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ERT103
MICROBIAL NUTRITION
AND GROWTH
BY
DR ZARINA ZAKARIA
School of Bioprocess
Engineering
Topic Outline
Growth Requirement For Microbes
1. Nutrients:Chemical and Energy
Requirements
2. Physical Requirements
3. Ecological Associations
1.
2.


Nutrients: Chemical and
Energy Requirements
Objectives:
To describe the roles of C, H, O, N,
trace elements and vitamins in
microbial growth and reproduction.
To compare the microbes based on
their C and energy sources.
i. Carbon (C) requirements
From inorganic source (i.e CO2)
that called autotrophs.
From organic molecules (proteins,
carbohydrates) they acquire from
other organisms.We called as
heterotrophs.
ii. Source of energy
- From redox reactions involving inorganic
and organic chemicals that called
chemotrophs.
- From light source that called as
phototrophs.
-Therefore based on carbon and energy
sources, microbes an be categorized into
one of four basic groups:
i. Photoautotrophs
ii. Chemoautotrophs
iii. Photoheterotrophs
iv. Chemoheterotrophs
iii. Electron requirements or H atoms
for hydrogen bonding and electron
transfer
- From same organic molecules that
provide C and energy are called
organotrophs.
- From inorganic sources (H2, NO2-,
H2S) are called lithotrophs.
iv) Oxygen requirements
1. Oxygen is essential for obligate
aerobe to serve as final electron
acceptor.
2. Oxygen is prohibited for obligate
anaerobes.
3. Various concentration of oxygen is
unaffected within the two extremes.
Microbes in such conditions are
called facultative anaerobe.
4. Oxygen tolerance. Do not use aerobic
metabolisme but having enzymes that
detoxify oxygen’s poisonous forms. Known
as aerotolerant anaerobes.
5. Oxygen is required between 2%-10% as
found in the stomach. Can be damaged by
the 21% of oxygen in the atmosphere.
Known as microaerophiles.
v) Nitrogen requirements
- Nitrogen makes up about 14% of the
dry weight of microbial cells.
- Bacteria such as cyanobacteria and
Rhizobium capable to reduce
nitrogen gas (N2) to ammonia (NH3)
via a process called nitrogen
fixation.
- The bacteria (nitrogen-fixers)
provide nitrogen in a useable form to
other organisms.
vi) Other Chemical Requirements
- Elements such as phosphorus, sulfur,
calcium, manganese, magnesium,
copper,iron make up 5% of the dry
weight of cells.
- Other elements, called as trace
elements are required in very small
amounts. They are such as
molybdenum, nickel, selenium and
silicon.
2. Physical Requirements
(Prescott et al., page 132)
Objective:
To explain how extremes of
temperature, pH and osmotic and
hydrostatic pressure limit microbial
growth.
i.
-
-
-
Temperature
Give effects on the three-dimensional
configurations of biological molecules
(exp. Proteins and lipids).
Hydrogen bonds are temperature
sensitive and easily break at higher
temperatures caused to proteins
denature and lose function.
At too low tempt. membranes become
rigid and fragile and too high lipids
become too fluid and membrane cannot
contain the cell or organelle.
The effects of tempt. On microbial
growth:
i.
Minimum growth tempt.
The lowest tempt. at which microbes are
able to conduct metabolism.
ii. Maximum growth tempt.
The highest tempt. at which microbes
continues to metabolize
iii. Optimum growth tempt.
The tempt. at which microbes’ metabolic
activities produce the highest growth
rate.
-
Draw figure
Microbes can be categorized into 4
groups based on their preferred
tempt. ranges:
i. Psychrophiles
o
o
- Between 0 C-20 C. Optimized at
15oC.
- In nature, this group live in
snowfields, ice and cold water.
- Can cause food spoilage in
refrigerators.
ii. Mesophiles
- Between 20oC to  40oC.Optimized
-
o
- Pathogens to human ( 37oC).
- Caused food spoilage with inadequate
heating during pasteurization.
iii. Thermophiles
o
- Grow at tempt. above 45 C. in habitats
such as compost piles and hot springs.
- Above 80oC are called hyperthermophiles
and can live up to 100oC.
- Record-holder is Geogemma barossii that
can grows and reproduces between 85oC
and 121oC.
- Do not cause disease to human (why?).
ii. pH
- Microbes can be categorized into 3
groups based on their preferred pH
ranges:
Neutrophiles
- Grow best between pH 6.5 and pH
7.5 and coincidentally the pH range
of most tissues and organs in human
(what would you expect from this
bacteria)
a.
b. Acidophiles
- Grow best in acidic habitats. Some can go
as low as 0.0 i.e chemoautotrophic
bacteria that oxidize sulfur to sulfuric acid.
- Obligate acidophiles die if pH approaches
7.0.
- Acid-tolerant microbes can survive without
preferring it.
c. Alkalinophiles
- Grow best in alkaline soils and water up to
pH 11.5.
- i.e Vibrio cholerae, the causative agent of
cholera grow best at pH 9.0.
iii. Physical effects of water
In metabolic processes, water is needed
to dissolve enzymes and nutrients and as
important reactant.
The effects of water on microbes can be
discussed in two conditions:
a.
Osmotic pressure (OP)
The pressure exerted on a
semipermeable membrane by a solution
containing solute.
Related to the concentration of dissolved
molecules and ions in solution.
Hypertonic, hypotonic and isotonic (T).
Effects of OP on cells:
i. Cell placed in hypotonic solution (fresh
water) will swell and burst.
ii. Cell placed in hypertonic solution
(seawater) will die from crenation of its
cytoplasm.
- Microbes grow under high osmotic
pressure are called obligate halophiles.
- Microbes can tolerate high salt
concentrations are called facultative
halophiles.
-
24 of 42 :
07_13WaterBalance_L.jpg
b. Hydrostatic pressure
- The pressure exerted on a
semipermeable membrane by the
depth of water level.
- Every additional 10m of depth, water
pressure increases 1 atm. If 100m
below, the pressure would be 10
atm.
- Microbes live under extreme
pressure are called barophiles.
- Their membrane and enzymes
depend on pressure to maintain their
3. Ecologial Associations
Objectives:
To describe how quorum sensing can lead to
formation of biofilms.
- Biofilms: complex relationship among
numerous individuals, different species,
attach as a group to surfaces and display
metabolic and structural traits different
from those expressed by any of the
microorganisms alone. About 65% of
bacterial diseases are caused by biofilms.
(What is the impacts on scientists?).
Quorum sensing: A process that give
result to formation of biofilms in which
one bacteria respond to the density of
nearby bacteria.
Growth of bacterial
population
Growth Terminology and the
concept of exponential growth




