Chapter 2

Microbial physiology

1
At the end of this unit, you should be able to:
 Define the term bacterial growth
 Draw and explain the growth curve of microorganisms in a batch
culture
 Identify chemical and physical conditions required for microbial
growth.
 Explain what means by culture medium, types of culture medium
and their purpose.
 Describe staining techniques used in microbiology laboratory.
 Explain the concepts of microbial metabolism.
 Describe the methods of measuring microbial growth
 State the advantage and disadvantages of the different methods of
microbial growth measurement

2 12/19/2023
2.1. Microbial growth
 Microbial growth is an increase in the amount of protoplasm the
formation of new structures, and eventually the formation of new
cells

 Microbial growth is an increase in number of cells, not cell size, or

emergence of new individuals (reproduction)

 Bacteria normally reproduce by binary fission (one cell’s equally

divided into two cells)
 Reproduction of prokaryotes( Binary fission, Budding,
Conidiospores (for actinomycetes), and Fragmentation of
filaments)

3
Binary Fission

4
Figure 6.11
• Batch Culture
• Comprises a liquid growth medium in a closed
container
• Microbial inoculums is allowed to grow with no net
input into the culture except for exchange of gases
• No waste products are removed
• Growth stops after sometime
• Continuous Culture
• The culture divides continuously due to supply of
fresh medium and removal of spent medium and toxic
products
• A microbial population can be maintained in the
exponential growth phase for extended periods
5
 Phases of growth
 Bacterial population growth in a closed system (batch
culture).
 Growth curve: plots of logarithmic cell number versus
incubation time.
 The time required for a cell to divide and its population
to double is called the generation (doubling) time.
 Arithmetic number of cells in each generation is
expressed as a power of 2, the exponent tells the number
of doublings that have occurred.
 Generation time differs in different species, for e.g., The
average generation time of E.Coli is 20 minutes and that
of M. Tuberculosis is about 15-20 hours.

6
 There are four basic phases of growth.

7
Figure 6.14
1. Lag phase
 When bacteria are inoculated in to a new medium, reproduction
usually doesn’t begin immediately, means No cell division but
cells increase in size very little
 The lag phase is a period of adaptation to the environment, and its
duration may vary from a few minutes to several days.
 The cells are not dormant only lag in multiplication. The
microbial population has intense metabolic activity in particular,
new enzyme and several molecules are synthesized.
 The length of time depends on the -kind of bacteria, the age of the
culture, and the available nutrients in the medium provided (if the
same medium used)
 Time of recovery from effects of toxic products

8
2. Logarithmic Growth phase (Log phase)
 The log phase of growth is the period of most rapid
reproduction.
 During the log phase, cells begin to show their visible
characteristics – the shape, color, density and grouping of
their colonies

 The population are at their metabolic peak and are

therefore most sensitive to inhibition by antibiotics or
other agents that disrupt metabolic functions.

9
 The generation time (doubling time) is constant for a
given environment for each species.

 The generation time of an organism can be determined

during this phase.

 Each generation results in a doubling of the cell

number. Thus, plotting the logarithm of the number of
organisms against time gives you a straight line.

10
 3. Stationary phase
 The overall number of bacteria remains constant due to
the rate of reproduction equals the rate of cell death
 As the culture grows older and approaches the
maximum population of bacteria the medium can
support, the rate of reproduction slows and some cells
die.
 The reason for cessation of the phase:
Accumulation of toxic waste products
Exhaustion of required nutrients
Changes in pH, temperature and oxygen
11
4. Death phase (logarithmic decline phase)

 Once the rate of death exceeds the rate of reproduction,

the actual number of bacteria declines. In this death

phase, reproduction will usually have stopped.

 Bacterial death is classically defined as the inability to

reproduce when transferred to a nutritionally supportive

medium under suitable environmental conditions.

 With certain species, it may take weeks, months and even

longer before the end of this phase is reached.

12
2.2. Growth requirements of microorganisms
There are two types of growth requirements (Physical
requirement & Chemical )
1. Physical requirements : The effect of environmental
conditions on microbial growth.
A.Temperature: When rises to some extent, there is an
increase in the growth rate due to increasing the rates
of enzyme reactions.
- As the temperature becomes too high,
microorganisms are damaged by enzymes and nucleic
acid denaturation, membrane disruption etc.

