Introduction Web

Microbial physiology and metabolism
These are lecture notes from a series of lectures given in the Virology
department of Sri Venkateshwara University in Tirupati, Andhra Pradesh,
South India.
I was the guest of Professor Sai Gopal

Funded by Society for General Microbiology (UK) and SVU
Microbial physiology and metabolism
Nitrogen: N2 Nitrate, Ammonium, Amino acids
Carbon: Almost all carbon compounds are used by bacteria, yeasts or fungi. Some microbes
use only one or 2 carbon sources while others use a wide range. We will be dealing mainly
with bacterial metabolism.
Methylotrophs: Grow on compounds with only one carbon atom or with no C-C bonds; they
differ from most other microbes in having special pathways for Carbon assimilation and for
energy production.
Environment
Polymers
Polysaccharides
Lipids
Proteins Exocellular enzymes
Nucleic acids (hydrolases) Bacteria
Monomers
Carbohydrates
Alcohols N2 Nitrate,
Fatty acids
Aromatics
Glycerol Ammonium, Amino acids
Methane
Amino acids
CO2
Purines/pyrimidines
Carbon Metabolism B Central metabolism
a)Further oxidation for energy

Catabolism Central metabolic
Carbon (ATP + NADH)
Substrat intermediates
e
A + Energy b)Redistribution to get correct
(ATP + NADH) proportions of precursors for
biosynthesis
C Biosynthesis (anabolism)
New microbe
Polysaccharides [membranes and storage]
Lipids [membranes and storage]
Proteins [structural and enzymes]
Nucleic acids [genes and gene expression] NOTE:
This is Primary metabolism; it is
essential for life
Microbes are all the same for C (Biosynthesis)
They are distinguished by their Catabolic routes Secondary metabolism; it is
And by their energy metabolism ‘extra’ like production of
antibiotics, spores etc
Bacterial Energy Metabolism
Four main headings:
1.Carbon source: Organic or Inorganic [CO2]
2.ATP source: Chemical oxidation or Light
3.NADH source: Organic or Inorganic
4.Energy production: Aerobic or Anaerobic
1. Carbon source
biosynthesis
a) Inorganic carbon [CO2] Autotrophs CO2 CELL
Light or Oxidation of inorganic By Calvin cycle
[ATP + NADH]
mols: hydrogen, nitrite, Ferrous ion
This process involves electron tpt & oxid. phosphorylation
b) Organic carbon source Heterotrophs (including Methylotrophs )
Organic carbon CO2 (Aerobes; ATP by oxid. Phosph.)

OR
[ATP + NADH] Fermentation products (Anaerobes ATP
by Substrate level phosphorylation SLP)
CELL
‘Classification’ of microbes according to their nutrition (Carbon, ATP, NADH)
1. Carbon source Inorganic (carbon dioxide) Organic
2. ATP source a) Chemical oxidations Chemotrophs

b) Light Phototrophs
3. NADH source a) Organic Organotrophs

b) Inorganic Lithotrophs
ATP NADH Carbon Other name
ChemoOrganotroph Oxid. Organic Oxid. Organic Organic ChemoHeterotroph
ChemoLithotroph Oxid. Inorganic Oxid. Inorganic Inorganic ChemoAutotroph
PhotoOrganotroph Light Organic carbon Organic PhotoHeterotroph

PhotoLithotroph Light Inorganic Inorganic PhotoAutotroph
ChemoOrganotrophs: many of the common bacteria; Pseudomonas, Bacillus, Lactobacillus, Ecoli. Aerobes
and anaerobes. Animals
ChemoLithotrophs: Oxidise hydrogen (hydrogen bacteria), nitrite (nitrobacter), ammonia (Nitrosomonas),
ferrous ions (Iron bacteria). Strict aerobes. Very important in soil and oceans
PhotoOrganotrophs; Anaerobic photosynthetic bacteria that use ethanol or acetate. Anoxygenic
photosynthesis. They can also grow aerobically on ethanol or acetate as typical chemorganotrophs. Eg
Rhodopseudomonas Rhodobacter
Photo Lithotrophs; plants and blue green algae (Cyanobacteria) Aerobic (oxygenic); AND some use hydrogen
sulphide instead of water (anaerobic)
Oxidation of organic carbon substrates
a) Anaerobic oxidation (Fermentation)
95-98% Fermentation All ATP by substrate level
Carbon substrate
products phosphorylation
2-5% [ATP + NADH]
CELL
b) Aerobic oxidation
50% Krebs’ TCA cycle
Carbon substrate C2 (Acetyl-CoA)
ATP Electron transport chain

NADH
50%
CELL Almost all ATP by Oxidative phosphorylation
Note: methylotrophs are special as the substrates are oxidised directly to CO2
Some special cases of aerobic / anaerobic oxidation of organic carbon substrates
1. Acetic acid bacteria

Strictly aerobic but do not have a TCA cycle. All ATP is by oxidative phosphorylation
They accumulate reduced end products but this is not by fermentation
Acetobacter: oxidise ethanol to acetic acid
Gluconobacter: oxidise glucose to gluconic acid
Similar to methylotrophs in having special systems for oxidation of growth substrates (quinoproteins)
2. Anaerobic respiration
Exactly same as typical aerobes, using TCA cycle etc but in the absence of oxygen they can use
alternatives final electron acceptors: Nitrate (reduced to nitrite, nitrogen); sulphate (reduced
to sulphide)
All ATP is by oxidative phosphorylation. Typical example is Pseudomonas
Hyphomicrobium is a methylotroph that can grow anaerobically on methanol or methylamine
with nitrate
3. Methylotrophs (Methylotrophs that use methane are Methanotrophs)

