Chapter 4 - Metabolisms PDF

Metabolism of Microorganisms
1. Introduction
2. Oxidation-reduction & energy-rich compounds
 Electron donors & electron acceptors
 Energy-rich compounds & energy storage
3. Essentials of catabolism
 Glycolysis
 Cellular respiration & Krebs cycle
 Electron transport chain
 Catabolic diversity
>> CO3: Describe cell growth, and major metabolic pathways such as
glycolysis and citric acid cycle.
Overview
Energy source? Carbon source?

Free Energy
Chemical Organic
Light ATP Inorganic
Photosynthesis Cellular respiration Fermentation
O2 Glycolysis No O2 Glycolysis
Krebs cycle
Electron
transport chain
Dept of Chem Eng, UM Dr. Adeline Chua S.M. 2

Introduction
 A major challenge in bioprocess development is to select an organism that can
efficiently make a product.
 A bioprocess developer must understand the metabolic capabilities of the

organism either to use them directly or to know how to metabolically engineer
them to make a desired, perhaps novel, product.
 Thus, knowledge about some essential metabolisms is necessary.
 What causes the differences in microbial metabolism?

 ?
 ?
 The control of metabolic pathways by nutritional and environmental regulation

has become an important consideration in bioprocess engineering.

Metabolism
 Before a cell can replicate, it must coordinate many different chemical
reactions and organize many different molecules into specific structures.
Collectively, these reactions are called metabolism.
 Metabolic reactions are either catabolic, which means energy releasing,

or anabolic, which means energy requiring.
 Catabolism breaks down the molecular structures of substrates,

releasing energy in the process.
 Anabolism uses energy to build larger molecules from smaller ones.
 The process of metabolism takes place via sequences of consecutive,

enzyme-catalyzed reactions, which is called metabolic pathway.

Oxidation-Reduction & Energy-Rich Compounds
 Redox reactions are common in cellular biochemistry. The energy
released during metabolism is conserved in cells by the simultaneous
synthesis of energy-rich compounds, such as ATP.
 We will look at:

 Electron donors & e- acceptors
 Redox reactions
 Reduction potential and redox tower
 Electron carriers
 Energy-rich compounds
 ATP
 Energy storage

Redox reactions
 An oxidation is the removal of electron(s) from a substance, and a
reduction is the addition of electron(s) to a substance.
 Redox reactions occur in pairs. E.g. hydrogen gas (H2) can release
electrons and protons and become oxidized.
 However, electrons cannot exist alone in solution; they must be part
of atoms or molecules. The reaction is only a half reaction.
 The oxidation of H2 can be coupled to the reduction of many
different substances, including O2, in a second half reaction.
 The substance oxidized (H2) is the e- donor.
 The substance reduced (O2) is the e- acceptor.
 This concept is very important in energy metabolism.

Reduction potential
 Substances differ in their tendency to be e- donors or e-

acceptors. This tendency is expressed as their reduction
potential (E0’), measured in volts (V).
 A substance can be either an e- donor or an e- acceptor under

different circumstances, depending on the substances with which
it reacts.
 The redox tower represents the range of reduction potentials

possible for redox couples in nature.
 By convention, E0’ are given for half reactions written as

reductions, with reactions at pH 7 because the cytoplasm of most
cells is neutral, or nearly so.
Strongest
Redox tower e donor
 The constituents on each side in the

half reactions are called a redox
couple. E.g. ?
 By convention, when writing a redox

couple, the oxidized form of the couple
is always placed on the left, before the
forward slash, followed by the reduced
form after the forward slash.
 The reduced substance in the redox Strongest

couple at the top of the tower (E0’ more e acceptor
negative) has the greatest tendency to
donate e-, whereas the oxidized
substance in the redox couple at the
bottom of the tower (E0’ more positive)
has the greatest tendency to accept e-.
 Example?

Redox tower

Redox Tower and its relationship to G0’
 The difference in E0’ between the donor and acceptor redox couples is
expressed as E0’.
 The further the e- drop from a donor before they are caught by an
acceptor, the greater the amount of energy released, that is, E0’ is
proportional to G0’.
 Oxygen, at the bottom of the tower, is the strongest e- acceptor of any

significance in nature. What about those in the middle of the tower?
 Many potential e- donors exist in nature, including a wide variety of

organic and inorganic compounds.
 E- donors used in energy metabolism are also call energy sources

because energy is released when they are oxidized.

