CIVL 3420 Water and Wastewater Engineering

Lecture 11

Chapter 3.2 Bacterial Metabolism in Wastewater

Treatment

Instructor: Prof. CHEN Guanghao

Dr. DENG Yangfan
Michaelis-Menten kinetics

◆ Enzymes are biological catalysts which act to increase the rate of a reaction
without being used up or changed themselves.
◆ They are specific to one type of reaction and one, or a small number of,
closely related reactants known as substrates.
◆ Enzymes are a vital component of the cell as without them, many biological
reactions would be too slow to sustain life.

Active Site: The active site is the region on the surface of an enzyme molecule to which a specific substrate
will bind and where it will undergo a chemical reaction.
Substrate: The substrate is the molecule, or combination of molecules, that has a specific and
complementary shape for a particular enzyme’s active site.
Product: The product is the molecule, or combination of molecules, that is released from the enzyme’s active
site following an enzyme-controlled reaction.
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Michaelis-Menten kinetics
Michaelis-Menten kinetics is a model of enzyme kinetics which
explains how the rate of an enzyme-catalyzed reaction depends on
the concentration of the enzyme and its substrate.

E: Enzyme
𝐸 [𝑆] S: Substrate
= P: Product
𝐾𝑚
ES: Enzyme-substrate complex
KM: Michaelis–Menten Constant

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Michaelis-Menten kinetics

𝑺
V= Vmax
𝑲𝒎+𝒔

✓ Km implies that half of the active

½ Vmax sites on the enzymes are filled.
✓ Different enzymes have different
Km values.
4 ✓ They typically range from 10-1 to
Km 10-7 M.

Reaction Order：
𝑺
◼ When S<<Km, V= Vmax
𝑲𝒎
◼ When S>>Km, V=Vmax

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Michaelis-Menten kinetics
Determine Vmax and Km

The general approach is to add a known concentration of substrate to the

enzyme and to determine the initial reaction rate for that concentration of
substrate.

Use linear plot and intercepts to determine Km and Vmax.

𝟏 𝐊𝐦 𝟏
= +
𝐕 𝐕𝐦𝐚𝐱[𝐒] 𝐕𝐦𝐚𝐱
Experimental approach
◼ Reaction rates are typically given as moles (or
micromole) of product produced per unit of
time (sec or min) per mole (or micromole) of
enzyme
◼ The experiment is repeated for a wide range
of substrate concentrations
◼ A table of [S] versus V datapoints are
collected
◼ These datapoints are plotted (V versus S) and
should fit a curve that agrees with the
Michaelis-Menten equation
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Example

β-glucosidase has KM = 6.0 x 10–5 M, what value of [S]

is needed to get V = 0.75 Vmax and V = 4.0 x 10–9 M·s–
1 given V = 2.0 x 10–8 M·
s –1
max

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 Michaelis-Menten kinetics
The factors that affect reaction ◼ When enzymes are exposed to high
rate are： temperatures and extreme pHs, the
structure of an enzyme changes, which
 Temperature
means the active site also changes shape
 pH irreversibly.
 ionic strengths ◼ This is called denaturing and it means that
 the nature of the substrate the active site is no longer a
complementary shape to the substrate
molecule.

Each individual enzyme has a specific optimum temperature and pH value.

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Monod Kinetics
As substrate utilization rate reaches its maximum in the log phase, mathematical models are
used to study the kinetics.
The rate expression is
ⅆ𝐗
= 𝛍𝐗
ⅆ𝐭
where
X = concentration of biomass, mg/L
𝜇 = specific growth rate, time-1
Integrated expression from above equation is given by

𝑋𝑡 = 𝑋0 ⅇ𝜇𝑡
where
𝑋𝑡 = concentration of cell at time t
𝑋0 = concentration of cell at time 0

Doubling time of the cell can be calculated from above equation.

ln 2
2𝑋0 = 𝑋0 ⅇ𝜇𝑡𝑑 or 𝑡𝑑 =
𝜇
where 𝑡𝑑 represents time to double the cell mass
𝛍 for AOB and NOB at 20 ℃ are 0.74 and 0.65 d−1, respectively. HB can be as high as 6 d−1.
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Monod Kinetics
The effect of substrate concentration (S) on microbial growth was formulated by Jacques Monod.
The equation describing the relationship between 𝜇 and S is given by

