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Microbial Metabolism
I.


Metabolism
Metabolism is all of an organism's chemical processes (an emergent property that arises from
interactions of molecules in the orderly environment of the cell)
Metabolism is very important for the management of cellular material and energy resources
Metabolic reactions
 Metabolic reactions are organized into pathways of enzyme controlled chemical reactions.
 Cells need a supply of molecules and energy
 Cells need to get rid of waste products
Catabolic pathways
 Break down complex molecules into simple molecules
 Energy stored in complex molecules is made available to do work or transformed into readily
usable chemical forms (i.e., ATP)
 small molecules resulting from the catabolism of complex energy rich molecules may be used by
the cell to build new molecules

e.g., cellular respiration
Energy stored in compounds can be used to perform cellular work
 mechanical - movement of cilia, chromosomes, organelles
 transport - movement of substances across membranes
 chemical - endergonic reactions
Anabolic pathways
 Use energy for the biosynthesis of complex molecules from simple molecules.
 Energy is obtained from usable chemical forms of energy (i.e., ATP) produced during catabolic
processes or from energy released during catabolic processes

e.g., synthesis of macromolecules
Note: some pathways may function both catabolically and anabolically – these pathways are
known as amphibolic pathways
Review the Enzymes Section on your own (Pages 115 to 121)
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II.





Metabolic diversity among microorganisms
Life is based on organic molecules made of carbon skeletons
Oxygen and hydrogen are important elements of organic compounds
Electrons are needed i) for processes that provide energy (e.g., movement of electrons along
energy transport chains and during oxidation reduction reactions) for cellular work and ii) to
reduce molecules during biosynthesis
Molecules that serve as a source of carbon may also provide a source of oxygen and hydrogen
Microbes show an incredible ability to use organic molecules as carbon sources
Organisms can be classified based on their sources of carbon, energy and electrons
Carbon Source
o Autotroph – CO2 is the sole or principal carbon source
o Heterotroph – reduced, preformed, organic molecules from other organisms
Energy Source
o Phototrophs – Light
o Chemotrophs – oxidation of organic or inorganic compounds
Electron Source
o Lithotrophs – reduced inorganic chemicals
o Organotrophs – Organic molecules
Major Nutritional Types of Microorganisms
Carbon
Energy
Source
source
Electron
source
Examples
1. Photolithoautotrophy
 aka - Photoautotrophs
CO2
Light
Inorganic
e- donor
Cyanobacteria,
Purple sulfur
bacteria
2. Photoorganoheterotrophy
 aka - Photoheterotrophs
Organic
carbon but
CO2 may be
used
Light
Organic
e- donor
Purple nonsulfur
bacteria and Green
nonsulfur bacteria
3. Chemolithoautotrophy
 aka - Chemoautotrophs
CO2
Inorganic
chemicals
Inorganic
e- donor
Sulfur oxidizing
bacteria, methanogens,
nitrifying bacteria
4. Chemolithoheterotrophy
Organic
carbon but
CO2 may be
Used
Inorganic
chemicals
Inorganic
e- donor
Some sulfur
oxidizing bacteria
5. Chemoorganoheterotrophy
 aka – Chemoheterotrophs
Organic
carbon
Organic
chemicals
often the
same as
C-source
Organic
e- donor
often the
same as
C-source
Most nonphotosynthetic
microbes including
most pathogens, fungi,
many protist and many
archaea
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Tortora et al- Chap 5
III Heterotrophic (Chemoorganotrophic) Metabolism
 Conversion of organic substrate molecules to end products by a metabolic pathway that releases
sufficient energy for it to be coupled to the formation of ATP.
 Chemoorganotrophs have three options for generating ATP from organic molecules; the electron
acceptor used differentiates these processes: i) aerobic respiration, ii) anaerobic respiration and
iii) fermentation
i.
Respiration
 An external terminal electron acceptor is present and is not derived from the organic
substrate
 Involves the activity of an electron transport chain, proton motive force (PMF) is generated
and ATP produced predominantly by oxidative phosphorylation
a) Aerobic - O2 is the terminal electron acceptor.
C6H12O6 + 6 O2  6 CO2 + 6 H2O + (ATP + Heat)
b) Anaerobic - compounds other than O2 serve as electron acceptor (e.g., NO3-, SO42-, CO2,
fumarate,…)
*Some microbes can carry out both aerobic and anaerobic respiration – dependent upon the
conditions
ii.
Fermentation
 An external terminal electron acceptor is absent
 Fermentation does not use an electron transport chain or the generation of a PMF
 Fermentations are internally balanced oxidation-reduction reactions – i.e., the terminal
electron acceptor is derived from the initial substrate or electron donor (e.g., glucose)
 The terminal electron acceptor is required to balance redox reactions
 Net result is energy production and an internally balanced redox reactions
 ATP produced predominantly by substrate-level phosphorylation
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A.
Respiration
1. Glycolytic pathways
 Breakdown of sugars to pyruvate and similar intermediates
 Some production of ATP (substrate-level phosphorylation) and reducing power (reduced
coenzymes; NADH)
 Several pathways by which a cell can break down a sugar (sugars are the major substrates of
catabolic energy releasing reactions used in heterotrophic metabolism).
 Glycolytic pathways are typically anoxic processes that do not require oxygen