Electron carriers
 Electron (e-) and/or proton (H+) released in redox reactions in microbial
cells are carried by nucleotide derivatives such as:
 Coenzyme nicotinamide adenine dinucleotide (NAD+).
 NAD+ and NADH is a coenzyme found in all living cells:

 Consists of two nucleotides joined through their phosphate groups:
with one nucleotide containing an adenine base, and the other
containing nicotinamide.
 Another coenzyme is NADP+/NADPH.

NAD+/NADH

Adenosine triphosphate (ATP)
 Energy released from redox reactions must be conserved by the cell
if it is to be used later to drive energy-requiring cell functions.
 In living organisms, chemical energy released in redox reactions is

conserved primarily in phosphorylated compounds.
 The most important energy-rich phosphate compounds in cells is

adenosine triphosphate (ATP).
 ATP consists of the ribonucleoside adenosine to which three phosphate

molecules are bonded in series.
 ATP is the prime energy currency in all cells, being generated during
exergonic reactions and consumed in endergonic reactions.

ATP
 Phosphoester bond joins the first phosphoryl group to the 5-carbon

sugar ribose
 Second and third groups are joined by phosphoanhydride bonds

(high-energy bonds).

Energy storage (important for industrial applications)
 ATP is a dynamic molecule in the cell; it is continuously being broken down to
drive anabolic reactions and resynthesized from catabolic reactions. For longer-
term energy storage, microorganisms produce insoluble polymers that can be
catabolized later for the production of ATP.
 Examples of energy storage polymers;

 In prokaryotes include glycogen, polyhydroxyalkanoates (PHA),
polyphosphate and elemental sulfur (stored from the oxidation of H2S by
sulfur chemolithotrophs). These polymers are deposited within the cell as
large granules (cell inclusions) that can be seen with the light or electron
microscope.
 In eukaryotes, polyglucose in the form of starch, and lipids in the form of fats
are the major reserve materials.
 In the absence of an external energy source, a cell can break down these
polymers to make new cell materials or to supply the very low amount of energy,
called maintenance energy, needed to maintain cell function when it is in a non-
growing state.
Catabolism
- Fermentation
- Cellular Respiration
Catabolism
 Fermentation and cellular respiration – are two important catabolisms.
 Fermentation is the form of anaerobic catabolism in which an organic

compound is both an electron donor and an electron acceptor, and ATP is
produced by substrate-level phosphorylation.
 Respiration is the catabolism in which a compound is oxidized (electron donor)

with O2 (or an O2 substitute) as the terminal electron acceptor, usually
accompanied by ATP production by oxidative phosphorylation.
 They are alternative metabolic choices available to some microorganisms. Some

organisms can both ferment and respire, e.g. yeast. Please read an article about
“Yeast Fermentation” posted at Reading corner, Spectrum.
 Many microbial habitats lack O2 or other electron acceptors, thus in such habitats,
fermentation is the only option for energy conservation by chemoorganotrophs.

Fermentation
 In fermentation, ATP is produced by a mechanism called substrate-level
phosphorylation. In this process, ATP is synthesized directly from energy-rich
intermediates during steps in the catabolism of the fermentable substrate.
 The fermentable substrate in a fermentation is both the e- donor and e- acceptor.
 Not all compounds can be fermented, but sugars, especially hexoses such as
glucose, are excellent fermentable substrates.
 Involved two main steps:

 First step is glycolysis, also called the Embden-Meyerhof-Parnas (EMP)
pathway. Glycolysis results in the breakdown of glucose to two pyruvate
molecules. A small amount of energy in the form of ATP and NADH is
generated during glycolysis.
 Second step is formation of fermentation products.

Reaction stages in fermentation
 Glycolysis occurs in two stages:
 Stage I comprises ‘preparatory” reactions; these are not redox reactions and
do not release energy but instead lead to the production of a key
intermediate of the pathway.
 In Stage II, redox reactions occur, energy is conserved in the form of ATP,
and two molecules of pyruvate are formed. The reactions of glycolysis are
finished at this point. However, redox balance has not yet been achieved.
 In Stage III, redox reactions occur once again and fermentation

products are formed.

