Microbial physiology.
Microbial metabolism.
Enzymes. Nutrition.
Bioenergetics. Bacterial
growth and multiplication.
Dr. Elena Romancenco
Department of Microbiology
Microbial physiology.
Microbial metabolism.
Enzymes. Nutrition.
Bioenergetics. Bacterial
growth and multiplication.
Microbial metabolism
the Greek metabole, meaning change.
Metabolism - the sum of the biochemical
reactions required for energy generation
AND the use of energy to synthesize cell
material from small molecules in the
environment.
Why do we must know the
metabolism of bacteria?
Because we want to know how to inhibit or
stop bacteria growth and want to control
their metabolism.
Metabolism
Two components:
 Anabolism - biosynthesis


building complex molecules from simple ones
requires ENERGY (ATP)
 Catabolism - degradation


breaking down complex molecules into simple ones
generates ENERGY (ATP)
 3 Biochemical Mechanisms Utilized



Aerobic Respiration
Anaerobic Respiration
Fermentation
 Catabolic reactions or sequences produce
energy as ATP adenosine triphosphate ,
which can be utilized in anabolic reactions to
build cell material from nutrients in the
environment.
METABOLIC DIVERSITY
 Bacterial metabolism is classified into
nutritional groups on the basis of three major
criteria:
1. Source of energy, used for growth
2. Source of carbon, and
3. Sours of electron donors used for growth.
1. ENERGY SOURCE
 a. Phototrophs —can use light energy
 b. Chemotrophs —must obtain energy
from oxidation-reduction of external
chemical compounds
2. CARBON SOURCE
a. Autotrophs —can draw carbon from
carbon dioxide
b. Heterotrophs —carbon from organic
compounds
c. Mixotrophic – carbon is obtained from both
organic compounds and by fixing carbon dioxide
These requirements can be
combined:
1. Photoautotrophs - light energy, carbon
from
2. Photoheterotrophs —light energy, carbon
from organic compounds
3. Chemoautotrophs —energy from chemical
compounds, carbon from CO2
4. Chemoheterotrophs —energy from
chemical compounds, carbon from organic
compounds
CHEMOHETEROTROPHS
 Energy and carbon both come from organic
compounds, and the same compound can provide
both. Specifically, their energy source is electrons
from hydrogen atoms in organic compounds.


Saprophytes—live on dead organic matter
Parasites—nutrients from a living host
 This group (more precisely chemoorganoheterotrophic)
includes most bacteria as well as all protozoa,
fungi, and animals. All microbes of medical
importance are included in this group.
Microbial physiology.
Microbial metabolism.
Bioenergetics. Enzymes.
Nutrition. Bacterial growth
and multiplication.
Energy – capacity to do work or
cause change
 Endergonic reactions – consume energy
 Exergonic reactions – release energy
Energy Production

3 Biochemical Mechanisms Utilized



Aerobic Respiration
Anaerobic Respiration
Fermentation
Aerobic and anaerobic
respiration
Aerobic respiration – terminal electron acceptor is
oxygen
Anaerobic respiration – terminal electron acceptor
is an inorganic molecule other than oxygen (e.g.
nitrogen)
Aerobic Respiration

Molecular Oxygen (O2) serves as the final eacceptor of the ETC



O2 is reduced to H2O
Energy-generating mode used by aerobic
chemoheterotrophs
 General term applied to most human pathogens
 Energy source = Oxidation of organic compounds
 Carbon Source = Organic Carbon
3 Coupled Pathways Utilized



Glycolysis
Kreb’s Cycle or Tricarboxylic Acid Cycle or Citric Acid
Cycle
Respiratory Chain or Electron Transport Chain (ETC)
1. Glycolysis (splitting of sugar)
 Carbohydrate (CHO) Catabolism
 Oxidation of Glucose into 2 molecules of Pyruvic
acid
 CHO’s are highly reduced structures (thus, H-donors);
excellent fuels
 Degradation of CHO thru series of oxidative reactions
 End Products of Glycolysis:
 2 Pyruvic acid
 2 NADH2
 2 ATP
Glycolysis
2. Krebs Cycle (Citric Acid
Cycle,TCA)

Series of chemical reactions that begin and end with citric
acid
1.
Initial substrate – modified end product of Glycolysis
•
2 Pyruvic Acid is modified to 2Acetyl-CoA, which enters the
TCA cycle
Circuit of organic acids – series of oxidations and reductions
•
Eukaryotes – Mitochondrial Matrix
•
Prokaryotes – Cytoplasm of bacteria & Cell Membrane
2.

