Chapter 2: Microbial Metabolism and

Growth

Environmental Microbiology (EnEg2102)// By : Dr. Tsedekech G/mesekel// Year:2024/25

 Outline

 Overview of microbial growth

 Microbial growth curve

 Growth Kinetics

 Unlimited growth

Growth at limited nutrient level

 Learning outcomes
At the end of this lesson, students able to
`Describe the four steps in microbial growth
 Explain the importance of understanding microbial growth
under the natural environment
 Compute the abundance and generation time of the microbial
community in a given culture media
Discuss the microbial growth kinetic
 Microbial growth
Provided with the right conditions (food, correct temperature, etc…)
microbes can grow very quickly.

Microbial growth is measured by the increase in population, either by

measuring the increase in cell number or the increase in overall mass.

Bacterial Division
Bacteria and archaea reproduce asexually only, while eukartyotic
microbes can engage in either sexual or asexual reproduction.
binary fission: where a single cell splits into two equally sized cells
 less common processes can include multiple fission, budding, and the
production of spores.
Microbial growth

Saccharomyces cerevisiae
reproducing by budding
 Importance of understanding microbial growth
under the natural environment
To predict rates of nutrient cycling

Microbial response to anthropogenic perturbation of the

environment

Microbial interaction with organic and metal

contaminants

Survival and growth of pathogens in the environment

 Microbial growth
Growth Curve
A growth curve composed of four distinct phases of growth: the lag phase,
the exponential or log phase, the stationary phase, and the death or decline
phase.
The lag Phase
Is an adaptation period, where the bacteria are adjusting to their new
conditions.
Cells in the lag period are synthesizing RNA, enzymes, and essential
metabolites that might be missing from their new environment, as well as
adjusting to environmental changes.
 The length of the lag phase can vary considerably based on
• how different the conditions are from the conditions that the bacteria
came from,
• the condition of the bacterial cells themselves.
 Microbial growth
Exponential or Log Phase
Growth is marked by predictable doublings of the population,
where 1 cell become 2 cells, becomes 4, becomes 8 etc…
This phase can be used to mathematically calculate the time it
takes for the bacterial population to double in number, known as
the generation time (g).
X= X0(2n ), where X0 is the initial number of cells, n is the
number of generations, and X is the number of cells after n
generations.
Generation time (g) can be represented by t/n, with t being the
specified period of time in minutes, hours, days, or months.
Microbial growth
 Microbial growth

Example 1
• If one starts with 10,000 (10 4 ) cells in a culture that has a generation
time of 2 h, how many cells will be in the culture after 4, 24, and 48 h?
.
After 4 h, n = 4 h/2 h per generation = 2 generations:
X= X0(2n ) 10 4(22)= 4 x 10 4 cells
After 24 h, n = 12 generations
10 4(212)= 4.1 x 10 7 cells
After 48 h, n= 24 generations
10 4(224)= 1.7 x 10 11 cells
 Microbial growth
Example 2
What is a generation time of a cell population that increases from
10,000 cells to 10,000,000 in 4 hr growth.

Solution:
g = t/n, where g is generation time, t is a specific of time, n is
numbers of generation.
X= X0(2n )
logX = logX0 + nlog2
n= logX- logX0 = logX- logX0 = 3.3logX/X0
log2 0.301
 Microbial growth
3.3log107
104
3.3 x 3= 9.96 ~ 10
g = t/n, 4 hrs
10
= 0.4 hr = 24 minutes
 Microbial growth
Stationary Phase
The number of new cells being produced is equal to the number of cells
dying off or growth has entirely ceased.
Although there is no net growth in stationary phase, cells still grow and
divided.
When a carbon source is used up, dying cells can lyse and provide a source
of nutrients.
 Growth on dead cells is called endogenous metabolism.

Death or Decline phase

 The death or decline phase, the number of viable cells decreases in a
predictable (or exponential) fashion.
The steepness of the slope corresponds to how fast cells are losing viability.
 Growth kinetics
Kinetics: is the study of reaction rates

To employ microorganisms for pollution control, environmental engineers must take
in to consideration two interrelated principles
1. The rate of pollutant removal depends on the concentration of the active
biomass (metabolically active microbes are the one that catalyse the
pollutant removing reactions)
2. The active biomass is grown and sustained through the utilization of its
energy and electron generating primary substrates, (which are electron donor)
and electron acceptor.
• chemical resources of the microorganisms usually are the pollutants that
the engineers must control
 Growth kinetics
To study the microbial growth Kinetics: There are two types of growth
mode (based on type of reactor)

1. Batch mode: when there is no addition of culture medium and no

removal of reactor liquid.

