MIcrobial Nutrition and Growth_ 13_12_2023.pptx

Microbial Nutrition
and Growth
GSBTM Sponsored
CRASH WORKSHOP
On M.Sc. Entrance Examination
1
GAT- B JNU – LS IIT – JAM TIFR CUET
Dr. Chitra Bhattacharya
Assistant Professor,
Department of Microbiology,
Atmiya University, Yogidham Gurukul, Rajkot
Email: chitra.bhattacharya@atmiyauni.ac.in

Microbial Cell composition shows that 95% of cell dry
weight is made up of few major elements:
Carbon, Oxygen, Hydrogen, Nitrogen, Sulfur,
Phosphorous, Potassium, Calcium, Magnesium and Iron
Nutritional Requirement
To obtain energy and construct new cellular components,
organisms, must have a supply of raw materials or
nutrients.
Nutrients- are substances used in biosynthesis and
energy production.
Ever Imagine What do Microbes Eat???

3
Introduction
Process by
which chemical
substances
(nutrients) are
required from
the
environment
and utilized for
cellular
activities
Substances in
the
environment
used by
organisms/
body for their
metabolic
activities
Nutrition
Nutrients

4
Importance of Nutrients
Microbial
Growth
Metabolic
Activity
Nutrients
Nutrients is necessary for Microbial growth
plays and vital role in their proper cultivation
Activate their Cellular and
metabolic activity.

5
Classification of Nutrients
Nutrients
Essential
Micro-
nutrient
Macro-
nutrient
Non-
Essential

6
Nonessential nutrients can be synthesized by the human
body, so they need not be obtained directly from food.
Macronutrients are nutrients that are needed in relatively
large amounts.
Essential Nutrients
Nonessential Nutrients
Essential nutrients cannot be synthesized by the human
body, so they must be consumed in food.
Ex: vitamins, minerals, protein, fats, water, and
carbohydrates.
Ex: biotin, cholesterol, vitamin K, and vitamin D.

7
Micronutrients
Micronutrients Cellular Function
Cobalt Vitamin B12; transcarboxylase (propionic acid
bacteria)
Copper Respiration (Cytochorme c oxidase);
photosynthesis (plastocyanin, some superoxide
dismutases)
Manganese Acts as Activator of various enzymes
Molybdenum Present in flavin-containing enzymes, nitrogenase
nitrate reductase, sulphide oxidase, some formate
dehydrogenase
Nickel Present in most hydrogenase enzyme
Tungsten In some formate dehydrogenase enzyme
Zinc In carbonic anhydrase; alcohol dehydrogenase;
RNA and DNA polymerases

8
Macronutrients
Macronutrients Function
Carbon Constituent of all organic cell material
Hydrogen Constituent of cellular water, organic cell
materials
Oxygen Molecular oxygen serves as an electron receptor
in aerobic respiration
Nitrogen Constituent of proteins, nucleic acid and
coenzyme
Phosphorus Constituent of nucleic acid, phospholipids,
coenzymes
Sulphur Constituent of some amino acids (cysteine &
methionine) and some coenzymes ) CoA &
Cocarboxylase)

9
Source of Elements
Hydrogen source:
• Major elements of
organic and
inorganic
compounds (water,
Salt and gases).
Roles of Hydrogen:
• Maintaining pH
• Forming the H-
bond

10
Source of Elements
Oxygen source:
• Major component of
carbohydrate, lipids,
nucleic acids and
proteins.
Roles of Hydrogen:
• Structural and
enzymatic functions
of cell.
Nitrogen source:
• It is a part of structure
of proteins, DNA, RNA
and ATP, these are the
primary source of
nitrogen heterotrophs.
Roles of Hydrogen:
• Bacteria, algae are
utilize inorganic
nitrogen sources (NO3,
NO2, NH3)

11
Source of Elements
Phosphorus source:
• Constituent of sugar
phosphate, nucleic
acids, phosphate
esters such as
ATP/ADP/AMP
system of the cellular
energy transfer.
Roles of Hydrogen:
• Terminal electron
acceptor in the
absence of sulphate,
nitrate and oxygen.
Sulphur source:
• It is found in living
organisms in the form
of compound such as
amino acids,
coenzymes and
vitamins.
Roles of Hydrogen:
• Available ad Sulphate
(SO4
-), or Sulfide (S2-)

12
Based on Nutrition
 All forms of life, from microorganisms to human
beings, share certain nutritional requirements for
growth and normal functioning. The great
diversity of nutritional types found among
bacteria:
1. All organisms require a source of energy. Some
rely on chemical compounds for their energy
and are designated as “Chemotrophs”.
2. Others can utilize radiant energy (light) and are
called as “Phototrophs”. Both Chemotrophs and
Phototrophs exists among bacteria.

