Microbial kinetics

Dr Ana Soares
Senior Lecturer in Biological
Engineering

Cranfield Water Science Institute

SWEE

1
 Why do we need to know about biological
reaction kinetics?

Settled wastewater: STW effluent :

500 mg/LCOD Biological conversions 50 mg/LCOD
375 mg/L BOD 10 mg/L BOD
15 mg/L TN 5 mg/L TN
7 mg/L P 2 mg/L P
All substrates for biological reactions

Dependant on reaction rates

Key to design treatment process
Predict performance
2
How fast does
a bacteria
grow?

(or a population of
microbes)

3
 Bacteria reproduction

Bacteria multiply by binary fission,

Single cell reproduction:

a.  Growth - Bacterial cells enlarge
or elongate
b.  Membrane formation - creation
of transverse membrane and
new cell wall.
c.  The new membrane and cell
wall grow inward from the outer
layers.
d.  Division - cell divided into two
daughter cells

4
 Measure bacterial growth

Common methods include:

•  Turbidity: to measure the total
bacteria (live and dead) in liquid
cultures (600-680 nm)

•  Colony counting method: counting

colony numbers on a petri dish

•  Automated methods (e.g.: flow

cytometer)

•  Measuring surrogates for growth

(O2 depletion, DNA, RNA, etc.)

5
 Microbial growth measurement and
interpretation

Abs 660 12000 Microbial growth

Time
(h) nm
(χ) 10000
0.1 60
8000

Abs 660 nm
3 60
6000
7 65 1 2 3
10 70
4000

12 122 2000

24 245 0
50 812 0 50 100 150

75 2697 Time (hours)

1 - Lag phase: microbes are adjus5ng to the new substrate and
100 8103 environment (substrate, pH, temp etc.)
110 8955 2 - Exponen5al growth phase: microbes have acclimated to the
condi5ons
120 9897
3 - Sta5onary phase: limi5ng substrate or electron acceptor limits
130 10938 the growth rate
6

 Microbial growth measurement and
interpretation

Time Abs 660

(h) nm Ln (χ) 10 Linearised Microbial growth profile
(χ) 9
0.1 60 8 1 2 3
4.09
7

ln(Abs 660 nm)

3 60 4.09
6
7 65 4.17 5
4
10 70 4.25 3
12 122 4.80 2
24 245
1
5.50 0
50 812 6.70 0 50 100 150

75 2697 Time (hours)

7.90
100 8103 9.00 1 - Lag phase: microbes are adjus5ng to the new substrate and
110 8955 environment (substrate, pH, temp etc.)
9.10
2 - Exponen5al growth phase: microbes have acclimated to the
120 9897 9.20 condi5ons
130 10938 3 - Sta5onary phase: limi5ng substrate or electron acceptor limits
9.30 7
the growth rate

 Taking a close look at the exponential
phase of growth….
1st order kine4cs
10 microbial growth profile
Linearised Microbial
ln (x/x0) versus 4me is linear
9
8 reac4on rate = growth rate =µ
7
nm)
660 nm)

6 y = 0.05x + 4.31
Slope of the linear phase
ln(Abs 660

5 (exponen4al phase of growth)

R2 = 1.00
ln(Abs

4
3
2
Calculate slope
µ = 0.05 h-1
1 (ln X2 - ln X1)
0 µ=
0 50 100 150 (t2 - t1)
Time (hours)

dX Xt µt
= µX ⇔ ln( ) = µt ⇔ X t = X 0 e
dt X0
8
 Doubling time - td

Time required for the popula4on to double , Xt=2 X0

Xt 2 ln 2
ln = µt ⇔ ln = µt d ⇔ ln 2 = µt ⇔ t d =
X0 1 µ

9
Specific growth rates and doubling
times

10
 General equations

dX
= µX
dt µ - growth rate
X – biomass concentra4on
µt
Xt = X 0 e T – 4me
X0 - biomass concentra4on start
Td - duplica4on 4me

ln 2
td =
µ
11
 Microbial growth rate: Summary

1.  Microbial growth rate can be used for a single species of

microorganisms, or a group, as in the activated sludge process

2.  Growth can be described by using a growth rate constant, µ,

which is analogous to the chemical reaction rate constant, k.

3.  The maximum specific growth rate for an organism or group of

organisms, µmax, describes its maximum possible growth under
one set of environmental conditions (temperature, pH).

4.  As with other kinetics constants, the units are in inverse time, e.g.
h-1

12
 How does
microbial
growth and
substrate
uptake relate?

13
 Modeling substrate depletion –
Monod kinetics

µmax

µ max Relates specific growth rate, µ, to substrate concentra4on

Empirical: no theore4cal basis - it just “fits”!
Have to determine µmax and Ks in the lab
2 Each µ is determined for a different star4ng S

S
Ks µmax – maximum specific growth rate
Ks – satura4on coefficient for the substrate
14
 Monod kinetics
Maximum specific growth rate Substrate conc.
(BOD, COD etc)
µ max × S
µ=
Half saturation constant Ks + S
1.  Microbial growth is described by Monod kine5cs where the growth
rate ceases to increase as substrate concentra5on increases, i.e.:
!  ini4ally the rate or growth increases as the substrate concentra4on
increases,
!  then the growth rate remains the same as the substrate concentra4on
increases further.
2.  Monod kine5cs apply to microbes whether in batch or con5nuous
culture
15
 What
happens in
continuous
culture?

