Nutrition, Culture, and

Metabolism of Microorganisms
01-22-2015

I. Laboratory Culture of
Microorganisms
3.1 Cell Chemistry and Nutrition
3.2 Media and Laboratory Culture

3.1 Cell Chemistry and Nutrition

Metabolism
The sum total of all chemical reactions that
occur in a cell

Catabolic reactions
Energy-releasing metabolic reactions

Anabolic reactions
Energy-requiring metabolic reactions

Most knowledge of microbial metabolism is

based on study of laboratory cultures

3.1 Cell Chemistry and Nutrition

Nutrients
Supply of monomers (or precursors of) required by cells for
growth

Macronutrients
Nutrients required in large amounts

Micronutrients
Nutrients required in trace amount

3.1 Cell Chemistry and Nutrition

Carbon

Required by all cells

Typical bacterial cell ~50% carbon (by dry weight)
Major element in all classes of macromolecules
Heterotrophs use organic carbon
Autotrophs use inorganic carbon

Nitrogen
Typical bacterial cell ~13% nitrogen
(by dry weight)
Key element in proteins, nucleic acids, and many
more cell constituents

3.1 Cell Chemistry and Nutrition

Other Macronutrients
Phosphorus (P)
Synthesis of nucleic acids and phospholipids

Sulfur (S)
Sulfur-containing amino acids (cysteine and methionine)
Vitamins (e.g., thiamine, biotin, lipoic acid) and coenzyme A

Potassium (K)
Required by enzymes for activity

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)

3.1 Cell Chemistry and Nutrition

Micronutrients: Trace metals
Iron
Key component of cytochromes and FeS proteins
involved in electron transport
Cells produce siderophores (iron-binding agents)
to obtain iron from insoluble mineral form

Growth Factors
Organic compounds required in small
amounts by certain organisms
Examples: vitamins, amino acids, purines,
pyrimidines

Vitamins
Most commonly required growth factors
Most function as coenyzmes

3.2 Media and Laboratory Culture

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)

Which has greater biosynthetic capacity: E. coli or L. mesenteriodes?

3.2 Media and Laboratory Culture

Enriched media
Contain complex media plus additional
nutrients

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
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

3.2 Media and Laboratory Culture

Pure culture: culture containing only a
single kind of microbe
Contaminants: unwanted organisms in a
culture
Cells can be grown in liquid or solid culture
media
Solid media are prepared by addition of a
gelling agent (agar or gelatin)
When grown on solid media,
cells form isolated masses
(colonies)

3.2 Media and Laboratory Culture

Microbes are everywhere
Sterilization of media is critical

Aseptic technique should be followed

Animation: Aseptic Transfer and the Streak Plate Method

3.2 Media and Laboratory Culture

Pure culture technique
Streak plate
Pour plate
Spread plate

Isolated colonies
at end of streak

Confluent growth at
beginning of streak

II. Energetics, Enzymes, and Redox

3.3 Energy Classes of Microorganisms
3.4 Bioenergetics
3.5 Catalysis and Enzymes
3.6 Electron Donors and Electron
Acceptors
3.7 Energy-Rich Compounds

3.3 Energy Classes of Microorganisms

Metabolism
The sum total of all of the chemical reactions that
occur in a cell

Catabolic reactions (catabolism)

Energy-releasing metabolic reactions

Microorganisms grouped into energy classes

Chemorganotrophs
Chemolithotrophs
Phototrophs
Heterotrophs
Autotrophs

Energy Sources
Chemicals

Light

Chemotrophy

Phototrophy

Organic
chemicals

Inorganic
chemicals

(glucose, acetate, etc.)

(H2, H2S, Fe2+, NH4+, etc.)

Chemoorganotrophs Chemolithotrophs Phototrophs

(glucose O2

CO2 H2O)

(H2 O2

H2O)

(light)

3.4 Bioenergetics
Energy is defined in units of kilojoules (kJ),
a measure of heat energy
In any chemical reaction, some energy is
lost as heat
Free energy (G): energy released that is
available to do work
The change in free energy during a
reaction is referred to as G0

3.4 Bioenergetics
Exergonic
Reactions with a negative G0 release free
energy

Endergonic
Reactions with a positive G0 require energy

To calculate free-energy yield of a

reaction, we need to know the free energy
of formation (Gf0; the energy released or
required during formation of a given
molecule from the elements)

3.4 Bioenergetics
For the reaction A + B C + D
G0 = Gf0 [C+D] - Gf0[A+B]

G0 not always a good estimate of actual freeenergy changes

G: free energy that occurs under actual
conditions
G = G0 + RT ln K
where R and T are
physical constants and
K is the equilibrium
constant for the reaction
in question