13
14
Each bacterial species grows at a particular minimum,
optimum, and maximum temperatures which can be referred
as cardinal temperatures.

 The minimum growth temperature -the lowest

temperature at which the species will grow

 The optimum growth temperature – the temperature at

which the species grows best

 The maximum growth temperature - the highest

temperature at which growth is possible

15
 Temperature and Microbial Growth

 Cardinal
temperatures
 minimum
 optimum
 maximum
 Temperature is a major
environmental factor
controlling microbial
growth.

16
16
Three groups of microbes on the basis of their preferred
range of temperature
 Psychrophiles :cold loving microbes

 Psychrotrophs (faculitative psychrophiles): they grow slowly

in cold but have an optimum temperature above 20Oc. E.g.
S.aureus
 Mesophiles :moderate temperature loving microbes.

 Majority of medically important microorganisms are

mesophiles.
 Thermoduric microbes: can survive short exposure to high
temperatures but are normally mesophiles. Common
contaminants of heated and pasteurize food.
17 Thermophiles: heat loving microbes
18
B. pH : The degree of acidity or alkalinity of a solution

- Has three cardinal points:

 The minimum pH- below which the organism cannot

grow

 The maximum pH- above which the organism cannot

grow

 The optimum pH- at which the organism grows best

 Most bacteria grow best in a narrow range of pH near

neutrality, between pH 6.5 and 7.5

19
  Three groups of microbes on the basis of their
preferred range of pH

 Neutrophiles- microorganisms that grow best at

neutral pH.
 Acidophiles- microorganisms that grow best at
acidic pH.
 Alkalophiles- microorganisms that grow best
under alkaline conditions

20
 C. Osmotic Pressure

 Microbes obtain almost all of their nutrients in

solution from the surrounding water. They need

about 80% to 90% water.

 High Osmotic pressures have the effect of removing

necessary water from a cell. This osmotic loss of

water causes plasmolysis, or shrinkage of cells

 Three types osmotic pressure: hypotonic, hypertonic

and Isotonic
21
The growth of the cell is inhibited as the cytoplasmic
membrane pulls away or turgid into cell wall

22
  The only common solute in nature that occurs over
a wide concentration range is salt [NaCl], and
microorganisms are named based on their growth
response to salt as:
 Halophiles- organisms that require some salt
(NaCl) for growth.
 Extreme halophiles- organisms that require high
salt concentrations for their growth.
 Halotolerants- organisms that are able to grow at
moderate salt concentrations.

23
  Osmophiles- organisms that are able to grow in
environments high in solutes.
 Xerophiles- organisms which live in dry
environments.
2. Chemical requirement
A. Oxygen
 It is an important respiratory gas, and it is also a
powerful oxidizing agent that exist in many toxic
forms.
 Bacteria can be divided into several general groups
on the basis of their oxygen requirements.

24
1. Obligate aerobes
 They require free oxygen in order to grow
 They use oxygen as a final electron acceptor in aerobic
respiration.

2. Obligate anaerobes
 They will not grow in the presence of free oxygen
 Oxygen is a toxic substance for them which either kills
or inhibits their growth
 All cells contain enzymes capable of reacting with
oxygen.

25
 In the presence of oxygen, all organisms normally
produce small amounts of a free radical of oxygen
called superoxide (O2-)
 Superoxide is very toxic to the cell, and complete
conversion of superoxide ion into harmless oxygen
requires two step process and two enzymes.
Step 1.
2O2- + 2H+ superoxide dismutase H2O2 + O2

 H2O2 is also very toxic and will kill the cells if it is not
destroyed.
 Aerobic bacteria break H2O2 down by producing the
enzyme catalase.
26
 Step 2.
2 H2O2 catalase 2H2O +O2

 Most obligate anaerobes lack enzymes for processing toxic

oxygen, therefore they cannot tolerate the presence of free
oxygen .
 Aerobes can use o2 in their metabolism and posses the
enzymes needed to process toxic oxygen products.

