Physiology and biochemistry of

microorganisms
When bacteria find themselves in a
suitable environment, they grow and
eventually divide. Growth is defined as an
increase in cellular constituents. Microbial
cells simply become longer and/or larger. It
leads to a rise in cell number when
microorganisms reproduce by processes like
budding or binary fission. The time it takes for
a bacterium to become two is called
generation time or doubling time.
 Generation time (doubling time)
for different bacteria in rich nutrient media
Escherichia coli
& 20 minutes
Staphylococcus aureus

Mycobacterium tuberculosis 14-16 hours

Treponema pallidum 33 hours
Mycobacterium leprae 21 day
 Closed system
(periodic culture, batch culture)
Binary fission and other cell divisions processes
bring about an increase in number of cells in a
population. When microorganisms are cultured in liquid
medium, they are grown in a batch culture (periodic
culture) or closed system. They are incubated in a
closed culture with a single batch of medium.
Because no fresh medium is provided during incubation,
nutrient concentrations decline and concentration of
wastes increase. The growth of m/o reproducing by
binary fission can be plotted as the logarithm of the
number of viable cells. The resulting curve has four
distinct phases.
Microbial growth curve
in closed system
Lag phase (phase of adaptation)
When microorganisms are introduced into fresh
culture medium, usually no immediate increase in cell
numbers occurs. Initially there is the phase of adaptation
to nutrient media. A lag phase is significant for a variety
of reasons. The cells may be old and depleted of ATP,
essential cofactors, and ribosomes; these substances
must be synthesized before growth can begin. Also
bacteria synthesize all the necessary enzymes. The
biochemical activity is maximal. The number of
ribosomes increase intensively. Usual number of
ribosome inside one bacterium is 10000-18000 per
bacterium, but total count of ribosomes increase to
90000-100000 per cell at the end of the lag period.
 Lag phase (continuation)
If we compare prokaryotes & eukaryotes, we must
remember that 7-8 aminoacids are incorporated in
polypeptide chain during 1 second in eukaryotic cell, but
15-16 aminoacids are included into protein chain of
prokaryotic cell during the same period of time.
Bacterial cell don’t divide during lag phase and can
contain even number of nucleoids (2, 4, 8 or more
nucleoids).
The log phase varies considerably in length with the
condition of the m/o and the nature of the medium. If the
culture is old or from chemically different medium, the
lag phase is long. If the culture is young (from fresh
medium of the same composition), the lag phase is short
or absent.
 Log phase (exponential phase)
Microorganisms are growing and dividing at
the maximal rate possible given their genetic
potential, the nature of the medium, and the
conditions under which they are growing. The
rate of growth is constant during the log phase.
The m/o are dividing and doubling at regular
intervals. The population of m/o is most uniform
in terms of chemical and physioloical properties
during this phase. Exponential growth is
balanced growth.
 Log phase (exponential phase)
continuation
Balanced growth can be described mathematically as follows.
Initial number of bacteria is No.
Nt is the number of bacteria at time t.
The process of bacterial division can be described by a geometric
progression during this phase:
n
No= Nt x 2 ; where n is the number of total divisions.
n= (lg Nt – lg No) : lg 2
ʋ= (lg Nt – lg No) : lg 2(t - t0), where ʋ is the count of divisions
during 1 hour.
= 1 / ʋ.
Thus, generation time is g
Generation time means doubling time.
Generation time of each m/o in nature is usually much
longer than in closed culture.
 Log phase (exponential phase)
continuation