Classification of Microorganisms based on
Nutrition
Nutrition Requirement based on the Source:
Microorganisms
Carbon
Autotrophs
Heterotrophs
Energy
Phototrophs
Chemotrophs
Electron &
Hydrogen
Lithotrophs
Organotrophs
Hypotrophs

Based on
Carbon Source
Autotrophs Heterotrophs
Photoautotrophic
Chemoautotrophic Chemoheterotrophic
Photoheterotrophic
Saprophytic Parasitic Holozoic

16
Autotrophs
A living thing that can feed itself using
simple chemical substances such as
carbon dioxide

17
Heterotrophs
The organisms obtain readymade
organic food from outside sources.

18
Based on Energy
Hypotrophs
Can’t utilize
energy
Based on Energy Source

19
Based on requirements for Sources of Energy
Bacteria which obtain energy by using
radiant energy i.e. light. These bacteria
possess photosynthetic pigments and
photosynthetic apparatus.
1. Phototrophs
2. Chemotrophs
3. Hypotrophs
Cyanobacteria, Green sulfur
bacteria, Purple sulfur
bacteria and Purple non
sulfur bacteria
Bacteria which obtain energy by oxidizing
chemicals. Upon oxidation of chemicals,
chemical energy is released.
Thiobacillus and other sulfur
oxidizers (Inorganic compound) and
E.coli, Bacillus and various other
bacteria(Organic compound)
Organisms, which cannot utilize any
external source of energy. This is because
of their inability to synthesis ATP. They
require ready made ATP for growth. It may
be obtained from other living host cells
Viruses and Rickettsiae.

Basis of source of Electron Donor
Based on type of electron donor utilized
Electron Donor
Lithotrophs Organotrophs
Reduced sulfur compounds, ferrous salts,
ammonia, ammonium compounds and
molecular hydrogen
These are the bacteria,
which utilize Inorganic
substances as electron
donor. They oxidize
selective Inorganic
substances and generate
necessary reducing power
required for biosynthesis
Bacteria generate their
reducing power from
oxidation of various organic
compounds.
Heterotrophs
Autotrophs
Paratrophs

21
Light
(Phototroph)
Chemical
(Chemotroph)
Organic
(Organotroph)
Inorganic
(Lithotroph)
Organic
(Organotroph)
Inorganic
(Lithotroph)
Organic (Heterotroph)
Inorganic (Autotroph)
Energy
Source
Electron
Source
Carbon
Source
Note: Make Names of the Organisms by employing three nutritional sources

23
Bacteria can use the same inorganic
chemical substances as the sources of
energy and electron donor.
Ferrobacillus, Nitrobacter,
Hydrogenbacteria
Bacteria, which obtain their energy
and reducing power through
oxidation of same organic
compound.
Photolithotrophs
H₂S ➞ S + 2e + 2H
Green sulfur bacteria and purple sulfur
bacteria belong to this category.
Photoorganotrophs
Succinate ➞ Fumarate + 2e + 2H
Purple non sulfur bacteria belong to
this category
Chemolithotrophs
Chemoorganotrophs

24
Gallery
Glimpse
Cyanobacteria
Autotroph Heterotroph
Rhizobium
Phototroph
Euglena
Nitrosomonas
Chemotroph
Purple Sulfur Bacteria
Lithotroph Organotroph
Pseudomonas

The organisms which can use reduced inorganic compounds as
electron donors are known as _________
a. Chemotrophs
b. Organotrophs
c. Lithotrophs
d. Phototrophs
Which of the following is the nutritional characterization of
Escherichia coli?
a. Chemotrophic
b. Organotrophic
c. Autotrophic
d. Chemotrophic, Organotrophic, Heterotrophic
Questions from Microbial Nutrition
Answer: c
Explanation: Organisms that can use reduced inorganic
compounds as electron donors are termed as lithotrophs.
Some organisms which use organic compounds as electron
donors are called organotrophs
Answer: d
Explanation: Escherichia coli are chemotrophic, organotrophic, and heterotrophic organisms. This means they rely
on chemical compounds for their energy and uses organic compounds as electron donors. They also require organic
compounds as their carbon source and are hence heterotrophic.

