Microbial physiology Unit 2
Bacterial Respiration

• When pyruvate is oxidized to CO2, a far higher
yield of ATP is possible.
• Oxidation using O2 as the terminal electron
acceptor is called aerobic respiration;
oxidation using other acceptors under anoxic
conditions is called anaerobic respiration.

Oxidation-Reduction Reactions
• Oxidation is the removal of electrons (e-) from an atom or molecule, a
reaction that often produces energy.
• An example of an oxidation in which molecule A loses an electron to
molecule B. Molecule A has undergone oxidation (meaning that it has lost
one or more electrons), whereas molecule B has undergone reduction
(meaning that it has gained one or more electrons).
• Oxidation and reduction reactions are always coupled; in other words,
each time one substance is oxidized, another is simultaneously reduced.
The pairing of these reactions is called oxidation-reduction or a redox
reaction.
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The Generation of ATP
• Much of the energy released during oxidation-
reduction reactions is trapped within the cell
by the formation of ATP. Specifically, a
phosphate group, is added to ADP with the
input of energy to form ATP:
2

• The symbol ~ designates a "high-energy" bond-
that is, one that can readily be broken to release
usable energy.
• The high-energy bond that attaches the third P in
a sense contains the energy stored in this
reaction.
• When this P is removed, usable energy is
released. The addition of P to a chemical
compound is called phosphorylation.
• Organisms use three mechanisms of
phosphorylation to generate ATP from ADP.

Substrate-level Phosphorylation
• In substrate-level phosphorylation. ATP is
usually generated when a high-energy P is
directly transferred from a phosphorylated
compound (a substrate) to ADP.
• Generally, the P has acquired its energy during
an earlier reaction in which the substrate itself
was oxidized.
• The following example shows only the carbon
skeleton and the P of a typical substrate:

Oxidative Phosphorylation
• In oxidative phosphorylation, electrons are transferred from
organic compounds to one group of electron carriers (usually to
NAD+ and FAD). Then, the electrons are passed through a series
of different electron carriers to molecules of oxygen (02) or other
oxidized inorganic and organic molecules.
• This process occurs in the plasma membrane of prokaryotes and
in the inner mitochondrial membrane of eukaryotes.
• The sequence of electron carriers used in oxidative
phosphorylation is called an electron transport chain (system).
• The transfer of electrons from one electron carrier to the next
releases energy, some of which is used to generate ATP from
ADP through a process called chemiosmosis.

Cellular Respiration
• After glucose has been broken down to pyruvic
acid, the pyruvic acid can be channeled into the
next step Cellular respiration or simply
respiration;
• Defined as an ATP-generating process in which
molecules are oxidized and the final electron
acceptor is (almost always) an inorganic
molecule.
• An essential feature of respiration is the
operation of an electron transport chain.

Aerobic Respiration
• An electron transport chain (system) consists of
a sequence of carrier molecules that are capable
of oxidation and reduction.
• As electrons are passed through the chain, there
occurs a step vise release of energy, which is used
to drive the chemiosmotic generation of ATP.
• The final oxidation is irreversible.
• In prokaryotic cells, the electron transport chain
is contained in the plasma membrane.

Carrier molecules in ETS
• There are three classes of carrier molecules in electron transport
chains.
• The first are Flavoproteins.
• These proteins contain flavin, a coenzyme derived from riboflavin
(vitamin B2) and are capable of performing alternating oxidations
and reductions. One important flavin coenzyme is flavin
mononucleotide (FMN).
• The second class of carrier molecules are cytochromes, proteins
with an iron-containing group (heme) capable of existing alternately
as a reduced form (Fe+2) and an oxidized form (Fe+3).
• The cytochromes involved in electron transport chains include
cytochrome b (cyt b), cytochrome c1 (cyt c1), cytochrome c (cyt c) ,
cytochrome a (cyt a), and cytochrome a (cyt a3).
• The third class is known as ubiquinones, or coenzyme Q.
symbolized Q; these are small nonprotein carriers.