16
 Continuous cultivation
Dilution rate and hydraulic retention time

Q, S0
Q, S, X

V, S, X

V 1 Q
τ = HRT = D= D=
Q HRT V
17
Dilution rate vs growth rate

18
 Dilution rate

1.  In a continuous culture system, dilution rate (D) is the inverse

of hydraulic retention time (HRT), i.e. D = 1/HRT.
2.  Dilution rate therefore has units of h-1 etc.
3.  Thus in a continuous culture reactor without recycle, HRT is
equal to the biomass or solids retention time (SRT).
4.  Therefore at steady state:
1.  the dilution rate = the growth rate of the microorganisms,
2.  D = µ.

19
 Critical dilution rate Dc

Steady state: D = µ
µm S
DC =
Ks + S
Because Ks << S: Dc ≈µm

DC (bacteria) = 1 h-1
DC (Fungi) = 0.1 h-1

20
How much
biomass is
formed?

21
 Biomass concentration, x

Q, Si

Q, S, X

V, S, X

In ac4vated sludge -
X = MLSS or
more accurately MLVSS
22
 Yield coefficient (Y)

• Tells us how much biomass is generated by the removal

of organics
• This is merely an experimentally derived ratio to describe
the relationship between a substrate and the product of a
microbial reaction (S→X) - biomass produced to mass of
substrate consumed - therefore Y is dimensionless
• Most commonly used for biomass yield compared to
influent BOD

dX
Y=
dS
23
Bacterial synthesis yield coefficients

24
How much
substrate
is utilized?

25
 Substrate depletion kinetics

dX dS
Since = µX = −Y
dt dt
µ max S
and µ=
Ks + S

dS µX µ max SX
then − = =
dt Y (K s + S)Y
µ max
dS kSX Where k =
and − = Y
dt K s + S
26
 Substrate depletion kinetics

• The rate of biodegradation or biotransformation is a

focus of environmental studies
• Substrate consumption rates have often been
described using ‘Monod kinetics’

dS kSX
•  S is the substrate concentration [mg/L] − =
•  X is the biomass concentration [mg/ L]
dt K s + S
•  k is the maximum substrate utilization rate [sec-1]
•  KS is the half-saturation coefficient [mg/L]

27
 What
happens
when the
bacteria dye?

28
 Endogenous respiration

1.  At steady state, in addi4on to cells growing, cells are

also dying.
2.  Just as there is a growth rate constant, there is also a
specific death rate constant, ke.
3.  In describing the amount of biomass in a system, or
the removal of substrate, the death of cells
(biomass) has to be taken into account.

29
Endogenous Metabolism (ke)

30
Endogenous metabolism (for
carbon)

dX
= [µ − ( D + ke )]X
dt

Ke - specific death rate constant,

31
 Endogenous respiration

1.  Values of ke are around 10% of µm

1.  0.1 h-1 (bacteria)
2.  0.01 h-1 fungi

2.  At low dilu4on rate ke is similar to µ and and

YS reduce.

32
To summarise….

33
 So what do we want to know

KS D
Effluent concentration S=
µm − D

( " D %+
Sludge produced X = Y *S − K S $ '-
) # µ m − D &,
34
 Typical values

µ (d-1) -1
Substrate Y m Ks (ppm) ke (d ) Basis

Domestic 0.5 0.055 BOD

0.67 3.7 22 0.070 COD

Skim milk 0.48 2.4 100 0.045 BOD

Glucose 0.42 1.2 355 0.087 BOD

Peptone 0.43 6.2 65 - BOD

35
…..How to avoid wash out?

Q, Si

Q, S, X

V, S, X

36
 So what is the problem

To use con4nuous culture without recycle for

wastewater treatment
• large reactor volumes are needed to get
sufficient substrate removal
• because micro-organisms grow slowly
• and their growth rate is controlled by the
reten4on 4me in the reactor

37
 Solu5on

Keep the micro-

organisms in the reactor
longer than the aqueous
phase
• by adding a
separa4on stage
with recycle and
• directly control the
4me the micro-
organisms stay in the
reactor system
38
The ac5vated sludge process

39 solids
Keeping the sludge

SRT ≠ HRT
SRT > HRT
SRT is the sludge age
θX
40
 Continuous culture with recycle

•  The ac4vated sludge process is considered to be a

con4nuous culture process with biomass recycle
operated at steady state (this is not oben strictly true but
is a good enough approxima4on).
•  Adding a biomass recycle to the con4nuous culture
reactor means that:
•  Biomass reten4on is independent of HRT
•  Gives beder control
•  Selects for flocculent microbes that will separate
from the effluent
•  Key point: SRT>HRT in a con4nuous culture with recycle

41
 Tradi5onal diagram of con5nuous culture with
recycle
Q, So, Xo
QE =(Q-Qw)
XE
S

Qr, Xw, S QW, Xw, S

42
Control MLSS
(waste rate)

SRT controlled

Microbial population
growth controlled

Performance
43
defined
 Process engineering

Get the process engineering

right and the biology will
work
•  Sludge age (secondary
sedler)
• Aera4on
Understand the microbial
kine4cs and you can
understand the process
44