Which of the following bacteria can grow both as chemolithotrophs
or as chemoorganotrophs?
a. Nitrosomonas sp.
b. Pseudomonas pseudoflava
c. Rhodospirillum rubrum
d. Chromatium okenii
An organism that can synthesize all its required organic
components from CO2 using energy from the sun is a:
a. Photoautotrophs
b. Photoheterotrophs
c. Chemoautotrophs
d. Chemoheterotrophs
Answer: b
Answer: a

27
Growth Factors
Organic compounds required because they are essential cell
components and cannot be synthesized by the organisms.
3 major classes of growth factors
Amino Acids Purines & Pyrimidines
Vitamins
Needed
for
protein
synthesis
For
nucleic
acid
synthesis
Enzyme cofactors,
common vitamins;
Biotin, Folic acid ,
Riboflavin (B2)

29
Uptake of Nutrients by Cell
A cell must bring in nutrients from the external environment
across the cell membrane. In bacteria and archaea, several
different transport mechanisms exist.
Passive Diffusion
• Passive or simple diffusion allows for the passage across
the cell membrane of simple molecules and gases, such as
CO2, O2, and H2O.
• In this case, a concentration gradient must exist, where there
is higher concentration of the substance outside of the cell
than there is inside the cell.
• As more of the substance is transported into the cell the
concentration gradient decreases, slowing the rate of
diffusion.

30
• Facilitated diffusion also involves the use of a
concentration gradient, where the concentration of the
substance is higher outside the cell, but differs with the
use of carrier proteins (sometimes called permeases).
• These proteins are embedded within the cell membrane
and provide a channel or pore across the membrane
barrier, allowing for the passage of larger molecules.
• If the concentration gradient dissipates, the passage of
molecules into the cell stops. Each carrier protein
typically exhibits specificity, only transporting in a
particular type of molecule or closely related molecules.
Facilitated Diffusion

31
• Many types of nutrient uptake require that a
cell be able to transport substances against a
concentration gradient (i.e. with a higher
concentration inside the cell than outside).
• In order to do this, a cell must utilize
metabolic energy for the transport of the
substance through carrier proteins embedded
in the membrane.
• This is known as active transport. All types
of active transport utilize carrier proteins.
Active Transport

33
Primary Active Transport
Primary active transport involves the use of chemical energy,
such as ATP, to drive the transport. One example is the ABC
system, which utilizes ATP-Binding Cassette transporters.
Each ABC transporter is composed of three different
components:
1) membrane-spanning proteins that form a pore across the
cell membrane (i.e. carrier protein),
2) an ATP binding region that hydrolyzes ATP, providing the
energy for the passage across the membrane, and
3) a substrate-binding protein, a peripheral protein that binds
to the appropriate substance to be transporter and ferries it to
the membrane-spanning proteins. In gram negative bacteria
the substrate-binding protein is located in the cell’s
periplasm, while in gram positive bacteria the substrate-
binding protein is attached to the outside of the cell
membrane.