2. Continuous mode (Chemostat): there is a continues addition and

removal of substrate

 Bioreactors : is vessel in which a biological reaction is takes place.

(Bioactive element (M.O) and favourable growth environment)
 Growth kinetics

Batch mode growth:

1. Unlimited growth
Nothing limits the growth of the bacteria.
2. Growth at limited nutrient level
There is a growth limiting condition (nutrient).
 Growth kinetics
Batch mode growth: with unlimited growth (growth rate kinetics)
The change in the amount of cells per unit time is the growth rate of
the population.
The growth rate is directly proportional to cell concentration or
biomass
dX/dt ⁓ X
dX/dt=µX (where; X is the number or mass of cells
(mass/volume), µ= growth rate constant (1/h).
 Growth kinetics

Where X0 is the original bacteria biomass concentration, 𝑒 is the base of the

natural logarism, X is the biomass concentration after the time interval, t
hours
 Growth kinetics
The doubling time td and the biomass growth rate (µ), is related as
follows; This describe the exponential phase
𝑋𝑡 = 𝑋𝑜𝑒 𝜇𝑡
𝑋𝑡 = 2𝑋𝑜 where t = td
2𝑋𝑜 = 𝑋𝑜𝑒 𝜇𝑡𝑑
2 = 𝑒 𝜇𝑡𝑑

td=ln2/µ
 Growth kinetics
Linear form of the equation
𝑋𝑡 = 𝑋𝑜𝑒 𝜇𝑡
lnXt= lnXo+ µt

 Therefore, a plot of natural logarithm of a cell or biomass

concentration against time should yield a straight line, the slop of
which would equal to µ
 Growth kinetics
t(h)
 Growth kinetics

Batch mode growth: death rate kinetics

The decrease /change in the amount of cells per unit time in the
decline growth curve part is mathematically expressed as;
dX/dt ⁓ X
dX/dt= - KbX

A decrease in number per unit time is known as death rate

 Kb the death rate constant

 Growth kinetics

dX/dt=−𝐾𝑑X (where; 𝐾𝑑= death rate constant (h-1) )

𝑋𝑡 = 𝑋𝑜𝑒 −𝐾𝑑𝑡 (boundary conditions X (Xo – Xt) and t (to-t)
 Linearization to determine 𝐾𝑑
lnXt= lnXo- 𝐾𝑑 t
 Growth kinetics
Net growth rate constant (µnet)
µnet= µ- 𝐾𝑑
 Growth kinetics
 Grow that limited nutrient condition: in the vast majority of
cases, the rate limiting substrate is the electron donor. Because of
this convention, the term substrate here refers to the electron
donor.
 The most commonly used equation/model used to relate growth rate
with the limiting substrate concentration is known as the Monod‟s
kinetic equation(Jacques Monod)
 Growth kinetics

Where μ = specific growth rate (h-1)

S = substrate concentration (g/L)
Ks = substrate affinity (g/L)
μmax= maximum growth rate that the organism can attain (h-1)
 Monod equation can be expressed in terms of cell number or cell mass ( X )
 Growth kinetics
 The Monod equation has two limiting cases
1. High substrate concentration where S >> Ks (Zero order )

 Under these conditions, growth will occur at the maximum growth rate.
 under the initial conditions found in pure culture
 under continuous culture conditions
 Unlikely to be found under natural conditions in a soil or water
environment, where either substrate or other nutrients are commonly
limiting.
 Growth kinetics
2. low substrate concentrations where S<< Ks (first order )

 In this case there is a first order dependence on substrate concentration

 Typically found in
 batch flask systems at the end of the growth curve as the substrate is nearly all
consumed.

 under conditions in a natural environment where substrate and nutrients are

limiting.
 Growth kinetics
 Biomass yield(Y):- In biological treatment process, cell growth
occurs concurrent with oxidation of organic compounds.

 The ratio of biomass produced to the amount of substrate

consumed (g biomass/g substrate) is defined as the biomass
yield.
 Y typically defined relative to the electron donors used

 Y =g biomass produced / g substrate utilized (consumed)

 Growth kinetics

 Example; for aerobic heterotrophic reaction with organic

substrate, the yield is expressed as

g biomass produced / g organic substrate

For nitrification: g biomass/ g NH4-N oxidized

 Example; In wastewater substrate concentration is defined in

terms of its oxygen equivalence (COD and BOD).
 Growth kinetics
 Although the exact stoichiometry involved in the biological oxidation
of mixture of wastewater compounds is not known. We use the
following expressions for the purpose of illustration;

