OVERVIEW OF MICROBIAL METABOLISM

Metabolism refers to the sum of all chemical reactions within a living organism. Chemical reactions
either release or require energy. Metabolism can be viewed as an energy-balancing act.

Catabolism – enzyme-regulated chemical reactions that release energy. Complex organic compounds
are broken down into simpler ones. These reactions are called catabolic or degradative reactions. They
are generally hydrolytic reactions (reactions that use water and in which chemical bonds are broken),
and they are exergonic (produce more energy than they consume). Ex. Cells break down sugars into CO2
and H2 O.

Anabolism – enzyme-regulated energy requiring reactions. The building of complex organic molecules
from simpler ones. These reactions are called anabolic or biosynthetic and they are generally dehydration
synthesis reactions (reactions that release water), and they are endergonic (consume more energy than
they produce). Ex. Formation of proteins from amino acids, nucleic acids from nucleotides,
polysaccharides from simple sugars)

These reactions generate the materials for growth. This coupling of energy requiring and energy-
releasing reactions is made possible through the molecule adenosine triphosphate (ATP). ATP stores
energy derived from catabolic reactions and releases it later to drive anabolic reactions and perform other
cellular work.

Fig. 1. ATP molecule

ATP – an adenine, a ribose and 3 phosphate groups. When terminal phosphate group is split from ATP,
ADP is formed, and energy is released to drive anabolic reactions.

ATP ADP + Pi + energy

Then, energy from catabolic reactions is used to combine ADP and a P to resynthesize ATP.
ADP + Pi + energy ATP
Anabolic reactions – coupled to ATP breakdown
Catabolic reactions – ATP synthesis.
ENZYMES:

• • Substances that can speed up a chemical reaction without being permanently altered themselves
are called catalysts.

• In living cells, enzymes serve as biological catalysts.

• Enzymes are specific and act on specific substances called the enzyme substrate(s) and each
catalyzes only one reaction.

Ex. Sucrose is the substrate of the enzyme sucrose, which catalyzes the hydrolysis of sucrose to glucose
and fructose.

Enzyme specificity and efficiency:

Enzymes are large globular proteins that range in MW from about 10,000 to several million. Each
enzyme has a characteristic three-dimensional shape with a specific surface configuration as a result of
its primary, secondary and tertiary structures. This enables it to find the correct substrate from among
the large number of diverse molecules in the cell. Enzymes are extremely efficient. Under optimum
conditions can catalyze reactions at rates 108 to 1010 times higher than those of comparable reactions
without enzymes. Turnover number (maximum number of substrate molecules an enzyme molecule
converts to product each second) is between 1 and 10,000 and can be high as 500,000.

Enzyme components:

• • Some enzymes consist entirely of proteins.

• Most consist of both a protein portion called an apoenzyme and a nonprotein component called
a cofactor.

• Ions of iron, zinc, magnesium or calcium are examples of cofactors. If the cofactor is an
organic molecule, it is called a coenzyme. organic cofactor
apoenzyme(protein compo of enzyme) + cofactor/Coenzyme = holoenzyme
• Together, the apoenzyme and cofactor form a holoenzyme, or whole active enzyme. If the
cofactor is removed, the apoenzyme will not function.

• Coenzymes assist the enzyme by accepting atoms removed from the substrate or by donating
atoms required by the substrate. Some act as electron carriers, removing electrons from the
substrate and donating them to other molecules in subsequent reactions.

• Many coenzymes are derived from vitamins. Two of the most important coenzymes in cellular
metabolism are

Nicotinamide adenine dinucleotide (NAD+)

Nicotinamide adenine dicucleotide phosphate (NADP+)
These contain derivatives of B vitamin nicotinic acid (niacin)

NAD+ - involved in catabolic (energy-yielding reactions)

 NADP+ - involved in anabolic (energy-requiring reactions)

• Flavin coenzymes, such as flavin mononucleotide (FMN) and flavin adenine dinucleotide
(FAD), contains derivatives of the B vitamin riboflavin and are also electron carriers.

• Coenzyme A (CoA) contains derivatives of pantothenic acid, another B vitamin. This plays an
important role in the synthesis and breakdown of fats and in a series of oxidizing reactions called
the Krebs cycle.

• Some cofactors are metal ions, including Fe, Cu, Mg, Mn, Zn, Ca and Co. They form a bridge
between the enzyme and a substrate. Ex. Mg2+ is required by many phosphorylating enzymes
(enzymes that transfer a phosphate group from ATP to another substrate).

