Microbial Growth:

- Types; Physical factors

- Kinetics; Systems

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Microbial Reproductive Strategies
• The reproductive strategies of eukaryotic
microbes
– asexual and sexual, haploid or diploid
• Bacteria and Archaea
– Asexual - binary fission, budding
– All must replicate and segregate the
genome prior to division

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 Bacterial Cell Cycle
• Cell cycle is a sequence of events from
formation of new cell through the next cell
division
– most bacteria divide by binary fission
• Two pathways function during cycle
– DNA replication and partition
– cytokinesis

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4
 The Influence of Environmental
Factors on Growth

• Most organisms grow in fairly moderate

environmental conditions
• Extremophiles
– grow under harsh conditions that would kill
most other organisms

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Microbial response to environmental
conditions

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 Extremely Adapted Microbes
• Halophiles
– grow optimally in the presence of NaCl or
other salts at a concentration above about
0.2M
• Extreme halophiles
– require salt concentrations of 2M and 6.2M
– extremely high concentrations of potassium
– cell wall, proteins, and plasma membrane
require high salt to maintain stability and
activity

7
 Effects of NaCl on Microbial
Growth

• Halophiles
– grow optimally at
>0.2 M
• Extreme halophiles
– require >2 M

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 Hydrogen ion concentration (pH)
The enzymes, electron transport systems and nutrient
uptake found in the cell membrane are sensitive to the
concentration of hydrogen ions.
Most organisms grow best when H+ and OH- ions are
present in approximately equal concentrations (pH 7).

Many bacteria prefer a slightly alkaline medium, while

many fungi prefer slightly acidic medium.

The maintenance of a constant pH during microbial

growth is especially important for organisms that
produce acid to avoid self-poisoining
 pH

• Acidophiles
– growth optimum between pH 0 and pH 5.5
• Neutrophiles
– growth optimum between pH 5.5 and pH 7
• Alkaliphiles (alkalophiles)
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– growth optimum between pH 8.5 and pH 11.5
 Temperature
• Microbes cannot regulate their internal temperature
• Enzymes have optimal temperature at which they
function optimally
• High temperatures may inhibit enzyme functioning
and be lethal
• Organisms exhibit distinct cardinal growth
temperatures
– minimal
– maximal
– optimal
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 Temperature

The range of temperature for microbial growth can be expressed as three

cardinal temperatures:

- The minimum temperature is the lowest temperature that permits a

microbe’s growth and metabolism, below this temperature its activities are
inhibited.
- The maximum temperature is the highest temperature at which growth
and metabolism can proceed. If the temperature rises slightly above
maximum, growth will stop, but if it continues to rise beyond that point, the
enzymes and nucleic acids become permanently inactivated and the cell
will die.
- The optimum temperature covers a small range, intermediate between
the minimum and maximum. It promotes the fastest of growth and
metabolism during a short period.

Depending on their natural habitats, some microbes have a narrow

cardinal range, others have a broad one.
 Temperature

Bacteria which can survive exposure to temperature

above the maximum temperature of growth are
termed thermoduric bacteria, they are mostly spore
formers.
 Temperature Ranges for
Microbial Growth
• psychrophiles – 0o C to 20o C- optimum
15 or below
• psychrotrophs – 0o C to 35o C
• mesophiles – 20o C to 45o C
• thermophiles – 55o C to 85o C
• hyperthermophiles – 85o C to 113o C

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 Effect of high temperatures
Killing effect of high temperatures (lethal temperature) is

the lowest temperature, at which under certain time are all

microorganisms killed (70 °C/10 min) denaturation of

proteins, enzyme inactivation, DNA and cytoplasmatic

membrane disruption is dependent on:

- species of microorganisms
-physiologic status
 Thermoresistance
Degree of microorganisms resistance depends on:

• physiologic status of bacteria

• their genetic properties
• water content in substrate
• quantity of protective compounds (lipids, proteins,
saccharides)
 Adaptations of Thermophiles
• Protein structure stabilized by a variety of
means
– e.g., more H bonds
– e.g., more proline
– e.g., chaperones
• Histone-like proteins stabilize DNA
• Membrane stabilized by variety of means
– e.g., more saturated, more branched and
higher molecular weight lipids
– e.g., ether linkages (archaeal membranes)
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 Oxygen Concentration