The end-product of glycolysis is commonly pyruvate
COOH
|
C=O
|
CH3
Glycolytic pathways
i). Embden-Meyerhof pathway (EMP)
 Most common pathway - Central metabolic pathway for eukaryotic cells and many bacteria
Net reaction
10 enzymatic steps
Glucose + 2 ADP + 2 Pi + 2 NAD+ 2 pyruvate + 2 ATP + 2 NADH + 2 H2O


ATP production - Substrate level phosphorylation
Phosphofructokinase is key enzyme in regulating this process, catalyzing the conversion of
fructose – 6-P to fructose 1,6-bisphosphate
ii). Entner-Doudoroff pathway
 Mainly used by Gram negative soil bacteria and a few other Gram-negative bacteria as well
as some Archaea
 Lacks 6-phosphofructokinase (i.e., a key enzyme in Embden-Meyerhof pathway)
Net reaction
9 enzymatic steps
Glucose + ADP + Pi + NADP+ + NAD+  2 pyruvate + 1 ATP + NADPH + NADH + 1 H2O
*NADPH is usually used in biosynthetic pathways
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iii).
Pentose Phosphate pathway
 Can occur at the same time as the Embden-Meyerhof or the Entner-Doudoroff pathways
 connects the metabolism of 6-C and 5-C sugars
 Consumes 1 ATP
 Products = reducing power (NADPH) and small molecules required for biosynthesis
 5-C sugars produced (e.g., ribose 5-phosphate; xylulose 5-phosphate)
 Erythrose 4-phosphate is used to synthesize aromatic amino acids and vitamin B6
 Intermediates of the pathway may be fed into the EMP to produce ATP
iv). Methylglyoxal pathway
 Alternative to Embden-Meyerhof pathway during conditions of low phosphate availability
 Consumes 2 ATP but produces pyruvate that can be used to generate ATP
2. Oxidation of pyruvate to 3 CO2.
i) Initial Step
Pyruvate + NAD+ + CoA  Acetyl-CoA + NADH + CO2




Three step process mediated by multienzyme pyruvate dehydrogenase complex
Acetyl CoA is a very unstable and reactive product.
Acetyl CoA feeds it's acetate  TCA cycle
Carbohydrates, fatty acids and amino acids may be converted into acetyl CoA during aerobic
respiration
ii) Tricarboxylic acid cycle (TCA cycle; also known as Citric acid cycle, Krebs cycle)
 Discovered by Hans Krebs - 1930s
 8 steps involved in the TCA cycle
 2 C enter in a relatively reduced form – acetate and two different C leave in a completely
oxidized form (CO2)
 acetate joins the cycle by enzymatic addition to oxaloacetate (4 C)  formation of citrate
 cyclical process resulting in the regeneration of oxaloacetate by the decomposition of citrate
and evolution of 2 CO2 per acetate


+
oxidation steps (transfer of electrons) of one acetate results in reduction of 3 NAD to 3
NADH and 1 FAD to FADH2 (like NADH it donates its e- to the electron transport chain but
at a lower energy level)
One step for the production of GTP (substrate level phosphorylation; GTP can be converted
to ATP)
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Tortora et al- Chap 5
Review with Figure 5.13
pyruvate + 4 NAD++ FAD  3 CO2 + 4 NADH + 1 FADH2 + 1 GTP

TCA cycle source of key biosynthetic intermediates
e.g., oxaloacetate and -ketoglutarate are precursors to a number of
amino acids
acetyl-CoA  starting material for fatty acid biosynthesis
3.
Oxidative phosphorylation
 Reducing power (NADH and FADH2) is used to generate a proton gradient (proton motive
force)
 NADH - FADH2 are oxidized – electron transport carrier proteins are reduced and in the
process H+ are moved across the plasma membrane (prokaryotes) or inner mitochondrial
membrane (eukaryotes). This results in a proton gradient or proton motive force across the
membrane. The movement of H+ across the membrane is not completely understood
Chemiosmosis
 Proton Gradient driving ATP synthesis
 Peter Mitchell (Nobel prize in Chemistry in 1978)
ATP synthase (ATPase)
 F0 subunit – multimeric membrane spanning proton conducting channel
 F1 – multimeric headpiece  inside of the membrane
 Catalyze ADP + Pi  ATP (oxidative phosphorylation)
 Highly conserved throughout all domains of life