𝐒
𝛍 = 𝝁𝐦𝐚𝐱
𝐊𝐬 + 𝐒
where
𝜇 = specific growth rate of a considered microorganism, time-1
S = concentration of growth limiting substrate, mg/L
𝜇𝑚ax = maximum specific growth rate, time-1
K s = half saturation constant, mg/L
(i.e. concentration of substrate when 𝜇 = 0.5 𝜇𝑚ax )

➢ Ks is an important factor as it describes the

outcome of competition between different
microorganisms competing for the growth-
limiting substrate.
➢ A lower value of Ks suggests a greater affinity
for the substrate by the organism. In an
environment with low growth-limiting
substrate concentration, the organism with
the lowest Ks will possess greater capacity to
grow rapidly.

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Kinetics
Substituting the value of 𝜇 into growth equation, we get:

ⅆ𝑿 𝑺
= 𝝁𝒎𝒂𝒙 𝑿
ⅆ𝒕 𝑲𝒔 + 𝑺

For S >> Ks :
ⅆX
= 𝜇𝑚ax 𝑋
ⅆt

i.e. the organism growth rate is a zero-order kinetics

For S << Ks :
ⅆX S
= 𝜇𝑚ax 𝑋
ⅆt Ks

i.e. the organism growth rate is a first-order kinetics

For many wastewater treatment applications, the substrate concentrations are low with
organisms having high Ks values, thus, becomes quite applicable.

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Kinetics
Endogenous respiration constant kd must be considered. The factor accounts
for basal metabolism of cell which causes a decrease in cells mass due to
metabolism of cellulose stored products.
ⅆX 𝜇𝑚ax S
= 𝑋 − kd𝑋
ⅆt K s + S

where kd = cell endogenous respiration constant, t-1

Only a fraction of the substrate is converted to cells while the remaining

fraction is used to produce energy (chemical energy in the form of high
energy phosphate bonds, ATP)
dX d𝑆
Y = yiⅇlⅆ coⅇfficiⅇnt = /
dt dt
dX 𝑑𝑆 𝜇𝑚ax S
or =Y = 𝑋
dt 𝑑t Ks +S

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 Bacterial Growth Patterns in a Batch Reactor

1. Lag phase represents the acclimation of the

cell to the experimental environment,
substrate, etc. The length of the lag phase
depends on the contrast between the
previous and new environmental conditions.
It ranges from half an hour to days.
2. Log (exponential) phase is a period
characterized by cell doubling. Once cells
have accumulated all that they need for
growth, they proceed into cell division. The
exponential or log phase of growth is
marked by predictable doublings of the
population, where 1 cell become 2 cells,
becomes 4, becomes 8 etc.
3. Stationary phase refers to stop of cellular growth as the substrate has been exhausted and new
cellular growth occurs at the expense of the death of other cells.
4. Death phase is a period when cellular decay/death rate is much higher than new cell growth, and
results in reduced number of cells.

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Temperature, pH, and Osmotic Pressure
Temperature
➢ Most microorganisms grow within a specified range of temperature with
well-defined upper and lower limits.
➢ The optimum growth temperature is generally close to the upper limit.
➢ The growth falls off drastically after temperature exceeds the upper limit.
➢ The growth rate will decrease when the temperature is below the
optimum value.
Psychrophiles – growth range : -5 - 30C
Mesophiles – growth range : 15 - 45C
Thermophiles – growth range : 45 - 70C
➢ Most disease-causing bacteria grow in the range of 35 – 40C, while
wastewater treatment bacteria may have a wide growth range from 5 –
35C.

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Temperature, pH, and Osmotic Pressure
pH Range
➢ Most microorganisms grow best at pH=7, although they may tolerate a pH range from 5 to
8.5.
➢ Natural alkalinity of waters can provide a buffer to maintain the pH but for specific
situations when proper buffer is not present, external chemicals may need to be added to
keep the system functioning properly.
➢ There are special bacteria that can tolerate very acidic conditions, e.g., Thiobacillus can
grow at pH1 or below.

