nitrogen fixation

2. NITROGEN
• Essential dietary element- derived from animal &
plant protein
• Role of N
• Nitrogen fixation, natural and synthetic, is
essential for all forms of life because nitrogen is
required to biosynthesize basic building blocks of
plants, animals and other life forms,
e.g., nucleotides for DNA and RNA and amino
acids for proteins

3. Role of nitrogen in plants
» Major substance in plants next to water
» Building blocks
» Constituent element of
» Chlorophyll
» Cytochromes
» Alkaloids
» Many vitamins
» Plays important role in metabolism, growth,
reproduction and heredity

4. Sources of nitrogen
• Atmospheric Nitrogen
–78% of atmosphere
–Plants cannot utilize this form
–Some Bacteria, Blue Green Algae,
leguminous plants
• Nitrates, Nitrites and Ammonia
• Amino acids in the soil
• Organic Nitrogenous compounds in insects

6. Nitrogen Fixation
• Is the removal and reduction of atm.
Molecular N2 & its subsequent incorporation
with other elements to form nitrogenous
compounds.

7. • “Nitrogen Fixation” is the process that causes the
strong two-atom nitrogen molecules found in the
atmosphere to break apart so they can combine with
other atoms.
Nitrogen gets “fixed” when it is combined with oxygen
or hydrogen.

8. Factors affecting N2 fixation
1. Presence of nitrate or ammonium :
2. Presence of certain inorganic substances
Ca, Co, Mo – influence N2 fixation along with P
3. Availability of energy source – addn. of C
source increase N2 fixation
4. pH : Neutral – favours Azotobacter – Acidic-
Beijerinkia
5. Soil moisture : Adequate is good for fixation
6. Temperature: Mesophilic – 30°C.

9. There are three ways that nitrogen
gets “fixed”
• Industrial N2 fixation
• Non biological N2 fixation
• biological N2 fixation

10. Industrial N2 fixation
• Accomplished by Haber -Bosch process
• Developed in Germany 1914 by Fritz haber & Karl
bosch
• Process- N2 and H2 react with each other in presence
of
 Industrial catalyst( nickel / iron)
 High temperature about 500 ͦ c
 High pressure – 200 atm
To form NH3
• Source of H2 - is methane (natural gas)
• Industrial production of fertilizers and explosives
N2 + 3 H2 2NH3
500C,Ni
200atm

11. Non biological N₂ fixation
• Non biological/ physico chemical N2 fixation
involves the photochemical & electro chemical
conversion of atm.N₂ to soil NO2,NO3, NH3.
• It is brought about by ionizing phenomena such
as cosmic radiations,meteor trails ,lightning,
thunderstorms, volcanic eruptions etc.
• These provides high energy for breaking N≡N &
also for the formation of free N₂ with oxygen or
hydrogen of atm H₂0

12. • combination of N₂ with o₂ forms nitrous and nitric
oxides.
• Combination of N₂ with hydrogen forms NH3
• Nitrous and nitric oxides get hydrated with atm. Water
vapour and forms nitrous and nitric acids
• Rain water bring these acids and NH3 to soil surface
• There ,the acids react with metallic ions and form
metallic nitrates.
• These nitrates and NH3 enrich the surface soil
• Non biological N₂ fixation amounts to only ˂ 10% of
the natural N₂ fixation
• It is common in some tropical regions, where thunder
bolt are frequent.

13. • 1. N2 + O2 lightning 2 NO
(Nitric oxide)
2. 2NO + O2 oxidation 2NO2
( Nitrogen per oxide)
3. 2NO2 + H2O HNO2 + HNO3
4. 4NO2 + 2H2O + O2 4HNO3
(Nitric acid)
5. CaO + 2HNO3 Ca (NO3)2 + H2O
(Calcium nitrate)
6. HNO3 + NH3 NH4NO3
(Ammonium nitrate)
7. HNO2 + NH3 NH4NO2
(Ammonium nitrite)

14. Biological Nitrogen Fixation
• Biological nitrogen fixation was discovered by the
German agronomist Hermann Hellriegel and
Dutch microbiologist Martinus Beijerinck.
• Biological nitrogen fixation (BNF) occurs when
atmospheric nitrogen is converted to ammonia by
an enzyme called nitrogenase
• The reaction for BNF is:
• N2+16ATP+8H++8 e− → 2 NH3+H2+16ADP+16Pi

15. • BNF is mostly accomplished by microorganisms
called diazotrophs or N₂ fixers
• They include some species of bacteria, fungi,
blue green algae,lichens etc.