Products:




2
6
2
4
ATP
NADH2
FADH2
CO2
TCA cycle
3. Electron Transport System
 Occurs within the cell membrane of
Bacteria
 Chemiosomotic Model of Mitchell

34 ATP
Electron transport system
Overview of aerobic respiration
Anaerobic respiration
Utilizes same 3 coupled pathways as Aerobic Respiration
Used as an alternative to aerobic respiration
Final electron acceptor something other than oxygen:
NO3- : Pseudomonas, Bacillus.
SO4-: Desulfovibrio
CO3-: methanogens
In Facultative organisms
In Obligate anaerobes
Lower production of ATP because only part of the TCA
cycle and the electron transport chain operate.
Fermentation
 Incomplete oxidation of glucose or other
carbohydrates in the absence of oxygen
 Uses organic compounds as terminal electron
acceptors
 Effect
- a small amount of ATP
 Production of ethyl alcohol by yeasts acting on
glucose
 Formation of acid, gas & other products by the
action of various bacteria on pyruvic acid
Fermentation
Fermentation may result in
numerous end products
1. Type of organism
2. Original substrate
3. Enzymes that are present and active
Fermentation End Products
Metabolic strategies
Pathways
involved
Final eacceptor
ATP yield
Aerobic
respiration
Glycolysis,
TCA, ET
O2
38
Anaerobic
respiration
Glycolysis,
TCA, ET
NO3-, So4-2,
CO3-3
variable
Organic
molecules
2
Fermentatio Glycolysis
n
 Many pathways of metabolism are bi-directional or
amphibolic
 Metabolites can serve as building blocks or
sources of energy



Pyruvic acid can be converted into amino acids
through amination
Amino acids can be converted into energy sources
through deamination
Glyceraldehyde-3-phosphate can be converted into
precursors for amino acids, carbohydrates and fats
Redox reactions
 Always occur in pairs.
 There is an electron donor and electron
acceptor which constitute a redox pair.
 Released energy can be captured to
phosphorylate ADP or another compound.
 Basic reaction
: electron uptake
: electron removal
 Biological reaction
35
ATP
 3 part molecule consisting of
 adenine – a nitrogenous base
 ribose – a 5-carbon sugar
 3 phosphate groups
 Removal of the terminal phosphate releases
energy
 Adenosine Tri Phosphate
ADP + energy + phosphate
 ATP contains energy that can be easily released
(high-energy or unstable energy bond)
 Required for anabolic reactions

ATP
Formation of ATP
1.
2.
3.
substrate-level phosphorylation
oxidative phosphorylation, (reduced chemicals)
Photophosphorylation (reduced chlorophyll
molecules)
Uses of ATP:

Energy for active transport

Energy for movement

Energy for synthesis of cellular components
ALL SYNTHESIS REACTIONS INVOLVE USE OF
ENERGY
Substrate-level phosphorylation
Phosphorylation of glucose
by ATP
Lipid Metabolism
 Lipids are essential to the structure and function of
membranes
 Lipids also function as energy reserves, which can be
mobilized as sources of carbon
 90% of this lipid is “triacyglycerol”
triacyglycerol
lipase
glycerol + 3 fatty acids
 The major fatty acid metabolism is “β-oxidation”
Lipid catabolism
Lipids are broken down
into their constituents of
glycerol and fatty acids
Glycerol is oxidised by
glycolysis and the TCA
cycle
Lipids are broken down to
2 carbon acyl units where
they enter the TCA cycle
Protein Catabolism
PROTEIN CATABOLISM

Intact proteins cannot cross bacterial plasma membrane, so bacteria
must produce extracellular enzymes called proteases and peptidases
that break down the proteins into amino acids, which can enter the
cell.