An electron transport chain
3

Steps involved
• The first step in the mitochondrial electron transport chain involves
the transfer of high-energy electrons from NADH to FMN, the first
carrier in the chain.
• This transfer actually involves the passage of a hydrogen atom with
two electrons to FMN, which then picks up an additional H+ from
the surrounding aqueous medium.
• As a result of the first transfer, NADH is oxidized to NAD+, and FMN
is reduced to FMNH2.
• In the second step in the electron transport chain, FMNH2 passes
2H+ to the other side of the mitochondrial membrane and passes
two electrons to Q.
• As a result, FMNH2 is oxidized to FMN. Q also picks up an additional
2H+ from the surrounding aqueous medium and releases it on the
other side of the membrane.

• The next part of the electron transport chain involves
the cytochromes.
• Electrons are passed successively from Q to
cytochrome b (cyt b), cytochrome c1 (cyt c1),
cytochrome c (cyt c) , cytochrome a (cyt a), and
cytochrome a (cyt a3).
• Each cytochrome in the chain is reduced as it picks up
electrons and is oxidized as it gives up electrons.
• The last cytochrome, cyt a3, passes its electrons to
molecular oxygen (02), which becomes negatively
charged and then picks up protons from the
surrounding medium to form H20.

• An important feature of the electron transport chain is the
presence of some carriers, such as FMN and Q, that accept
and release protons as well as electrons, and other carriers,
such as cytochromes, that transfer electrons only.
• Electron flow down the chain is accompanied at several
points by the active transport (pumping) of protons from
the matrix side of the inner mitochondrial membrane to
the opposite side of the membrane.
• The result is a buildup of protons on one side of the
membrane. Just as water behind a dam stores energy that
can be used to generate electricity, this build up of protons
provides energy for the generation of ATP by the
chemiosmotic mechanism.

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The Chemiosmotic Mechanism of
ATP Generation
• The mechanism of ATP synthesis using the electron
transport chain is called chemiosmosis.
• Substances diffuse passively across membranes from areas
of high concentration to areas of low concentration; this
diffusion yields energy.
• And the movement of substances against such a
concentration gradient requires energy and that, in such an
active transport of molecules or ions across biological
membranes, the required energy is usually provided by ATP.
• In chemiosmosis, the energy released when a substance
moves along a gradient is used to synthesize ATP. The
"substance" in this case refers to protons.

The steps of chemiosmosis
1. As energetic electrons from NADH pass down the electron transport
chain, some of the carriers in the chain pump- actively transport-
protons across the membrane. Such carrier molecules are called proton
pumps.
2. The phospholipid membrane is normally impermeable to protons, so this
one-directional pumping establishes a proton gradient (a difference in
the concentrations of protons on the two sides of the membrane). In
addition to a concentration gradient, there is an electrical charge
gradient. The excess H+ on one side of the membrane makes that side
positively charged compared with the other side. The resulting
electrochemical gradient has potential energy, called the proton motive
force.
3. The protons on the side of the membrane with the higher proton
concentration can diffuse across the membrane only through special
protein channels that contain an enzyme called ATP synthase. When this
flow occurs, energy is released and is used by the enzyme to synthesize
ATP from ADP and Pi.

5

A Summary of Aerobic Respiration
6

A summary of aerobic respiration
in prokaryotes. Glucose is broken
down completely to carbon dioxide
and water, and ATP is generated.
This process has three major phases:
glycolysis, the Krebs cycle, and the
electron transport chain. The
preparatory step is between
glycolysis and the Krebs cycle. The
key event in aerobic respiration is
that electrons are picked up from
intermediates of glycolysis and the
Krebs cycle by NAD+ or FAD and
are carried by NADH or FADH2 to the
electron transport chain. NADH is
also produced during the conversion
of pyruvic acid to acetyl CoA Most of
the ATP generated by aerobic
respiration is made by the
chemiosmotic mechanism during the
electron transport chain phase: this
is called oxidative phosphorylation.
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inhibition of electron
transport chain
Electron transport inhibitors:
• Rotenone
• Antimycin
• Cyanide
• Malonate
• Carbon monoxide (CO)
• Oligomycin (inhibitor of oxidative
phosphorylation)

