5ch05 Lecture

Brock Biology of Microorganisms
Twelfth Edition
Madigan / Martinko Dunlap / Clark
Chapter 5
Nutrition, Culture, and Metabolism of Microorganisms
Lectures by Buchan & LeCleir
Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings
I. Nutrition and Culture of Microorganisms

5.1 Microbial Nutrition
5.2 Culture Media

5.3 Laboratory Culture of Microorganisms

Metabolism
The sum total of all of the chemical reactions that occur in a cell Catabolic reactions (catabolism)
Energy-releasing metabolic reactions
Anabolic reactions (anabolism)

Energy-requiring metabolic reactions
Most of what is known of microbial metabolism is based on study of laboratory cultures

Nutrients
Supply of monomers (or precursors of) required by cells for growth
Macronutrients
Nutrients required in large amounts
Micronutrients
Nutrients required in trace amounts
A Microbial Periodic Table of the Elements
Figure 5.1 Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Macronutrients
Carbon
Required by all cells
Typical bacterial cell ~50% carbon (by dry weight) Major element in all class of macromolecules Heterotrophs use organic carbon Autotrophs use inorganic carbon
Nitrogen
Typical bacterial cell ~12% nitrogen (by dry weight)
Key element in proteins, nucleic acids, and many more cell constituents
5.1 Microbial Nutrients

Other Macronutrients
Phosphorus (P)
Required by cell for synthesis of nucleic acids and phospholipids
Sulfur (S)
Plays structural role in S-containing amino acids (cysteine and methionine) Present in several vitamins (e.g. thiamine, biotin, lipoic acid) and coenzyme A
Potassium (K)
Required by enzymes for activity

Other Macronutrients (contd)
Magnesium (Mg)
Stabilizes ribosomes, membranes and nucleic acids Also required for many enzymes
Calcium (Ca)
Helps stabilize cell walls in microbes Plays key role in heat stability of endospores
Sodium (Na)
Required by some microbes (e.g., marine microbes)

Iron
Plays major role in cellular respiration; key component of cytochromes and FeS proteins involved in electron transport Under anoxic conditions, generally ferrous (Fe2+) form; soluble Under oxic conditions: generally ferric (Fe3+) form; exists as
insoluble minerals
Cells produce siderophores (iron-binding agents) to obtain iron from insoluble mineral form
Iron-Chelating Agents Produced by Microorganisms
Figure 5.2a
Iron-Chelating Agents Produced by Microorganisms
Figure 5.2b and c
Micronutrients (Trace Elements) Needed by Microbes
Micronutrients (Trace Elements) Needed by Microbes
Growth Factors
Organic compounds required in small amounts by certain
organism
E.G., vitamins, amino acids, purines, pyrimidines
Vitamins
Most commonly required growth factors
Most function as coenyzmes
Growth Factors: Vitamins and their Functions
Growth Factors: Vitamins and their Functions
5.2 Culture Media
Culture Media
Nutrient solutions used to grow microbes in the laboratory
Two broad classes

Defined media: precise chemical composition is known
Complex media: composed of digests of chemically undefined substances (e.g., yeast and meat extracts)
5.2 Culture Media
Selective Media
Contains compounds that selectively inhibit growth of
some microbes but not others
Differential Media
Contains an indicator, usually a dye, that detects particular chemical reactions occurring during growth
Culture Media for Simple and Demanding Nutritional Requirements
5.2 Culture Media

For successful cultivation of a microbe it is important to
know the nutritional requirements and supply them in

proper form and proportions in a culture medium
II. Energetics and Enzymes

5.4 Bioenergetics
5.5 Catalysis and Enzymes

Free energy calculations do not provide information on
reaction rates
Activation energy: energy required to bring all molecules in a chemical reaction into the reactive state
A catalysis is usually required to breach activation energy barrier
Activation Energy and Catalysis
Figure 5.6
Catalyst: substance that

Lowers the activation energy of a reaction
Increases reaction rate Does not affect energetics or equilibrium of a reaction

Enzymes
Biological catalysts Typically proteins (though some RNAs) Highly specific Generally larger than substrate
Typically rely on weak bonds