Bacterial cells are decreasing in size during

log phase. There are normal count of ribosomes
in bacterium, usually 10000 – 18000 per cell.
Bacterial cells are dividing intensively. Only one
nucleoid is present in each bacterium. Cell wall
is very thin and delicate in such bacteria. Thus,
the bacterial cells are very sensitive to
antibiotics, temperature & pH during this period.
Such microbial cells are very immunogenic,
consequently, they are widely used for
production of vaccines.
 Stationary phase
Microbial population enters the stationary phase for
several reasons. When bacteria grow to a certain
density, they either exhaust required nutrients, or they
accumulate toxic levels of metabolites. They may runof
carbon source, a required inorganic compound, or
essential aminoacids or vitamins. For aerobic bacteria
crowding leads to exhaustion of oxygen since it is poorly
soluble in water. Toxic metabolites may be hydrogen
peroxide for some anaerobes that lack catalase, or acid
formed by fermentation, which result in a pH to be too
low to be compared with growth. The maximal count of
viable microbial cell can be obtained during this
phase.
 Phase of decline (“death phase”)
Nutrient deprivation and buildup of toxic
wastes cause irreparable harm resulting in loss
of viability. Starving cells show an exponential
decline in density. Bacteria are destroyed by
their own enzymes (autolysis). Some bacteria
form as survival mechanism. Maybe “death
phase” is programmed cell death (Bacterial
population is genetically programmed to commit
suicide).
 Mathematics of growth
I. Total count
1) Collection of the samples of microbial cells
at different times & calculation of the number
of bacteria in each sample under a
microscope using a hamocytometer chamber.
2) Electronic particle analysis which detect
bacteria as tiny semiconducters in an electric
field.
3) Measurement of the turbidity of liquid
culture (microbial density in broth is
proportional to the number of bacteria).
 Mathematics of growth

I. Count of viable (living) bacteria

This number is determined by a colony count,
which is carried out by placing an appropriate
dilution on solid growth medium. Since
colonies arise from living bacteria, the
number of colonies multiplied by the dilution
factor is a number of colony-forming units
originally present.
Effects of temperature on growth
– Psychrophiles (Yersinia spp.) -- optimum
temp. typically 15 deg C or lower. Note:
some organisms are psychrotolerant --
optimum temperature is 20-40 deg, but can
grow as low as 0 deg. These are not
considered psychrophiles.
– Mesophiles (most bacterial spp.)-- optima
from 20-45 deg, minimum around 15-20
deg.
– Thermophiles -- optima 55 deg or higher.
Some (hyperthermophiles) have optima of
80 deg or higher (mostly Archaea in this
group). Found in hot springs, deep-sea
hydrothermal vents, other locations.
 Effects of oxygen on growth

• Obligate aerobes -- grow only when oxygen is

present (Neisseria meningitidis, Mycobacterium
tuberculosis, Corynebacterium diphtheriae)
• Facultative anaerobes -- grow with or without oxygen,
grow better in oxygen (respire) (family
Enterobacteriaceae)
• Aerotolerant anaerobes -- ignore oxygen, grow
equally well with or without (Lactobacillus spp.)
• Obligate anaerobes -- die in presence of oxygen
(Clostridium, Bacteroides, Peptococcus, Eubacterium)
• Microaerophiles -- won't grow at normal atmospheric
oxygen (20%), but require some oxygen for growth (2-
10%) (Campylobacter, Helicobacter, Brucella spp.)
Effects of pH on growth
• Acidophiles = acid pH optimal (1 to 5.5)
• Neutrophiles = pH 5.5 to 8 optimal
• Alkaliphiles = pH 8.0 to 11.5
• Extreme alkaliphiles = optimum pH 10 or
greater
• Note: most bacteria are neutrophiles
(Exceptions: some bact in hot springs have
optimum of 1-3)
• But most fungi prefer slight acid (pH 4 to 6)
• Saboraud's Medium (in lab this week) -- uses
low pH to stop bacterial growth, selective for
fungal growth.
 Metabolism
The total of all chemical reactions that occur
in the cell is called metabolism; almost all are
enzyme catalyzed. It is divided into two major
parts:
catabolism (energy conserving reactions that
release and conserve the energy provided by an
organisms energy source);
and anabolism (the reactions that consume
energy in order to built large, complex molecules
from smaller, simpler molecules)
Anabolism is the general term for the
synthesis of cell structures.
 Catabolism
In catabolism (energy-conserving reactions, or
fueling reactions), the energy provided to the cell
by its energy source is released and conserved
as ATP. Energy is necessary for three major
types of work:
chemical work ( synthesis of complex biological
molecules from much simple precursors);
transport work (across cell membranes against
the electrochemical gradient);
mechanical work (for cell motility and movement
of structures within cells).
 Definition of Frequently Used Terms
Carbon (C) plays the most significant role in
anabolism. All organisms are classified into:
1. Autotrophs: organisms that use CO2 as there sole or
principle sorce of carbon;
2. Heterotrophs: organisms that use reduced, preformed
organic molecules as there principle carbon source.
All m/o need such chemical elements as H, N, C, P,
S, and microelements (K, Ca, Na, Mg, Mn, Fe et al.) &
growth factors. Organic components that are essential
cell components or precursors of such components but
cannot be synthesized by the organism are called growth
factors. There are three main classes of growth factors:
(1) aminoacids; (2) purines and pyrimidines; and (3)
vitamins.
 Definition of Frequently Used Terms
Organisms that can’t ensure themselves by own
synthesis by all essential nutrients and need growth
factors from their surrounding are called auxotrophs;
they are said to be auxotrophic to the molecules they
cannot synthesize. If the organism can grow on the
minimal medium containing only salts (to supply
needed elements such as phosphorus) and carbon
source are called prototrophs.
By energy sources all m/o are divided into;
• Phototrophs use light as there energy sources;
• Chemotrophs obtain energy from oxidation of chemical
compounds (either organic or inorganic).
 Definition of Frequently Used Terms
By electron sources all m/o are classified into:
1. Lithotrophs use reduced inorganic substances as their
electron source;
2. Organotrophs extract electrons from reduced organic
components.