Compounds
Use
Effect on oxidative
phosphorylation
Cyanide
Carbon monoxide
Azide
Hydrogen sulfide
Poisons Inhibit the electron transport chain by
binding more strongly than oxygen to
the Fe–Cu center in cytochrome c
oxidase, preventing the reduction of
oxygen.
Oligomycin Antibiotic Inhibits ATP synthase by blocking the
flow of protons through the Fo
subunit.
CCCP
2,4-Dinitrophenol
Poisons This ionophore uncouples proton
pumping from ATP synthesis because
it carries protons across the inner
mitochondrial membrane
Rotenone Pesticide Prevents the transfer of electrons
from complex I to ubiquinone by
blocking the ubiquinone-binding site.
Malonate and oxaloacetate Poisons Competitive inhibitors of succinate
dehydrogenase
Antimycin A Piscicide Binds to the Qi site of cytochrome c
reductase, thereby inhibiting the
oxidation of ubiquinol.

heterotrophic and
chemolithotrophic bacteria
• Organisms able to use inorganic chemicals as
electron donors are called chemolithotrophs.
• Examples of relevant inorganic electron donors
include H2S, hydrogen gas (H2), Fe2+, and NH3.
• Chemolithotrophic metabolism is typically
aerobic and begins with the oxidation of the
inorganic electron donor.
• Electrons from the inorganic donor enter an
electron transport chain and a proton motive
force is formed inexactly the same way as for
chemoorganotrophs.

• However, one important distinction between chemolithotrophs and
chemoorganotrophs, besides their electron donors, is their source
of carbon for biosynthesis.
• Chemoorganotrophs use organic compounds (glucose, acetate, and
the like) as carbon sources. By contrast, chemolithotrophs use
carbon dioxide (CO2) as a carbon source and are therefore
autotrophs.
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Sources of Energy for Microorganisms
9

The Three Stages of Catabolism. A
general diagram of aerobic
catabolism in a
chemoorganoheterotroph showing
the three stages in this process
and the central position of the
tricarboxylic
acid cycle. Although there are
many different proteins,
polysaccharides, and lipids, they
are degraded through the
activity of a few common
metabolic pathways. The dashed
lines show the flow of electrons,
carried by NADH
and FADH2, to the electron
transport chain.
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Anaerobic Respiration
• Electrons derived from sugars and other organic
molecules are usually donated either to endogenous
organic electron acceptors or to molecular O2 by way
of an electron transport chain.
• However, many bacteria have electron transport chains
that can operate with exogenous electron acceptors
other than O2. This energy-yielding process is called
anaerobic respiration.
• The major electron acceptors are nitrate, sulfate, and
CO2, but metals and a few organic molecules can also
be reduced.

• Some bacteria can use nitrate as the electron
acceptor at the end of their electron transport
chain and still produce ATP.
• Often this process is called dissimilatory
nitrate reduction.
• Nitrate may be reduced to nitrite by nitrate
reductase, which replaces cytochrome
oxidase.

• However, reduction of nitrate to nitrite is not a particularly
effective way of making ATP, because a large amount of
nitrate is required for growth.
• The nitrite formed is also quite toxic. Therefore nitrate often is
further reduced all the way to nitrogen gas, a process known
as denitrification. Each nitrate will then accept five electrons,
and the product will be nontoxic.
• There is considerable evidence that denitrification is a
multistep process with four enzymes participating: nitrate
reductase, nitrite reductase, nitric oxide reductase, and
nitrous oxide reductase.

