Nitrogen Fixation Nitrogen Fixation by Cyanobacteriaby Cyanobacteria

KSU StudentsKSU StudentsFaculty of ScienceFaculty of Science

Botany & Microbiology Dept.Botany & Microbiology Dept.SupervisorSupervisor

Prof .Dr. Ibraheem IBN Prof .Dr. Ibraheem IBN

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 (NN)

• 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 (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

In biological nitrogen fixation two moles of ammonia are produced from one mole of nitrogen gas, using 16 moles of ATP and a supply of electrons and protons (hydrogen ions):

N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi

This reaction is performed exclusively by prokaryotes (the bacteria and related organisms), using an enzyme complex termed Nitrogenase. This enzyme consists of two proteins - an iron protein and a molybdenum-iron protein.

A point of special interest is that the nitrogenase enzyme complex is highly sensitive to oxygen

Nitrogen fixing bacteria

Some nitrogen fixing organisms

• Free living aerobic bacteria– Azotobacter– Beijerinckia– Klebsiella– Cyanobacteria (lichens)

• Free living anaerobic bacteria– Desulfovibrio– Purple sulphur bacteria– Purple non-sulphur bacteria– Green sulphur bacteria

• Free living associative bacteria– Azospirillum

• Symbionts– Rhizobium (legumes)– Frankia (alden trees)

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.

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.

CyanobacteriaCyanobacteria

• Gram negativeGram negative

• Contain photosytem I and II (fix COContain photosytem I and II (fix CO22, , produce Oproduce O22))

– Note:Note:

•Photosystem I provides the cells with Photosystem I provides the cells with ATPATP

• Photosystem II breaks water down to Photosystem II breaks water down to OO22

CyanobacteriaCyanobacteria

• Diversity: Most diverse photosynthetic Diversity: Most diverse photosynthetic bacteriabacteria– Split into five subsectionsSplit into five subsections

• I: Unicellular rods or cocciI: Unicellular rods or cocci

• II: Unicellular, aggregate with use of outer wall, from II: Unicellular, aggregate with use of outer wall, from reproductive baeocytesreproductive baeocytes

• III: Vegetative unbranched III: Vegetative unbranched trichomestrichomes

• IV: Filamentous unbranched trichomes withIV: Filamentous unbranched trichomes with heterocycst heterocycst formationformation

•V: Filamentous branched trichomes with heterocycst V: Filamentous branched trichomes with heterocycst formationformation

CyanobacteriaCyanobacteria

•TrichomesTrichomes•HeterocystsHeterocysts

– When the cell is deprived of When the cell is deprived of fixed inorganic nitrogen fixed inorganic nitrogen (ammonia)…(ammonia)…•Thick cell wall formationThick cell wall formation

•Photosystem I for ATP Photosystem I for ATP productionproduction

•Degredation of photosysten IIDegredation of photosysten II– Involved in OInvolved in O22 production production

– OO22 inhibits inhibits nitrogenasenitrogenase

Nitrogen

Fixation

Anabaena with heterocysts

The other cells have both photosystem I and photosystem II, which generates oxygen when light energy is used to split water to supply H2 for

synthesis of organic compounds.

Anabaena sp. with symbiont bacteria (possibly Zoogloea) around heterocysts

In some cyanobacteria nitogen fixation occurs in heterocycts. These cells only have Photosystem I

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

Nitrogenase (encoded by Nitrogenase (encoded by nifHnifH gene) gene)

Fe ProteinFe Protein

FeMoFeMoProteinProtein

2ATP2ATP 2ADP + 2P2ADP + 2Pii

NN22 NHNH33

O2 Inhibition

Heterocyst

Characteristic features of Heterocyst:

1. The Heterocyst is the site for cyanobacterial nitrogen fixation which is an enlarged cell, and may be present terminally or intercalary in the filamentous cyanophycean algae.

2. In the process of cyanobacterial nitrogen fixation, hydrogen gas (H2) is also evolved as a by product and 40% of it is recycled by the hup gene (hydrogen uptake gene), whereas remaining 60% hydrogen gas can be used by biotechnologists as a source of future clean fuel.

3. The Heterocyst is made up of three (3) different cell wall layers- the outer fibrous and middle homogenous layers are made up of non-cellulose polysaccharide. Whereas, the inner laminated layer is made up of glycolipids.

Characteristic features of Heterocyst: Cont.

4. On one hand, these special cell wall layers permit the atmospheric N2

(g) to diffuse inside, whereas on the other hand they stop the atmospheric O2

(g) to come inside.

5. This is a damage-control mechanism for the enzyme

nitrogenase, as the nitrogenase is sensitive to O2 and

cold, and cannot function in the presence of O2 (g).