• growth in oxygen correlates with microbes

energy conserving metabolic processes and
the electron transport chain (ETC) and nature
of terminal electron acceptor

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 Basis of Different Oxygen
Sensitivities
• Oxygen easily reduced to toxic reactive
oxygen species (ROS)
– superoxide radical
– hydrogen peroxide
– hydroxyl radical
• Aerobes produce protective enzymes
– superoxide dismutase (SOD)
– catalase
– peroxidase

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 Strict Anaerobic Microbes
• All strict anaerobic microorganisms lack or
have very low quantities of
– superoxide dismutase
– catalase
• These microbes cannot tolerate O2
• Anaerobes must be grown without O2
– work station with incubator
– gaspak anaerobic system

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 Pressure

• Microbes that live on land and water surface

live at 1 atmosphere (atm)
• Some Bacteria and Archaea live in deep sea
with very high hydrostatic pressures

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 Pressure
• Barotolerant
– adversely affected by increased pressure, but
not as severely as nontolerant organisms
• Barophilic (peizophilic) organisms
– require or grow more rapidly in the presence
of increased pressure
– change membrane fatty acids to adapt to
high pressures

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 Radiation Damage
• Ionizing radiation
– x-rays and gamma rays
– mutations → death (sterilization)
– disrupts chemical structure of many
molecules, including DNA
• damage may be repaired by DNA repair
mechanisms if small dose
– Deinococcus radiodurans
• extremely resistant to DNA damage
(prevents oxidative damage, and has
very potent mechanisms of DNA repair)
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 Radiation Damage

• Ultraviolet (UV) radiation

– mutations → death
– causes formation of thymine dimers in DNA
– requires direct exposure on microbial surface
– DNA damage can be repaired by several
repair mechanisms

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 Microbial Growth in Natural
Environments

• Microbial environments are complex, constantly

changing, often contain low nutrient
concentrations (oligotrophic environment).

Microbial growth is an autocatalytic process:

no growth will occur without the presence of at least
one viable cell and the rate of growth will increase with
the amount of viable biomass present.
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 How do bacteria divide

• Growth refers to population growth rather than growth

of individual cells
29
 Study of growth
• Growth - increase in cellular constituents
that may result in:
increase in cell number
e.g., when microorganisms reproduce by budding or
binary fission
increase in cell size
e.g., coenocytic microorganisms have nuclear divisions
that are not accompanied by cell divisions

Microbiologists usually study population growth

rather than growth of individual cells.
Scheme of
division
 Lag phase
• Short period immediatelly after inoculation.
• Organisms are synthesising the enzymes
needed to exploit the new medium.
• Cell may grow in size but not usually in
number.
• If organisms have been transferred from
an identical medium, at the same
temperature, the lag phase may be very
short.
 Log growth phase

• The log phase (the logarithmic phase or the

exponential phase) is a period characterized by cell
doubling.
• The number of new bacteria appearing per unit time is
proportional to the present population.
• If growth is not limited, doubling will continue at a
constant rate so both the number of cells and the rate of
population increase doubles with each consecutive time
period.
• Rate of growth and division is constant and maximal.
• Population is mostly uniform in terms of chemical and
physical properties during this phase.
 Stationary growth phase
• The stationary phase is often due to a growth- limiting
factor such as the depletion of an essential nutrient,
and/or the formation of an inhibitory product such as an
organic acid.
• Stationary phase results from a situation in which
growth rate and death rate are equal.
• The number of new cells created is limited by the growth
factor and as a result the rate of cell growth matches the
rate of cell death.
• Closed system population growth eventually ceases, total
number of viable cells remains constant
– active cells stop reproducing or reproductive rate is
balanced by death rate
 Death phase
• At death phase (decline phase), bacteria
die. This could be caused by lack of
nutrients, environmental temperature
above or below the tolerance band for the
species, or other injurious conditions.
 The Growth Curve
• Observed when microorganisms are cultivated in
batch culture
• Usually plotted as logarithm of cell number versus
time
• Has four distinct phases

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 Balanced Growth

• During log phase, cells exhibit

balanced growth
– cellular constituents manufactured at
constant rates relative to each other.

