Nitrogen Cycle

• • 79% atmosphere is N as gaseous N2.

• • Atmospheric N2 is recycled, but slowly
• • N2 unavailable to most life forms
• • Large but essentially unavailable reservoirs of N are
• present in rocks and are not recycled
• – N is essential part of protein, DNA, and many other
• organic molecules
• – Nitrogen has stable valence states ranging from –3 , as
• ammonia (NH3), to +5, as nitrate(NO3
• -), occurs in
• numerous oxidation states.
• Nitrogen Fixation
• • N2 must be converted to another forms
• • Fixation of Nitrogen happens in two ways:
• – High energy fixation
• – Cosmic radiation, meteorite trails and lightning combined N with O and H
• – < 8.9 kg N/ha come to earth annually this way
• – 2/3 as ammonia; 1/3 as nitric acid
• – Industrial Fixation
• – Used to make fertilizer (NH4+)
• – Requires H2, High Temp (400-650*C), High Pressure (200-400 atm)
• – Consumes ~1% worlds annual energy
• – High cost
• – Biological Fixation
• – Biological Temperature and Pressure
• – Microbially mediated
• – Symbionts and Free-Living
 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.
 Nitrogen Fixation
• The nitrogen molecule • Three processes are
(N2) is quite inert. To responsible for most
break it apart so that of the nitrogen
its atoms can combine fixation in the
with other atoms biosphere:
requires the input of • atmospheric fixation
substantial amounts of • biological fixation
energy.
• industrial fixation
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).
 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.
 Some nitrogen fixing organisms
• Free living aerobic bacteria • Free living associative bacteria
– Azotobacter – Azospirillum
– Beijerinckia
– Klebsiella
– Cyanobacteria (lichens)

• Free living anaerobic bacteria • Symbionts

– Clostridium – Rhizobium (legumes)
– Desulfovibrio – Frankia (alden trees)
– Purple sulphur bacteria
– Purple non-sulphur bacteria
– Green sulphur bacteria
 Some nitrogen fixing organisms
Free living Symbiotic
Aerobes Anaerobes Leguminous Non
Heterotrophs Phototrophs Heterotrophs Phototrophs plants leguminous plants
Azotobacter spp. Various Clostridium spp Chromatium soybeans, Alnus, Myrica
Klebsiella Cyanobacteria Desulfovibrio Chloribium clover, Ceanthus
Beijerinckia Disulfoto- Rhodospirillum locust, etc Comptorinia
Bacillus maculum Rhodopseudo- In association Casurina
polymyxa monas with a bacterium in assocation
Mycobacterium Rhodo- of the genus with
flavum microbium Rhizobium or actinomycetes
Azospirillium Rhodobacter Bradyrhizobium of the genus
lipoferum Heliobacterium Frankia
Citrobacter
freundii
Some
Methylotrophs
Rank of Biological Nitrogen Fixation

N2 fixing system Nitrogen Fixation (kg

N/ha/year)
Rhizobium-legume 50 - 600

Cyanobacteria- moss 10 - 300

Rhizosphere 5 - 25
associations
Free- living 0.1 - 25
 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
 Nitrogenase
Fd(ox) FeMo Cofactor

Fd(red) N2 + 8H+
8e-

nMgATP 2NH3 + H2

nMgADP + nPi 4C2H2 + 8H+ 4C2H2

Dinitrogenase
Dinitrogenase
reductase

N2 + 8H+ + 8e- + 16 MgATP  2NH3 + H2 + 16MgADP

Nitrogenase
 Types of Biological Nitrogen Fixation

Free-living (asymbiotic)
• Cyanobacteria
• Azotobacter
Associative
• Rhizosphere–Azospirillum
• Lichens–cyanobacteria
• Leaf nodules
Symbiotic
• Legume-rhizobia
• Actinorhizal-Frankia
Heterocyst
Formation of a Root Nodule
 Leghemoglobin:

• Serves as electron carrier

• Supply oxygen to bacteroids
• Protects the oxygen sensitive nitrogenase
• The heme portion is coded by Rhizobia
• The globin portion is coded by the plant
• Leghemoglobin is unique and occurs in nowhere
• else in plant kingdom
• Specific expression of plant and bacterial genes
• accompanies the development of the symbiosis