Microbial Metabolism

Metabolic Reactions
Enzymology
Catabolism
Phototrophy
Anabolism
 Metabolism Overview:

Reduction;
e- gain
from donor

Oxidation;
e- loss to
acceptor
Metabolic Pathways
• Although we can recognize substrate and product of
individual enzymatic reactions; metabolic functions are
often performed by several enzymatic reactions in a
series or “pathway”.

• Pathways can be linear, branched, cyclic or even spiral.

• Pathway activity is controlled in three ways:

– Metabolites and enzymes may be localized in different parts of
the cell; called metabolic channeling. (important in eukaryotes)

– The total amount of enzymes in a pathway can vary (gene

expression).

– Pathway activity is controlled by critical regulated enzymes.

These “pacemaker enzymes” are often the rate-limiting step in
the pathway.
 “Pacemaker” Enzyme Activity
• Enzyme activity may change due to inhibitor or activator
molecules called effectors.
• Inhibitors can be competitive (bind at substrate active site)
• Noncompetitive inhibitors and activators bind to allosteric
(regulatory) sites; separate from the active site; These effectors
change the shape of the protein and its activity as a catalyst.
Metabolic Pathway Control Strategies
Feedback Inhibition:
(“end-product inhibition”; red)

• rate limiting enzyme is first in

pathway and is allosteric.

• end-product is a negative effector

(inhibitor) of first enzyme

Feed Forward Activation:

(“earlier-substrate activation”;
blue) +

• rate limiting enzyme of a

branch point is allosteric.

• earlier-substrate is a positive Arrows =

effector (activator) of a forward enzymes
reaction enzyme.
 Reversible Metabolic Pathways
• Amphibolic Pathways:
– Catabolic direction
– Anabolic direction

• Separate regulatory enzymes each

way function as “check valves” for
flow control.

• Other pathway enzymes are

reversible; their equilibrium shifts
based on concentration of reactants
& products.

• Gycolysis / Gluconeogenesis is a
good example. Catabolic
breakdown of glucose for energy
versus the its anabolic formation,
respectively.
 Microorganisms – Prokaryotic/Eukaryotic

• Main agents responsible for biogeochemical transformations of

carbon, nitrogen, sulfur, iron, & other elements.

• Importance to the environmental engineer: by Bioremediation, water

purification. Microbes work is essential in all aspects of
Environmental Engineering.

• Range of carbon sources as substrates used by prokaryotes greater

than eukaryotes.

• 2nd Prokaryotes perform types of energy generation unknown to

eukaryotes.
1. Denitrification: (Reduction of nitrate to dinitrogen and other
gaseous nitrogen),
2. Nitrification: (oxidation of ammonium ions to nitrate, nitrite),
3. Reduction of sulfate to badly smelling H2S
4. Acidic wastewater (yellow-brown: Fe (OH) near coal mines
5. Greenhouse gas (Methane): Anaerobic degradation of organic
matter.
6. Environmental biotech: Understand before prevention of such
processes