Energy production:

There are two general aspects of energy production; the concept of oxidation-reduction and the
mechanism of ATP generation.

Oxidation – reduction reactions:

Oxidation – is removal of electron from an atom or molecule, a reaction that often produces energy.

Reduction – is addition of one or more electrons to an atom or molecule.

Oxidation and reductions reactions are always coupled. The pairing of these reactions is called oxidation-
reduction or redox reactions. Most biological oxidation reactions involve the loss of hydrogen atoms,
they are also dehydrogenation. Hydrogen atom contains both electrons and protons and in cellular
oxidations, electrons and protons are removed at the same time. An organic molecule is oxidized by the
loss of two hydrogen atoms, and a molecule of NAD+ is reduced by accepting two electrons and one
proton. One proton is left over and is released into the surrounding medium. The reduced coenzyme
contains more energy than NAD+. This energy can be used to generate ATP in later reactions.

Cells use oxidation- reduction (biological) reactions in catabolism to extract energy from nutrient
molecules. Ex. Cell oxidizes a molecule of glucose to CO2 and H2O. The energy in the glucose molecule
is removed in stepwise manner and ultimately is trapped by ATP, which can then serve as an energy
source for energy requiring reactions. Thus glucose is a valuable nutrient for organisms.

The generation of ATP:

Much of the energy released during oxidation – reduction reactions is trapped within the cell by the
formation of ATP. A phosphate group is added to ADP with the input of energy to form ATP. Addition
of a phosphate to a chemical compound is called phosphorylation. Organisms use three mechanisms of
phosphorylation to generate ATP from ADP.

Substrate – level phosphorylation: ATP is generated when a high energy phosphate is directly
transferred from a phosphorylated compound (a substrate) to ADP. Generally the phosphate has acquired
its energy during an earlier reaction in which the substrate itself was oxidized.

C-C-C~ P + ADP C-C-C + ATP

 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 O2 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. 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.

Photophosphorylation: Occurs only in photosynthetic cells which contain light-trapping pigments such
as chlorophylls. In photosynthesis, organic molecules, especially sugars, are synthesized with the energy
of light from the energy-poor building blocks CO2 and H2O. Photophosphorylation starts this process by
converting light energy to the chemical energy of ATP and NADPH, which in turn, are used to synthesize
organic molecules. As in oxidative phosphorylation, an electron transport chain is involved.

All microbial metabolisms can be arranged according to three principles:

1. How the organism obtains carbon for synthesizing cell mass?

• • autotrophic – carbon is obtained from carbon dioxide (CO2)

• heterotrophic – carbon is obtained from organic compounds

• mixotrophic – carbon is obtained from both organic compounds and by fixing carbon dioxide

2. How the organism obtains reducing equivalents used either in energy conservation or in
biosynthetic reactions:

• • lithotrophic – reducing equivalents are obtained from inorganic compounds

• organotrophic – reducing equivalents are obtained from organic compounds

3. How the organism obtains energy for living and growing:

• • chemotrophic – energy is obtained from external chemical compounds

• phototrophic – energy is obtained from light.

• chemolithoautotrophs obtain energy from the oxidation of inorganic compounds and carbon
E from chem compounds
Red Eq - inorganic comp
from the fixation of carbon dioxide. Examples: Nitrifying bacteria, Sulfur-oxidizing bacteria,
C - CO2
Iron-oxidizing bacteria, Knallgas-bacteria

• photolithoautotrophs obtain energy from light and carbon from the fixation of carbon
dioxide, using reducing equivalents from inorganic compounds. Examples: Cyanobacteria
(water (H2O) as reducing equivalent donor), Chlorobiaceae, Chromatiaceae (hydrogen sulfide
(H2S) as reducing equivalent donor), Chloroflexus (hydrogen (H2) as reducing equivalent
donor)

• chemolithoheterotrophs obtain energy from the oxidation of inorganic compounds, but

cannot fix carbon dioxide (CO2). Examples: some Thiobacilus, some Beggiatoa, some
 Nitrobacter spp., Wolinella (with H2 as reducing equivalent donor), some Knallgas-bacteria,
some sulfate-reducing bacteria

• chemoorganoheterotrophs obtain energy, carbon, and reducing equivalents for biosynthetic

reactions from organic compounds. Examples: most bacteria, e. g. Escherichia coli, Bacillus
spp., Actinobacteria

