Chair of Microbiology, Virology, and Immunology

Physiology of Bacteria.
Growth and reproduction of
Bacteria
 Lecture schedule
1. Chemical composition of Bacteria
2. Cell metabolism
3. Contstructive metabolism метаболізм
4. Types of microbial nutrition
5. Bacterial transport systems
6. Types of respiration
7 Growth and reproduction of microbes
8. Nutrient media
Metabolism refers to all the
biochemical reactions that
occur in a cell or organism.
The study of bacterial
metabolism focuses on the
chemical diversity of substrate
oxidations and dissimilation
reactions (reactions by which
substrate molecules are broken
down), which normally
function in bacteria to generate
energy.
Chemical composition of bacteria
Protein 55 %
Total RNA 20.5 %
DNA 3.1 %
Phospholipid 9.1 %
Lipopolysaccharide 3.4 %
Murein 2.5 %
Inorganic ions 1.0 %
 Bacterial cell consists of:

Water – 70-90 % Dry weight – 10-30 %

Proteins – 55 %, 2,35 million of molecules, 1850 different

types of molecules
RNA – 20,5 %, 250000 molecules, 660 different types of
molecules
DNA – 3,1 %, 2 molecules
Lipids – 9 %, 22 million of molecules
Lipopolysaccharides –3,4 %, 1,5 million of molecules
Peptidoglycan – 1 molecule
 Microbial metabolism
1. Catabolism (Dissimilation)
- Pathways that breakdown
organic substrates
(carbohydrates, lipids, &
proteins) to yield metabolic
energy
for growth and maintenance.
2. Anabolism (Assimilation)
- Assimilatory pathways for the
formation of key
intermediates and then to end
products (cellular
components).
4. Intermediary metabolism -
Integrate two processes
 Catabolism

Substrate-level
phosphorylation Fermentation

Glycolysis
(EMP pathway)

Aerobic
respiration

Pyruvate: universal intermediate

The bacterial cell is a highly specialized energy
transformer. Chemical energy generated by substrate
oxidations is conserved by formation of high-energy
compounds such as adenosine diphosphate (ADP)
and adenosine triphosphate (ATP) or compounds
containing the thioester bond
O
║
(R –C ~ S – R), such as acetyl ~ S-coenzyme A
Another form of energy -
transmembrane potential - ΔμН+
 Chemiosmosis

• Production of ATP in Electron

Transport
• Electrochemical Gradient
Formed between membranes
• H+ (Protons) generated from
NADH
• Electrical Force (+) & pH Force
(Acid)
• Gradient formed
• ATPase enzyme that channels
H+ from High to Low
concentration
– 3 ATP/NADH
– 2 ATP/NADH
 Sources of metabolic energy
Substrate-level phosphorylation
Fermentation: metabolic
process in which the final
electron acceptor is an organic
compound.
Respiration: chemical
reduction of an electron
acceptor through a specific
series of electron carriers in
the membrane. The electron
acceptor is commonly O2,
but CO2, SO42-, and NO3-
are employed by some
microorganisms.
Photosynthesis: similar to
respiration except that the
reductant and oxidant are
created by light energy.
Respiration can provide
photosynthetic organisms
with energy in the absence
of light.
The Krebs cycle intermediate compounds serve as
precursor molecules (building blocks) for the
energy-requiring biosynthesis of complex organic
compounds in bacteria. Degradation reactions that
simultaneously produce energy and generate
precursor molecules for the biosynthesis of new
cellular constituents are called amphibolic.
 Energy Requirements
Oxidation of organic compounds - Chemotrophs
Sunlight - Phototrophs
 Metabolic Requirements
Carbon source
- Autotrophs (lithotrophs): use CO2 as the C source
Photosynthetic autotrophs: use light energy
Chemolithotrophs: use inorganics
- Heterotrophs (organotrophs): use organic carbon (eg.
glucose) for growth.
- Clinical Labs classify bacteria by the carbon sources
(eg. Lactose) & the end products (eg. Ethanol,…).

