NITROGEN FIXATION

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NITROGEN FIXATION
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Nitrogen Fixation

• The growth of all organisms depend on the
  availability of Nitrogen (e.g. amino acids)
• Nitrogen in the form of Dinitrogen (N2) makes up
  80 of the air we breathe but is essentially
  inert due to the triple bond (N?N)
• In order for nitrogen to be used for growth it
  must be "fixed" (combined) in the form of
  ammonium (NH4) or nitrate (NO3) ions.

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Nitrogen Fixation

• The nitrogen molecule (N2) is quite inert. To
  break it apart so that its atoms can combine with
  other atoms requires the input of substantial
  amounts of energy.

• Three processes are responsible for most of the
  nitrogen fixation in the biosphere
• atmospheric fixation
• biological fixation
• industrial fixation

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Industrial Fixation

• Under great pressure, at a temperature of 600oC,
  and with the use of a catalyst, atmospheric
  nitrogen and hydrogen (usually derived from
  natural gas or petroleum) can be combined to form
  ammonia (NH3).
• Ammonia can be used directly as fertilizer, but
  most of its is further processed to urea and
  ammonium nitrate (NH4NO3).

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Changing Nitrogen Cycle
Humans have doubled the N fixation rates over
natural levels
Haber-Bosch 3CH4 6H2O --gt 3CO2 12H2
4N212H2 --gt 8NH3 (high T,press)
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Nitrogen Fixation Process

• Energetics
• N?N
• Haber-Bosch (100-200 atm, 400-500C, 8,000 kcal
  kg-1 N)
• Nitrogenase (4,000 kcal kg-1 N)

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Biological Fixation

• The ability to fix nitrogen is found only in
  certain bacteria.
• Some live in a symbiotic relationship with plants
  of the legume family (e.g., soybeans, alfalfa).
• Some establish symbiotic relationships with
  plants other than legumes (e.g., alders).
• Some nitrogen-fixing bacteria live free in the
  soil.
• Nitrogen-fixing cyanobacteria are essential to
  maintaining the fertility of semi-aquatic
  environments like rice paddies.

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Biological Fixation cont.

• Biological nitrogen fixation requires a complex
  set of enzymes and a huge expenditure of ATP.
• Although the first stable product of the process
  is ammonia, this is quickly incorporated into
  protein and other organic nitrogen compounds.
• Scientist estimate that biological fixation
  globally adds approximately 140 million metric
  tons of nitrogen to ecosystems every year.

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Some nitrogen fixing organisms

• Free living aerobic bacteria
• Azotobacter
• Beijerinckia
• Klebsiella
• Cyanobacteria (lichens)
• Free living anaerobic bacteria
• Clostridium
• Desulfovibrio
• Purple sulphur bacteria
• Purple non-sulphur bacteria
• Green sulphur bacteria

• Free living associative bacteria
• Azospirillum
• Symbionts
• Rhizobium (legumes)
• Frankia (alden trees)

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Some nitrogen fixing organisms
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Estimated Average Rates of Biological N2 Fixation
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Rank of Biological Nitrogen Fixation
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Nitrogen Fixation

• All nitrogen fixing bacteria use highly conserved
  enzyme complex called Nitrogenase
• Nitrogenase is composed of of two subunits an
  iron-sulfur protein and a molybdenum-iron-sulfur
  protein
• Aerobic organisms face special challenges to
  nitrogen fixation because nitrogenase is
  inactivated when oxygen reacts with the iron
  component of the proteins

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Nitrogenase
FeMo Cofactor
Fd(ox)
N2 8H
Fd(red)
8e-
2NH3 H2
nMgATP
nMgADP nPi
4C2H2 8H 4C2H2
Dinitrogenase reductase
Dinitrogenase
N2 8H 8e- 16 MgATP ? 2NH3 H2 16MgADP
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Nitrogenase
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Genetics of Nitrogenase
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Types of Biological Nitrogen Fixation

• Free-living (asymbiotic)
• Cyanobacteria
• Azotobacter
• Associative
• RhizosphereAzospirillum
• Lichenscyanobacteria
• Leaf nodules
• Symbiotic
• Legume-rhizobia
• Actinorhizal-Frankia