Most of known microorganisms are

chemoorganoheterotrops.
 Uptake of nutrients by microbial cell
• Passive diffusion ( such substances as glycerol, H2O,
O2, CO2).
• Facilitated diffusion is greatly increased diffusion
across selectively permeable membranes by using
carrier proteins (permeases). If concentration gradient
disappears, the process stops. Facilitated diffusion is
used for transport of sugars and aminoacids.
• Active transport is the transport of solute molecules
with the input of metabolic energy and involves
permeases (e.g., ATP-binding casette transporters) .
But solute molecules move across membrane without
modification. Active transport is used for transport of
sugars and aminoacids.
 Uptake of nutrients by microbial cell
continuation

4. Group translocation chemically modifies the molecule as

it is brought into the cell. The most famous group
translocation system is:
Phosphoenolpyruvate + sugar (outside the bacterium) ->
-> pyruvate + sugarphosphate (inside).
 Types of Catabolic Pathways

1. Fermentation (substrate-level
phosphorylation)

2. Aerobic respiration

3. Anaerobic respiration
 Central pathways of energy production
In most cases energy generation begins with
splitting of carbohydrates (sugars). There are three
main pathways of energy production in m/o:
• Glycolysis converts glucose into two three-carbon
molecules of important compound pyruvate (universal
metabolic substrate) and generates 2 molecules of
ATP. It’s typical for most bacteria.
• The hexose monophosphate shunt (known also as
pentose phosphate pathway) serves as the source of
peptose units and other carbohydrate components
and source of energy, especially NADPH.
• Entner-Doudoroff pathway leads to catabolic
conversion of sugars to pyruvate only by 4 reactions
and found in some G- (Pseudomonas & Azotobacter)
 Fermentation
Fermentation (substrate level phosphorylation) is an
energy-yielding process in which an organic molecule is
oxidized without an exogenous electron acceptor. In
fermentation electrons are extracted from a relatively
reduced organic compound and eventually end up on a
more oxidized organic molecule. Thus, organic
compounds serve as both ultimate electron donors and
acceptors. Fermentation is the major source of energy
for those organisms that survive only in the absence of
air (obligate anaerobes). Other fermentative organisms
that can grow in presence or absence of air (facultative
anaerobes) use fermentation as a source of energy
when energy when oxygen is absent. Most of the energy
of substrates remains untapped. Energy gain is very low.
 Types of fermentation by end products
• Lactic acid fermentation:
• Homofermentive: pyruvate ->lactic acid
(energy gain = 2 ATP). It’s typical for Bacillus,
Lactobacillus, Streptococcus)
• Heterofermentive. Products are lactic acid,
acetic, formic & other acids, possible ethanol,
other alcohols, & acetone. It’s typical for
Bifidobacterium.
2. Ethanol fermentation: pyruvate -> ethanol
(energy gain = 2 ATP). It’s typical for
Saccharomyces.
 Types of fermentation by end products
continuation