35
• Secondary active transport utilizes energy from a proton
motive force (PMF). A PMF is an ion gradient that develops
when the cell transports electrons during energy-conserving
processes.
• Positively charged protons accumulate along the outside of the
negatively charged cell, creating a proton gradient between the
outside of the cell and the inside.
• There are three different types of transport events for simple
transport: uniport, symport, and antiport and each mechanism
utilizes a different protein porter.
• Uniporters transport a single substance across the membrane,
either in or out.
• Symporters transport two substances across the membrane at the
same time, typically a proton paired with another molecule.
• Antiporters transport two substances across the membrane as
well, but in opposite directions. As one substance enters the cell,
the other substance is transported out.
Secondary Active Transport

37
• Group translocation is a distinct type of active
transport, using energy from an energy-rich organic
compound that is not ATP.
• Group translocation also differs from both simple
transport and ABC transporters in that the substance
being transported is chemically modified in the process.
• One of the best studied examples of group translocation
is the phosphoenolpyruvate: sugar
phosphotransferase system (PTS), which uses energy
from the high-energy molecule phosphoenolpyruvate
(PEP) to transport sugars into the cell. A phosphate is
transferred from the PEP to the incoming sugar during
the process of transportation.
Group Translocation

39
Iron Uptake
• Iron is required by microbes for the function of their
cytochromes and enzymes, resulting in it being a
growth-limiting micronutrient.
• However, little free iron is available in environments,
due to its insolubility.
• Many bacteria have evolved siderophores, organic
molecules that chelate or bind ferric iron with high
affinity. Siderophores are released by the organism to
the surrounding environment, whereby they bind any
available ferric iron.
• The iron-siderophore complex is then bound by a
specific receptor on the outside of the cell, allowing
the iron to be transported into the cell.

Which of the following is true of passive transport?
a. it requires a gradient.
b. it uses the cell wall
c. it includes endocytosis
d. it only moves water
Active transport of a substance across a membrane
requires:
a. A Gradient
b. The expenditure of ATP
c. Water
d. Diffusion
Answer: a
Answer: b

Bacteriological-Media and their Types
• Most bacteria can be cultured artificially on culture
media containing required nutrients, pH and osmotic-
pressure.
• Each ingredient or the complete medium(powder) is
dissolve in the appropriate volume of distilled water.
• The pH of the fluid medium is determined with pH meter
or pH strip.
• If solid medium is desired, agar is added and medium is
boiled to dissolve the agar.
• The medium is sterilized generally by autoclaving.
• Heat-Labile components are sterilized by Filteration.

Composition of Culture-media
Basic Ingredients:
 Water
 Sodium-Chloride
 Peptones
 Beef Extract
 Yeast Extract ( Source of Vitamin B)
 Buffers
 Indicators
 Solidifying-agents
 Selective-agents
 Additive for enrichment
 Agar

• Agar:
 Agar is used to solidify culture media because-
a) It has high gelling capacity
b) It has setting temperature between 32-39°C
c) It has melting temperature between 90-95°C
d) It gives firm gel at a concentration of 1.5%(w/v)

Different Types of Culture-media
1. Basic-Media:
These support the growth of microorganisms that
do not have special nutritional requirements. They
are often used:
a. To maintain stock-cultures of control strains of
bacteria and
b. For sub-culturing pathogens from selective
media prior to performing biochemical and
serological identification tests.
c. Example:. Nutrient-Agar, Nutrient-Broth.

2. Enrichment-Media: These are enriched with :
a. Whole-blood
b. Serum
c. Extra Peptones
d. Vitamins
e. Sterol
 Example: Blood-agar, Tryptone Soya Media,

3. Selective-Media:
 The media that provides nutritions that
enhances the growth of particular type
of bacterium and do not enhance or may
inhibit other types of organisms-known
as “Selective-Media”.
 For E.g. Use of typical
nutrients(Cellulose) , Antibiotics, etc.

4. Differential-Media:
 Certain Reagents/Indicator/Supplements when
incorporated into the culture media, that may allow the
differentiation of various types of bacteria.
 Example:. Eosin Methylene Blue (EMB) ,
MacConkey’s Agar.
 Both the media allows the growth of Gram-Negative
Bacteria only and inhibits the growth of Gram-Positive
Bacteria.
EMB agar shows Green Metallic Shine for E.coli while
MacConkey’s Agar shows Pink color colony (for
Lactose Fermenter E.g. E.coli) and Yellow color colony
(for Lactose Non-Fermenter E.g. Salmonella, Proteus)

5. Assay Media:
 Media of prescribed composition that is used for assay of
Vitamin, Amino-acids and Antibiotics.
6. Maintenance Media:
 The specific medium that maintain the viability and
physiological characteristics of the bacteria over the period
of time.
7. Minimal Media:
 Are those that contain the minimum nutrients possible for
colony growth, generally without the presence of Amino-
Acids.
8. Solid and Semi-solid Media:
 WIDELY USED for cultivation of bacteria; can be prepared
by agar. Can be used to study Motility of bacteria.