Many of the amino acids are used in building bacterial proteins, but
some may also be broken down for energy. If this is the way amino
acids are used, they are broken down to some form that can enter the
Kreb’s cycle. These reactions include:
1. Deamination—the amino group is removed, converted to an ammonium
ion, and excreted.
2. Decarboxylation—the ---COOH group is removed
3. Dehydrogenation—a hydrogen is removed

Tests for the presence of enzymes that allow various amino acids to
be broken down are used in identifying bacteria in the lab.
Catobolism of
organic food
molecules
Proteins and
carbohydrates are
degraded by secreted
enzymes – proteases
and amylases
Amino acids must be
deaminated for
further oxidation
Microbial physiology.
Microbial metabolism.
Enzymes. Bioenergetics.
Nutrition. Bacterial growth
and multiplication.
Growth and multiplication
mode: Binary fission
Bacterial Cell Division
1. Replication of chromosome
2. Cell wall extension
3. Septum formation
4. Membrane attachment of
DNA pulls into a new cell.
Growth
 It is an increase in all the cell components,
which ends in multiplication of cell leading
to an increase in population.
 It involves - an increase in the size of the
cell & an increase in the number of
individual cells.
 Bacteria divide by binary fission.
Generation time
 Interval of time between two cell divisions
OR
 The time required for a bacterium to give
rise to 2 daughter cells under optimum
conditions
 Also called population doubling time.
Generation time
 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
Growth form in Laboratory
 Colony – formed by bacteria growing on
solid media. (20-30 cell divisions)
 Each bacterial colony represents a clone of
cells derived from a single parent cell.
 Turbidity – liquid media
- 107-109 cells/ml
 Biofilm formation – thin spread over an
inert surface.
Solid medium
Colony
Liquid medium
Bacterial biofilm
Bacterial counts
 Cell Counts ... many ways
 2 methods – Total cell count
- Viable cell count
Total Count

Total number of cells in the sample = living
+ dead.
Can be obtained by :
 Direct counting under microscope using
counting chambers.

Counting in an electronic device – Coulter
counter.
Counting chambers
Over method

Direct counting using stained smears - by
spreading a known volume of culture over a
measured area of slide.

Opacity measurements using an
absorptiometer/ nephalometer.

Chemical assays of cell components.
Turbidity- a spectrophotometer
measures how much light gets through
Compared to known controls,
MacFarland controls
Viable Cell Count

Measures the number of living cells.

Methods – Surface colony count



Dilution method
Plating method
Number of colonies that develop after
incubation gives an estimate of the viable
count.
Plate counts
Bacterial Growth Curve
 When a 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 :
 Lag,
 Log or Exponential,
 Stationary
 Decline.
Phases of Growth Curve
 1. Lag phase – No increase in number
but there may be an increase in the size
of the cell.
 2. Log OR Exponential phase – cells
start dividing and their
increases exponentially.
number
Phases of Growth Curve
 3. Stationary phase – cell division stops
due to depletion of nutrients &
accumulation of toxic products.
- equilibrium exists between dying cells and
the newly formed cells, so viable count
remains stationary
 4. Phase of Decline – population decreases
due to the death of cells – autolytic
enzymes.
Morphological & Physiological
alterations during growth
 Lag phase – maximum cell size towards the end of
lag phase.
 Log phase – smaller cells, stain uniformly
 Stationary phase – irregular staining, sporulation
and production of exotoxins & antibiotics
 Phase of Decline –involution forms(with ageing)
Factors Affecting Bacterial
Growth
 Availability of Nutrients & H2O
 Temperature
 Atmosphere – O2 & CO2
 H-ion concentration
 Moisture & drying
 Osmotic effects
 Radiation
 Mechanical & sonic stress.
Bacterial Nutrition
 Water constitutes 80% of the total weight of
bacterial cells.
 Proteins, polysaccharides, lipids, nucleic acids,
mucopeptides & low molecular weight compounds
make up the remaining 20%.
Moisture & Drying
 Water – essential ingredient of bacterial
protoplasm. Hence drying is lethal to cells.
 Effect of drying varies :


