Microbial Metabolism—
Procuring Energy!
Metabolic diversity
Energy flow
For Energy
For Energy
Carbon Flow for Anabolism
Key Points
Dissimulative metabolism—Reduction
of chemicals for energy—much material
must be used to achieve sufficient energy for growth
Assimilative metabolism—Reduction
of chemicals for biomass—the cell only
uses as much starting material as required
Anaerobic Respiration—
molecular oxygen does NOT serve as an
electron acceptor but energy (ATP) is
produced via chemiosmosis
NO3-    N2 or SO4--  H2S or CO2   CH4
1. Nitrate    Nitrogen gas
2. Sulfate  Hydrogen Sulfide gas
3. Carbon dioxide    methane
1,2. Standard electron transport, 3. membrane bound enzymes
Both generate proton gradient required for PMF
In anaerobic
metabolism
Nitrate or
I
Sulfate serve
as terminal
FADH
electron
Co Q 
II
 FAD
acceptors at
the end of the
III
electron
Thus under anaerobic
transport
conditions some organisms
can still obtain high levels of energy chain




NADHNAD
IV
NO3-    N2
SO4--  H2S
Mixotrophs, Energy from oxidation of
organic chemicals inorganic chemicals
are reduced.
Denitrification (Nitrate reduction)
Some Pseudomonas, Bacillus and Thiobacillus spp.
Nitrate reduction is mediated by enzymes
1. nitrate reductase (NR)
2. nitric oxide reductase (NcOR)
3. nitrous oxide reductase (NsOR)
NR
NR
NcOR
NsOR
NO3-  NO2-  NO  N2O  N2
Gases to atmosphere
Significance of denitrification
1. Agriculture: soil nitrate that could be fixed to ammonia and
assimilated by plants are reduced to atmospheric nitrogen and lost
from the soil (however—nitrogen fixing bacteria can restore atmospheric nitrogen
to the soil as part of the overall nitrogen cycle)
2. Acid rain: atmospheric nitrous oxide is converted to nitric
oxide via sunlight. This combined with nitric oxide released via
denitrification reacts with ozone to form nitrite that returns to the
earth as acid rain.
3. Sewage treatment plants (water purification): Denitrifying
bacteria are added to the sewage to convert nitrate to atmospheric
nitrogen to remove nitrogen that would otherwise promote the
growth of algae
Significance of denitrification
1. Agriculture: soil nitrate that could be fixed to ammonia and
assimilated by plants are reduced to atmospheric nitrogen and lost
from the soil (however—nitrogen fixing bacteria can restore atmospheric nitrogen
to the soil as part of the overall nitrogen cycle)
NR
NR
NcOR
NsOR
NO3-  NO2-  NO  N2O  N2
Gases to atmosphere
Significance of denitrification
2. Acid rain: atmospheric nitrous oxide is converted to nitric
oxide via sunlight. This combined with nitric oxide released via
denitrification reacts with ozone to form nitrite that returns to the
earth as acid rain.
NR
NR
NcOR
NsOR
NO3-  NO2-  NO  N2O  N2
NO2sunlight
NR
N2O
NO
ozone
H2O
NO2
-
Acid
rain
Significance of denitrification
1. Agriculture: soil nitrate that could be fixed to ammonia and
assimilated by plants are reduced to atmospheric nitrogen and lost
from the soil (however—nitrogen fixing bacteria can restore atmospheric nitrogen
to the soil as part of the overall nitrogen cycle)
2. Acid rain: atmospheric nitrous oxide is converted to nitric
oxide via sunlight. This combined with nitric oxide released via
denitrification reacts with ozone to form nitrite that returns to the
earth as acid rain.
3. Sewage treatment plants (water purification): Denitrifying
bacteria are added to the sewage to convert nitrate to atmospheric
nitrogen to remove nitrogen that would otherwise promote the
growth of algae
Sulfate Reduction
Bacteria responsible for this are widespread in
aquatic environments
Sulfate reduction is mediated by enzymes
1. ATP sulfurylase (ATPS)
2. APS reductase (APSR)
3. sulfite reductase (SR)
ATPS APSR
SR
--SO4   SO3 
H2S
Excreted into environment
Significance of sulfate reduction
Pollution of waters:
Sulfate reducing bacteria are limited by the amount of organic
starting material available in the aquatic environment which when
metabolized by these bacteria provide the electrons/protons that drive
the sulfate to sulfide reaction.