E.G., hydrogen bonds, van der Waals forces, hydrophobic interactions Active site: region of enzyme that binds substrate

Enzymes (contd)
Increase the rate of chemical reactions by 108-1020 times the spontaneous rate
Enzyme catalysis: E + S
Catalysis dependent on
Substrate binding
E-S
E+P
Position of substrate relative to catalytically active amino acids in active site
The Catalytic Cycle of an Enzyme
Figure 5.7

Many enzymes contain small nonprotein molecules that
participate in catalysis but are not substrates Prosthetic groups

Bind tightly to enzymes Usually covalently and permanently (e.g., heme group in
cytochromes)
Coenzymes
Loosely bound to enzymes Most derivatives of vitamins (e.g., NAD+/NADH)
III. Oxidation-Reduction and Energy-Rich Compounds

5.6 Oxidation-Reduction
5.7 NAD as a Redox Electron Carrier

5.8 Energy-Rich Compounds and Energy Storage
Energy is conserved in cells from oxidation-reduction (redox) reactions
This energy is conserved in the synthesis of energy-rich

compounds (e.g., ATP)
Redox reactions occur in pairs (two half reactions)

Electron donor: the substance oxidized in a redox reaction Electron acceptor: the substance reduced in a redox reaction
Example of an Oxidation-Reduction Reaction
Figure 5.9
Reduction potential (Eo): tendency of a compound to donate
electrons
Measured under standard condition
Expressed as volts (V)
Substances can be either electron donors or acceptors under different circumstances (redox couple) Reduced substance of a redox couple with a more negative Eo donates electrons to the oxidized substance of a redox couple
with a more positive Eo
The redox tower represents the range of possible reduction
potentials for redox couples in nature
The reduced substance in the redox couple at the top of the

tower has the greatest tendency to donate electrons The oxidized substance in the redox couple at the bottom of the tower has the greatest tendency to accept electrons The farther the electrons drop from donor to acceptor, the greater the amount of energy released
The Redox Tower
Figure 5.10

Redox reactions usually involve reactions between
intermediates (carriers)
Electron carriers are divided into two classes
Prosthetic groups (attached to enzymes) Coenzymes (diffusible; e.g., NAD+, NADP)
The Oxidation-Reduction Coenzyme NAD+
Figure 5.11

NAD+ and NADH facilitate redox reactions without being
consumed; they are recycled
NAD+/NADH Cycling
Figure 5.12

Chemical energy released in redox reactions is primarily
conserved in certain phosphorylated compounds

ATP; the prime energy currency Coenzyme A
Some Compounds Important in Energy Transformation
Figure 5.13

Long-term energy storage involves insoluble polymers
that can be oxidized to generate ATP
Examples in prokaryotes Glycogen
Poly--hydroxybutyrate and other polyhydroxyalkanoates
Elemental sulfur
Examples in eukaryotes Starch

Lipids (simple fats)
IV. Essentials of Catabolism

5.9 Energy Conservation
5.10 Glycolysis as an Example of Fermentation

5.11 Respiration and Membrane-Associated Electron
Carriers
5.12 Respiration and the Proton Motive Force 5.13 Carbon Flow in Respiration: The Citric Acid Cycle 5.14 Catabolic Diversity
Microbial Metabolism
Heterotroph/Chemoorganotroph
Autotroph C source CO2
C source organic C Energy Source org C
Energy Source
Light [photoautotroph] Reduced inorganic cmpds [chemolithotrophs]
H2S, NH3, Fe++, H2, CO2
5.9 Energy Conservation

Two reaction series are linked to energy conservation in chemoorganotrophs: fermentation and respiration
Differ in mechanism of ATP synthesis

Fermentation: substrate-level phosphorylation; ATP directly synthesized from an energy-rich intermediate Respiration: oxidative phosphorylation; ATP produced from proton motive force formed by transport of electrons from
organic or inorganic electron donors
Energy Conservation in Fermentation and Respiration
Figure 5.14