• photoorganoheterotrophs obtain energy from light, carbon and reducing equivalents for
biosynthetic reactions from organic compounds. Some species are strictly heterotrophic, many
others can also fix carbon dioxide and are mixotrophic. Examples: Rhodobacter,
Rhodopseudomonas, Rhodospirillum, Rhodomicrobium, Rhodocyclus, Helioba

DIVERSITY OF ELECTRON ACCEPTORS FOR RESPIRATION

Most microorganisms oxidize carbohydrates as their main source of cellular energy. Microorganisms
use two general processes: Cellular respiration and fermentation. Microorganisms also use anaerobic
pathway to oxidize glucose. In case of aerobic respiration, the ultimate e-acceptor is O2 and the reduced
form is H2O. There are four stages of aerobic respiration:

Oxygen extracts chemical energy from glucose, the glucose molecule must be split up into two
molecules of pyruvate. This process also generates two molecules of adenosine triphosphate as an
immediate energy yield and two molecules of NADH.

• • The citric acid cycle begins with the transfer of a two-carbon acetyl group from acetyl-CoA
to the four-carbon acceptor compound (oxaloacetate) to form a six-carbon compound (citrate).

• The citrate then goes through a series of chemical transformations, losing two carboxyl
groups as CO2. The carbons lost as CO2 originate from what was oxaloacetate, not directly
from acetyl-CoA. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon
backbone after the first turn of the citric acid cycle. Loss of the acetyl-CoA-donated carbons as
CO2 requires several turns of the citric acid cycle. However, because of the role of the citric
 acid cycle in anabolism, they may not be lost, since many TCA cycle intermediates are also
used as precursors for the biosynthesis of other molecules.

• Most of the energy made available by the oxidative steps of the cycle is transferred as energy-
rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle,
three molecules of NADH are produced.

• Electrons are also transferred to the electron acceptor Q, forming QH2.

• At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle
continues.

Anaerobic respiration - Microbes are capable of using all sorts of other terminal electron accepters
besides oxygen. A few examples of anaerobic respiration;

• Final electron acceptor is an inorganic substance other than O2.

• Some bacteria such as Pseudomonas and Bacillus can use a nitrate ion (NO-3), in the
presence of an enzyme called nitrate reductase, as a final electron acceptor, the nitrate ion is
reduced to nitrite ion (NO2-).

• Nitrite ion can be converted to nitrous oxide (N2O), or nitrogen gas (N2) (denitrification
process) which helps in recycling of nitrogen.

• Other bacteria like Desulfovibrio use sulfate (SO42-) as the final electron acceptor and forms
hydrogen sulfide (H2S).

• Still other bacteria use carbonate (CO32-) to form methane (CH4).

• Anaerobic respiration by bacteria using nitrate and sulfate as final electron acceptors is
essential for the nitrogen and sulfur cycles that occur in nature.

• Amount of ATP generated varies with the organisms and the pathway. Because only a part of
the Krebs cycle operates and since not all the carriers in the electron transport chain participate,
ATP yield is less and accordingly anaerobes tend to grow more slowly than aerobes.

MICROBIAL FERMENTATION

Fermentation is a specific type of heterotrophic metabolism that uses organic carbon instead of oxygen
as a terminal electron acceptor. This means that these organisms do not use an electron transport chain
to oxidize NADH to NAD+ and therefore must have an alternative method of using this reducing
power and maintaining a supply of NAD+ for the proper functioning of normal metabolic pathways
(e.g. glycolysis). As oxygen is not required, fermentative organisms are anaerobic.

Many organisms can use fermentation under anaerobic conditions and aerobic respiration when oxygen
is present. These organisms are facultative anaerobes. To avoid the overproduction of NADH,
obligatory fermentative organisms usually do not have a complete citric acid cycle. Instead of using an
ATP synthase as in respiration, ATP in fermentative organisms is produced by substrate-level
phosphorylation where a phosphate group is transferred from a high-energy organic compound to
ADP to form ATP. As a result of the need to produce high energy phosphate-containing organic
compounds (generally in the form of CoA -esters) fermentative organisms use NADH and other
cofactors to produce many different reduced metabolic by-products, often including hydrogen gas (H2).
These reduced organic compounds are generally small organic acids and alcohols derived from
pyruvate, the end product of glycolysis. Examples include ethanol, acetate, lactate, and butyrate.
Fermentative organisms are very important industrially and are used to make many different types of
food products. The different metabolic end products produced by each specific bacterial species are
responsible for the different tastes and properties of each food.