3.5 Catalysis and Enzymes

Free energy

Free-energy calculations do not provide

information on reaction rates
Activation energy:
energy required to
bring all molecules Activation
energy
in a chemical
no enzyme
Activation
reaction into the
energy with
enzyme
reactive state
G = G (C + D)
A catalysis is usually
required to breach
activation energy
barrier

Substrates
(A + B)
0

0
f

Gf0(A + B)

Products (C + D)

Progress of the reaction

3.5 Catalysis and Enzymes

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 (some RNAs)
Highly specific
Generally larger than substrate
Typically rely on weak bonds
Examples: hydrogen bonds, van der Waals
forces, hydrophobic interactions

Active site: region of enzyme that binds substrate

3.5 Catalysis and Enzymes

Enzymes (contd)

Increase the rate of chemical reactions by 108 to 1020 times the spontaneous
rate
Enzyme catalysis: E + S E S E + P
Catalysis dependent on
Substrate binding
Position of substrate relative to catalytically active amino acids in active site
Products

Substrate

1. Substrate is
bound to
enzyme active
site.

Active site

4. Products
are released.

2. Enzyme
substrate
complex
forms.

3. Strain is
placed on
bond.

5. Enzyme is ready to
begin new catalytic
cycle.

3.5 Catalysis and Enzymes

Many enzymes contain small nonprotein
molecules that participate in catalysis but are
not substrates
Prosthetic groups
Bind tightly to enzymes
Usually bind covalently and permanently
(e.g., heme group in cytochromes)

Coenzymes
Loosely bound to enzymes
Most are derivatives of vitamins
(e.g., NAD+/NADH)

3.6 Electron Donors and Electron Acceptors

Energy from oxidationreduction (redox) reactions is
used in 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
Half reaction
donating e

Half reaction
accepting e

Electron
donor

Formation
of water

Electron
acceptor

Net reaction

3.6 Electron Donors and Electron Acceptors

Reduction potential (E0): tendency to
donate electrons
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 E0 donates electrons to
the oxidized substance of a redox couple
with a more positive E0

Redox couple

3.6 Electron Donors and

Electron Acceptors
The redox tower
represents the range of
possible reduction
potentials
The reduced substance
at the top of the tower
donates electrons
The oxidized substance
at the bottom of the
tower accepts electrons
The farther the electrons
drop, the greater the
amount of energy
released
G = -nFE

E0 (V)
0.60
0.50
0.40
0.30

(1)

0.20
0.10

0.0
+0.10
(2)

+0.20
+0.30
+0.40
+0.50
+0.60

+0.70
(3)

+0.80
+0.90

(1) H2 fumarate

succinate

G0 86 kJ

(2) H2 NO3

NO2 H2O

G0 163 kJ

(3) H2

1
2 O2

H2O

G0 237 kJ

3.6 Electron Donors and Electron

Acceptors
Redox reactions usually involve reactions
between intermediates (carriers)
Electron carriers are divided into two
classes
NAD
NADH + H
+

Prosthetic groups
(attached to
enzymes)
Coenzymes
(diffusible)
Examples: NAD+,
NADP

Nicotinamide

Adenine

NAD+/ NADH
E0 = 0.32 V

In NADP+, this OH has a

phosphate attached.

3.6 Electron Donors and Electron Acceptors

NAD+ and NADH facilitate redox reactions
without being consumed; they are recycled
NAD+ reduction

NAD+

Active
binding site
site

1. Enzyme
and oxidized form of
coenzyme, NAD+.

I reacts with e donor

Enzymesubstrate
complex

Enzyme I

NAD+

4. NAD+ is
released.

Substrate
(e donor)

NADH

+
Product
Product

NADH
binding
site

Activ
e
site
Enzyme Il

3. Enzyme
acceptor and reduced
form of coenzyme, NADH.
II reacts with e

NADH oxidation

Enzymesubstrate
complex

Substrate
(e acceptor)

2. NADH and
reaction
product are
formed.

3.7 Energy-Rich Compounds

Chemical energy released in redox reactions is primarily
stored in certain phosphorylated compounds
ATP: the prime energy currency
Phosphoenolpyruvate
Glucose 6-phosphate

Chemical energy also stored in coenzyme A

Anhydride bonds

Ester
bond
Ester bond

Anhydride bond

Phosphoenolpyruvate

Anhydride
bond

Thioester
bond

Acetyl

Adenosine triphosphate (ATP)

Coenzyme A

Acetyl-CoA

Acetyl phosphate

Glucose 6-phosphate

3.7 Energy-Rich Compounds

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)