7. Microorganisms are both natural & manipulated by the

environmental engineer (imp: Rapid growth + high metabolic
rates).
8. Small community of microorganisms has a huge impact on
ecosystem (Terrestrial/ aquatic) essential for the environmental
engineer.
Metabolic Properties of Microorganisms: Thermodynamic Approach
1. Cell has to obtain building blocks (new cellular components), for
growth & multiplication.
2. Building blocks need to contain all the cell is built (Carbon, hydrogen,
oxygen, nitrogen, sulfur, phosphorus, iron, trace elements.
3. Proteins, nucleic acids, cell walls (From simple inorganic comp e.g.
Co2 as carbon source, Nitrate/Ammonium ions as nitrogen source,
sulfate, phosphate (sulfur, phosphorus).
4. Such cells that do not req any organic carbon compounds for growth
are designated autotrophic.
5. Green plants, eukaryotic algae (autotrophs): Types of autotrophic
microorganisms: phototrophic purple & green sulfur bacteria (Use
sulfide than water as electron donor to fix CO2).
6. Aerobic chemoautotrophic (chemolithotrophic) bacteria: Energy
from oxidation of inorganic compounds e.g. ammonium/nitrite ions
/sulfide.
Types of microorganisms, classified according to energy & carbon sources
1. Anaerobic prokaryotes: (Methanogenic Archaea): Energy from
reduction of CO2 to methane (hydrogen as electron donor), CO2:
Sole carbon source for growth.
2. Many bacteria, fungi, protozoa, higher animals are heterotrophs:
require organic carbon compounds, organic sources of nitrogen and
sulfur as well, as building blocks for cell material production.
3. Biosynthesis of proteins, NAs, cell macromolecules from simple
precursors is energetically expensive process.
4. Autotrophic organism produce all its chemical components from CO2
and other simple, generally oxidized components (energetically
expensive); then heterotrophic org.
5. Heterotrophic org takes sugars, AAs, from the medium and needs to
assemble components to proteins, NAs, cell wall.
6. New biomass formed, depends on availability of building blocks
organic/inorganic and on generation of energy by the cells to enable
the biosynthesis.
Based on the known G of values of common substrates and metabolic
products, the free energy yield of microorganisms can easily be
calculated.
For example, the free energy change associated with the alcoholic
fermentation of yeast:
 Glucose (aerobic)

Catabolism
• ATP as the cellular energy
storage unit, can be formed
during respiration (R) or
fermentation (F).
• Both contain the Glycolysis
pathway; which produces ATP,
the electron carrier molecule
NADH, and pyruvate from
glucose.
• Aerobic Respiration will
proceed via Krebs Cycle and an (anaerobic)
ETC if there is oxygen to react
as a terminal electron acceptor. • Fermentation
• Oxygen is not the only proceeds when
possible terminal electron there is no
acceptor in some bacteria (e.g. terminal electron
NO3 or SO4 can be used); acceptor for
called Anaerobic Respiration. respiration.
(ETC)
 Glycolysis:

6C glucose goes to 2x 3C pyruvate plus 2 ATP net, and 2 NADH. ATP must first be
invested to then yield energy from oxidation and substrate level phosphorylation of ATP.
 Products of Fermentation
Without any form of respiration, glycolysis products, pyruvate and NADH,
will accumulate. To keep making any more ATP by glycolysis, fermenting
cells must convert NADH (red.) back to NAD+ (ox.) by passing its
electrons to pyruvate. Reaction pathways that do this convert pyruvate
to many other compounds, depending on the organism.
Lactate Fermentation
2. Alcoholic Fermentation

COO- CO2 CH2OH

H O
C=O C + NADH CH3 +
CH3  CH3  NAD+

pyruvate acetaldehyde ethanol

pyruvate decarboxylase-irreversible
 alcohol dehydrogenase- reversible
Note: NADH used up
Zn+2 at the active site polarizes the carbonyl oxygen of acetaldehyde, allowing transfer of a
hydride ion (red) from the reduced cofactor NADH. The reduced intermediate acquires a
proton from the medium (blue) to form ethanol.
Fermentations Yield a Variety of Common Foods, Industrial Chemical
• Fermentation in production, preservation of foods. Microorganisms present in raw
food products ferment carbohydrates and yield metabolic products (foods their
characteristic forms, textures, and tastes)
• Unsweetened Yogurt, Lactobacillus bulgaricus: ferments carbohydrate in milk,
producing lactic acid; drop in pH: milk proteins precipitate, produce thick texture,
sour taste
• Propionibacterium freudenreichii: Ferments milk to produce propionic acid and
CO2, propionic acid ppt. milk proteins, and bubbles of CO2 cause the holes
(characteristic of swiss cheese)
• Pickles, sauerkraut (Ferm cabbage), sausage, soy sauce (Ferm soybeans, roasted
grain, brine), kimchi (Korea) (Ferm vegetables), tempoyak (Indonesia) (Ferm Fruit),
kefir (Russia) (Ferm milk), dahi (India), pozol (Mexico) (Ferm corn dough).
• Drop in pH associated with fermentation helps to preserve foods, MO (Cause food
spoilage cannot grow at low pH)
• Agriculture: Corn stalks (Byproduct), animal feed by packing them into a large air
tight microbial fermentation produces acids that lower the pH (Silage long shelf life)