3. Microaerophile
 An aerobes that grow best with reduced oxygen tension. Does
not grow under anaerobic conditions.
 Most organisms in this category live in a habitat such as soil,
water, or human body that provides small amounts of oxygen
but is not directly exposed to the atmosphere.
27
4. Facultative aerobes
 Can switch between aerobic and anaerobic types of metabolism.
 Grow by fermentation or anaerobic respiration, but in the
presence of oxygen they switch to aerobic respiration.
 They possess catalase and superoxide dismutase.
5. Aerotolerant
 These anaerobes are not harmed by oxygen, mainly because
they possess alternative mechanisms for breaking down
peroxide and superoxide. e.g. lactobacilli uses manganese ions
or peroxidases.
 They grow in the presence of air but do not possess an oxidative
metabolism
 They do not use oxygen in their metabolism but carry out a
fermentative degradation of carbohydrates even in the presence
of oxygen.
28
  Oxygen (O2)

obligate Facultative Obligate Aerotolerant

Microaerophiles
aerobes anaerobes anaerobes anaerobes

29
30
30
 B. Carbon source
 Carbon is the structural backbone of living matter; it is
needed for organic compounds that make up a living cell
(energy source)

 Autotrophic bacteria –use CO2 as the principal carbon

source

 Heterotrophic bacteria- use organic compounds (such

as proteins, carbohydrates, and lipids) as the principal
carbon source

31
C. Nitrogen source
 Nitrogen is used primarily to form the amino group of
the amino acids of proteins and for syntheses of DNA
and RNA

 Most bacteria decompose proteins organically

bound nitrogen, in the form of glutamine, asparagin,
peptide digests

• Some bacteria use NH4+ or NO3 (inorganic

sources)
• A few bacteria use N2 in nitrogen fixation
32
D. Inorganic ions
 All organisms require some inorganic ions
 Phosphorus, phosphate (PO43-) is essential to
synthesize nucleic acids and the phospholipids
of cell membranes. It is also found in the energy
bonds of ATP
• Sulfur, sulfate (SO42-) - used to synthesize sulfur
containing amino acids and vitamins such as
thiamine and biotin. Some bacteria also use H2S
 Potassium, magnesium, and calcium are
elements that microorganisms require as
33 cofactors for enzymes
E. Organic growth factors
 They are essential organic compounds that the
organism is unable to synthesize and must be
provided as a nutrient (directly obtained from the
environment)
 They fall into three classes, in terms of chemical
structure and metabolic functions
1. Amino acids, required as constituents of proteins
2. Purines and pyrimidines , required as constituents
of nucleic acids
3. Vitamins, required as cofactors for the activity of
enzymes

34
F. Trace elements (micronutrients)
 Nutrients (mineral elements) required by microbes
in very small amounts
 Example: Iron, copper, molybdenum, and zinc
 Essential for activity of certain enzymes, usually as
cofactors
G. Water
 Water accounts for 80 to 90 percent of the total
weight of cells. It is therefore the major essential
nutrient for cells

35
 Nutritional types of microorganisms
 Most microorganisms can be categorized as belonging to
one of four major nutritional types depending on their source
of carbon and energy
Nutritional type Carbon Energy source Examples
source
Photoautotrophs CO2 Light Photosynthetic bacteria

(green sulfur bacteria, purple sulfur bacteria,

Cyanobacteria), algae, plants

Photoheterotrophs Organic Light Purple nonsulfur and green nonsulfur bacteria
compounds

Chemoautotrophs CO2 Inorganic Hydrogen, sulfur, iron and nitrifying bacteria

compounds
Chemoheterotrophs Organic organic Most bacteria and all fungi, protozoans, and animals
compounds compounds
36
 Culturing Microbes
 Inoculation: introduction of a sample into a container of
media to produce a culture of observable growth
 Isolation: separating one species from another
 If an individual bacterial cell is separated from other cells and
has space on a nutrient surface, it will grow into a mound of
cells - a colony.
 A colony consists of one species.
 Isolation techniques include:
 Streak plate technique
 Pour plate technique
 Spread plate technique

37 12/19/2023
  Incubation: growing microbes under proper conditions

 Inspection: Observation of characteristics and Systematic

recording of data. Macroscopic and microscopic appearance,
biochemical tests, genetic characteristics, immunological testing

 Identification: Correlating data from all observations to ID

38
organism to species 12/19/2023
 Culture media
 A nutrient material prepared for the growth of
microorganisms in a laboratory
 Some microorganisms can grow well on any culture
medium
 Other require special media
 Still others can not grow on any non living medium
 Microbes that are introduced in to a culture to initiate
growth known as inoculums
 The microbes that grow and multiply in or on a
culture medium are known as culture

39 12/19/2023
 The culture medium must meet the following to grow
microorganisms:
 It must contain the right nutrients for the specific
microorganism
 It should also contain sufficient moisture
 A properly adjusted pH, and a suitable level of O2
 The medium must initially be sterile
 Finally, the growing culture should be incubated at the proper
temperature.

