Nitrogen cyclenitrogen fixation

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Nitrogen cycle/nitrogen fixation Nitrogen is an
important constituent of biological molecules.
The availability of N can affect plant growth
and thus primary Production. Microbes are
intimately involved in this process.
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Nitrogen is a very stable and common molecule. A
lot of energy is required to break the N?N bond.
Most organic nitrogen is recycled from the more
easily available forms nitrate and NH4. But N
fixation is a critically important process in
the environment and in agriculture
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Denitrification   Dissimilatory (anaerobic
process), nitrate is used as an electron
acceptor, producing N2. It is beneficial in waste
water treatment, removing nitrate, thus reducing
algal growth (blooms), eutrophication. Note they
will grow aerobically using O2 as an electron
acceptor if it is available (THIS IS NOT
FERMENTATION).
Pseudomonas denitrificans   2NO3- 10e- 2H ?
N2 6H2O The electrons come from metabolism of
carbohydrates etc.   NO3- ? NO2- ? NO ? N2O
? N2 Thought to be an enzyme for each stage.
Nitric and nitrous oxide can be released into the
atmosphere causing potential problems.
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Nitrification It is oxidation of ammonia to
nitrate (via nitrite). Occurs in well drained,
aerated soils by two nitrifying bacteria,
Nitrosomas and Nitrobacter together (example of
syntrophism). Manure and sewage promote
nitrification. Nitrate is rapidly absorbed by
plants but as it is very soluble it is easily
leached out by rain, so it is not always of
benefit. Ammonia at neutral and acid pH, is
cationic and is absorbed by clay
minerals. Anhydrous ammonia is used as a
fertiliser, chemicals are added to inhibit
nitrification. This (nitrapyrin) increases
efficiency of the fertiliser and reduces run off
water pollution.
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Nitrification is a two stage process The process
in energetically fairly inefficient, generating
few ATP molecules the bacteria grow slowly.
  Nitrosomas NH4 3/2O2- NO2- 2H
energy   Nitrobacter NO2- 3/2O2 NO3-
energy   The energy generated is used to fix
CO2
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Assimililatory is is the conversion of nitrate
(or NH3) to NH3 and then to nitrogenous
compounds like amino acids.
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Nitrogen Fixation This reaction is very
important. N2 is very stable and there is a large
a reservoir of N in the atmosphere. It requires
a lot of energy to break the triple bond, and
only a small number of organisms can do it, all
prokaryotes. Both free living and in symbiotic
associations.
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Examples of nitrogen fixers Free living aerobes
Azotobacter, Azomonas, Cyanobacteria (some) Free
living anaerobes Closridium, Rhodobacter
etc   Symbiotic Rhizobium and Bradyrhizobium with
legumes (Soya, peas, clover etc) Frankia with
woody shrubs Anabaena with azolla (fern) in
paddy fields
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Biochemistry of Nitrogen fixation   Nitrogen has
a triple bond and this requires a lot of energy
to break it. 940 kj compared to 493kj for O2. 6
electrons are required to reduce N2 to 2NH3. This
reduction process is catalysed by Nitrogenase.
Made up of two proteins dinitrogenase and
dinitrogen reductase. Both contain Fe and DR
also has Mo. In DR the Fe and Mo are contained in
a cofactor FeMo-co and the reduction of N2
occurs here. The formula is MoFe7S9. Two
molecules of FeMo-co per molecule. FeS forms a
cage.
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FeMo co complex
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Properties of Nitrogen fixation Nitrogen
fixation is inhibited by O2. As it is a highly
reducing process. Both enzymes are rapidly and
irreversibly inactivated by O2. In aerobic
bacteria, nitrogenase is protected from O2
either by rapid respiration, production O2
retarding slime or production of special cells
(heterocysts), or O2 is removed by special
chemicals. For every molecule of N2 fixed. 16-24
molecules ATP are used.
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Electron Flow   Ferredoxin, flavodoxin or low
potential iron-sulphur protein are the electron
donors. They transfer electrons to dinitrogen
reductase. For each cycle of e- transfer,
dintrogen reductase binds two ATP, which is then
able to interact with dinitrogenase and transfer
electrons to it. ATP is hydrolysed and the two
proteins disassociate to begin another cycle of
reduction. Only 6 electrons used in the useful
reduction, another two are wasted to make H2,
which can back react withN2H2.
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Steps in Nitrogen Fixation
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Assay Nitrogenase activity by acetylene to
ethylene Artificial substrate HC?CH ? H2CCH2
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Soybean Root Nodules
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Leguminous plants are at an advantage in poor
soils. These bacteria are unable to fix, N2
alone, they need the plant. In the nodule O2
levels are controlled by leghaemolobin. The
bacteria and plant form this iron containing
compound which binds O2. Bound free O2 is
10,0001
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Stages in Root Nodule Formation 1 Recognition
and attachment of bacterium to root
hair 2 Invasion of root hair, by formation of an
infection thread. 3 Travel to main
root 4 Formation deformed cells, bacteroids to
get to N fixing state 5 Formation of Nodule
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Rhizobia grow well in the rhizopsphere,
responding well to plant secretions. Rhicadhesin
on the surface of bacterium may bind calcium
complexes on the root hair surface. Invasion of
the root hair is via the tip as a result of the
action of bacterial encoded nod factors,
inducing formation of a cellulosic tube, the
infection thread. Root cells adjacent to this
thread also become infected. Nod factors
stimulate plant cell division. Bacteria multiply
rapidly in the root. Bacterial cells become
swollen into bacteroids, become surrounded
singly, or in groups by plant cell membrane to
become symbiosome. Only then does nit fix take
place. When the plant dies, nodule deteriorate,
bacteroid cannot divide, but some dormant rods
always there which proliferate on the products
released from the dying nodules. The fixed N is
released to the soil
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Stem nodulating legumes in tropics Sesbania
(water plant). Soils get leached because of
high microbial activity.
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Azolla pinnata (left) 1cm. Anabaena from crushed
leaves Of Azolla.
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