. If Ks << S, =

m S
S

= m
the model would become a zero order expression

f. 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/um and an intercept of 1/ m
on the 1/ axis.
Lineweaver-Burk plot

Slope = Ks/m

1/

Intercept = =1/m

1/S

2-Bacterial Growth Kinetics_F11.doc

The Eadie Plot

Eadie plot
m S
= ------------

Ks + S

m/Ks

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

m -

/S
Slope = 1/Ks

-- = ------------ = ------- ----Ks

Km
S
Ks
1
m
-- = - ---- + -----Ks
S
Ks

dX
Rate of Bacterial Growth, rg ,

dt g
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 S
dX
rg =
= X =
X
dt g
Ks + S
where rg = rate of bacterial growth, M L-3 T-1 (mg/Ld)
X = concentration of viable bacteria (in VSS) at time t, ML-3 (mg/L)
= specific growth rate, T-1 (d-1)

m S
Ks + S

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

1
X

dX

dt g

 dS
Rate of Substrate Utilization, rsu ,
dt u
dS
rsu , = rate of substrate utilization, ML-3 T-1 (mg/L-d)
dt u
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.
X

t g

t u

Xo

X, S

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.

So

X2 X1
-------- = Y = yield coefficient
S1 S2

2
Time, t

X1 X2
---------- = Y
S1 S2
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
X2 = concentration of viable bacteria at time t = 2, M L-3
X1 = concentration of viable bacteria at time t = 1, M L-3
S2 = concentration of growth limiting substrate at time t = 2, M L-3
S1 = concentration of growth limiting substrate at time t = 1, M L-3
dX

dt g

dS

dt u

dX

= Y
dt g

dS

dt u

2-Bacterial Growth Kinetics_F11.doc

( dX dt )
( dS dt )

= Y or Y

dS
where rsu = = rate of substrate utilization, M L-3 T-1
dt u
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:

dX

= Y
dt g

dS

dt u

or

rg = - Y rsu

1 dX
1 m SX
kSX
dS
rsu = =
=
=
dt u Y dt g Y K s + S K s + S
where k = maximum rate of substrate utilization per unit mass of organisms, T-1 (d-1)

or
m = kY
Y
dX
m S
kYS
rg =
= X =
X =
X
dt g
Ks + S
Ks + S
m SX
dS
dX
Y =
=
Ks + S
dt u
dt g
k=

Note:
when (dS/dt)u has positive value,

kSX
dS
rsu = =
dt u K s + S
This is used in mass balance on S.
9

In a continuous flow reactor, CSTR,

So S
dS
rsu = =

dt u
dS

So S
dt
U = u =
X
X

Other Rate Expressions

The substrate utilization rate, rsu
1) When S >> Ks, rsu =
-

rsu =

kSX
Ks + S

kSX
= kX
Ks + S

.. 1st order expression

If the system is in endogenous phase and excess microbes,

rsu = - k .. zero order expression (8-15), ME, p 373

2) When S << Ks, rsu =

kSX
= k ' SX
Ks + S

where k = k/Ks

. 2nd 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

rsu = k ' X

S
So

(8-18)

Exponential growth phase

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

dX
rg =
= X
dt g

m S
Ks + S

(1)

(2)

dX
dS

= Y
dt g
dt u

(3)

X
YS X o X
(Q) ln o

YSo
Xo

mt = ( P) ln

(4)

10

2-Bacterial Growth Kinetics_F11.doc

where

P=

Q=

KsY + SoY + X o
YSo + X o
K sY
YSo + X 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.

dX
Bacterial Death Rate, Endogenous Death (Decay) Rate, rd ,

dt d
dX
rd =
= kd X
dt d
where
rd = endogenous decay rate, , ML-3 T-1(mg L-1 d -1)
kd = death rate constant = engogenous decay coefficient, T-1 (d -1)

Endogenous Phase - near starvation phase

dX
Net rate of bacterial growth, rg , (-----)g net
dt
Net Growth Rate = Growth Rate - Death Rate
net

dX
dX dX
r =
=

dt g
dt g dt d
'
g

Where

dX

= X
dt g

dX

= kd X
dt d

net

dX
r =
= ( kd ) X = net X
dt g
'
g

in which

11

net =

m S
Ks + S

kd =

YkS
kd
Ks + S

where k = m / Y
= maximum substrate utilization rate constant
net

YkS

dX
rg' =
kd X
= net X =
dt g
Ks + S

dX
Endogenous Phase Net rate of bacterial growth, rg , (-----)g net
dt
dX
dX
dX
rg= (----)g net = (----)g (-----)d = X kd X = ( kd) X = net X = X
dt
dt
dt

dS
= Y kd X
dt u
dX
rg= (----)g net = net rate of bacterial growth, ML-3 T-1
dt
where = net = net specific growth rate, T-1
dS
dX
m S X
since (----)g = ----------- = Y (----)u
dt
Ks + S
dt
dX
m S X
dS
net
rg = (----)g = ----------- - kd X = Y (----)u - kd X = - Y rsu - kd X
dt
Ks + S
dt
dX
m S
YkS
rg = (----)g net = (---------- kd ) X = net X = (------------ kd) X
dt
Ks + S
Ks + S
where k = m /Y = maximum substrate utilization rate constant

12

2-Bacterial Growth Kinetics_F11.doc

Note:
m S
Y ( m /Y) S
= net = ----------- kd = ---------------- kd
Ks + S
Ks + S
YkS
= ----------- kd
Ks + S

since m /Y = k
m = Y k

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

Observed Growth Yield, Yobs

(dX/dt)g net
Yobs = -------------(dS/dt)u

Substrate Utilization Rate, rsu

1 dX
1 S
dS
rsu = =
= m X

dt u Y dt g Y K s + S
kSX
=
Ks + S
Specific Substrate Utilization Rate, U
dS

dt u
U=
X

(1)

where (dS/dt)u has positive value

kSX
dS
=
dt u Ks + S

(2)

Dividing (2) by X yields

dS

kS
dt
U = u =
X
Ks + S

13

U=

kS
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
S=

U Ks
k U

This is an expression for S as a function of U.

- as U increases, S decreases

In a CSTR,

dS

dt u So S So( So S )
U=
=
=
X
X
X So
QSo So S F
=

= E
VX So M
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 U =

kS
Ks + S

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

UKs
k U

14

2-Bacterial Growth Kinetics_F11.doc

As U increases, S decreases

Evaluation of k and Ks
Linearlize Equation (3), Specific Substrate Utilization Rate eqn.
U=

kS
Ks + S

(1)

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

Slope = Ks/k

1/U

Or, dividing Eq. (1) by S gives

Intercept = =1/k

U
k
=
S Ks + S

1/S

Inverse the eqn

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

Slope = 1/k

S/U

Intercept = =Ks/k

15