 Organic mater is presented by (C6H12O6) and new cells can be

represented (C5H7NO2 or C2H87O23N12P )
3C6H12O6+8O2+ 2NH3=2C5H7NO2 + 8CO2+14 H2O
3(180) +8(32) +2(17) = 2(113)
 Growth kinetics
Y =g biomass produced / g substrate utilized (consumed)

= g (C5H7NO2 )/g (C6H12O6)= 2*(113g/mol)/3*(180g/mol)

= 0.42 g cell/g glucose used

 The mass balance equation has a number of practical applications. It can be used to
estimate the amount of oxygen or nitrogen required for growth and utilization of a
particular substrate. This is useful for
– wastewater treatment,
– for production of high value microbial products (e.g., antibiotics or vitamins),
– remediation of contaminated sites
Factors affecting the growth/development of
microbes
Physical factors
Temperature
Osmotic pressure

Chemical factors
Oxygen
pH
Nutrients (C, N, P, S…)
 Factors affecting the growth of microbes
Temperature
 High temperature damages microbes by
denaturing enzymes, transport carriers, &
other proteins.

 Microbial membrane are disrupted by

extreme temperature.
• At very low temperatures membranes solidify
and enzymes do not function properly
Temperature conditions which has to be maintained in
microbes growth
Classification of bacteria based on temperature

 Psychrophiles: are the cold lovers, with an optimum of 15oC or lower

and a growth range of -20oC to 20oC.
Enzymes function in low temperatures.
Plasma membrane has increased amount of unsaturated and
shorter-chain fatty acids.
Produce cryoprotectants that prevent the development of ice
crystals.

 Mesophiles, microbes with a growth optima of 37oC and a range of

20-45oC.
Classification of bacteria based on temperature
 Thermophiles (“heat lovers”), the microbes that like high temperatures.
Thermophiles typically have a range of 45-80oC, and a growth optimum of
60oC.

 Hyperthermophiles, those microbes that like things extra spicy. These

microbes have a growth optima of 88-106oC, a minimum of 65oC and a
maximum of 120oC
 Specialized heat-stable enzymes that are resistant to denaturation and
unfolding.

 The plasma membrane contains more saturated fatty acids, with increased
melting points
Classification of bacteria based on temperature
 Factors affecting the growth of microbes
Oxygen
 The oxygen requirement of an organism relates to the type of
metabolism that it is using.
• Energy generation process require oxygen or a non-oxygen molecule as a
final acceptor of electrons.

 Obligate aerobes: they require the presence of atmospheric oxygen

(20%) for their metabolism
 Microaerophiles: require oxygen, but at a lower level than normal
atmospheric levels – they only grow at levels of 2-10%.
 Factors affecting the growth of microbes

 Facultative anaerobes: being able to grow in the presence or absence

of oxygen but prefer to grow in the presence of oxygen.

 Aerotolerant anaerobes: can also grow in the presence or absence of

oxygen, exhibiting no preference.

 Obligate anaerobes: can only grow in the absence of oxygen and find
an oxygenated environment to be toxic.
Oxygen and Bacterial Growth
 Factors affecting the growth of microbes
Osmolarity
Osmotic relationships: relative concentration of the solutions on
either side of the cell membrane

Isotonic – equal concentration of solute in the environment and

In the cell
 No change in the cell volume

 The most stable condition for the cell

 Factors affecting the growth of microbes
Hypertonic: the environment has a higher solute concentration
than that of the cytoplasm (cell)
 water will diffuse out of the cell

Cell will shrink (dry)

 Factors affecting the growth of microbes
 Hypotonic: when external environment has low solute concentration
than the cell.
 Water flows to the cell
Cells with out cell wall will burst out
 Factors affecting the growth of microbes

 Cells in a hypotonic solution need to reduce the osmotic concentration of

their cytoplasm.

 Remove solute from the cell via mechanosensitive (MS) channels.

 Cells in a hypertonic solution needing to increase the osmotic concentration

of their cytoplasm can take up additional solutes from the environment.

 Halophilic („salt-loving‟) forms are adapted to grow best in conditions of

high salinity. Example, halophiles bacteria living in salty lakes
 Factors affecting the growth of microbes
pH
Neutrophiles (“neutral lovers”), preferring a pH in the range of 5.5 to 8.0.

Acidophiles (“acid lovers”), preferring an environmental pH range of 0 to 5.5

• These organisms transport cations (such as potassium ions) into the cell,
thus decreasing H+ movement into the cell.
• Utilize proton pumps that actively pump H+ out.

Alkaliphiles (“alkaline lovers”), preferring an environmental pH range of 8.0

to 11.5
• Employ antiporters, which pump protons in and sodium ions out.