The interval for the formation of two cells from
one is called a generation
The time required for this to occur is called the
generation time.
Generation time is the time required for the cell
population to double (the cell mass doubles
during this period as well).
Because of this, the generation time is also called
the doubling time.



In nature, microbial doubling times may
be much longer than those obtained in
laboratory culture.
This is because in nature, ideal growth
conditions for a given organism may exist
only intermittently.
Depending on resource availability,
physiochemical conditions (temperature,
pH, and the like), moisture availability,
and seasonal changes, bacterial
populations in nature double only once
every few weeks, or even longer.
A mathematical relationship exists
between the number of cells present in a
culture initially (start) and the number
present after a period of exponential
growth:
N = N02n
where
N is the final cell number,
No is the initial cell number,
n is the number of generations that have
occurred during the period of exponential
growth.
The Mathematics of Exponential
Growth


As one cell divides to become two
cells,
2° --'> 21.
As two cells become four,
21 --'> 22, and so on
The generation time (g) of the exponentially
growing population is (t / n),
where
t
is the duration of exponential growth
expressed in days, hours, or minutes,
depending on the organism and the growth
conditions.
From a knowledge of the initial and final cell
numbers in an exponentially growing cell
population, it is possible to calculate
n, and
n and knowledge of t, the generation
time g.
from
Relation equation of N and No to n
The equation N = No2n can be expressed in
terms of n as follows:
N = No2n
log N = log No + n log 2
log N – log No = n log 2
n = log N – log No = log N – log No
log 2
0.301
= 3.3 (log N – log No)
example
N = 108, No = 5 X 107, and t = 2
n = 3.3 (log N – log No)
n = 3.3 [log(108) - log(5 X 107)]
= 3.3(8 - 7.69)
= 3.3(0.301)
=1
generation time, g = t/n = 2 / 1 = 2 h
Related growth parameter

The generation time g of an exponentially
growing culture can also be calculated from the
slope of the line obtained in the semilogarithmic
plot of exponential growth.