• Ferm= Feremented
• Clostridium acetobutyricum ferments starch to butanol and acetone
• Carbohydrates (Corn starch/Molasses), supplied to a pure culture of specific
microorganism, which ferments it into a product of greater value.
• Methanol used to make “gasohol” by microbial fermentation, (like formic, acetic,
propionic, butyric, and succinic acids, glycerol, ethanol, isopropanol, butanol, and
butanediol).
• In huge closed vats in which temperature and access to air are adjusted to favor the
multiplication of the desired microorganism (Exclude contaminating organisms)

Industrial fermentations
• Immobilize cells in an inert support, to pass starting material continuously through
the bed of immobilized cells, collect the desired product.
Pyruvate Decarboxylation:
(Preparatory Step Before Kreb Cycle)
• Pyruvate loses a carbon in the form of
CO2 ; an electron is removed to convert
NAD+ to NADH, and coenzyme-A (CoA)
binds to the 2C acetyl group.
• Acetyl CoA enters the Kreb Cycle by
binding with 4C oxaloacetate to form 6C
citric acid.

Krebs Cycle:
• The cycle converts a citric acid back to
oxaloacetate; losing 2 CO2 ; releasing
electrons to yield 3 NADH plus 1 FADH,
and one ATP by substrate level
phosphorylation.
• For one glucose the cycle runs twice.
Maximum yield
per glucose = 38
ATP
• Only achieved by
aerobic respiration of
mitochondria in
eukaryote cells.
• Aerobic respiration
by bacteria is less
efficient (< 24 ATP).
• Anaerobic
respiration is even
less efficient.
• Fermentation least
efficient (2 ATP)
 Energy Perspective on the Electron
Transport Chain (ETC) Function
The ETC is a series of membrane bound electron carriers
that transports electrons from high to low energy state,
ending with oxygen accepting electrons to water.
Energy release is first used to pump protons
(H+) across the membrane; a proton motive
force (PMF) then drives ATP synthesis.

Each NADH will make 3 ATP.

Each FADH will make 2 ATP

Energy
State
Each electron
PMF= more protons on
transport step
this side of membrane.
releases energy

FADH

Only 2
ATP per
FADH
The Aerobic Respiratory System of E. coli. NADH is the electron source.
Ubiquinone-8 (Q) connects the NADH dehydrogenase with two terminal oxidase
systems. The upper branch operates when the bacterium is in stationary phase and
there is little oxygen. At least five cytochromes are involved: b558, b595, b562, d, and o.
The lower branch functions when E. coli is growing rapidly with good aeration.
Hydrolysis of Major Biomolecules

Enyzymes of Hydrolysis:
• Proteins by proteases.
• Polysaccharide and
other carbohydrates by
glycosidase.
•Nucleic acids (DNA or
RNA) by nucleases.
• Lipids by lipases.
 Amphibolic Nature of Metabolism

Most catabolic pathways

have anabolic
counterparts, so not all
compounds are used to
generate ATP, but rather
shunted to make new
cell biomass.
1. Energy is also available to living systems in the form of gradients of
protons (H+) across biological membranes.
2. In many dissimilatory (energy-yielding) processes in prokaryotes,
energy is conserved in the form of proton gradients generated by
transport of protons from the cytoplasmic side of the membrane to
the extracellular environment.
3. Such electrochemical gradients of protons involve both a pH
difference (alkaline inside, acidic outside) and a membrane potential
(negative inside, positive outside).
4. In eukaryotes, similar processes take place across the membranes of
mitochondria & chloroplasts.
5. Controlled entry of protons through the enzyme ATP synthase located
within the membrane is coupled with of ATP from ADP and
inorganic phosphate.
6. ATP & proton electrochemical gradients across membranes as fully
interconvertible forms of energy, to be used to drive energy.
7. Biosynthesis of new cellular components enabling growth.
1. Dissimilatory, assimilatory metabolism reactions, electron transfer
processes in which electrons flow from an electron donor to an
electron acceptor.