First Five Reactions of Glycolysis – Investing Energy
 Net reactions:
Glucose + 2 ATP  2 glyceraldehyde-3-phosphate + 2 ADP
 A 6-carbon substrate (glucose) is split into 2 molecules of a

3-carbon metabolite (what is the name?).
 For each glucose entering glycolysis, 2 ATP molecules are

consumed for activation purposes during investment stage.

Last Five Reactions of Glycolysis – Obtaining Energy
 Net reactions: write yourself (refer Table 15.1)
 Transform each 3-carbon metabolite (glyceraldehyde-3-

phosphate) to another 3-carbon metabolite, pyruvate.
 Here, we recover the initial investment of 2 ATPs plus a
dividend of 2 more ATPs.
 In addition, 2 NADH are generated.

Fermentation (first step – glycolysis)
Glucose
e-
NAD+
ATP Glycolysis
Pyruvate Pyruvate NADH

We have a problem here!
Glucose
e-
Depletion of
ATP Glycolysis X
NAD+ NAD+ 
Glycolysis stops
To solve the problem, pyruvate needs to be

reduced to fermentation products to recycle
NADH back to NAD+ so that glycolysis can
proceeds
Fermentation products

Second step to complete fermentation – lactic
acid as end product
Glucose
e-
ATP Glycolysis X
NAD+

e-
Lactate Lactate
NAD+

Second step to complete fermentation – ethyl
alcohol as end product
Glucose
e-
ATP Glycolysis X
NAD+

e-
CO2
Ethanol NAD+

Industrial fermentation products
Yoghurt (lactic acid)
Bioethanol from yeast from milk fermentation
fermentation of corn syrup

Cellular Respiration
 If O2 are present, pyruvate can be oxidized to CO2 instead of being
reduced to fermentation products and excreted. When pyruvate is
oxidized to CO2, a far higher yield of ATP is possible.
 Oxidation of substrate such as glucose using O2 as the final/terminal e-

acceptors is called cellular respiration.
 It consists of 3 basic steps:

 Glycolysis
 Krebs cycle/Citric acid cycle/Tricarboxylic acid cycle (TCA cycle)
 Electorn transport chain
 In cellular respiration, a total of 36-38 ATP is produced – much higher

than that in fermentation.

Respiration (Glycolysis → → )
Glucose
NADH
Glycolysis
Pyruvate
2
ATP
Respiration (Glycolysis → Krebs cycle → )
Glucose
NADH
Glycolysis
NADH
Acetyl Krebs
Pyruvate
CoA cycle
FADH2
CO2
2 2
ATP
Dept of Chem Eng, UM ATP
Dr. Adeline Chua S.M. 36
Respiration (Glycolysis → Krebs cycle → ETC)
ETC
Glucose H+
e-
NADH
H+
e- H+
Glycolysis
NADH H+
e- H+
Acetyl Krebs H+
Pyruvate
CoA cycle +
H+H
FADH2
H+
CO2
O2 + 4e- + 4H+
H2O
2 2 32-34
ATP
Dept of Chem Eng, UM ATP
Dr. Adeline Chua S.M. ATP
37
Krebs Cycle / Citric Acid Cycle / TCA Cycle
 In cellular respiration, pyruvate is oxidized to CO2. This pathway is called citric
acid cycle (CAC), also known as Krebs cycle or TCA cycle --- a key pathway in
virtually all cells.
 For each pyruvate molecule oxidized through the CAC, three CO2 molecules are
released. Electrons released during the oxidation of intermediates in the CAC are
transferred to NAD+ to form NADH, or to FAD to from FADH2. Instead of being
used in the reduction of pyruvate as in fermentation; in respiration, electrons from
NADH and FADH2 are fuel for the electron transport chain, ultimately resulting in
the reduction of an electron acceptor (O2) to H2O. This is where respiration and
fermentation differ in a major way.
 This allows for the complete oxidation of glucose to CO2 along with a much
greater yield of energy.

Electron transport chain
 In aerobic or anaerobic respiration, electrons carried by NADH

and FADH2 are transferred to oxygen via a series of electron
carriers (such as cytochrome, ubiquinone ) – which is called the
electron transport chain.
 The energy released from this electron transport results in the

formation of ATP molecules. The process of forming ATP from
the electron transport chain is known as oxidative
phosphorylation.