The slope is equal to 0.301 n/t (log 2n/t) and in
the above example would be 0.301(1)/2, or 0.15.

Since g is equal 0.301/slope, we arrive at the
same value of 2 for g.

The term 0.301nlt is called the specific growth
rate, abbreviated k.



index of growth is the reciprocal of the
generation time, called the division rate,
abbreviated v.
The division rate is equal to 1/g and has units of
reciprocal 1 (h-1). While the term g is a measure
of the time it takes for a population to double in
cell number, v is a measure of the number of
generations that occur per unit time in an
exponentially growing culture.
The slope of the line relating log cell number to
time is equal to v /3.3
The Growth cycle or phases of
microbial growth

observed when microorganisms
are cultivated in batch culture
• culture incubated in a closed vessel
with a single batch of medium


usually plotted as logarithm of cell
number versus time
usually has four distinct phases
Lag phase



the cells are adjusting to their new environment
most cells do not reproduce immediately, but instead
actively synthesize enzymes to utilize novel nutrients in
the medium.
bacteria inoculated from a medium containing glucose
as a carbon source into a medium containing lactose
must synthesize two types of proteins:
• membrane proteins to transport lactose into the cell
• the enzyme lactase to catabolize the lactose.
Log phase

bacteria synthesize the necessary chemicals for conducting metabolism in
their new environment, and they then enter a phase of rapid chromosome
replication, growth, and reproduction.

population increases logarithmically

reproductive rate reaches a constant as DNA protein syntheses are
maximized.

more susceptible to antimicrobial drugs that interfere with metabolism

preferred for Gram staining because most cells' walls are intact – an
important characteristic for correct staining.

the metabolic rate of individual cells is at a maximum during log phase

this phase is sometimes preferred for industrial and laboratory purposes.
Stationary phase





If bacterial growth continued at the exponential rate
of the log phase, bacteria would soon overwhelm the
earth.
does not occur because as nutrients are depleted
and wastes accumulate, the rate of reproduction
decreases.
the number of dying cells equals the number of cells
being produced, and the size of the population
becomes stationary
During this phase the metabolic rate of surviving
cells declines.
The onset of the stationary phase can be postponed
indefinitely by a special apparatus called a
chemostat, which continually removes wastes (along
Stationary Phase

total number of viable cells remains
constant
• may occur because metabolically active
cells stop reproducing
• may occur because reproductive rate is
balanced by death rate
Possible reasons for entry into
stationary phase




nutrient limitation
limited oxygen availability
toxic waste accumulation
critical population density reached
Starvation responses

morphological changes
• e.g., endospore formation




decrease in size, protoplast
shrinkage, and nucleoid
condensation
production of starvation proteins
long-term survival
increased virulence
Death phase




If nutrients are not added and wastes are not removed,
a population reaches a point at which cells die at a
faster rate than they are produced.
Such a culture has entered the death phase (or decline
phase).
during the death phase, some cells remain alive and
continue metabolizing and reproducing, but the number
of dying cells exceeds the number of new cells
produced, so that eventually the population decreases
to a fraction of its previous abundance.
In some cases, all the cells die, while in others a few
survivors may remain indefinitely. The latter case is
especially true for cultures of bacteria that can develop
resting structures called endospore
Death Phase

two alternative hypotheses
• Cells are Viable But Not Culturable
(VBNC)


Cells alive, but dormant
programmed cell death
• Fraction of the population genetically
programmed to die (commit suicide)
Loss of Viability
Concept check

From knowledge of the initial and
final cell numbers and the time of
exponential growth, the generation
time and growth rate constant of a
cell population can be calculated
directly. Key parameters here are n,
g, v, k and t.
Concept check

If in 8 h an exponentially growing
cell population increase from 5x106
cells/ml to 5x108 cells/ml, calculate
g, n, v and k.
Concept check