40 12/19/2023
 Culture media can be used to:
 Grow a wide range of organisms in a particular group,
e.g. bacteria
 Maintain organisms in a culture collections
 Distinguish between different types of microorganisms
 Select specific groups or types of microorganisms from
an environment
 Help identify organisms

41 12/19/2023
 Media can be classified according to three
properties:
1. Physical state – liquid, semisolid and solid
2. Chemical composition –
 Synthetic (chemically defined)- contains pure
organic and inorganic compounds in an exact
chemical formula
Nonsynthetic (complex)- contains at least one
ingredient that is not chemically definable
3. Functional type – general purpose, enriched,
selective, differential, transport etc.,

42 12/19/2023
 Types of culture medium
• General purpose (non-selective) medium
have nutritional content that allow the growth of a wide
range of either bacteria or yeast and moulds,
Used extensively in the laboratory to maintain
cultures and carry out total viable counts.
E.g. nutrient agar/ broth, plate count agar for bacteria
MEA/ MEB, PDA for growing moulds/ yeasts
• Selective medium
Are often used to isolate a particular groups of
microbes.
Contains ingredient that inhibit or suppress the growth
43 of certain organisms but allow other to grow. 12/19/2023
 Cont…
 A wide variety of different chemicals can be added to media to

select groups of organisms or specific organism

 The media work by inhabiting unwanted organisms

 Selection can operate in a number of ways:- pH, temperature,

oxygen, moisture, etc.

 Enrichment medium

 Used to encourage the growth of a particular microorganisms in a

mixed culture.
 It is designed to increase very small numbers of the desired types

44
of organisms to detectable levels. 12/19/2023
 Cont…
 Differential medium

 Contains ingredient that are changed as a result of microbial

metabolism
 The change helps to distinguish colonies of one organism from

colonies of other organism on the same plate

 E.g in Eosin Methylene blue agar, E.coli produces colonies with

brilliant green metallic sheen while, Aerobacter aerogenes

produces pink colonies with dark centers

45 12/19/2023
 Cont…
 Living medium
Some microorganisms, e.g Viruses will only grow
in the living cells of their hosts
To grow them in a laboratory a living culture of
host cells needs to be provided, e.g. chick
embryos or tissue cultures
E.g. Treponema pallidum and Mycobacterim
leprae-in live animals
 Transport (carrying) medium
Used for the temporary storage of specimens
being transported to the laboratory for the
46
cultivation 12/19/2023
 Cont…
• Such medium ideally maintain the viability of all
organisms in the specimen with out altering their
concentration
• Contains only buffer and salt.
• The lack of carbon, nitrogen, and organic growth
factors prevents microbial multiplication
• Pure culture techniques
• Culture: population of microorganisms grown under
well defined conditions
Pure culture- one that contains one type of
microorganism
47
Mixed culture-more than one microorganism 12/19/2023
 Cont…
• Most microbiological works requires pure cultures
• Simpler methods for isolation of a pure culture
include: (i) pour plate method and (ii) streak
plating with a loop
• The isolation method most commonly used to get
pure cultures of bacteria is the streak plate method
• The streak plate method works well when the
organism to be isolated is present in a large
numbers
• The number must be greatly increased by selective
enrichment before it can be isolated with the streak
plate method
48 12/19/2023
Pure culture preservation and maintenance
methods
 Refrigeration can be used for short term storage of
microbial cultures
 Two common methods of preserving microbial cultures
for long periods are deep-freezing and lyophilization
 Deep-freezing:- is a process in which a pure culture of
microbes is placed in a suspension liquid and quickly
frozen at temperature ranging from -500c to -950c

49 12/19/2023
 Staining techniques
• Wet mount versus stained smear
 Many microorganisms are motile and colorless, they are
invisible because of their lack of contrast with the water in
which they may reside.
 Thus, staining is necessary in order to make them readily
visible.
 Staining means coloring a microorganisms with a dye to make
some structures more visible.
 Fixing uses heat or alcohol to kill and attach microorganisms to
a slide.
 Microbial smear is a dried preparation of microbial cells on a
glass slide.