Electron transport chain

Important concepts in metabolisms
1. What is the starting material for glycolysis and Krebs cycle?
2. What are the end product(s) for fermentation and respiration?
3. Compare the amount of ATP produced in fermentation and respiration.
4. Explain why much lesser ATP is produced in fermentation.
5. Why is pyruvate reduced to fermentation product in the absence of
oxygen?
6. How is NAD+ and NADH being recycled in fermentation and respiration?
7. What is the main role/function of the electron carriers?
8. Membrane-bound protein complexes are arranged in membrane in an
order of increasing reduction potential. Why? >>> use Redox tower to
explain.
9. Identify the electron donor(s) and electron acceptor(s) involved in
fermentation and respiration.
10. Differentiate substrate phosphorylation and oxidative phosphorylation.

Exercise 6
Name the major metabolism involved in each process. Use a

schematic diagram, show the major steps involved in the metabolism.
Label clearly the electron donor, electron acceptor, major
intermediates (if any), final product(s) and amount of ATP generated.
1. Activated sludge process
2. Biogas production from anaerobic digestion of waste
3. Removal of ammonium in wastewater by nitrification process
4. Removal of nitrate in wastewater by denitrification process

Electron donors and acceptors (Exercise)
 Suggest 3 organic compounds that can be the electron donors

under aerobic condition.
 Suggest 3 inorganic compounds that can be the electron donors

under aerobic condition.
 Suggest 3 organic compounds that can be the electron donors

under anoxic condition (presence of NO3- ions).

Electron donors and acceptors (Exercise)
 Identify the electron donor and acceptor in the following catabolic

reactions:
1. Bacteria growing on a media containing glucose and inorganic

nutrients under aerobic conditions.
2. Bacteria growing in the sediment of UM lake by consuming

sulfate ion (SO42-) and producing sulfur granules.
3. Bacteria growing in the media by converting ammonium (NH4+) to

nitrate (NO3-) under aerobic condition.

The End

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Metabolism of Microorganisms

Energy source? Carbon source?

Light ATP Inorganic

Photosynthesis Cellular respiration Fermentation

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 2

 A bioprocess developer must understand the metabolic capabilities of the

 Thus, knowledge about some essential metabolisms is necessary.

 What causes the differences in microbial metabolism?

 The control of metabolic pathways by nutritional and environmental regulation

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 4

 Metabolic reactions are either catabolic, which means energy releasing,

 Catabolism breaks down the molecular structures of substrates,

 Anabolism uses energy to build larger molecules from smaller ones.

 The process of metabolism takes place via sequences of consecutive,

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 5

 We will look at:

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 6

 This concept is very important in energy metabolism.

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 7

 Substances differ in their tendency to be e- donors or e-

 A substance can be either an e- donor or an e- acceptor under

 The redox tower represents the range of reduction potentials

 By convention, E0’ are given for half reactions written as

 The constituents on each side in the

 By convention, when writing a redox

 The reduced substance in the redox Strongest

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 9

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 10

 Oxygen, at the bottom of the tower, is the strongest e- acceptor of any

 Many potential e- donors exist in nature, including a wide variety of

 E- donors used in energy metabolism are also call energy sources

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 11

 NAD+ and NADH is a coenzyme found in all living cells:

 Another coenzyme is NADP+/NADPH.

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 12

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 13

 In living organisms, chemical energy released in redox reactions is

 The most important energy-rich phosphate compounds in cells is

 ATP consists of the ribonucleoside adenosine to which three phosphate

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 15

 Phosphoester bond joins the first phosphoryl group to the 5-carbon

 Second and third groups are joined by phosphoanhydride bonds

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 16

 Examples of energy storage polymers;

 Fermentation is the form of anaerobic catabolism in which an organic

 Respiration is the catabolism in which a compound is oxidized (electron donor)

 They are alternative metabolic choices available to some microorganisms. Some

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 20

 The fermentable substrate in a fermentation is both the e- donor and e- acceptor.

 Involved two main steps:

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 21

 In Stage III, redox reactions occur once again and fermentation

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 22

Glucose + 2 ATP  2 glyceraldehyde-3-phosphate + 2 ADP

 A 6-carbon substrate (glucose) is split into 2 molecules of a

 For each glucose entering glycolysis, 2 ATP molecules are

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 25

 Net reactions: write yourself (refer Table 15.1)

 Transform each 3-carbon metabolite (glyceraldehyde-3-

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 26

Pyruvate Pyruvate NADH

Dept of Chem Eng, UM Dr. Adeline Chua S.M. 28

Pyruvate Pyruvate NADH