Starting with four bacterial cells per
milliliter in a rich nutrient medium,
with a 1-h lag phase and a 20-min
generation time, how many cells will
there be in 1 liter of this culture after
1 h? After 2 h? After 2 h if one of the
initial four cells was dead?
Answer
n
g
v
k
=
=
=
=
6.6
73 min
0.0136
0.0041
answer
All cells are viable: After one hour, there
would still be only the four original cells
since the lag time is one hour.
After two hours, three divisions would have
occurred, since the generation time is 20
minutes; the population would be 4 x 23
= 32 cells/ml.
One of the initial cells is dead: After two
hours the population would be 3 x 23 =
24 cells/ml.
Concept check

Microorganisms show a characteristic
growth pattern when inoculated into a
fresh culture medium. There is usually a
lag phase, and then growth commences in
an exponential fashion. As essential
nutrients are depleted or toxic products
build up, growth ceases, and the
population enters the stationary phase. If
incubation continues, cells may begin to
die.



In what phase of the growth curve
are cells dividing in a regular and
orderly process?
When does a lag phase usually not
occur?
Why do cells enter the stationary
phase?
BATCH CULTURE


A batch culture is a fixed volume of culture
medium that is continually being altered by
the metabolic activities of growing organisms
and is therefore a closed system.
In the early stages of exponential growth in
batch cultures, conditions may remain
relatively constant, but in later stages when
cell numbers become quite large, drastic
changes in the chemical composition of the
culture medium occur.


A continuous culture is an open system
of constant volume which fresh medium
is added continuously and spent culture
medium removed continuously, both at
a constant rate.
Once such a system is in equilibrium,
the chemostat volume, cell number, and
nutrient status main constant, and the
system is said to be in steady state.
CONTINUOUS CULTURE: THE
CHEMOSTAT


The chemostat controls
both the growth rate and
the population density of
the culture simultaneously
Two factors are important
in such control:
• the dilution rate and the
• concentration of a limit
nutrient, such as a carbon or
nitrogen source.
batch culture

nutrient
concentration
can affect both
the growth rate
and the growth
yield of a culture
chemostat


growth rate and growth yield can be
controlled independently of each other
Growth rate by adjusting the dilution rate
and the growth yield by varying the
concentration of a nutrient present in a
limiting amount.



wide limits over which the
dilution rate controls growth
rate, although at both very
low and very high dilution
rates the steady state
breaks down.
At high dilution rates, the
organism cannot grow fast
enough to keep up with its
dilution, and the culture is
washed out of the
chemostat.
very low dilution rates, a
large fraction of the cells
may die from starvation



The cell density (cells/milliliter) is controlled by
the level of the limiting nutrient
If the concentration of this nutrient in the
incoming medium is raised, with the dilution rate
remaining constant, the cell density will increase.
by adjusting dilution rate and nutrient level, the
experimenter can obtain dilute, moderate or
dense populations growing at slow, moderate, or
rapid growth rates.
EXPERIMENTAL USES OF
THE CHEMOSTAT
applications such as

the study of a particular enzyme
• enzyme activities may be quite lower in stationary phase cells than in
exponential phase cells and thus chemostat-grown cultures are ideal.

in microbial ecology

enrichment and isolation of bacteria

constant supply of cells in exponential phase growing at a known rate

study of microbial growth at very low nutrient concentrations, close to
those present in natural environment

study of interactions of microbes under conditions resembling those in
aquatic environments

food and industrial microbiology
Concept check

Continuous culture devices
(chemostats) are a means of
maintaining cell populations in
exponential growth for long periods.
In a chemostat, the rate, and the
population size is governed by the
concentration of the growth-limiting
nutrient entering the vessel


Differentiate the definition of batch
cultures and continuous culture
What are the advantages of
chemostat compare to batch culture.


A batch culture is a fixed volume of culture
medium that is continually being altered by the
metabolic activities of growing organism and is
therefore a closed system. In the early stage of
exponential growth in batch culture, conditions
may remain relatively constant, but in later
stages when cell numbers become quite large,
drastic changes in the chemical composition of
the culture medium occur.
A continuous culture is an open system of
contrast volume to which fresh medium added
continuously and spent culture medium is added
continuously both at a constant rate . Once such
a system is in equilibrium, the chemostate
volume, cell number, and nutrient status remain
constant, and the system is said to be in steady
state.