The two main types of fermentation are alcoholic fermentation and lactic acid fermentation (Fig.2).
The two main types of fermentation are:

1) Alcoholic fermentation
2) Lactic acid fermentation

Fig. 2. Lactic acid and ethanolic fermentations

Both types have the same reactants: Pyruvic acid and NADH, both of which are products of glycolysis.

In alcoholic fermentation, the major products are alcohol and carbon dioxide. In lactic acid
fermentation, the major product is lactic acid.
For both types of fermentation, there is a side product: NAD+ which is recycled back to glycolysis so
that small amounts of ATP can continue to be produced in the absence of oxygen.

The chemical equations below summarize the fermentation of sucrose , whose chemical formula is
C12H22O11. One mole of sucrose is converted into four moles of ethanol and four moles of carbon
dioxide :

C12H22O11+H2O + invertase →2C6H12O6

C6H12O6+Zymase → 2C2H5OH + 2CO2

The process of lactic acid fermentation using glucose is summarized below. In homolactic
fermentation, one molecule of glucose is converted to two molecules of lactic acid:[3]

C6H12O6 → 2CH3CHOHCOOH

In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose converted
to one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon dioxide:

C6H12O6→ CH3CHOHCOOH + C2H5OH + CO2

CARBOHYDRATE CATABOLISM

Most microorganisms oxidize carbohydrates as their primary source of cellular energy. Glucose is the
most common carbohydrate energy source used by cells. To produce energy from glucose
microorganisms use two general processes: cellular respiration and fermentation. Anaerobic respiration
is another mode where the final electron acceptor is an inorganic substance other than oxygen.

Catabolism/Oxidation of carbohydrates or Aerobic respiration of carbohydrates:

-- Most efficient way to extract energy from glucose. Occurs in three principal stages:

1. Glycolysis
2. Kreb Cycle
3. Electron transport chain

Glycolysis – Oxidation of glucose to pyruvic acid with the production of some ATP and energy
containing NADH.

Krebs cycle – Oxidation of acetyl (a derivative of pyruvic acid) to Co2, with the production of some
ATP, energy containing NADH, and another reduced electron carrier, FADH2.

Electron Transport chain – NADH and FADH2 are oxidized, contributing the electrons, they have
carried from the substrate to a ‘cascade' of oxidation-reduction reactions involving a series of
additional electron carriers. Energy from these reactions is used to generate a considerable amount of
ATP. In respiration, most of the ATP is generated in this step.
Fermentation: Initial stage is also glycolysis which produces pyruvic acid. But pyruvic acid is
converted into one or more different products, depending on the type of cell. These products might
include alcohol and lactic acid. Unlike respiration, there is no Krebs cycle or electron transport chain.
Accordingly, the ATP yield is also much lower.

Glycolysis Or Embden-Meyerhof (EMF) pathway:

In glycolysis (from the Greek glykys, meaning “sweet,”and lysis, meaning “splitting”), a molecule of
glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-
carbon compound pyruvate. During glycolysis NAD+ is reduced to NADH and there is a net production
of 2 ATP molecules by substrate level phosphorylation. Glycolysis does not require oxygen and can
occur whether present or not.

Reactions in glycolytic pathway

Glycolysis involves 10 enzymatic reactions, summarized in Figure 1:

1.
2. 1. The phosphorylation of glucose at position 6 by hexokinase,
2. The isomerization of glucose-6-phosphate to fructose-6-phosphate by phosphohexose
isomerase,

3. The phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate by

phosphofructokinase,

4. The cleavage of fructose-1,6-bisphosphate by aldolase. This yields two different products,

dihydroxyacetone phosphate and glyceraldehyde-3-phos phate,

5. The isomerization of dihydroxyacetone phosphate to a second molecule of glyceraldehyde

phosphate by triose phosphate isomerase,

6. The dehydrogenation and concomitant phosphorylation of glyceralde- hyde-3-phosphate to

1,3-bis-phosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase,

7. The transfer of the 1-phosphate group from 1,3-bis-phosphoglycerate to ADP by

phosphoglycerate kinase, which yields ATP and 3-phosphoglycerate,

8. The isomerization of 3-phosphoglycerate to 2-phosphoglycerate by phos phoglycerate

mutase,

9. The dehydration of 2-phosphoglycerate to phosphoenolpyruvate by eno lase.

10. The transfer of the phosphate group from phosphoenolpyruvate to ADP by pyruvate
kinase, to yield a second molecule of ATP.
 Fig. 3. Glycolysis. (Source, Lehninger, Principles of Biochemistry, Fifth Edition)

Overall reaction of glycolysis

Because 2 moleucles of ATP were needed to get glycolysis started and four molecules of ATP are
generated by the process, there is a net gain of two molecules of ATP for each molecule of glucose that
is oxidised.