T. pallidum – highly sensitive
Staphylococci sp– stand for months
 Spores – resistant to desiccation, may
survive for several decades.
Nutrients
Functions
–
–
–
Generation of energy
Synthesis of cellular materials
Essential nutrients (basic bioelements needed for bacterial
cell growth)
–
–
–
–
–
–
H2O: universal solvent; hydrolyzing agent
Carbon: food & E* source; in form of prot., sugar, lipid
Nitrogen: for prot. syn; nucleic acid syn (purines &
pyrimidines)
Sulfur (sulfate): AA syn (i.e., Cystine)
Phosphate: key component of DNA & RNA, ATP, and inner
& outer membrane phospholipids
Minerals: assoc’d w/ PRO (i.e., Fe:PRO); common
component of enzymes.
Nutrients
2 types
1.
Macronutrients – needed in large quantities
for cellular metabolism & basic cell structure

2.
Micronutrients – needed in small quantities;
more specialized (enzyme & pigment
structure & function)

–
C, H, O, N
Mn, Zn
Fastidious Bacteria: microbes that
require other complex - nutrients/growth
factors ( i.e., Vitamins or AAs)
Temperature
 Vary in the temperature requirements.
 Temperature range – growth does not
occur above the maximum or below the
minimum.
 Optimum Temperature – growth occurs
best, 37ºC for most pathogenic bacteria.
Uptake of nutrients by
bacteria
o
Passive diffusion
o
o
o
simple diffusion
facilitated diffusion
Active transport
 Psychrophiles: -10 to 20C
 Psychrotrophs: 0 to 30 C
 Mesophiles: 10 to 48C
e.g. most bacterial pathogens
 Thermophiles: 40 to 72C
 Hyperthermophile: 65 to 110C
77
 Some pathogens can multiply in the
refrigerator: Listeria monocytogenes
78
H-ion Concentration
 Neutral or slightly alkaline pH (7.2 – 7.6) –
majority of pathogenic bacteria grow best.
 acidic pH – Lactobacilli
 alkaline pH -Vibrio cholerae
Osmotic Pressure or Osmolarity

Most bacteria require an isotonic environment or
a hypotonic environment for optimum growth.

Osmotolerant - organisms that can grow at
relatively high salt concentration (up tp 10%).

Halophiles - bacteria that require relatively high
salt concentrations for growth, like some of the
Archea that require sodium chloride
concentrations of 20 % or higher.
Similar effect with sugars
82
Radiation, stress
 Radiation
 X rays & gamma rays exposure – lethal
 Mechanical & Sonic Stress
 May be ruptured by mechanical stress.
Growth Factors
Some bacteria require certain organic
compounds in minute quantities – Growth
Factors OR Bacterial Vitamins.
It can be :
 Essential – when growth does not occur in
their absence.
 Accessory – when they enhance growth,
without being absolutely necessary for it.

Growth Factors
Identical with eukaryotic nutrition







Vitamin B complex –
thiamine
riboflavine
nicotinic acid
pyridoxine
folic acid &
Vit.B 12
Presence or Absence of Gases
 Primary gases = O2, N2, & CO2

O2 - greatest impact on microbial growth
(even if the microorganism does not require it)
 Aerobic respiration – terminal electron
acceptor is oxygen.
 Anaerobic respiration – terminal electron
acceptor is an inorganic molecule other
than oxygen (e.g. nitrogen).
Depending on the O2
requirement
Strict (Obligate) Aerobes – O2 present, require O2 for growth

e.g. Pseudomonas aeruginosa


Obligate aerobe – 20% O2: only grows with O2
Microaerophile – 4% O2: best growth with small amount O2

e.g. Campylobacter spp, Helicobacter spp
Strict (Obligate) Anaerobes – O2 depleted, grow in the absence
of O2 & may even die on exposure to O2
e.g. Bacteroides