3. Propionic acid, acetic acid, CO2 & H2.

(Propionibacterium).
4. Formic acid, ethanol, lactic acid, butandiol,
CO2 & H2. (Enterobacteriaceae).
5. Butiric acid, butanol, acetone, isopropanol,
alcohol, CO2. (Clostridium).
 Aerobic respiration
Aerobic respiration is metabolic
process, in which molecules, often
organic, are oxidized with oxygen as the
terminal electron acceptor. The organic
substrate is often completely oxidized to
H2O and CO2 and a large amount of
energy is extracted. Much more energy is
released than with fermentation.
Practically aerobic respiration may be
divided into three stages:
1. Larger nutrient proteins (proteins, polysaccharides,
and lipids are hydrolyzed into their constituent parts.
That chemical reactions do not release much energy.
• Aminoacids, monosaccharides, fatty acids, glycerol,
and other products are degraded to a few simpler
molecules (usually pyruvate or acetyl coenzyme A).
The second stage produces some ATP as well as
NADH and/or FADH2.
• Partially oxidized carbon is fed into the tricarboxylic
acid cycle and oxidized completely to CO2 with the
production of ATP, NADH and FADH2. Most of the
ATP derived from aerobic respiration comes from the
oxidation NADH and FADH2 by electron transport
chain which uses O2 as final electron acceptor.
 glucose
2 ATP
2 ATP 2 ATP
(total 4 ATP) ethanol  2 pyruvate  lactate (total 4 ATP)

2 acetyl Co A

tricarboxylic acid cycle (Krebs cycle) total 38 ATP

 Respiratory chain
Respiration (oxidative phosphorylation) means
transport of electrons and protons along the chain of
specific carrier molecules with fixed positions, leading to
storage of energy. Simplified scheme
Substrate
 NAD ATP
 FAD
 Quinone
 b ATP
 c1
c
aa3 ATP
O2
 Electron transport chain carriers
• Nicotinamide adenine dinucleotide (NAD+) &
nicotinamide adenine dinucleotide phosphate
(NADP+);
• Flavin adenine dinucleotide (FAD) and flavin
mononucleotide (FMN) are flavoproteins;
• Ubiquinone = Coenzyme Q (Co Q);
• Cytochromes b, c, c1, aa3.
ATP is generated if the energy level between
carriers decreases on more than 0.2 volt. There are
only three such points inside respiratory chain (see
scheme).
But the presented scheme-staircase is typical for
eukaryotic cell. Prokaryotes have several distinctive
characteristics of their respiratory chains.
 Comparison of respiratory chains
of prokaryotes and eukaryotes
1. Electron transport chain of eukaryotes is located
along internal membrane of mitochondria, but
into cytoplasmic membrane and mesosomes in
prokaryotes.
2. The first acceptor of prokaryotic chain can differ
for prokaryotes.
3. The are a lot of different quinone carriers
instead of CoQ (menoquinone, naftoquinone) for
prokaryotes.
 Comparison of respiratory chains
of prokaryotes and eukaryotes
continuation

4. The are a lot of different terminal cytochromes instead of

aa3 for prokaryotes (a2, c4, c5, d, etc.).
5. Energy gain of respiratory chain in prokaryotes is lower.
6. Several different respiratory chains can exist in one
prokaryotic cell simultaneously (depends on conditions
of cultivation)
7. Anaerobic respiration can occur in prokaryotes.
 Anaerobic respiration
In anaerobic respiration electrons are
extracted from organic (and sometimes
inorganic sources) and donated to electron
transport chain acceptor - an inorganic molecule
that is not oxygen. There are several different
types of anaerobic respiration, but the most
common terminal electron accepters are nitrate,
sulfate and carbonate. Anaerobic respiration
typically extracts more energy than fermentation,
but less than aerobic respiration.
 Example of anaerobic respiration

Many facultative anaerobic bacteria can reduce

nitrates to nitrite under anaerobic conditions. And many
species of Bacillus and Pseudomonas are continue
reduction of nitrite to gaseous nitrogen:
NO3-  NO2-  NO  N2O  N2

The organisms that reduce sulphate and carbonate

are strictly anaerobic.