Alternatives of Glycolysis:

Many bacteria have another pathway in addition to glycolysis for the oxidation of glucose. The most
common are i) pentose phosphate pathway and ii) Entner-Doudoroff pathway

1. Pentose Phosphate pathway (Hexose monophosphate shunt): This provides a means for the
breakdown of five-carbon sugars (pentoses) as well as glucose. A key feature is that it produces
important intermediates pentoses used in the synthesis of nucleic acids, glucose from Co2 in
photosynthesis and certain amino acids. The pathway is an important producer of the reduced
coenzyme NADPH from NADP+. This pathway yields a net gain of only one molecule of ATP for
each molecule of glucose oxidised. Bacteria that use this pathway include Bacillus subtilis, E.coli,
Leuconostoc mesenteroides and Enterococcus faecalis.

The Entner-Doudoroff pathway: For each molecule of glucose this pathway produces 2 molecules of
NADPH and one molecule of ATP for use in cellular biosynthetic reactions. Bacteria that have the
enzymes for this pathway can metabolize glucose without either glcolysis or the pentose phosphate
pathway. Found in some gram-negative bacteria, including Rhizobium , Pseudomonas and
Agrobacterium ; generally not found among gram-positive bacteria.

Cellular/Aerobic respiration

After glucose has been broken down to pyruvic acid, the pyruvic acid can be channeled into the next
step of either fermentation or cellular respiration.

Cellular respiration – is defined as an ATP generating process in which molecules are oxidized and
the final electron acceptor is an inorganic molecule. Two types of respiration occur, depending on
whether an organism is an aerobe or an anaerobe. In aerobic respiration – the final electron acceptor is
O2 and in anaerobic respiration – it is an inorganic molecule other than O2 or rarely an organic
molecule.

The Krebs cycle /Citric Acid Cycle/ Tricarboxylic Acid Cycle

The pyruvate produced by glycolysis is oxidized completely, generating additional ATP and NADH in
the citric acid cycle and by oxidative phosphorylation. However, this can occur only in the presence of
oxygen. Oxygen is toxic to organisms that are obligate anaerobes, and are not required by facultative
anaerobic organisms. In the absence of oxygen, one of the fermentation pathways occurs in order to
regenerate NAD+; lactic acid fermentation is one of these pathways.

In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion . Bacteria also use
the TCA cycle to generate energy, but since they lack mitochondria, the reaction sequence is
performed in the cytosol with the proton gradient for ATP production being across the plasma
membrane rather than the inner membrane of the mitochondrion

Fig. 4. Citric Acid cycle

•
• Pyruvic acid, the product of glycolysis, cannot enter the Krebs cycle directly. In a preparatory
step; it must lose one molecule of Co2 and become a two-carbon compound. This process is
called decarboxylation. The two carbon compound called an acetyl group, attaches to
Coenzyme a through a high-energy bond, the resulting complex is known as Acetyl Coenzyme
A. During this reaction, pyruvic acid is also oxidized and NAD+ is reduced to NADH.

• Oxidation of one glucose molecule produces 2 molecules of pyruvic acid, so for each
molecule of glucose, 2 molecules of Co2 are released in the preparatory step, 2 molecules of
NADH are produced, and 2 molecules of Acetyl Coenzyme A are formed.

• As Acetyl coenzyme A enters the Krebs cycle, CoA detaches from the acetyl group. The two
carbon acetyl group combines with a four carbon compound called oxaloacetic acid to form six
carbon compound, called citric acid. This synthesis reaction requires energy, which is provided
by the cleavage of the high energy bond between the acetyl group and CoA. The formation of
citric acid is the first step in the Krebs cycle.
 • Two decorboxylation reactions take place in the Krebs cycle while converting Isocitric acid
to α – Ketoglutaric acid and this to succinyl CoA.

• Altogether 3 decarboxylation reactions take place and hence all three carbon atoms in
pyruvic acid are eventually released as Co2 by the Krebs cycle. This represents the conversion
to Co2 by all 6 carbon atoms contained in the original glucose molecule.

• Oxidation-reduction reactions also occurs, where NAD+ and FAD picks up hydrogen atoms
to be reduced to NADH and FADH2.

