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 e-

acceptor 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
2. Circuit of organic acids – series of oxidations and reductions
• Eukaryotes – Mitochondrial Matrix
• Prokaryotes – Cytoplasm of bacteria & Cell Membrane

 Products:
 2 ATP
 6 NADH2
 2 FADH2
 4 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 Final e-
involved acceptor ATP yield
Aerobic Glycolysis, O2 38
respiration TCA, ET

Anaerobic Glycolysis, NO3-, So4-2, variable

respiration TCA, ET CO3-3

Fermentatio Glycolysis Organic 2

n molecules
 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. substrate-level phosphorylation
2. oxidative phosphorylation, (reduced chemicals)
3. Photophosphorylation (reduced chlorophyll molecul
es)

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 number
increases exponentially.
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
 C, H, O, N
2. Micronutrients – needed in small quantities;
more specialized (enzyme & pigment
structure & function)
 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 simple diffusion
o facilitated diffusion

o 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 Sucrase
 Lipids Lipase
 DNA DNase
 Proteins Protease
 removes a Hydrogen Dehydrogenase
 removes a phosphate phosphotase
Naming of Enzymes
 Grouped based on type of reaction they
catalyze
 1. Oxidoreductases oxidation &
reduction
 2. Hydrolases hydrolysis
 3. Ligases synthesis
 Types of enzymes
Oxidoreductase Oxidation reduction in Cytochrome oxidase,
which hydrogen or oxygen lactate dehydrogenase
are gained or lost
Transferase Transfer of functional Acetate kinase, alanine
groups, e.g. amino, acetyl deaminase
or phosphate groups
Hydrolase Hydrolysis – addition of Lipase, sucrase
water
Lyase Removal of atoms without Oxalate decarboxylase,
addition of water isocitrate lyase
Isomerase Rearrangement of atoms Glucose phosphate
within a molecule 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