fragilis


Obligate anaerobe: only grows in absence of O2
Aerotolerant anaerobe: anaerobes that “tolerate” +/or survive in O2, but
do NOT utilize O2 during E* metabolism


e.g. Clostridium perfringens
Facultative Anaerobe – grows both in presence & absence of O2;
but grows BEST under Aerobic conditions; considered to be
aerobic organism; O2 present – aerobic respiration for E*; O2 absent –
anaerobic pathways (fermentation)


e.g. Staphylococcus spps
Capnophilic organism – requires high CO2 levels eg Neisseria
spps
Oxygen-related growth zones in
a standing test tube
 Oxygen is readily converted into radicals
(singlet oxygen, superoxide, hydrogen
peroxide, hydroxyl radical)
 Most important detoxifying enzymes are
superoxide dismutase and catalase
 Cells differ in their content of detoxifying
enzymes and hence, ability to grow in the
presence of oxygen
90
 Classification of gram-positive cocci
 Staphylococci are catalase +
 Streptococci are catalase -
Staphylococci
Streptococci
91
pH
Majority of bacteria grow BEST at neutral or
slightly alkaline pH

pH 7.0 – 7.4 => this is near most normal body
fluids
•

Acidophiles: grow BEST at low pH (acid: pH 0
– 1.0)


T.B. - pH 6.5-6.8
Alkalophiles: grow BEST at high pH (alkaline:
pH 10.0)

V. cholerae - pH 8.4-9.2
Microbial physiology.
Microbial metabolism.
Enzymes. Nutrition.
Bioenergetics. Bacterial
growth and multiplication.
Enzymes
 Biological catalysts
 Highly specific
 Extremely efficient
 Increase reaction rates 108-1010 times
 High turnover numbers
 Proteins or RNA (ribozymes)
Uptake of nutrients by bacteria
Passive diffusion
simple diffusion
Facilitated diffusion
Active transport
Enzymes - catalysts that speed
up and direct chemical reactions
 A. Enzymes are substrate specific
 Lipases
Lipids
 Sucrases
Sucrose
 Ureases
Urea
 Proteases
Proteins
 DNases
DNA
Naming of Enzymes - most are named
by adding “ase” to the substrate
 Sucrose
 Lipids
 DNA
 Proteins
 removes a Hydrogen
 removes a phosphate
Sucrase
Lipase
DNase
Protease
Dehydrogenase
phosphotase
Naming of Enzymes
 Grouped based on type of reaction they
catalyze
 1. Oxidoreductases
reduction
 2. Hydrolases
 3. Ligases
oxidation &
hydrolysis
synthesis
Types of enzymes
Oxidoreductase
Oxidation reduction in
Cytochrome oxidase,
which hydrogen or oxygen lactate dehydrogenase
are gained or lost
Transferase
Transfer of functional
groups, e.g. amino, acetyl
or phosphate groups
Acetate kinase, alanine
deaminase
Hydrolase
Hydrolysis – addition of
water
Lipase, sucrase
Lyase
Removal of atoms without
addition of water
Oxalate decarboxylase,
isocitrate lyase
Isomerase
Rearrangement of atoms
within a molecule
Glucose phosphate
isomerase, alanine
racemase
Ligase
Joining of two molecules
Acetyl-CoA synthetase,
DNA ligase
Enzyme Components
2 Parts
1. Apoenzyme - protein portion
2. Coenzyme (cofactor) - non-protein
Holoenzyme - whole enzyme
Coenzymes
 Many are derived from vitamins
 1. Niacin

NAD (Nicotinamide adenine dinucleotide)
 2. Riboflavin

FAD (Flavin adenine dinucleotide)
 3. Pantothenic Acid

CoEnzyme A
Enzyme components
 Cofactors may be metal ions
 Cofactors may accept or donate atoms removed from the
substrate or donated to the substrate
 Cofactors may act as electron carriers
 Often derived from vitamins
 e.g. NAD and NADP – electron carries derived from nicotinic
acid