• On the whole, for every two molecules of acetyl CoA that enter the cycle, 4 molecules of Co2
and 6 for pyruvic acid are liberated by decorboxylation, 6/8 moelucles of NADH and 2
moleucles of FADH2 are produced by oxidation-reduction reactions, and two molecules of ATP
are generated by substrate- level phosphorylation. Many of the intermediates in the Krebs cycle
also play a role in other pathways, especially in amino acid biosynthesis.

• Reduced coenzymes NADH and FADH2 are the important products of the Krebs cycle
because they contain most of the energy originally stored in glucose. During the next phase of
respiration, a series of reductions indirectly transfers the energy stored in those coenzymes to
ATP. These reactions are collectively called Electron transport chain.

The Electron Transport Chain:

• • Consists of a sequence of carrier molecules that are capable of oxidation and reduction.

• As electrons are passed through the chain, there is a stepwise release of energy, used to drive
the chemiosmotic generation of ATP.

• In eukaryotic cells, it is contained in the inner membrane of mitochondria.

• In prokaryotes, it is found in the plasma membrane.

Fig. 5. Electron Transport Chain

Three classes of carrier molecules are involved:

1. Flavoproteins – these contain flavin, a coenzyme derived from riboflavin (Vitamin B2). One
important flavin coenzyme is flavin mononucleotide (FMN).

2. Cytochromes – proteins with an iron-containing group capable of existing alternately as a reduced

form (Fe2+) and an oxidized form (Fe3+). The cytochormes include cytochrome b, C1, a, a3.

3. Ubiquinones or Coenzyme Q – these are small non-protein carriers.

• • Electron transport chains of bacteria are somewhat diverse, and the particular carriers and the
order in which they function may differ from those of other bacteria and from those of
eukaryotic mitochondrial systems. Much is known about the electron transport chain in the
mitochondria of eukaryotic cells.

1. Transfer of high energy electrons from NADH to FMN, the first carrier in the chain. This
transfer involves at the passage of a hydrogen atom with 2e- to FMN, which then pick up an
additional H+ from the surrounding aqueous medium. NADH is oxidised to NAD+ and FMN
reduced to FMNH2.

2. FMNH2 passes 2H+ to the other side of the mitochondrial membrane and passes 2e- to Q. As
a result FMNH2 is oxidized to FMN. Q picks up an additional 2H+ from the medium and
releases it on the other side of the membrane.

3. Electrons are passed successively from Q to Cyt b, cyt c1, cyt c, cyt a and cyt a3. Each
cytochrome in the chain is reduced as it picks up e-and is oxidised as it gives up electrons. The
last cyt a3 passes it electrons to molecular O2, which becomes negatively charged and then
picks up protons from the medium to form H2O.

• • FADH2 adds its electrons to the electron transport chain at a lower level than NADH.
Because of this, the electron transport chain produces about one-third less energy for ATP
generation when FADH2 donates electrons than when NADH is involved.

• FMN and Q accept and release protons as well as electrons and other carrier cytochromes
transfer only electrons.

• 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 build up of protons on one side of the membrane, which
provides energy for the generation of ATP by the chemiosmotic mechanism.

Chemiosmotic mechanism of ATP generation:

• • 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. In chemiosmosis, the energy released when a
substance moves along a gradient is used to synthesize ATP.
 1. As energetic electrons from NADH (or chlorophyll) 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. 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 one side of the membrane can diffuse across the membrane only
through special protein channels that contain an enzyme called adenosine triphosphate
(ATP synthase). When this flow occurs, energy is released and is used by the enzyme to
synthesize ATP from ADP and Pi.

• Electron transport chain also operates in photophosphorylation and is located in the thylakoid
membrane of cyanobacteria and eukaryotic chloroplasts.

Summary of Aerobic respiration:

• • Electron transport chain regenerates NAD+ and FAD+ which can be used again in glycolysis
and Krebs cycle.

• Various electron transfers in the electron transport chain generates about 34 molecules of
ATP from each molecule of glucose oxidized, 10 NADH and 2 FADH2 .

• A total of 38 ATP molecules can be generated from one molecule of glucose in prokaryotes.