Osmotic Pressure
➢ Most bacteria grow over a broad range of salt concentration as the cell is capable of
maintaining a constant internal salt concentration.
➢ Cell growth is inhibited if the salt concentration outside the cell is too high. Water will flow
out of the cell.
➢ Halophiles actually require high salt for their survival and can grow in salt saturated systems
(up to 30%).

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Oxygen, Nutrients, and Toxic Compounds
Oxygen
➢Aerobes
✓ These organisms have an absolute requirement for oxygen.
✓ They use molecular oxygen as their electron (e-) acceptor.
✓ e.g. pseudomonas species found in soil.
➢Anaerobes
✓ These organisms can only grow in the absent of oxygen.
✓ They use some molecules other than molecular oxygen as their e- acceptor.
✓ They are unable to break down toxic derivative of oxygen induced reactions due to
lack of certain enzymes.
➢Facultative
✓ These organisms can grow with or without oxygen.
✓ Their growth in oxygen is more rapid.
✓ e.g., coliform group including E-coli.

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Oxygen, Nutrients, and Toxic Compounds
Nutrients
➢ C is for cell tissue synthesis and as energy source through oxidation of organic compounds.
➢ N and P are required in large quantities. N is for protein and nucleic acid synthesis; P is for
nucleic acid synthesis and play a vital role in energy transfer.
➢ BOD : N : P = 100 : 5 :1 is desirable on a mass basis to ensure proper metabolism.
➢ Micronutrients:

for activating exzymes during

S for proteins synthesis phosphate transfer, Zn hold protein subunits in proper
Mg configuration for enzyme activity
Ca stability of cell wall stabliser for cell membrane and
nucleic acids Cu for redox reaction

Toxic Compounds
➢ Some toxic compounds are possibly discharged into industrial wastewater, which may lead to
toxicity to microorganisms, such as heavy metals, cyanides, phenols, chloro-organic, etc.
➢ Pretreatment is needed if such industrial wastewater is discharged into municipal sewage
system.

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 Summary of Lecture 11
1. What is the bacterial growth curve?
The bacterial growth curve represents the number of live cells in a bacterial population
over a period of time. There are four distinct phases of the growth curve: lag, log
(exponential), stationary, and death. The initial phase is the lag phase where bacteria are
metabolically active but not dividing. The log phase is a time of exponential growth. In
the stationary phase, growth reaches a plateau as the number of dying cells equals the
number of dividing cells. The death phase is characterized by an exponential decrease in
the number of living cells.
2. What is the Monod equation?
The Monod equation is a mathematical model for the growth of microorganisms.
The empirical Monod equation is:
S
𝜇 = 𝜇𝑚ax
Ks + S
3. What are the growth temperature ranges of Psychrophiles, Mesophiles and Thermophiles?
Psychrophiles: -5 - 30C; Mesophiles: 15 - 45C; Thermophiles: 45 - 70C
4. What are the aerobes, anaerobes, and facultative microbes?
Aerobes have an absolute requirement for oxygen. Anaerobes can only grow in the
absent of oxygen. Facultative microbes are able to grow either with or without free
oxygen.
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Summary of Lecture 11
5. What are the major roles of C, N, P in the cells growth?
C is for cell tissue synthesis and as energy source through oxidation of organic
compounds. N is for protein and nucleic acid synthesis. P is for nucleic acid synthesis
and play a vital role in energy transfer.
6. What are the aerobic respiration and anaerobic fermentation of glucose in microbes?
• Aerobic respiration of glucose is the process which cells uses oxygen as electron
accepter and convert glucose into CO2, H2O and energy.
• Anaerobic fermentation is a metabolic process by which glucose is converted into
cellular energy and the metabolite lactate, which is lactic acid in solution.
7. What are typical types of reactors used in biological wastewater treatment?
• Plug flow reactor - The liquid passes along the length of the reactor without any
longitudinal mixing between succeeding elements.
• Completely mixed reactor - The influent flow is immediately mixed with the
reactor contents and the concentration is constant throughout the reactor.
• Arbitrary flow reactor - The flow behavior is in between plug flow and completely
mixed reactors.

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