50 12/19/2023
 Cont…
 Bacteria are slightly negatively charged at pH 7.0
Basic dye stains bacteria
Acidic dye stains background
 Simple stain
 The use of a single stain or dye to create contrast
between the bacteria and background
 It is aqueous or alcohol solution of single basic dye
 It is used to make cellular shapes and
arrangement visible
 The basic dye such as crystal violet, carbol fuchsin
or methylene blue are of the used
51 12/19/2023
 Cont…
 Differential stains
Differential stains such as the Gram stain and
acid-fast stain differentiate bacteria according to
their reactions to the stains
 Gram stain procedure uses
Crystal violet: primary stain
Lodine: modant
Alcohol or acetone- alcohol: decolorizer
Safranin: counterstain

52 12/19/2023
53 12/19/2023
 Cont…
 Gram positive retain the puple stain after decolorization
step
 Gram negative do not and thus appear pink from the
counter stain.
 Acid-fast stain
Type of differential stain that has important medical
applications
The acid-fast staining procedure is based on the
presence of a waxy lipid in the cell wall of some
bacteria called mycolic acid
If this substance is present the primary dye in the acid-
fast procedure, carbol fuchsin, will bind very tightly to
the mycolic acid and stain the cells a very bright
purple-pink color
54 12/19/2023
 Cont…
 If there is no mycolic acid present the cell will not
retain the dye and will stain the color of the
counter stain which is blue
 Special stain
Spore and capsule stain
By simple stain the spore appears as unstained
structure in the stained cell
o They are difficult to stain by ordinary methods
The spore can be stained with a hot dye of
contrasting color, the spore appear as green
where as vegetative cell stained with counter
stain and appear as pink.
55 12/19/2023
 Capsule staining

 Some bacteria posses a gummy layer which

surrounds the cell called capsule
 Composed of polysaccharides
 Capsule when observed under microscope
appears as an envelope around the cell
 Dyes such as india ink are incapable of
penetrating capsules while other dyes can
penetrate and stain the cell but not capsule
 Thus the capsule appears as a halo (clear)
zone in dark back ground while the cells are
stained by the dye.
56 12/19/2023
 Cont.,
 Capsule
 2.3.Measurement of growth
 Some methods measure cell numbers; other
methods measure the population’s total mass,
which is often directly proportional to cell
numbers
 Population numbers are usually recorded as the
number of cells in a milliliter of liquid or in a gram
of solid material
 Because bacterial populations are usually very
large, most methods of counting them are based on
direct or indirect counts of very small samples

58
Mainly two methods of bacterial counting
1. Direct counting methods
2. Indirect counting methods
1. Direct counts( total counting)
A. Direct microscopic count
For this purpose there are specially designed slides, Petroff
Hauser counter (slides).
 A shallow well of known volume is indented in to the
surface of a microscope slide and covered with a thin
glass inscribed with squares of known area. The well is
filled with the bacterial suspension.

59
  The average number of bacteria in each of a series
of these squares is calculated, and then multiplied by
the factor that produces the count per milliliter.

 Important for immediate estimates of how many

microorganisms are present in a solution with time

 Good for grading the quality of milk.