• Interestingly, one of the intermediates is nitric oxide (NO). In mammals this
molecule acts as a neurotransmitter, helps regulate blood pressure, and is used by
macrophages to destroy bacteria and tumor cells.
• Two types of bacterial nitrite reductases catalyze the formation of NO in bacteria.
One contains cytochromes c and d1 (e.g., Paracoccus and Pseudomonas
aeruginosa), and the other is a copper protein (e.g., Alcaligenes).
• Nitrite reductase seems to be periplasmic in gram-negative bacteria.
• Nitric oxide reductase catalyzes the formation of nitrous oxide from NO and is a
membrane-bound cytochrome bc complex.
• A well studied example of denitrification is gram-negative soil bacterium
Paracoccus denitrificans, which reduces nitrate to N2 anaerobically.
• Its chain contains membrane-bound nitrate reductase and nitric oxide reductase,
whereas nitrite reductase and nitrous oxide reductase are periplasmic.
• The four enzymes use electrons from coenzyme Q and c-type cytochromes to
reduce nitrate and generate PMF.

• Denitrification is carried out by some members of
the genera Pseudomonas, Paracoccus, and
Bacillus. They use this route as an alternative to
normal aerobic respiration and may be
considered facultative anaerobes.
• If O2 is present, these bacteria use aerobic
respiration (the synthesis of nitrate reductase is
repressed by O2).
• Denitrification in anaerobic soil results in the loss
of soil nitrogen and adversely affects soil fertility.

• Two other major groups of bacteria employing
anaerobic respiration are obligate anaerobes.
• Those using CO2 or carbonate as a terminal
electron acceptor are called methanogens
because they reduce CO2 to methane.
• Sulfate also can act as the final acceptor in
bacteria such as Desulfovibrio. It is reduced to
sulfide (S2- or H2S), and eight electrons are
accepted.

• Anaerobic respiration is not as efficient in ATP
synthesis as aerobic respiration—that is, not as
much ATP is produced by oxidative
phosphorylation with nitrate, sulfate, or CO2 as
the terminal acceptors.
• Reduction in ATP yield arises from the fact that
these alternate electron acceptors have less
positive reduction potentials than O2.
• The reduction potential difference between a
donor like NADH and nitrate is smaller than the
difference between NADH and O2.

• Because energy yield is directly related to the
magnitude of the reduction potential difference,
less energy is available to make ATP in anaerobic
respiration.
• Nevertheless, anaerobic respiration is useful
because it is more efficient than fermentation
and allows ATP synthesis by electron transport
and oxidative phosphorylation in the absence of
O2.
• Anaerobic respiration is very prevalent in oxygen-
depleted soils and sediments.

• Often one will see a succession of microorganisms in an
environment when several electron acceptors are present.
• For example, if O2, nitrate, manganese ion, ferric ion,
sulfate, and CO2 are available in a particular environment, a
predictable sequence of oxidant use takes place when an
oxidizable substrate is available to the microbial population.
• Oxygen is employed as an electron acceptor first because it
inhibits nitrate use by microorganisms capable of
respiration with either O2 or nitrate.
• While O2 is available, sulfate reducers and methanogens
are inhibited because these groups are obligate anaerobes.

• Once the O2 and nitrate are exhausted, competition for use
of other oxidants begins.
• Manganese and iron will be used first, followed by
competition between sulfate reducers and methanogens.
• This competition is influenced by the greater energy yield
obtained with sulfate as an electron acceptor. The sulfate
reducer Desulfovibrio grows rapidly and uses the available
hydrogen at a faster rate than Methanobacterium.
• When the sulfate is exhausted, Desulfovibrio no longer
oxidizes hydrogen, and the hydrogen concentration rises.
• The methanogens finally dominate the habitat and reduce
CO2 to methane.

References
• Reading
• Brock biology of
microorgamism (13th
edition) by Madigan,
Martinko, Stahl, Clark
• Microbiology (10th
edition) by Tortora,
Funke and Case
• Microbiology (5th
edition) by Prescott
• Images
• 1-10: Microbiology (10th
edition) by Tortora,
Funke and Case