Disposal of sewage and garbage into waters provides the organic
material required for this process
Sulfides are toxic to living organisms as these sulfides combine with
iron centers of cytochromes and hemoglobin thus inhibiting their
function
N.B. Fe can serve as a detoxifying agent as they react with sulfides to produce
insoluble FeS—black sediments found in aquatic environments are
good indicators of pollution!!!)
Carbon dioxide reduction
Methanogenesis
(methanogens: anaerobic archaebacteria)
Complex set of reactions that take place in the membranes of these
bacteria: protons for CO2 reduction come from fermentation,
methanogenesis (somewhat different from electron transport)
provides proton motive force that drives the production of ATP
Glycolysis  pyruvate  FERMENTATION
Formate, Acetate
Lactate, Proprionate
Butyrate, etc
Membrane associated
reactions of methanogenesis
   
CH4
excreted
CO2 and protons (H2)
Significance of methanogenesis
Sewage treatment plants
Insoluble sludge from
primary treatment is
degraded by a variety of
anaerobic bacteria using
catabolic pathways and
fermentation.
(we drink)
Anoxic digestion of sludge
1. Methanogens use protons from
catabolism and fermentation to reduce
CO2 from fermentation to methane.
2. The methane is collected as natural gas to fuel and heat the
sewage plant or burned off.
Significance of methanogenesis
Digestive processes:
Methanogens are found in the rumen of cows, sheep, deer etc.
The caecum of horses and rabbits, the large intestines of humans,
cats, dogs etc. and the hindgut of termites.
Herbivores: cows/horses/rabbits/termites
bacteria degrade cellulose to cellobiose to glucoseglycolysis
fermentation  organic acids are assimilated and CO2 and H2
are reduced to methane by methanogens
Omnivores: humans/cats/dogs/pigs vast catabolic processes
fermentation methanogenesis of CO2
Methane waste: Cows belch, humans expel gas and I don’t know what the termites do!!!
Stop here
Chemolithotrophy—obtaining energy from
inorganic chemicals
1. inorganic chemicals are oxidized as coenzymes in the
electron transport chain are reduced.
2. Oxygen serves as the terminal electron acceptor in
electron transport
3. Reducing power is not derived from the catabolism of
organic matter to produce NADH and FADH therefore
these cofactors are usually not re-oxidized in
chemolithotropy
4. The chemicals that are oxidized have lower energy
potential than NADH, therefore more of these chemicals
must be oxidized to generate equivalent proton motive
force to produce ATP
Chemolithotrophs tend to grow more slowly than chemo-organotrophs
X


NADHNAD
Chemolithotrophy


Co Q 
O2  H2 O
X
FADH
 FAD
Fe2+ Fe3+
NH4 NO2- /NO3H2S/S2O32- H2SO4
Sulfur and Iron oxidation
(Thiobacillus thiooxidans, Thiobacillus ferrooxidans
and others.)
1. Fe2+ Fe3+
ferrous iron to ferric iron
2. H2S  elemental
sulfurH2SO4
hydrogen sulfide to sulfuric acid
3. S2O32- H2SO4
thiosulfate to sulfuric acid
N.B. Ferric iron can often serve as an oxidizing agent
Sulfuric acid greatly decreases the pH of the surrounding environment
Sulfates can also be assimilated as a food source for bacteria/plants
Significance of sulfur and iron oxidation
Hydrothermal
vents::symbiotic
relationships between
animals and
bacteria dwelling in these
niches (Thiobacillus,
Thiomicrospora, Thiotrix)—
clams, mussels, tube worms
1. Basalt/magma rich in minerals
beneath ocean floor produce
cracks in ocean floor. 2. Minerals
mix with sea water and are
expelled from ocean floor.
Black smokers: precipitated
minerals mixed with seawater/
270-380oC
Life in hydrothermal vents
There is no sunlight at these depths in the ocean yet niches
around the vents are robust with life. Chemolithotrophs serve
as primary producers
1. Bacteria live in the GI tract of tube worms/ the gills of mussels
and clams.
2. Tube worms/mussels/clams provide CO2 as a carbon source for
the bacteria.
3. The bacteria oxidize hydrogen sulfide and thiosulfate to H2SO4
for energy and reducing power and use this for the assimilation
of CO2.
4. Wastes from bacterial metabolism feed the larger animals
Significance of sulfur and iron oxidation
Pollution/Acid mine Drainage (Thiobacillus and Metallogenium spp.)