60
 Advantage of the method
1. It is a quick method since no incubation time is required
to count the cells
2. It provides an idea of the total number of organisms and
the diversity with in the population.
Disadvantage of the method:
1. It is difficult to count motile bacteria
2. Since direct counts do not permit differentiation between
living and dead cells, they usually yield higher number
than a count of viable cells.
3. High concentration of cells is required, about 10
million bacteria per milliliter

61
 B. Electronic cell counters
 Bacteria can be counted electronically by passing a
culture through a very small opening in a Coulter
particle counter
Advantage
1. For grading milk because it is quickest
2. For diagnosis of urinary tract infections
3. Used to estimate the microbial populations which
are not cultivated in lab media
Disadvantages
1. Very expensive
2. Counts the debris as one of the living bacteria
62
 C. Plate count (viable count)

 The most frequently used method for the measurement

of bacterial populations

 It measures the number of viable cells

 based on the assumption that each bacterium grows and

divides to produce a single colony on a solid medium

 A plate count is done by either the pour plate or the

spread plate method

63
 • Plate Counts: Perform serial dilutions of a sample

64
After incubation, count colonies on plates that have 25-300 colonies
(CFUs), When too many colonies are present, cells are
overcrowded and cause inaccuracies in the count

65
  Pour plate
 Dilutions of the bacterial suspension in the amounts of 1
ml or 0.1ml are introduced into a Petri dish.
 The nutrient medium, in which the agar is kept liquid by
holding it in a water bath at about 45 - 50oC, is poured
over the sample, then mix it by gentle agitation of the
plate.
 When the agar solidifies, the plate is incubated
 colonies grow on the surface the agar plate as well as
deep (beneath) into the nutrient agar

66
67
 Limitation of the method
1. Some relatively heat sensitive microorganisms may
be damaged by the melted agar

2. When using certain differential media, Colonies that

form beneath the surface of a pour plate are not
show distinctive appearance of the colony for
diagnostic purposes

 To avoid these problems, the spread plate method

is frequently used instead

68
 Spread plate
 A 0.1 ml inoculum is added to the surface of a prepoured, solidified
agar medium. spread inoculum uniformly over the surface of the
medium with a specially shaped sterilized glass

69
 Disadvantages
1. It is not effective because a single cell may bring a
number of colonies if the bacterium is motile
2. This method fails for organisms that can’t be cultured
in artificial lab media
3. No single medium or incubation condition will allow
growth of all bacteria
4. Organisms that form colonies that swarm together
can’t be counted with this technique.
5. It fails for organisms that multiply very slowly

70
 D. Filtration
 This method is used to quantitate the number of
bacteria present in a sample of water.
 Membrane filters are usually employed for such
purposes.
 The membrane will be placed (after filtering the
sample through it) directly onto suitable agar
medium.
 The plate is then incubated, and each viable
bacterium able to grow under the conditions used
will produce a visible colony

71
2. Indirect methods
A. Turbidity
 Measuring turbidity (cloudiness) of a growing culture
using a colorimeter or spectrophotometer
 Depends on the fact that the amount of light transmitted

through a bacterial suspension is diminished in

proportion to the number of bacteria present.

 The amount of light striking the light sensitive detector

is inversely proportional to the number of bacteria

under standardized conditions. The less light
72
transmitted, the more bacteria in the sample.
73
  Disadvantage

About 10 to 100 million cells per

milliliter are needed to make a
suspension turbid enough to be read on a
spectrophotometer.
Thus, is not a useful measure of
contamination of liquids by relatively
small number of bacteria
74
B. Dry weight
 One of the better measurements of growth of filamentous
organisms such as fungus
 In this procedure, the fungus is removed from the growth
medium, filtered to remove extraneous material, placed in
a weighing bottle, and dried in a desiccator.
 For bacteria, the same basic procedure is followed.
 It is needed to remove the bacteria from the culture
medium by centrifugation

75
 C. Metabolic activity

 In this method the amount of a certain metabolic

product of the population is measured

 Assumed to be directly proportion to the number of

bacteria present

 The metabolic product might be acid or CO2

76
Microbial Metabolism: Basic Concepts
Definition: Metabolism is the sum of the chemical
reactions in an organism.
1) Types of Metabolism
A) Anabolism: The enzyme-regulated energy-
requiring reactions involving the building
of complex organic molecules from
simpler ones.
• These reactions are called anabolic, or
biosynthetic, reactions
o Often involve dehydration synthesis reactions
(reactions that release water).
77
o Anabolic reactions are endergonic (consume
more energy than they produce).
E.g. formation of proteins from amino acids, nucleic
acids from nucleotides, and
polysaccharides from simple sugars.
B) Catabolism: Enzyme-regulated chemical
reactions that release energy by
breaking down complex organic
compounds into simpler ones.
• These reactions are called catabolic, or
degradative, reactions.
o Are generally hydrolytic reactions (reactions
which use water and in which chemical bonds
are broken),
o Are exergonic (produce more energy than they
consume).
  Catabolism provides the building blocks and furnish
the energy needed for anabolism