• A total of 36 molecules of ATP are produced in eukaryotes. Some energy is lost when
electrons are shuttled across the mitochondrial membranes that separate glycolysis (in the
cytoplasm) from the electron transport chain. No such separation exists in prokaryotes.
Fig. 6. Generation of ATPs and NADH/FADH2 during Aerobic Respiration

ANAEROBIC RESPIRATION AND FERMENTATION

Anaerobic Respiration

Respiration in some prokaryotes is possible using electron acceptors other than oxygen (O2). This type
of respiration in the absence of oxygen is referred to as anaerobic respiration. Electron acceptors used
by prokaryotes for respiration or methanogenesis (an analogous type of energy generation in archaea
bacteria) are described in the table below.
Biological methanogenesis is the source of methane (natural gas) on the planet. Methane is preserved as
a fossil fuel (until we use it all up) because it is produced and stored under anaerobic conditions, and
oxygen is needed to oxidize the CH4 molecule.

Methanogenesis is not really a form of anaerobic respiration, but it is a type of energy-generating

metabolism that requires an outside electron acceptor in the form of CO2.

Sulfate reduction is not an alternative to the use of O2 as an electron acceptor. It is an obligatory process
that occurs only under anaerobic conditions. Methanogens and sulfate reducers may share habitat,
especially in the anaerobic sediments of eutrophic lakes such as Lake Mendota, where they crank out
methane and hydrogen sulfide at a surprising rate.

Nitrate reduction
Some microbes are capable of using nitrate as their terminal electron accepter. The ETS used is
somewhat similar to aerobic respiration, but the terminal electron transport protein donates its electrons
to nitrate instead of oxygen. Nitrate reduction in some species (the best studied being E. coli) is a two
electron transfer where nitrate is reduced to nitrite. Electrons flow through the quinone pool and the
cytochrome b/c1 complex and then nitrate reductase resulting in the transport of protons across the
membrane as discussed earlier for aerobic respiration.

Fig. 7. Nitrate reduction

Steps in the dissimilative reduction of nitrate

Some organisms, for example Escherichia coli , can carry out only the first step. All enzymes involved
are derepressed by anoxic conditions. Also, some prokaryotes are known that can reduce NO3- to NH4+
in dissimilative metabolism.

Denitrification

Denitrification is an important process in agriculture because it removes NO3 from the soil. NO3 is a
major source of nitrogen fertilizer in agriculture. Almost one-third the cost of some types of agriculture
is in nitrate fertilizers. The use of nitrate as a respiratory electron acceptor is usually an alternative to the
use of oxygen. Therefore, soil bacteria such as Pseudomonas and Bacillus will use O2 as an electron
acceptor if it is available, and disregard NO3. This is the rationale in maintaining well-aerated soils by
the agricultural practices of plowing and tilling. E. coli will utilize NO3 (as well as fumarate) as a
respiratory electron acceptor and so it may be able to continue to respire in the anaerobic intestinal
habitat.

Nitrite, the product of nitrate reduction, is still a highly oxidized molecule and can accept up to six more
electrons before being fully reduced to nitrogen gas. Microbes exist (Paracoccus species, Pseudomonas
stutzeri, Pseudomonas aeruginosa , and Rhodobacter sphaeroides are a few examples) that are able to
reduce nitrate all the way to nitrogen gas. The process is carefully regulated by the microbe since some
of the products of the reduction of nitrate to nitrogen gas are toxic to metabolism. This may explain the
large number of genes involved in the process and the limited number of bacteria that are capable of
denitrification. Below is the chemical equation for the reduction of nitrate to N2.

Denitrification takes eight electrons from metabolism and adds them to nitrate to form N2

Fig. 8. Denitification by Pseudomonas stutzeri

• Four terminal reductases involved in denitrification steps;

- Nar : Nitrate reductase (Mo-containing enzyme)
- Nir : Nitrite reductase
- Nor : Nitric oxide reductase
- N2 Or : Nitrous oxide reductase
• All can function independently but they operate in unison

Fermentation:
Fermentation is the process of extracting energy from the oxidation of organic compounds, such as
carbohydrates, using an endogenous electron acceptor, which is usually an organic compound. In
contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen,
via an electron transport chain. Fermentation is important in anaerobic conditions when there is no
oxidative phosphorylation to maintain the production of ATP (adenosine triphosphate) by glycolysis.

During fermentation, pyruvate is metabolised to various compounds. Homolactic fermentation is the

production of lactic acid from pyruvate; alcoholic fermentation is the conversion of pyruvate into
ethanol and carbon dioxide; and heterolactic fermentation is the production of lactic acid as well as
other acids and alcohols.