Grow on one-carbon compounds – methane, methanol methylamine. They cannot be
Oxidised to acetyl-CoA. They are oxidised by special enzymes to carbon dioxide. ATP is by
oxidative phosphorylation. Some can use nitrate or sulphate instead of oxygen [anaerobic
respiration
Bioenergetics; thermodynamic principles in biology
1st Law: Total energy of a system plus surroundings is constant

Energy changes involve heat absorbed/released and work done
2nd Law: A process only occurs spontaneously (no energy input is required) only if ENTROPY (S) of a system and
its surroundings INCREASES. S is a measure of Disorder For reaction A B then ΔS Must be +ve
.
Problem: Cannot measure ΔS and anyway we are interested in ENERGY changes during
reactions. So we use Gibbs Free Energy Change ΔG. This is the energy available to do work
ΔG = ΔE – TΔS So ΔG includes change in energy and change in entropy
Relationship between ΔG, Equilibrium constant (Keq), direction of reaction and energy change
Reaction equilibrium A B At equilibrium [B]/[A] is Keq, the equilibrium constant
A+B C+D Keq = [c] x [d] / [a] x [b] Keq tells Direction Not Energy change
ΔG = ΔGo + RTln [c] x [d] / [a] x [b] At equilibrium ΔG = 0 So: ΔGo = -RTlnKeq
ΔGo is Standard free energy change when reactants and products are 1.0 M
Example: If Keq = 1.0 then ΔGo = 0
If Keq = 105 then ΔGo = about – 30kj/mole
If Keq = 10-5 then ΔGo = about + 30kj/mole

For a reaction to occur ΔG must be –ve (an exergonic reaction); If +ve it is endergonic
If for reaction A → B ΔGo = + 10 then energy must be put in to enable the reaction to occur
a) Increase concentration of A
ΔG = ΔGo + RTln B/A If [A] is increased then ln of B/A becomes more –ve until eventually ΔG is-ve
b) Put energy into the system by coupling to an exergonic reaction
e.g. couple to a 2nd reaction B → C where ΔGo = - 20 Now A → B → C ΔGo = - 10
NOTE:
1. The rate of reaction is not related to the ΔG value; this could be very large and negative but a slow reaction.
2. The free energy change ΔG for a reaction is the same regardless of the route taken; the oxidation of glucose
to carbon dioxide is same if glucose is metabolised by the TCA cycle or if it is burnt.
The importance of ATP and other energy rich compounds
Energy-rich compounds have a high free energy change on hydrolysis; about -30 kj/mol
ATP is an energy currency; it is not a store.
Ad-rib-P-P-P → AdRib-P-P + Pi → AdRib –P + Pi → AdRib = Pi
ATP ADP AMP
ΔGo - 30 -30 -10
If more energy is needed then ATP may be hydrolysed to AMP plus pyrophosphate which is
hydrolysed rapidly to phosphate (Δgo = -30); this hydrolysis drives the reaction.
As in Amino acid + tRNA → AminoacyltRNA ATP → AMP + PPi → 2 Pi
Recycling of AMP Myokinase: AMP + ATP → 2 ADP
Phosphorylation of ADP to ATP
Substrate level phosphorylation, oxidative phosphorylation or photophosphorylation
Free energy changes in redox reactions
When reduced compounds are oxidised energy is released; this can be harnessed as energy-rich compounds
such as ATP or phosphoglycerate [in SLP]
Oxidation: removal of electrons Fe2+ → Fe3+ + e Reduction: addition of electrons (reverse)
In oxidation of organic mols 2 electrons are removed at a time together with 2 protons
CH3OH → HCHO + [2H] ( = 2e + 2 H+ )
A Redox couple is a mixture of the oxidised and reduced forms of a compound eg HCHO / CH 3OH
Redox potential (E) tells how good an oxidising or reducing agent a redox couple is
A good oxidising agent has a high + potential and a good reducing agent has a high –ve redox
pot. o
E is the standard E value when the concn. of oxidised and reduced forms are equal
The actual of E depends on the concns of ox and red E = E o + RT/nF x ln OX/RED
If [OX] form is increased then E is more +ve That is, the couple is a better oxidiser.
If [RED] form is increased then E is more -ve That is, the couple is a better reducer.
Note: electrons flow from –ve to +ve. The difference in E (ΔE) tells how much energy is available
Ascorbate (red) + Fe (ox) → ascorbate (ox) + Fe (red)
E = +260V
o
+440mV So e flow from ascorbate to iron ΔE = OX – RED = + 180mV
For reaction to occur ΔE must be +ve (different from ΔGo )
How much energy is available from a redox reaction? Convert ΔE to ΔG: ΔG = -nFΔE
NADH2 + O → NAD + H2O

ΔE = 880 - (-320) = + 1200 mV
-320mV +880mV
ΔG = - 180 kj/mol enough for 6 ATP

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Microbial physiology and metabolism

I was the guest of Professor Sai Gopal

a)Further oxidation for energy

b) Organic carbon source Heterotrophs (including Methylotrophs )

Organic carbon CO2 (Aerobes; ATP by oxid. Phosph.)

2. ATP source a) Chemical oxidations Chemotrophs

3. NADH source a) Organic Organotrophs

ChemoLithotroph Oxid. Inorganic Oxid. Inorganic Inorganic ChemoAutotroph

PhotoOrganotroph Light Organic carbon Organic PhotoHeterotroph

ATP Electron transport chain

CELL Almost all ATP by Oxidative phosphorylation

1. Acetic acid bacteria

3. Methylotrophs (Methylotrophs that use methane are Methanotrophs)

1st Law: Total energy of a system plus surroundings is constant

Example: If Keq = 1.0 then ΔGo = 0

If Keq = 105 then ΔGo = about – 30kj/mole

If Keq = 10-5 then ΔGo = about + 30kj/mole

NADH2 + O → NAD + H2O

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