79
Figure 5.1
2) Energy Production
 Oxidation-Reduction
 Nutrient molecules have energy associated with the electrons
that form bonds between them
 To extract energy from organic compound and store it in chemical
forms, organisms pass electrons from one compound to another
through a series of oxidation – reduction reaction
 This energy which extracted from organic compound is
concentrated into ATP bonds and serve as energy source for
energy requiring reactions
o Oxidation is the removal of electrons.
o Reduction is the gain of electrons.
 Redox reaction is an oxidation reaction paired with a reduction
reaction.

80
Figure 5.9
 In biological systems, the electrons are often
associated with hydrogen atoms.
 Biological oxidations are often dehydrogenations
 Cells use oxidation reduction reactions in
catabolism to extract energy from nutrient
molecules

Figure 5.10
81
 The Generation of ATP
Much of the of the energy released during
oxidation- reduction reaction is trapped within the
cell by formation of ATP
ATP is a “currency of energy” for many cellular
reactions
ATP is generated by the phosphorylation of ADP
Phosphorylation – addition of P04- to a
chemical compound

82
ATP + H2O  ADP + Phosphate + Energy (7.5 kcal/mol)
Definition: Enzymes are organic or biological catalysts produced by all
living cells. Specific for a chemical reaction
 Enzyme Components
 Apoenzyme: protein portion
 Cofactor: Nonprotein component
 Coenzyme: Organic cofactor. Assist the enzymes by accepting
Hydrogen atoms removed from the substrate
 Holoenzyme: Apoenzyme + cofactor
 Important Coenzymes
 NAD+= Nicotine Amide Adenine Dinucleotide
 NADP+= Nicotine Amide Adenine
Dinucleotide Phosphate
 FMA: Flavine Mono nucleotide
 FAD: Flavine Adenine Dinucleotide
 CoA: Coenzyme A
83
 Energy production (catabolism) in microbes
 Microbes are distinguished by their great metabolic diversity
 Some can sustain themselves on inorganic substances by using
pathways that are unavailable to either plants or animals
 This is why they can live in diverse environments
 They can catabolize various organic molecules including
carbohydrates, lipids and proteins for energy production
 However, most microorganisms oxidize carbohydrates as their
primary source of cellular energy
 Glucose is the most common carbohydrate energy source used
by the cells
 To produces energy from glucose microorganism use two
general process:-
 Cellular respiration and Fermentation
 Both start with the same first step glycolysis but follow different
subsequent path way
 1. Respiration
 Respiration is ATP generating process in which molecules are
oxidized and the final electron acceptor is inorganic molecule
 The respiration of glucose typically occurs in three principal
stages: Glycolysis, the Tricarboxylic acid cycle and the Electron
transport chain (system)
 There are two types of respiration, depending on whether an
organism is an aerobe or anaerobe
1.1. Aerobic Respiration
 It is characteristics of many bacteria, fungi ,protozoa and algae
 Aerobic respiration is a series of enzyme catalyzed reactions in
which electrons are transferred from fuel molecules such as
glucose to oxygen as a final electron acceptor
 Aerobic respiration in microorganisms can be summarized by an
equation
 Glucose (C6H12O6) + 6o2+38ADP+38pi  6CO2+6H2O+38ATP
 1.1.2. Glycolysis
 The first stage in respiration of glucose
 Glycolysis is oxidation of glucose molecule to Pyruvic
acid
 This pathway is a series of ten chemical reactions each
catalyzed by a different enzymes
 During glycolysis NAD+ is reduced to NADH and there is
net production of two ATP molecules by substrate level
phosphorylation
 Glycolysis does not require O2
 It also called Embden-Meyerhof-Parnas pathway
 Alternatives to glycolysis
1.Hexose monophosphate pathway
o Also found in most organisms
o Responsible for synthesis of pentose sugars used in nucleotide
synthesis
o Involve two unique enzymes: Transketolase and Transaldolase
o Operates simultaneously with glycoysis
2. Entner-Doudoroff pathway
o Found in Pseudomonas and related genera
o Does not involve glycolysis
3. Phosphoketolase pathway
o Found in Bifidobacterium and Leuconostoc
 1.1.2. Tricarboxylic acid cycle (TCA)
 The TCA is the oxidation of acetyl COA (a derivative of pyruvic
acid) to CO2, with the production of some ATP, energy-containing
NADH and another reduced electron carrier, FADH2
 TCA has eight steps, beginning with citric acid formation and
ending with oxaloacetic acid
 In TCA four molecules of CO2 liberated by decarboxylation, six
molecules of NADH and two molecules of FADH2 formed by
oxidation –reduction reactions, and two molecules of ATP are
generated by substrate level phosphorylation
1.1.3. Election transport chain
 ETC consists of a chain of special redox carriers that receive
electron from reduced carriers (NADH,FADH2) generated by
glycolysis and the TCA cycle
 In eukaryotic cells the ETC is contained in the inner membrane of
mitochondria while, in prokaryotic cells, it is found in the plasma
membrane
 The ETC regenerates NAD+ and FAD, which can be used again in
glycolysis and krebs cycle
 The various electron transfers in the electron transport chain
generate about 34 molecules of ATP from each molecule of
glucose oxidized
 In aerobic respiration among prokaryotes, a total of 38 molecules
of ATP can be generated from one molecule of glucose
1.2. Anaerobic Respiration
 Some bacteria have evolved anaerobic respiratory system that
functions like aerobic cytochrome system except that it utilizes
oxygen-containing ions rather than free oxygen as the final
electron acceptor
 Electron acceptor in an aerobic respiration are : NO3,NO2,
carbonates and sulfates
 Pathway Eukaryote Prokaryote