Fermentation does not necessarily have to be carried out in an anaerobic environment. For example,
even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative
phosphorylation, as long as sugars are readily available for consumption (a phenomenon known as the
Crabtree effect). Yeast cells preferring fermentaion over aerobic respiration despite readily available O2

Fig. 9. Respiration and Fermentation pathways

Lactic acid fermentation is the simplest type of fermentation. In essence, it is a redox reaction. In
anaerobic conditions, the cell's primary mechanism of ATP production is glycolysis. Glycolysis
reduces – transfers electrons to – NAD+, forming NADH. However there is a limited supply of NAD+
available in any given cell.
 • • For glycolysis to continue, NADH must be oxidized – have electrons taken away – to
regenerate the NAD+ that is used in glycolysis. In an aerobic environment (Oxygen is
available), reduction of NADH is usually done through an electron transport chain in a process
called oxidative phosphorylation; however, oxidative phosphorylation cannot occur in
anaerobic environments (Oxygen is not available) due to the pathways dependence on the
terminal electron acceptor of oxygen.

• Instead, the NADH donates its extra electrons to the pyruvate molecules formed during
glycolysis. Since the NADH has lost electrons, NAD+ regenerates and is again available for
glycolysis. Lactic acid, for which this process is named, is formed by the reduction of pyruvate.

In heterolactic acid fermentation, one molecule of pyruvate is converted to lactate; the other is
converted to ethanol and carbon dioxide.

In homolactic acid fermentation, both molecules of pyruvate are converted to lactate. Homolactic
acid fermentation is unique because it is one of the only respiration processes to not produce a gas as a
byproduct.

• • Homolactic fermentation breaks down the pyruvate into lactate. It occurs in the muscles of
animals when they need energy faster than the blood can supply oxygen.

• It also occurs in some kinds of bacteria (such as lactobacilli) and some fungi. It is this type
of bacteria that converts lactose into lactic acid in yogurt, giving it its sour taste. These lactic
acid bacteria can be classed as homofermentative, where the end-product is mostly lactate, or
heterofermentative, where some lactate is further metabolized and results in carbon dioxide,
acetate, or other metabolic products.

C6H12O6 -----→ 2CH3CHOHCOOH.

or one molecule of lactose and one molecule of water make four molecules of lactate (as in some
yogurts and cheeses):

C12H22O11+H2O ----→ 4CH3CHOHCOOH.

In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose being
converted to one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon dioxide:

C6H 12O6 -------→ CH3CHOHCOOH + C2H5OH + CO2

Before lactic acid fermentation can occur, the molecule of glucose must be split into two molecules of
pyruvate. This process is called glycolysis.
 Fig. 10. Fate of pyruvate in Fermentation

Mixed fermentations

Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition, 2,3 butanediol
from the condensation of 2 pyruvate. The use of the pathway decreases acid formation (butanediol is
neutral) and causes the formation of a distinctive intermediate, acetoin. Water microbiologists have
specific tests to detect low acid and acetoin in order to distinguish non fecal enteric bacteria
(butanediol formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid fermenters,
such as E. coli, Salmonella and Shigella).

Butyric acid fermentations, as well as the butanol-acetone fermentation (below), are run by the
clostridia, the masters of fermentation. In addition to butyric acid, the clostridia form acetic acid, CO2
and H2 from the fermentation of sugars. Small amounts of ethanol and isopropanol may also be
formed.

Butanol-acetone fermentation. Butanol and acetone were discovered as the main end products of
fermentation by Clostridium acetobutylicum during the World War I. This discovery solved a critical
problem of explosives manufacture (acetone is required in the manufacture gunpowder) and is said to
have affected the outcome of the War. Acetone was distilled from the fermentation liquor of
Clostridium acetobutylicum, which worked out pretty good if you were on our side, because organic
chemists hadn't figured out how to synthesize it chemically. You can't run a war without gunpowder, at
least you couldn't in those days.

Propionic acid fermentation . This is an unusual fermentation carried out by the propionic acid
bacteria which include corynebacteria, Propionibacterium and Bifidobacterium . Although sugars can
be fermented straight through to propionate, propionic acid bacteria will ferment lactate (the end
product of lactic acid fermentation) to acetic acid, CO2 and propionic acid. The formation of
propionate is a complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of lactate are
converted to 2 moles of propionate + 1 mole of acetate + 1 mole of CO2, and 1 mole of ATP is
squeezed out in the process. The propionic acid bacteria are used in the manufacture of Swiss cheese,
which is distinguished by the distinct flavor of propionate and acetate, and holes caused by entrapment
of CO2.