Fermented substance is both an electron donor and
electron acceptor
Glycolysis (Embden-Meyerhof pathway): a common pathway for fermentation of glucose
Anaerobic process Three stages
My Definitions of Glycolysis & Fermentation

Glycolysis: glucose 2 pyruvate + 2 NADH & 2 ATP
Fermentation: pyruvate is reduced to regenerate NAD+ producing fermentation end products such as ethanol, CO2, lactate, etc. Fermentation of pyruvate results in a net gain of potential energy of 2 ATP
Embden-Meyerhof Pathway (Glycolysis)
Figure 5.15
Figure 5.15
Figure 5.15
Central Metabolic Pathways

Glycolysis
Primary pathway to convert one glucose to two pyruvate
10 step process
Pathway generates
Two 3 C pyruvate molecules Net gain of two ATP
2 ATP expended to break glucose 4 ATP harvested
Two molecules reducing power

NADH
6 different precursor metabolites

5 intermediates and pyruvates
Glycolysis
Glycolysis
Fermentation
Used by organisms that cannot respire Due to lack of suitable inorganic electron acceptor or lack of electron transport chain ATP produced only in glycolysis Other steps for consuming excess reducing power Recycles NADH, regenerating NAD+ Fermentation pathways use pyruvate or derivative as terminal electron acceptor

Glycolysis
Glucose consumed
Two ATPs produced Fermentation products generated
Some harnessed by humans for consumption
Figure 6.20a
Figure 6.20b
Fermentation
End products of fermentation include
Lactic acid Ethanol Butyric acid Propionic acid 2,3-Butanediol Mixed acids
All are produced in a series of reactions to produce appropriate terminal electron acceptors
Transition step
Links glycolysis to Tricarboxylic Acid Cycle Modifies 3-C pyruvate from glycolysis to 2-C acetyl CoA
CO2 is removed through decarboxylation NAD+ is reduced to NADH
Remaining 2-C acetyl group joined to coenzyme A Forms Acetyl CoA
Each pyruvate enters transition step

Reaction occurs twice for one glucose
Yield from transition step

Reducing power
NADH
Precursor metabolites
Acetyl CoA

Tricarboxylic Acid/Citric Acid/Krebs Cycle
Completes the oxidation of glucose Incorporates acetyl CoA from transition step
Releases CO2 in net reaction
Cycle turns once for each acetyl CoA

Two turns for each glucose molecule
Cycle produces
2 ATP 6 NADH 2 FADH2 2 precursor metabolites
The Citric Acid Cycle
Figure 5.22a
Tricarboxylic Acid Cycle
Krebs Cycle
The Citric Acid Cycle
Figure 5.22b
Figure 6.19
Respiration
Uses NADH and FADH2 to synthesize ATP
3 ATP/ NAPH, 2 ATP/FADH2

Oxidative phosphorylation Occurs in electron transport chain Generates proton motive force
5.11 Respiration and Membrane-Associated Electron Carriers
Aerobic Respiration
Oxidization using O2 as the terminal electron acceptor
Higher ATP yield than fermentations
ATP produced at the expense of the proton motive force which is generated by electron transport

Electron Transport Systems
Membrane associated Mediate transfer of electrons from primary donor to terminal acceptor Conserve some of the energy released during transfer and
use to synthesize ATP

Many oxidation-reduction enzymes are involved in electron transport (e.g., NADH dehydrogenases, flavoproteins, ironsulfur proteins, cytochromes)

NADH dehydrogenases: proteins bound to inside surface
of cytoplasmic membrane; active site binds NADH and

accepts 2 electrons and 2 protons that are passed to flavoproteins Flavoproteins: contains flavin prosthetic group (e.g. FMN, FAD) that accepts 2 electrons and 2 protons but only donates the electrons to the next protein in the chain
5.12 Respiration and the Proton Motive Force

Electron transport system oriented in cytoplasmic membrane so that as electrons are transported, protons are separated
Carriers in electron transport chain arranged in membrane

in order of their increasingly positive reduction potential
The final carrier in the chain donates the electrons and

protons to the terminal electron acceptor
Generation of the Proton Motive Force
Figure 5.20
5.12 Respiration and the Proton Motive Force