16. Special features of diazotrophs
• Free living –bacteria like azotobacter,
rhodopseudomonas fix atm.N₂ & also protects
nitrogenase enzyme – sensitive to 0₂
• They produce exopolysaccharide (slime) which
retains water and prevents diffusion of 0₂ inside
cell during N₂ fixation
• Azotobacter have exceedingly high rate of
respiratory metabolism thus preventing 0₂
retension inside the cell
• Blue green algae –are of 2 kinds
• One that possess heterocyst
• Other that devoid of it ( non heterocystous)

17. • Filamentous cyanobacteria contain pale , thick
walled , hollow cells called heterocyst
• Heterocyst are the site of N₂ fixation
• They lack PSII and photosynthetic bile proteins
• non heterocystous N₂ fixing BGA like
(oscillatoria) - the filaments are arranged clumps
and N₂ fixation takes place in internaly organised
cell having reduced conditions
• Azospirillum paspalum survive in microaerophilic
conditions associated with the rhizosphere ( area
surrounding the roots ) of paddy plants - fix atm.
N₂ in the rhizosphere.

18. Anabaena

20. • Soil N₂ fixers are of two categories
Non symbiotic N₂ fixers
symbiotic N₂ fixers

21. Non symbiotic or free living nitrogen fixers
• They inhabit both terrestrial & aquatic
habitats
o Fixation carried out by free living micro-
organisms
o Aerobic, anaerobic and blue green algae
Free living nitrogen fixers
oFree living aerobic : Azotobacter, Beijerenckia
oFree living anaerobic : Clostridium
oFree living photosynthetic : Chlorobium,
Rhodopseudomonas
oFree living chemosynthetic :Desulfovibro,Thiobacillus

22. o Free living fungi: yeasts and Pillularia
o Blue green algae:
o unicellular – Gloeothece , Synechococcus
o Filamentous (non heterocystous) – Oscillatoria
o Filamentous ( heterocystous) – Tolypothrix,
Nostoc , Anabaena

23. • Ability to fix N by MO was confirmed by-technique -
acetylene reduction to ethylene - diazotropic MO
• Conversion is controlled by an enzymatic complex (or
nitrogenous enzyme)-reduce gaseous N - ammonia.
• Nitrogenase – sensitive to O.
• Several groups of MOs fix N in presence of minute
quantities of O - Microaerobic fixers - Spirilla
(Aquaspirillum & Azospirillum).
• O-not only inhibits the activity of nitrogenase-but also
regulate biosynthesis.

24. SYMBIOTIC N₂ FIXERS
• Fixation of free nitrogen by micro-organisms
living in soil symbiotically inside the plants
• ‘Symbiosis’ – coined by DeBary
• Three categories
– Nodule formation in leguminous plants
– Nodule formation in non-leguminous plants
– Non nodulation

25. Nodule formation in leguminous plants
• 2500 sp. Of family leguminosae ( Cicer arientium,
Pisum, Cajanus, Arachis) produce root nodules with
Rhizobium spp.
• They fix Nitrogen only inside the root nodules
• Association provides-food and shelter to
bacteria
-bacteria supply fixed
nitrogen to plant
• Nodules may buried in soil even after harvesting –
continue nitrogen fixation

26. Nodule formation in non-leguminous
plants
• Some other plants also produces root nodules
– Causuarina equisetifolia – Frankia
– Alnus – Frankia
– Myrica gale – Frankia
– Parasponia – Rhizobium
• Leaf nodules are also noted
– Dioscorea, Psychotria
• Gymnosperms – root – Podocarpus,
- leaves – Pavetta zinumermanniana,
Chomelia