Nitrogen source
Ammonium (NH4+) is used as the sole N source by most
microorganisms. Ammonium could be produced from N2 by
nitrogen fixation, or from reduction of nitrate (NO3-)and nitrite
(NO2).
 Physiologic types of bacterial existence
Energy Source
Oxidation of organic Sunlight - Phototrophs
compounds - Chemotrophs

Carbon Source

Organic - Heterotrophs Inorganic - Autotrophs

Electrone donor

Оrganic -Organotrophs Inorganic - Lithotrophs

Chemoorganoheterotrophic bacteria
 Metabolic Requirements
Sulfur source
A component of several coenzymes and amino acids.
Most microorganisms can use sulfate (SO42-) as the S
source.

Phosphorus source
- A component of ATP, nucleic acids, coenzymes,
phospholipids, teichoic acid, capsular polysaccharides;
also is required for signal transduction.
- Phosphate (PO43-) is usually used as the P source.
Mineral source
- Required for enzyme function.
- For most microorganisms, it is necessary to provide sources
of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-.
- Many other minerals (eg., Mn2+, Mo2+, Co2+, Cu2+ and Zn2+)
can be provided in tap water or as contaminants of other
medium ingredients.
- Uptake of Fe is facilitated by production of siderophores
(Iron-chelating compound, eg. Enterobactin).

Growth factors: organic compounds (e.g., amino acids, sugars,

nucleotides, vitamines) a cell must contain in order to grow but
which it is unable to synthesize. Purines and pyrimidines: required
for synthesis of nucleic acids (DNA and RNA);
Amino acids: required for the synthesis of proteins;
Vitamins: needed as coenzymes and functional groups of certain
enzymes.
 Transport systems
The proteins that mediate the passage of solutes
through membranes are referred to as transport
systems, carrier proteins, porters, and
permeases. Transport systems operate by one of
three transport processes.
In a uniport process, a solute passes through the
membrane unidirectionally. In symport
processes (cotransport) two solutes must be
transported in the same direction at the same
time; in antiport processes (exchange diffusion),
one solute is transported in one direction
simultaneously as a second solute is transported
in the opposite direction.
Transport systems
 Diffusion systems

• passive diffusion
• facilitated diffusion
• ion-driven transport
• binding protein dependent transport
• group translocation
• Membrane is selectively permeable
– Few molecules pass through freely
– Movement involves both active and passive
processes
 Passive processes
– no energy (ATP) required
– Along gradient
– simple diffusion, facilitated diffusion, osmosis
• Simple diffusion

• Facilitated diffusion
Can reduce concentration
gradient but can’t create
one
Osmosis
• Osmotic pressure
Active processes
• energy (ATP)
required
– Active transport
– Group translocation
Facilitated diffusion
Active transport
Transport systems
 TEMPERATURE
• One of the most important
factors
• optimal growth temperature
– temperature range at which the
highest rate of reproduction
occurs
• optimal growth temperature
for human pathogens ????
 TEMPERATURE
• Microorganisms can be categorized
based on their optimal temperature
requirements
– Psychrophiles
• 0 - 20 ºC
– Mesophiles
• 20 - 40 ºC
– Thermophiles
• 40 - 90 ºC
• Most bacteria are mesophiles
especially pathogens that require 37 ºC
 BACTERIAL TEMPERATURE
REQUIREMENTS

100
Psychrophile Thermophile
% Max 50
Growth Mesophile

0
0 C 37 C 90 C
0 0 0

Variable
Effects of Temperature on
Growth
 Thermophiles
70o-110o

Mesophiles
10o-50o

For lecture only BC Yang

 TEMPERATURE
• Psychrophiles
– some will exist below 0 oC if liquid water is
available
• oceans
• refrigerators
• freezers

Pigmented bacteria
in Antarctic ice
 TEMPERATURE
• Mesophiles
– most human flora and
pathogens
 TEMPERATURE
• Thermophiles
– hot springs
– effluents from
laundromat
– deep ocean thermal
vents
 Respiration in Bacteria