During electron transfer, several protons are released causing
a slight acidification of the external surface of the membrane

Protons originate from NADH and the dissociation of water
Results in generation of pH gradient and an electrochemical

potential across the membrane (the proton motive force) The inside surface membrane becomes electrically negative and alkaline; the outside electrically positive and acidic
5.13 Carbon Flow in Respiration: The Citric Acid Cycle

Citric Acid Cycle (CAC): pathway through which pyruvate
is completely oxidized to CO2

Initial steps (glucose to pyruvate) same as glycolysis Per glucose molecule, 6 CO2 molecules released and 38 ATP generated Plays a key role in catabolism and biosynthesis
5.13 Carbon Flow in Respiration: The Citric Acid Cycle

The citric acid cycle generates many compounds available
for biosynthetic purposes
-ketoglutarate and oxalacetate (OAA): precursors of several amino acids; OAA also converted to phosphoenolpyruvate, a precursor of glucose Succinyl-CoA: required for synthesis of cytochromes, chlorophyll, and other tetrapyrrole compounds Acetyl-CoA: necessary for fatty acid biosynthesis
5.14 Catabolic Diversity

Microorganisms demonstrate a wide range of mechanisms
for generating energy

Fermentation Aerobic respiration Anaerobic respiration Chemolithotrophy
Phototrophy

Anaerobic Respiration
The use of electron acceptors other than oxygen
Examples include nitrate (NO3-), ferric iron (Fe3+), sulfate

(SO42-), carbonate (CO32-), certain organic compounds Less energy released compared to aerobic respiration Dependent on electron transport, generation of a proton motive force, and ATPase activity
Catabolic Diversity
Figure 5.23
Catabolic Diversity
Figure 5.23
Catabolic Diversity
Figure 5.23

Chemolithotrophy
Use of inorganic chemicals as electron donors Examples include hydrogen sulfide (H2S), hydrogen gas (H2), ferrous iron (Fe2+), ammonia (NH3) Typically aerobic
Begins with oxidation of inorganic electron donor

Uses electron transport chain and proton motive force Autotrophic; uses CO2 as carbon source

Phototrophy: metabolism that uses light as energy source
Photophosphorylation: light-mediated ATP synthesis
Photoautotrophs: use ATP for assimilation of CO2 for biosynthesis
V. Essentials of Anabolism
5.15 Biosynthesis of Sugars and Polysaccharides
5.16 Biosynthesis of Amino Acids and Nucleotides

5.17 Biosynthesis of Fatty Acids and Lipids
5.18 Regulation of Activity of Biosynthetic Enzymes

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Brock Biology of Microorganisms

Madigan / Martinko Dunlap / Clark

I. Nutrition and Culture of Microorganisms

5.2 Culture Media

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrition

Energy-releasing metabolic reactions

Anabolic reactions (anabolism)

Most of what is known of microbial metabolism is based on study of laboratory cultures

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrition

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

A Microbial Periodic Table of the Elements

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrition

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrition

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrients

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrients

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrients

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Iron-Chelating Agents Produced by Microorganisms

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Iron-Chelating Agents Produced by Microorganisms

Figure 5.2b and c

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Micronutrients (Trace Elements) Needed by Microbes

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Micronutrients (Trace Elements) Needed by Microbes

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.1 Microbial Nutrients

Most function as coenyzmes

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Growth Factors: Vitamins and their Functions

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Growth Factors: Vitamins and their Functions

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.2 Culture Media

Two broad classes

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.2 Culture Media

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Culture Media for Simple and Demanding Nutritional Requirements

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.2 Culture Media

know the nutritional requirements and supply them in

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

II. Energetics and Enzymes

5.5 Catalysis and Enzymes

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.5 Catalysis and Enzymes

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Activation Energy and Catalysis

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.5 Catalysis and Enzymes

Catalyst: substance that

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5.5 Catalysis and Enzymes

Typically rely on weak bonds

5.5 Catalysis and Enzymes

Position of substrate relative to catalytically active amino acids in active site