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Free-living N2 Fixation

• Energy
• 20-120 g C used to fix 1 g N
• Combined Nitrogen
• nif genes tightly regulated
• Inhibited at low NH4 and NO3- (1 µg g-1 soil,
  300 µM)
• Oxygen
• Avoidance (anaerobes)
• Microaerophilly
• Respiratory protection
• Specialized cells (heterocysts, vesicles)
• Spatial/temporal separation
• Conformational protection

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Heterocyst
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Associative N2 Fixation

• Phyllosphere or rhizosphere (tropical grasses)
• Azosprillum, Acetobacter
• 1 to 10 of rhizosphere population
• Some establish within root
• Same energy and oxygen limitations as free-living
• Acetobacter diazotrophicus lives in internal
  tissue of sugar cane, grows in 30 sucrose, can
  reach populations of 106 to 107 cells g-1 tissue,
  and fix 100 to 150 kg N ha-1 y-1

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Phototrophic N2-fixing Associations

• Lichenscyanobacteria and fungi
• Mosses and liverwortssome have associated
  cyanobacteria
• Azolla-Anabaena (Nostoc)cyanobacteria in stem of
  water fern
• C Gunnera-Nostoccyanobacteria in stem nodule of
  dicot
• C Cycas-Nostoccyanobacteria in roots of
  gymnosperm

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Azolla pinnata (left) 1cm. Anabaena from crushed
leaves Of Azolla.
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Simbiosis Anabaena-Azolla
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Frankia and Actinorhizal Plants

• Actinomycetes (Gram , filamentous) septate
  hyphae spores in sporangia thick-walled vesicles

Frankia vesicles showing thick walls that confer
protection from oxygen. Bars are 100 nm.
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(No Transcript)
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Actinorhizal Plant Hosts
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Legume-Rhizobium Symbiosis

• The subfamilies of legumes (Caesalpinioideae,
  Mimosoideae, Papilionoideae), 700 genera, and
  19,700 species of legumes
• Only about 15 of the species have been evaluated
  for nodulation
• Rhizobium
• Gram -, rod
• Most studied symbiotic N2-fixing bacteria
• Now subdivided into several genera
• Many genes known that are involved in nodulation
  (nod, nol, noe genes)

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Taxonomy of Rhizobia
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Rhizobium Root Nodules
The picture above shows a clover root nodule.
Available from Internet
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Rhizobium Root Nodules
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A few legumes (such as Sesbania rostrata) have
stem nodules as well as root nodules. Stem
nodules (arrows) are capable of photosynthesis as
well as nitrogen fixation.
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Formation of a Root Nodule
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Nodulation in Legumes
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Infection Process

• Attachment
• Root hair curling
• Localized cell wall degradation
• Infection thread
• Cortical cell differentiation
• Rhizobia released into cytoplasm
• Bacterioid differentiation (symbiosome formation)
• Induction of nodulins

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Role of Root Exudates

• General
• Amino sugars, sugars
• Specific
• Flavones (luteolin), isoflavones (genistein),
  flavanones, chalcones
• Inducers/repressors of nod genes
• Vary by plant species
• Responsiveness varies by rhizobia species

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nod Gene Expression
Common nod genes
Nod factorLCO (lipo-chitin oligosaccharide)
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Nodule Metabolism

• Oxygen metabolism
• Variable diffusion barrier
• Leghemoglobin
• Nitrogen metabolism
• NH3 diffuses to cytosol
• Assimilation by GOGAT
• Conversion to organic-N for transport
• Carbon metabolism
• Sucrose converted to dicarboxylic acids
• Functioning TCA in bacteroids
• C stored in nodules as starch

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Anaerobic Culture Methods

• Anaerobic jar

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Anaerobic Culture Methods

• Anaerobic chamber

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Enrichment Media

• Encourages growth of desired microbe
• Assume a soil sample contains a few
  phenol-degrading bacteria and thousands of other
  bacteria
• Inoculate phenol-containing culture medium with
  the soil and incubate
• Transfer 1 ml to another flask of the phenol
  medium and incubate
• Transfer 1 ml to another flask of the phenol
  medium and incubate
• Only phenol-metabolizing bacteria will be growing

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Selective Media

• Suppress unwanted microbes and encourage desired
  microbes.

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Streak Plate
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Plate Count

• After incubation, count colonies on plates that
  have 25-250 colonies (CFUs)