Glycolysis Cytoplasm Cytoplasm

Intermediate step Cytoplasm Cytoplasm

Krebs cycle Mitochondrial matrix Cytoplasm

ETC Mitochondrial inner Plasma

membrane membrane

• Bacterial ETC as compared to eukaryotic ETC:

• Vary in electron carriers (cytochromes)
• Extensively branched
• May be shorter

92
  ATP produced from complete oxidation of 1 glucose using
aerobic respiration
By oxidative
By substrate- phosphorylation
Pathway level
phosphorylation From From
NADH FADH
Glycolysis 2 6 0
Intermediate step 0 6
Krebs cycle 2 18 4

Total 4 30 4

 38 ATPs are produced in prokaryotes.

93
Anaerobic respiration

Electron acceptor Products

NO3– NO2–, N2 + H2O

SO4– H2S + H2O

CO32 – CH4 + H2O

94
2) Fermentation
Features of fermentation pathways
 Pyruvic acid is reduced to form reduced organic acids or
alcohols.
 The final electron acceptor is a reduced derivative of
pyruvic acid
 NADH is oxidized to form NAD: Essential for continued
operation of the glycolytic pathways.
 O2 is not required.
 No additional ATP are made.
 Gasses (CO2 and/or H2) may be released
 Does not use the Krebs cycle or ETC

95
 • It is useful as tools in biochemical identification
• It is used in industry ex. Synthesis of certain
organic compounds

96
Figure 5.18b
D) Types of fermentation pathways
i- Alcohol fermentation:
 Produces ethyl alcohol + CO2
 Carried out by a number of bacteria and yeasts
ii- Lactic acid fermentation:
 Produces lactic acid
e.g. Streptococcus cremoris, Lactobacillus acidophilus
a- Homolactic fermentation
o Produces lactic acid only
b- Heterolactic fermentation
o Produces lactic acid and other compounds
iii- Formic Acid Fermentation
 Produces formic acid and other products
o Formic acid -------- CO2 and H2
Chemolithotrophy
 Reduced inorganic compounds serve as electron
donors in energy production
 Ex. Hydrogen, reduced nitrogen compounds (nitrifying),
reduced sulfur compounds (sulfur oxidizing), and Ferrous
Iron (Fe 2+) (Iron oxidizing bacteria)
Lipid Catabolism

99
Figure 5.20
Protein Catabolism

Extracellular proteases
Protein Amino acids

Deamination, decarboxylation, dehydrogenation

Organic acid Krebs cycle

100