Obligate Aerobe
Microaerophile
Obligate Anaerobe
Facultative Anaerobe (Facultative Aerobe)
Aerotolerant Anaerobe
Capneic bacteria
Categories of Oxygen Requirement
Aerobe – utilizes oxygen and can detoxify it
 obligate aerobe - cannot grow without oxygen
(Mycobacterium tuberculosis, Micrococcus spp.,
Bacillus spp., Pseudomonas spp.
 facultative anaerobe – utilizes oxygen but can
also grow in its absence (Echericihia spp.,
Salmonella spp., Sta[phylococcus spp.)
 microaerophylic – requires only a small
amount of oxygen (Helycobacter spp.,
Lactobacillus spp.)
38
Categories of Oxygen Requirement
Anaerobe – does not utilize oxygen
• obligate anaerobe - lacks the enzymes to
detoxify oxygen so cannot survive in an
oxygen environment (Clostridium spp.,
Bacteroides spp.)
• aerotolerance anaerobes – do no utilize
oxygen but can survive and grow in its
presence (Streptococcus pyogenes)

39
 Carbon Dioxide Requirement
All microbes require some carbon dioxide in
their metabolism.
• capneic – grows best at higher CO2
tensions than normally present in the
atmosphere (Brucella abortus)

40
 OXYGEN
Obligate Facultative Obligate
Aerobe Anaerobe Anaerobe
 Four Toxic Forms of Oxygen

Toxic Oxygen Forms Are formed:

during photosynthesis as molecular oxygen with
Singlet oxygen
electrons are boosted to higher energy state
during incomplete reduction of oxygen in aerobic
Superoxide radicals
and anaerobic respiration
during reactions that neutralizes superoxide
Peroxide anion
radicals
from ionizing radiation and from incomplete
Hydroxyl radical
reduction of hydrogen peroxide
 Four Toxic Forms of Oxygen

Toxic Oxygen Forms Are neutralized by:

carotenoids that remove the excess energy of
Singlet oxygen
singlet oxygen
superoxide dismutases, enzymes that detoxify
Superoxide radicals
them
catalase or peroxidase, enzymes that detoxify
Peroxide anion
peroxide anion
catalase, peroxidase, and antioxidants such as
Hydroxyl radical vitamins C and E that protect against toxic oxygen
products
 Enzymes and Their Role in Metabolism

Enzymes, organic catalysts of a highly molecular

structure, are produced by the living cell. They are of a
protein nature, are strictly specific in action, and play an
important part in the metabolism of micro-organisms. Their
specificity is associated with active centres formed by a
group of amino acids.
Some enzymes are excreted by the cell into the
environment (exoenzymes) for breaking down
complex colloid nutrient materials while other
enzymes are contained inside the cell
(endoenzymes).
Bacterial enzymes are subdivided into some groups:
1. Hydrolases which catalyse the breakdown of the link between the
carbon and nitrogen atoms, between the oxygen and sulphur atoms,
binding one molecule of water (esterases. glucosidases, proteases.
amilases, nucleases, etc.).
2. Transferases perform catalysis by transferring certain radicals from
one molecule to another (transglucosidases, transacylases.
transaminases).
3. Oxidative enzymes (oxyreductases) which catalyse the oxidation-
reduction processes (oxidases, dehydrogenases, peroxidases, catalases).
4. Isomerases and racemases play an important part in carbohydrate
metabolism. Rearrangement atoms of a molecule.
5. Lyases (remove chemical groups from molecules without adding
water).
6. Lygases (join two molecules together and usually require energy from
ATP).
Enzymes
 Significance of the enzymes

With the help of amylase produced by mould fungi starch

is saccharified and this is employed in beer making,
industrial alcohol production and bread making.
Proteinases produced by microbes are used for removing
the hair from hides, tanning hides, liquefying the
gelatinous layer from films during regeneration, and for
dry cleaning.
Fibrinolysin produced by streptococci dissolves the
thrombi in human blood vessels. Enzymes which
hydrolyse cellulose aid in an easier assimilation of rough
fodder.
Due to the application of microbial enzymes, the medical
industry has been able to obtain alkaloids,
polysaccharides, and steroids (hydrocortisone,
prednisone, prednisolone. etc.).
Bacteria play an important role in the treatment of
caouichouc, coffee, cocoa, and tobacco.
Enzymes permit some species of microorganisms to
assimilate methane. butane, and other hydrocarbons,
and to synthesize complex organic compounds from
them.
With the help of the enzymatic ability of yeasts in
special-type industrial installations protein-vitamin
concentrates (PVC) can be obtained from waste products
of petroleum (paraffin’s).
 Metabolism Results in
Reproduction
• Microbial growth – an increase in a population
of microbes rather than an increase in size of
an individual