The chemostat controls both the growth rate and the population
density of the culture simultaneously . Two factors are important in
such control: the dilution rate and the concentration of a limiting
nutrient, such as a carbon or nitrogen source.
In a batch culture, nutrient concentration can affect both rate and
the growth yield of a culture. At very low concentrations of a given
nutrient, the growth rate is reduced, probably because the nutrient
cannot be transported into the cell fast enough to satisfy metabolic
demand. At moderate or higher nutrient levels, the growth rate
may not be affected while the cell yield continues to increase.
In contrast to a batch culture, in a chemostat, growth rate and
growth yield can be controlled independently of each other, the
former by adjusting the dilution rate and the latter by varying the
concentration of a nutrient present in a limiting amount. A
practical advantage to the chemostat is that a population may be
maintained in the exponential growth phase for long periods, thus,
experiments can be planned in detail and then performed
whenever most convenient.
Measuring microbial
growth
Direct methods





viable plate counts
membrane filtration
microscopic counts
the use of electronic counters
the most probable number
method.
Viable plate count

spread and pour plate techniques
• diluted sample of bacteria is spread over solid
agar surface or mixed with agar and poured
into Petri plate
• after incubation the numbers of organisms are
determined by counting the number of colonies
multiplied by the dilution factor
• results expressed as colony forming units
(CFU)
Viable plate count





What if the number of cells in even a very small sample is
still too great to count?
for example, a 1-milliliter sample of milk containing 20,000
bacterial cells per ml were plated on a Petri plate, there
would be too many colonies to count.
In such cases, we make a series of dilutions and count the
number of colonies resulting on a spread or pour plate
from each dilution.
We count the colonies on plates with 25-250 colonies and
multiply the number by the reciprocal of the dilution to
estimate the number of bacteria per ml of the original
culture.
This method is called a viable plate count

The accuracy of a viable plate count is
also dependent on
• the homogeneity of the dilutions,
• the ability of the bacteria to grow on the
medium used,
• the number of cell deaths, and
• the growth phase of the sample population.

Thoroughly mixing each dilution,
inoculating multiple plates per dilution,
and using log-phase cultures minimize
errors.
Membrane filtration


In this method, a large sample (perhaps
as large as several liters) is poured (or
drawn under a vacuum) through a
membrane filter with pores small enough
to trap the cells.
The membrane is then transferred onto a
solid medium, and the colonies present
after incubation are counted. In this case,
the number of colonies is equal to the
number of CFUs in the original large
sample.
Membrane filtration
Plating methods…



simple and sensitive
widely used for viable counts of
microorganisms in food, water, and
soil
inaccurate results obtained if cells
clump together
Microscopic count



easy,
inexpensive, and
quick
useful for
counting both
eucaryotes and
procaryotes
cannot
distinguish living
from dead cells
Electronic counters





useful for large microorganisms and blood cells,
but not procaryotes
microbial suspension forced through small orifice
movement of microbe through orifice impacts
electric current that flows through orifice
instances of disruption of current are counted
it is less useful for bacterial counts because of
debris in the media and the presence of filaments
and clumps of cells.
Flow cytometry


is a variation of counting with a
Coulter counter.
A cytometer uses a light-sensitive
detector to record changes in light
transmission through the tube as
cells pass.
Most Probable Number

a statistical estimation technique
based on the fact that the more
bacteria in a sample, the more
dilutions are required to reduce their
number to zero.
Indirect methods



Metabolic Activity
Dry Weight
Turbidity
Metabolic Activity


Under standard temperature conditions,
the rate at which a population of cells
utilizes nutrients and produces wastes is
dependent on their number.
Once they establish the metabolic rate of
a microorganism, scientists can indirectly
estimate the number of cells in a culture
by measuring changes in such things as
nutrient utilization, waste production, or
pH.
Dry Weight



The abundance of some microorganisms,
particularly filamentous microorganisms,
is difficult to measure by direct methods.
Instead, these organisms are filtered from
their culture medium, dried, and weighed.
The dry weight method is suitable for
broth cultures, but growth cannot be
followed over time because the organisms
are killed during the process
Turbidity


As bacteria reproduce in a broth
culture, the broth often becomes
turbid (cloudy)
An indirect method for estimating the
growth of a microbial population
involves measuring changes in
turbidity using a device called a
spectrophotometer
more cells

more light
scattered

less light
detected


dry weight
• time consuming and not very
sensitive
quantity of a particular cell
constituent
• e.g., protein, DNA, ATP, or
chlorophyll
• useful if amount of substance in
each cell is constant