Can also drive reverse reaction ATP  ADP + Pi
explains why some obligate fermenters have ATPase - creates proton gradients that can be
used to drive transport and flagella
Much evidence supporting the chemiosmotic theory comes from studies using chemicals that
inhibit the aerobic synthesis of ATP


inhibitors, (e.g., CO, cyanide) inhibit ATP synthesis by blocking the electron flow –
preventing oxidative phosphorylation
uncouplers (e.g., dinitrophenol, dicumarol) allow protons to cross the membrane without
activating ATP synthase and prevent ATP synthesis without affecting electron transport
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Net ATP Production in Aerobic Respiration
Substrate-level phosphorylation
Glycolysis (EMP)
4 ATP
-2 ATP
Citric acid cycle (2 GTP 2 ATP)
2 ATP
Oxidative phosphorylation
2 NADH (from Glycolysis)
6 ATP
if membrane impermeable to NADH
then e- relayed across membrane
at the expense of 2 ATP
0-2 ATP
8 NADH (from TCA cycle)
2 FADH2 (from TCA cycle)
24 ATP
4 ATP
36-38 ATP
Energy Storage?
4.
Anaerobic respiration
 terminal electron acceptors other than O2
e.g., NO3-, Fe3+, SO42-, CO2 – Table 9.1
 Used by many bacteria and archaea
 These compounds have reduction potentials (E0’) that are less electropositive than O2 and
therefore less energy is released during anaerobic respiration
 Some organisms can use both aerobic and anaerobic respiration depending if O2 is present
What if oxygen or other terminal electron acceptors are absent?
B
Fermentation
 Bacteria and Archaea can survive in environments lacking O2, and other types of terminal
electron acceptors (e.g., NO3- or SO42-)
 Alternative heterotrophic processes producing energy and molecules needed for growth
 ATP generated by substrate-level phosphorylation
 Coupled with glycolysis and involve the anaerobic metabolism of pyruvate
 Three important themes operating during microbial fermentation are:
1. NADH is oxidized back to NAD+
2. Substrate is electron donor and a derivative of substrate is the ultimate electron acceptor.
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3. Oxidative phosphorylation cannot operate
Unlike respiration, the substrate is only partially oxidized during fermentation and therefore:
 energy yields are much lower
 substrate use is less efficient.
e.g.,


Lactate production during lactic acid fermentation  G = -58 kcal/mole
The complete oxidation of glucose during aerobic respiration  G = -686 kcal/mole
Organisms capable of both respiration and fermentation will use the more energetically
favourable respiration if conditions permit. If there is no available external electron acceptor,
then they will use fermentation.
Some organisms are obligate fermenters (can only ferment carbon compounds) while others
are facultative fermenters (i.e., have citric acid cycle and electron transport chain)
Example Fermentation pathway
Glycolytic pathway
Substrate + 2 ADP + 2 Pi + 2 NAD+ 2 pyruvate + 2 ATP + 2 NADH endproduct + 2 NAD+
The fermentation pathway achieves the following
 Balances the redox reactions
 Regenerates oxidizing power (oxidized coenzymes) – NAD+
Different Fermentation Pathways occur in bacteria, archaea and eukaryotic cells and produce
different end products.
 Fermentation pathways are generally named after end products.
 The type of fermentation end product produced is often used to identify a particular
microorganism (see below and Pages 137 – 138 and Clincial focus on page 144)
Common fermentation pathways
i. homolactic acid fermentation
ii. heterolactic acid fermentation
iii. ethanol fermentation
iv. propionic acid fermentation
v. mixed acid fermentation
vi. butanediol fermentation
vii. butyric acid fermentation
viii. amino acid fermentation
ix. methane fermentation
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i)
Lactic acid fermentation
 pyruvate is reduced to lactic acid
 reduction of NADH to NAD+ is coupled to this process
 Lactic acid bacteria
If the Embden-Meyerhof pathway is used
then Homofermentative or homolactic fermentation occurs
Glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP
Lactic acid  CH3 - C(HOH) - COOH

lactate dehydrogenase catalyzes conversion of pyruvate to lactate and vice versa




NB in the dairy industry - dairy products - cheese, yogurt, acidophilus milk
silage production
dental caries - Streptococcus mutans
Streptococcus, Lactococcus, Pediococcus, Lactobacillus
If the Pentose phosphate pathway is used
then heterofermentative or heterolactic fermentation occurs
Glucose + ADP + Pi  lactate + ethanol + CO2 + ATP

ATP is produced during the conversion of pentose sugars to lactate


sausage and sauerkraut
Leuconostoc and Lactobacillus
ii)
Alcohol or ethanol fermentation
Overall reaction
Glucose + 2 Pi + 2 ADP  2 ethanol + CO2 + 2 ATP
Key steps
Glucose  pyruvate  acetaldehyde + CO2  ethanol
Enzymes involved
 pyruvate decarboxylase catalyzes conversion of pyruvate  acetaldehyde + CO2

alcohol dehydrogenase catalyzes conversion of acetaldehyde  ethanol
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