• Result of microbial growth is discrete colony

– an aggregation of cells arising from single
parent cell

• Reproduction results in growth

 BINARY FISSION
• division exactly in half
• most common means of bacterial reproduction
– forming two equal size progeny
– genetically identical offspring
– cells divide in a geometric progression doubling
cell number
 BINARY
FISSION
Doubling time is
the unit of
measurement of
microbial growth
 CULTURE GROWTH
• Growth of culture goes
through four phases with
time
• 1) Lag phase

• 2) Log or Logarithmic
phase

• 3) Stationary phase

• 4) Death or Decline
phase
BACTERIAL GROWTH CURVE
 LAG PHASE
• Organisms are adjusting to the
environment Mouse click for lag
phase adjustment

– little or no division
• synthesizing DNA, ribosomes and
enzymes
– in order to
breakdown
nutrients, and to
be used for growth
 LOGARITHMIC PHASE
• Division is at a constant rate (generation
time)
• Cells are most susceptible to inhibitors
 STATIONARY PHASE
• Dying and dividing organisms are at an
equilibrium
• Death is due to reduced nutrients, pH
changes, toxic waste and reduced oxygen
• Cells are smaller and have fewer ribosomes
• In some cases cells do not die but they are
not multiplying
STATIONARY PHASE
DEATH PHASE
 In bioreactors
in 37oC, pH 5.1 ; in 45oC, pH 6.2

For lecture only BC Yang

 ENUMERATION OF
BACTERIA
• 1) viable plate count
• 2) direct count
• 3) most probable number (MPN)
4
1 5 9
10
3 6 8
2

7
 VIABLE PLATE COUNT
• Most common procedure for assessing
bacterial numbers
– 1) serial dilutions of a suspension of bacteria
are plated and incubated
 VIABLE PLATE COUNT
– 2) the number of colonies developing are then
counted
• it is assumed that each colony arises from an
individual bacterial cell
 VIABLE PLATE COUNT
3) by counting the colonies
and taking into account the
dilution factors the
concentration of bacteria in
original sample can be
determined
4) only plates having
between 30 and 300 colonies
are used in the calculations

See next slide for

bigger diagram
VIABLE PLATE COUNT
 VIABLE PLATE COUNT
– 5) multiply the number of colonies times the
dilution factor to find the number of bacteria in
the sample
– Example
• Plate count = 54

• Dilution factor = 1:10,000 ml

• Calculation

– 54 X 10,000 = 540,000 bacteria/ml

 VIABLE PLATE COUNT
• “TNTC”
– if the number of colonies is too great (over 300)
the sample is labeled “TNTC”
– Too Numerous To Count
• limitation of viable plate count
– selective as to the bacterial types that will grow
given the incubation temperature and nutrient
type
 VIABLE PLATE COUNT
Dilution factor of Click to
1/1,000 (10 -3) incubate

417 colonies
“TNTC”
 VIABLE PLATE COUNT
Dilution factor of Click to
1/1,000,000 (10 -6)
incubate

22 colonies Too few the

count is less
than 30
 VIABLE PLATE COUNT
Dilution factor of Click to
1/100,000 (10 -5) incubate

42 colonies

Calculate the number

of bacteria per ml
 VIABLE PLATE COUNT
• Calculate:

– 42 colonies

– dilution factor of 100,000

• 42 X 100,000 = ???

• 4,200,000 bacteria/ml
 Nutrient media
• Ordinary (simple) media
• Special media (serum agar, serum broth,
coagulated serum, potatoes, blood agar, blood
broth, etc.).
• Elective media
• Enriched media
• Differential diagnostic media: (1) proteolytic
action;
• (2) fermentation of carbohydrates (Hiss media);
• (3) haemolytic activity (blood agar);
• (4) reductive activity of micro-organisms;
• (5) media containing substances assimilated only
by certain microbes.
Biochemical properties
Colonies
Colonies
Colonies
Pure Cultures Isolation
Isolated colonies obtaining