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 Possible Reasons for Stationary
Phase

• Nutrient limitation
• Limited oxygen availability
• Toxic waste accumulation

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 Stationary Phase and Starvation
Response
• Entry into stationary phase due to starvation
and other stressful conditions activates
survival strategy
– morphological changes
• e.g., endospore formation
– decrease in size, protoplast shrinkage, and
nucleoid condensation

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 Senescence and Death Phase
• Two alternative hypotheses
– cells are Viable But Not Culturable (VBNC)
• cells alive, but dormant, capable of new growth when
conditions are right.
• Programmed cell death
– fraction of the population genetically programmed to
die (commit suicide).

– Viable but non culturable (VBNC) bacteria refers as to

bacteria that are in a state of very low metabolic activity and
do not divide, but are alive and have the ability to become
culturable once resuscitated. 42
 The Mathematics of Growth
• Generation (doubling) time
– time required for the population to double in
size
– varies depending on species of
microorganism and environmental conditions
– range is from 10 minutes for some bacteria to
several days for some eukaryotic
microorganisms.
– Population is doubling every generation

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 The Batch Reactor
•Many biochemical processes involve batch growth of cell
populations. A limited supply of nutrients for growth is
provided; when these are used up, or another factor
becomes limiting, the culture declines. Cells, or products
that the organisms have made, can then be harvested from
the culture.
•After seeding a liquid medium with an inoculums of
living cell, nothing is added to the culture or
removed from it as growth proceeds.

•In such a reactor, concentrations of the nutrients, cells

and products vary with time as the growth proceeds.
Cultivation
vessels

Scale-up
Fed-batch culture (Semi-batch)
Fed-batch culture is, in the broadest sense, defined as an
operational technique in biotechnological processes where one or
more nutrients (substrates) are fed (supplied) to the bioreactor
during cultivation and in which the product(s) remain in the
bioreactor until the end of the run.
An alternative description of the method is that of a culture in which
"a base medium supports initial cell culture and a feed medium is
added to prevent nutrient depletion.
It is also a type of semi-batch culture. In some cases, all the
nutrients are fed into the bioreactor.

Fed-batch culture is superior to conventional batch culture when

controlling concentrations of a nutrient (or nutrients) affect the yield
or productivity of the desired metabolite.
Note: the volume of culture liquid is increasing.
 The Continuous Culture of
Microorganisms
• Growth in an open system
– continual provision of nutrients
– continual removal of wastes
• Maintains cells in log phase at a constant
biomass concentration for extended
periods
• Achieved using a continuous culture system

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 Importance of Continuous
Culture Methods
• Constant supply of cells in exponential
phase growing at a known rate.
• Commonly used in Food and industrial
microbiology

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Fermentor: an apparatus that maintains optimal conditions for the growth of
microorganisms, used in large-scale fermentation
 Chemostat – continuous culture
• Control flow rate and concentration of growth-
limiting nutrient of liquid medium entering and
exiting a growth chamber (bioreactor).
• – Control of:
• pH
• Temperature
• Concentration of terminal electron acceptor
• Concentration of toxic by-products of metabolism
• A completely mixed continuous stirred-tank
• reactor for the cultivation of cells are called
chemostats.
Batch/Fed-Batch and Continuous culture
 Primary and secondary metabolites are often used in
industrial microbiology for the production of food, amino
acids, and antibiotics
• Primary metabolites are considered essential
to microorganisms for proper growth.
• Secondary metabolites do not play a role in
growth, development, and reproduction, and are
formed during the end or near the stationary
phase of growth.
• These metabolites can be used in industrial
microbiology to obtain amino acids, develop
vaccines and antibiotics, and isolate chemicals
necessary for organic synthesis.
 Primary metabolites
• Small molecules of living cells.
• Intermediates or end products of the
pathway.
• Related to synthesis of microbial cells in
the growth phase.
• Include alcohols, amino acids, nucleotides,
organic acids, vitamins, and enzymes.
 Secondary metabolites
• Accumulate following active growth

• Have no direct relationship to synthesis of

cell material and natural growth

• Include antibiotics and toxins