2. Aerobic respiration in which organic substrates are oxidized, coupled

to the transfer of the electrons to molecular oxygen with the
formation of water is just one example.
3. The tendency of different compounds to gain electrons by
reduction/donate e- & become oxidized can be expressed in terms of
standard reduction potential of the redox couples.

4. The more negative standard reduction potential, stronger the

tendency of the reduced form to donate electrons to an oxidized
compound with a higher reduction potential.
5. The amount of energy to be invested/gained during such redox
reactions is directly proportional to the difference in standard
reduction potential of the reductant and the oxidant involved
Pentose Phosphate Pathway of Glucose Oxidation
Pentose Phosphate
Pathway.
The conversion of three
glucose 6-phosphate
molecules to 2 fructose 6-
phosphates and
glyceraldehyde 3-
phosphate is traced.
The fructose
6-phosphates are changed
back to glucose 6-
phosphate.
The glyceraldehyde 3-
phosphate can be
converted to pyruvate or
combined with a molecule
of dihydroxyacetone
phosphate (from the
glyceraldehyde 3-
phosphate formed by a
second turn of the pathway)
to yield fructose 6-
phosphate.
Product of oxidative phase is Ribose 5-ph + NADPH (reduce glutathione). Cells not using Ribose 5-ph for
biosynthesis, non-oxidative phase recycles 6 molecules of the pentose into five molecules of the (Hexose
Glucose 6-ph), allow continued production of NADPH and converting glucose 6-phosphate (in six cycles)
to CO2.
Nonoxidative reactions of the pentose phosphate pathway.
(a) These reactions convert pentose ph to hexose ph, allowing the
oxidative reactions to continue. Transketolase and transaldolase are
specific to this pathway; Other enzymes also serve in the glycolytic/
gluconeogenic pathways. Every reaction shown here is reversible.
In light-indep reactions of photosynthesis, direction of these reactions is
reversed
• Transketolase requires the cofactor TPP, stabilizes a 2-carbon
carbanion in this reaction, just as it does in pyruvate decarboxylase
reaction .
• Transaldolase uses a Lys side chain to form a Schiff base (R2C=NR)
with carbonyl group of its substrate, a ketose, stabilizing a carbanion;
central to the reaction mechanism.
Autotrophic growth in eukaryotic algae, cyanobacteria, purple sulfur bacteria, aerobic
chemoautotrophic bacteria (Nitrifiers (Nitrosomonas, Nitrobacter and relatives), colorless sulfur
bacteria (Thiobacillus, Beggiatoa, and related organisms).
Green sulfur bacteria (Chlorobium, Prosthecochloris) fix CO2 by reversing reactions of
Krebs cycle.
Light activation of several enzymes of the Calvin cycle. The light activation is
mediated by thioredoxin, (small, disulfide-containing protein). In the light, thioredoxin is
reduced by electrons moving from photosystem I through ferredoxin (Fd) (blue arrows),
then thioredoxin reduces critical disulfide bonds in each of the enzymes sedoheptulose
1,7-bisphosphatase, fructose 1,6-bisphosphatase, ribulose 5-phosphate kinase, and
glyceraldehye 3-phosphate dehydrogenase, activating these enzymes. In the dark,
the OSH groups undergo reoxidation to disulfides, inactivating the enzymes
Role of NADPH in regulating the partitioning of G 6-ph b/w
glycolysis & pentose phosphate pathway. When NADPH is forming
faster than it is being used for biosynthesis and glutathione reduction
[NADPH] rises & inhibits 1st enzyme in pentose phosphate pathway.
As a result, more G 6-ph is available for glycolysis
Energy Source
Overview:
• In addition to organisms
feeding on organic carbon for
energy (chemoorganotrophs).
• There are chemolithotrophs,
which gain energy from reduced
inorganic compounds (litho =
rock).
• There are phototrophs that
yield energy from sunlight and
do not depend on any chemical
energy sources.
• Also note how the terminal
(final) electron acceptor
determines which respiration
type or fermentation.