Alcohol fermentation is used in the production of ethanol for beverages, solvent and fuel
Many yeast such as Saccharomyces cerevisiae and a few bacteria such as Zymomonas
mobilis are capable of carrying out the alcohol fermentation pathway
iii)
Propionic Acid fermentation
 Utilize lactic acid and produce propionic acid (CH3CH2COOH)
 Propionibacterium
 Used in Swiss cheese production to give the cheese its distinct flavour
iv)
Mixed Acid Fermentation
 Members of the family Enterobacteriaceae carry out fermentation of pyruvate into a variety
of organic acids including acetate, lactate, succinate, formate.
 Other endproducts include H2, CO2, ethanol
 NADH is reoxidized to NAD+
 Formation of acetate is accompanied by substrate-level phosphorylation
 Mixed acid endproducts are detected via the Methyl Red test because of an overabundance of
organic acids produced relative to neutral endproducts
Methyl red is Yellow at neutral pH and red if pH is less than 5
v)





Butanediol fermentation
Butanediol (CH3-CHOH-CHOH-CH3) is a neutral product
NADH is oxidized to NAD+ and CO2 is also produced
No additional ATP is generated
Erwinia, Klebsiella, Enterobacter and Serratia
Acetoin intermediate can be detected by Voges Proskauer test
vi)
Butyric acid Fermentation
 Clostridial species
 Important process for solvent production (e.g., Acetone, Butanol, Ethanol, isopropanol)
Note: Fermentation yields little usable energy compared to aerobic and anaerobic
respiration
Why?
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IV.
Autotrophy
 Self sufficient organisms that are capable of growing by metabolizing simple inorganic
compounds or by photosynthesis
1.
Chemolithotrophy (Chemoautotrophy)
 Bacteria and Archaea that carry out respiration by oxidizing inorganic compounds
 May or may not couple oxidation of inorganic compounds with reduction of coenzyme
(NAD+) e.g., H2, H2S, FeS, NH3, NO2

These organisms play an important role in biogeochemical recycling
Use O2 or other compounds (NO3-) as the terminal electron acceptor
Examples
i) Hydrogen oxidation


Carry a unique enzyme called hydrogenase - Alcaligenes eutrophus
generates protons and electrons which are used in the membrane associated electron transport
chain
Homoacetogenic bacteria couple H2 oxidation with reduction of CO2 to form acetate
 final step involves conversion of acetyl-CoA to acetate results in sufficient free energy for
ATP formation - Clostridium aceticum
Extreme thermophilic Archaea - Pyrodictium - deep sea around thermal vents
 H2 oxidation coupled with sulfur reduction
ii) Nitrification = Ammonia and Nitrite oxidation
 Very important organisms in nitrogen cycling
 oxidize ammonia or nitrite (NO2-) to produce energy by chemiosmosis
Nitrosomonas
Nitrobacter
NH4+  NO2NO2-  NO3-
ii) Sulfur and Iron oxidation
Thiobacillus and Sulfolobus
 oxidize H2S, S, and thiosulfate (S2O3-) to produce ATP by substrate level phosphorylation
and chemiosmosis
Thiobacillus ferrooxidans
 oxidizes reduced sulfur & iron (Fe3+  Fe2+)
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
2.
Acid mine drainage problems and use in mineral recovery processes (acid leaching).
Photoautotrophy
Photophosphorylation - conversion of light energy into chemical energy (ATP)



Light sensitive pigments (Chlorophylls, bacteriochlorophyll, phycobilins and carotenoids)
capture light energy and release electrons.
Electrons are transferred through a series of membrane bound electron carriers
The whole system is called a photosystem
1.




Oxygenic phototrophy
Algae, cyanobacteria and prochlorobacteria
ATP and O2 are produced as a result of oxidative phosphorylation
Similar to green plants have two photosystems PS I and PSII
cyclic and noncyclic oxidative phosphorylation
2. Anoxygenic phototrophy
 Capture of light energy and production of ATP in the absence of O2.
 Anaerobic green and purple bacteria and heliobacteria (use reduced sulfur compounds like
H2S as a source of electrons)
 Only PSI - (photons excite bacteriochlorophyll and are cycled back through a series of
electron carriers) ATP produced by cyclic oxidative phosphorylation.
 No need for a external electron acceptor or donor
3. Rhodopsin-based phototrophy
 Oxygenic and anoxygenic phototrophy have not been observed in Archaea
 Archaea capture light energy using a membrane protein called bacteriorhodopsin, that is a
light driven proton pump – creates a proton gradient across the plasma membrane
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