1. Strip coal mining exposes the pyrite
(FeS2) in coal to oxygen.
2. Bacteria oxidize ferrous iron to ferric
iron
3. Oxidation of sulfides to sulfuric acid
greatly reduces the pH of the water
4. Ferric iron reacts with water to form
iron III hydroxide (Fe(OH)3) which
further lowers the pH of the water
5. Fe(OH)3 precipitates to form slimy
orange coating that covers the stream bed
(indicator for pollution)
6. Acid soon kills the aquatic life at the
bottom of stream bed
Significance of sulfur and iron oxidation
Microbial Bioremediation—
Bio-leaching  use of bacteria
to extract pure metal from ores
with low metal content
Can be used to isolate almost any
divalent metal: copper, uranium
nickel, cobalt, tin, zinc, etc.
Insoluble metal sulfides are
oxidized to soluble metal
sulfates
i.e. Copper required for electricity
is in short supply
Bioremediation
Similarly, sulfur/iron oxidizing bacteria can be used to isolate
important fuel sources.
Uranium usually found naturally as the low grade ore uranium oxide
(UO2) . Thiobacillus spp. oxidize ferrous iron to ferric iron which
In turn oxidizes UO2 to soluble UO2SO4
Oil recovery: recovery of petroleum and hydrocarbons from oil shales
1. Oil shales contain large amounts of carbonates and pyrites
2. Thiobacillus oxidizes the sulfur and iron in the pyrites to
produce acids
3. Acids dissolve the carbonates thus increasing the porosity of
the oil shales
4. Oil can be more easily recovered from these shales.
Nitrification/Nitrogen oxidation by
nitrifying bacteria
A two step process mediated by two genera of bacteria
NH4 NO2Nitrosomonas spp. ammonium to nitrite
NO2-  NO3Nitrobacter spp. nitrite to nitrate
N.B. 35 moles of ammonium and/or 100 moles of nitrate are required
to generate enough reducing power and ATP to convert
one mole of carbon dioxide into organic carbon
Significance of nitrifying bacteria
Agriculture: nitrifying bacteria leach nitrogen required by
plants from the soil.
1. Positively charged Ammonium ions are absorbed by negatively
charged clay particles present in the soil, thus retaining nitrogen
2. Negatively charged nitrites and nitrates are not absorbed by these
clay particles and are leached into the groundwater
A. loss of nitrogen from soil
B. nitrites in the water supply are toxic
1. nitrites combine with hemoglobin to block the
exchange of oxygen
2. nitrites react with amino compounds to form
carcinogenic nitrosamines
Oxidative and Anaerobic Photophosphorylation
(obtaining energy from sunlight)
Algae –takes place in chloroplasts
Cyanobacteria –occurs in thylakoid membranes
Purple bacteria/sulfur bacteria and Heliobacteria –occurs in
lamellar membranes
Web sites for better understanding
http://www-micro.msb.le.ac.uk/video/photosynthesis.html
http://www.biologie.uni-hamburg.de/b-online/chimes/photo/ebacphot.htm
Photosynthetic membranes of the bacteria
Thylakoid membranes in the
cytoplasm of cyanobacteria.
Chloroplasts would be somewhat
analogous to cyanobactria being
present in the cytoplasm of algae and
plant cells
Lamellar membranes in a purple
bacterium
These membranes also arise from
invagination of the cytoplasmic
membrane, but instead of forming
vesicles, they become arranged as
membrane stacks, similar to the
thylakoids of cyanobacteria
Photo-phosphorylation
Is similar to electron transport in that:
1. a proton gradient is generated to provide PMF for ATP
synthesis
2. it involves a series of membrane bound electron acceptors
(known collectively as photosystems)
3. the membranes involved in photosynthesis contain an
ATPase that is responsible for ATP synthesis when a proton
gradient is established
Is different in that:
1. it produces reduced cofactors in the form of NADPH
2. H2O is split into O2 to provide electrons and protons.
Oxidative Photophosphorylation
Synopsis
1. Carbon dioxide (to be used in Carbon fixation) enters the outer and inner membranes
of the chloroplast or Cyanobacteria.