Which of the following is a characteristic of beef extract?
a. product resulting from the digestion of proteinaceous
materials
b. aqueous extract of lean beef tissue
c. aqueous extract of yeast cells
d. complex carbohydrate obtained from certain marine algae
Which of the following is used as a solidifying agent for
media?
a. Beef extract
b. Peptone
c. Agar
d. Yeast extract
Questions from Nutrient Media
Answer: b
Explanation: Beef extract, a complex raw
material used as ingredient for preparing
bacteriological media is an aqueous
extract of lean beef tissue concentrated
to a paste.
Answer: c
Explanation: Agar is used as a solidification agent for media and is not considered a source of nutrient
to the bacteria. Agar dissolved in aqueous solutions, gels when the temperature is reduced below 45
degrees Celsius.

Which of the following is a rich source of B vitamins?
a. Peptone
b. Yeast extract
c. Beef extract
d. Agar
Nutrient broth, a liquid media contains beef extract and
peptone respectively in how much amounts?
a. 0.2%, 0.4%
b. 0.1%, 0.6%
c. 0.3%, 0.5%
d. 0.7%, 0.3%
Answer: b
Explanation: Yeast extract which is an aqueous extract of yeast cells
is a very rich source of the B vitamins and it also contains apart from
it organic nitrogen and carbon compounds.
Answer: c
Explanation: Nutrient broth which is the most widely used media in general
bacteriological work, contains 0.3 percent beef extract and 0.5 percent peptone. It
may also contain if required 0.8 percent NaCl to maintain the salt concentration.

EMB agar is a medium used in the identification and
isolation of pathogenic bacteria. It contains digested meat
proteins as a source of organic nutrients. Two indicator
dyes, eosin and methylene blue, inhibit the growth of gram-
positive bacteria and distinguish between lactose
fermenting and nonlactose fermenting organisms. Lactose
fermenters form metallic green or deep purple colonies,
whereas the nonlactose fermenters form completely
colorless colonies. EMB agar is an example of which of the
following?
a. selective medium only
b. differential medium only
c. selective medium and a chemically defined medium
d. selective medium, a differential medium, and a complex
medium
Answer: d

Pseudomonas aeruginosa is a common pathogen that
infects the airways of patients with cystic fibrosis. It
does not grow in the absence of oxygen. The bacterium
is probably which of the following?
a. an aerotolerant anaerobe
b. an obligate aerobe
c. an obligate anaerobe
d. facultative anaerobe
Answer: b
Resource: https://www.labxchange.org/library/pathway/lx-pathway:c7c497d0-b358-3ddb-
825e-fa11c02f129f/items/lx-pb:c7c497d0-b358-3ddb-825e-fa11c02f129f:html:bfa3cdd4

60
Microbial Growth
 Microbial growth defined as an increase in cellular
constituents result an increase in a microorganism size,
population number or both.
 Growth of bacterial cell characterize via several changes
such as total population numbers using different
analysis method such as growth curve of microbial
culture.
 The growth of microorganisms reproducing by binary
fission can be plotted as the logarithm of the number
of viable cells versus the incubation time, resulting in
curve has four distinct phases.

62
Bacterial Growth Curve
Bacterium is added to a suitable liquid medium and incubated, its
growth follows a definite course.
If bacteria counts are made at intervals after inoculation & plotted
in relation to time, a growth curve is obtained Shows 4 phases:
Growth
Lag
Log
Stationary
Decline

Logarithmic (Exponential) phase: In logarithmic phase the bacterial cell start
dividing and their number increaseby geometric progression withtime.
During this period…
a. Bacteria havehigh rate ofmetabolism
b. Bacteria are more sensitive to antibiotics andradiation during this period.
Lag Phase
Log Phase
Making new enzymesin responseto new medium. Thelength of lagphase
dependupon
a. Typeof bacteria.
b. Better the medium, shorter thelagphase.
c. Thephaseof culture from which inoculation istaken.
d. Sizeor volume of inoculum.
e. Environmental factors liketemperature.