2. For the Calvin cycle, CO2 is fixed in the stroma of the chloroplast or the cytoplasm
of cyanobacteria
3. Oxidative photophosphorylation occurs in the thylakoid membranes that are present
A. The photosystems and electron carriers are present in these membranes
4. H20 is split into O2 inside the thylakoid space of thylakoids.
5. Oxygen is released during oxidative photophosporylation and exits through the inner
and outer membranes of the chloroplasts/cyanobacteria
6. NADPH and ATP is released into the stroma of chloroplasts/cytoplasm of
cyanobacteria and used in CO2 fixation
7. Chemiosmotic theory—shuttling of protons to produce the PMF required for ATP
synthesis.
A. During the transfer of electrons through the e- carriers in the thylakoid
membrane a proton gradient is achieved.
B. Protons are pumped into the thylakoid space during electron transfer
C. An ATP synthase complex is embedded in the thylakoid membrane
D. When the protons flow through the ATP synthase complex from the thylakoid
space into the stroma /cytoplasm—ATP is synthesized!!!!!
Enlarged thylakoid in a chloroplast or in Cyanobacteria
Photosynthesis
Light dependent reactions
vs.
Light independent reactions (Dark Reactions)
Summary Rxn:
OR
6CO2 + 12H2O  C6H12O6 + 6O2 + 6H2O
6CO2 + 6H2O  C6H12O6 + 6O2
chlorophyll
Light reaction: Water + ADP + Pi + NADP
Oxygen + ATP + NADPH
light
Dark reaction: Carbon dioxide + ATP + NADPH
Glucose
enzymes
The Z pathway is used by the aerobic
micro-organisms
1.algae
2. cyanobacteria.
Oxidative phosphorylation involved 2 photosystems that require
two separate photo absorption acts.
1. Photosystem I (PSI)Chlorophyll reaction center
absorbs light at 700 nm
2. Photosystem II (PSII)Chlorophyll reaction center
absorbs light at 680 nm
The Z pathway allows
the noncyclic flow of
electrons seen in
oxidative
photophosphorylation
1. ATP production
2. NADP reduced to
NADPH for biosynthesis
3. Electrons/protons from
splitting water or exciting
photo reaction centers
Step by step actions in noncyclic
oxidative photo-phosphorylation
1. Chlorophyll Rxn center of PSII absorbs light at 680 nm
2. The chlorophyll Rxn center becomes energetically excited and loses
an electron (e-)
3. The e- is transferred through a series of membrane bound e- carriers
until the e- is transferred to the chlorophyll Rxn center of PSI
4. The transfer of e-s from the Rxn center of PSII to PSI creates a proton
gradient such that ATP is generated via chemiosmosis
5. Light excites the chlorophyll Rxn center of PSI such that an e- is
released from that Rxn center
6. e-s are transferred through a series of membrane bound e- carriers in
PSI
7. The protons generated through this e- transfer is used to reduce NADP
to NADPH (NADPH is used in biosynthetic pathways)
8. The Rxn center of PSII meanwhile has lost an e- that must be replaced
9. This is accomplished when H2O is split to form O2 and protons.
10. The e-s are transferred thru membrane bound carriers to the Rxn center
Of PSII. This also generates PMF for ATP synthesis.
Anaerobic Photosynthesis (cyclic) is used by
the halophilic purple bacteria, the
Heliobacteria and the Green sulfur bacteria.
Only one photosystem is employed (PSI)
The Rxn center of PSI absorbs light at 840 nm
NADPH is usually not produced
The bacteria get reducing power from other sources (external or
internal) besides water
Cyclic Anaerobic photo-phosphorylation
H2 S
S2O3
H2 S
S2 O 3
1. ATP production 2. In some cases NADP reduced to NADPH by reverse
electron flow 3. Reducing equivalents for biosynthesis can also come
from external sources besides water or from FeS centers within membranes.
Step by step actions in cyclic anaerobic
photo-phosphorylation
1. Light is absorbed by the Rxn center in PSI
2. e-s are transferred through membrane bound carriers to generate
PMF.
3. The excited e- is returned to the Rxn center of PSI
Where do the bacteria get the reducing equivalents for biosynthesis?
1. Purple bacteria: reverse e- flow through the membrane requires
ATP but can be used to reduce NADP to NADPH
2. Green Sulfur bacteria/Heliobacteria: FeS of the PSI can transfer
e-s and protons to molecules to be reduced (i.e. CO2)
3. Purple bacteria/Green sulfur bacteria: Sulfide in the form of H2S
or S2O3 serve as proton/electron donors that enter the chain at
cytochrome c2 for extra reducing power
(N.B. H2S or S2O3 are oxidized to elemental sulfur that can be stored in
bacteria or expelled)