In decline (death) phase, death exceeds division. During this phase
population decreases due to death of cells. The factors
responsible are:
a. Nutritional exhaustion
b. Toxicaccumulation
c. Autolysinenzymes
Stationary Phase
Decline Phase
Nutrients becoming limiting orwaste products becomingtoxic.
death rate =divisionrate
In stationary phase after some time a stage comes when rate of
multiplication and death becomesalmost equal. It maybe dueto:
a. Depletion of nutrients.
b. Accumulation of toxic products and sporulation may occur during this
stage.

Morphological & Physiological alterations
during growth
• Log phase – smaller cells, stain uniformly.
• Stationary phase – irregular staining, sporulation and
production of exotoxins & antibiotics.
• Phase of Decline –involution forms (with ageing).
Potential Importance of the Growth Curve
• Implications in microbial control, infection, food microbiology,
and culture technology.
• Growth patterns in microorganisms can account for the stages
of infection.
• Understanding the stages of cell growth is crucial for working
with cultures.
• In some applications, closed batch culturing is inefficient, and
instead, must use a chemostat or continuous culture system.

67
Generation Time
During the exponential phase each M.O is dividing at
constant intervals, thus the population will double in
number during a specific length of time called the
generation time or doubling time
Measurement
of microbial
Growth
Cell Number Cell Mass

69
• Coliform bacilli like
E.coli & other medically
important bacteria/ 20
mins.
• Staphylococcus aureus/
27-30 mins.
Mycobacterium
tuberculosis/ 792-932
mins.
• Treponema pallidum/
1980 mins.
Types of bacteria with generation times

How Can We Calculate Generation
Time???
70
Let’s Solve

If the generation time of a bacterium is 40
minute and a culture containing 107 cell/ml. is
grown for 4 hours. Then calculate its population
after the period.
a. 64×107
b. 32×107
c. 6×107
d. 40×107
Questions from Microbial Growth
Answer: a

• Let’s Calculate,
Total population formed from 1 bacterium we have 4 hours given,
Doubling time = 40 min.
Total no. of bacteria = 1 (initially)
T = 4 hrs. converted into seconds = 4 × 60 = 240 min.
Now, the bacterium formed after 40 min. = 2 (21)
After
40 × 2 = 80 min. so population will be = 2 ×2 = 4 (22)
40 × 3 = 120 min. so, population will be = 2 ×2 ×2 = 8 or (23)
40 × 4 = 160 min. so, population will be = 2 ×2 ×2 ×2 = 16 or (24)
40 × 5 = 200 min. so, population will be = 2 ×2 ×2 ×2 ×2 = 32 or (25)
40 × 6 = 240 min. so, population will be = 2 ×2 ×2 ×2 ×2 ×2 = 64 or (25)
Answer : 64×107
72

If a culture starts with 50 cells, how many cells will be
present after five generations with no cell death?
a. 200
b. 400
c. 1600
d. 3200
The portion of the growth curve where rapid growth of
bacteria is observed is known as ____________
a. Lag phase
b. Logarithmic phase
c. Stationary phase
d. Decline phase
Answer: c
Answer: b

74
Answer and Explanation:
There will be c. 1600 cells in the culture after 5 generations with no cell
death.
Given:
Number of cells at the beginning of the culture = 50
Since the cells are grown in culture, they will most likely divide by binary
fission. In binary fission, two daughter cells are produced after each
division (in the next generation). This means the number of cells in the
culture doubles, or is multiplied by 2, with each generation.
Number of cells produced after each generation = 2n
The variable n represents the number of generations, and using it as an
exponent with the base of 2 allows us to double the number of cells at each
generation.
Therefore, the number of cells produced in the given culture, assuming that
no cell death occurred, is calculated as follows.
The no. of cell after n generation = 50 × 25 cells = 1600 cells
Solution

Which of the following is the best definition of
generation time in a bacterium?
a. the length of time it takes to reach the log phase
b. the length of time it takes for a population of cells to
double
c. the time it takes to reach stationary phase
d. the length of time of the exponential phase
Answer: b

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76

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