27. Non-nodulation
• Lichens - cyanobacteria
• Anthoceros - Nostoc
• Azolla – Anabaena azollae
• Cycas – Nostoc and anabaene
• Gunnera macrophylla - Nostoc
• Digitaria, Maize and Sorghum – Spirillum
notatum
• Paspalum notatum – Azotobacter paspali

28. Nodule formation
• Root nodules formed due to infection of
Rhizobium
• Free living bacteria growing , near the root of
legumes unable to fix nitrogen in free condition
• Roots of the legumes secrete some growth
factors helps in fast multiplication of bacteria
• (E.g.) Pisum sativum secretes homo serine &
also carbohydrate containing protein Lectins over
their surface

29. • Rhizobia are chemotactically attracted to root
hair
• Medited by lectins, some attach to root hair cell
wall
• Tryptophan is a component of the root hair
exudate
• Trytophan is transformed by rhizobia to indole
acetic acid (IAA)
• This plant hormone causes the root hair to curl or
branch around the attached rhizobia

31. • Polygalacturonase , secreted by rhizobia or
possibly by the plant, depolymerizes & softens
root hair cellwall
• Rhizobia gain entry into the root hair cell
• The root hair cell nucleus directs the
development of infection thread
• Infection thread ,a tube consisting of cell
membrane & surrounding cellulosic wall,
• Grows into root cortex & infects some tetraploid
cells that proliferate & form nodule tissue
• Rhizobia are released from infection thread ,lose
their rod shape

32. • Infected root cells swell and cease dividing.
• Bacteria within the swollen cells change
form to become endosymbiotic bacteroids,
which begin to fix nitrogen
• The nodule provides an oxygen-controlled
environment
• (leghemoglobin = pink nodule interior)
structured to facilitate transport of
reduced nitrogen metabolites from the
bacteroids to the plant vascular system, and
of
• photosynthate from the host plant to the
bacteroids.

36. 

37. Nitrogen fixing organisms found in
nodules
• Rhizobium
 Rod shaped,motile.etc.
 Non sporing, non-acid fast.
 Rhizobia are susceptible to antibiotics, bacteriophages,
fungicides, herbicides etc.
• Named after the host plant
– Pea – Rhizobium leguminosarum
– Beans – R. phaseoli
– Soyabeans – R. japonicum
– Lupins – R. lupini
• Two types of Rhizobium-
– Bradyrhizobium – slow growing spp.
– Rhizobium - fast growing spp.

38. Structure and function of nodule
• Outermost layer of nodule-bacteriod zone-enclosed by
cortical cells.
• Rate of N- fixation of nodule directly proportional to
the volume of the nodule.
• Nodules are small-contain leghaemoglobin
• Leghaemoglobin
• Effective nodules are larger- pink in colour -due to the
presence of red coloured leghaemoglobin.
• This pigment is similar to haemoglobin of blood.
• Found in nodules between bacteriods & membrane
envelops, enclosing them.

40. • It is heme protein.
• Contain heme moiety attached to a peptide chain-
represent globin part.
• The amount of leghaemoglobin in nodules has direct
relationship between amount of atm N fixed by
legumes.
• MW-16,000-17,000 daltons.
• Functions:
-Represents an active site of N absorption and reduction.
-Acts as a specific electron carrier.
-Regulate the O –supply in the nodule.
-O-carrier.

42. Site and mechanism of nitrogen
fixation in nodules
Site
• Bacteroids-site of N-fixation.
Mechanism
• 1) Theory of Virtanen
 N fixation in roots appear immediately after nodule
formation.
 -young plants fix N than the old plants.
 A great part of N- converted to-L-aspartic acid and L-
glutamic acid.
 Apart from this alpha-alanine present in nodule-produced
from L-aspartic acid by decarboxylation.
 small amount of Oxime-N and nitrite-N are also present.

43. • 2) Theory of Burris and Wilson –
hydroxylamine is the central compound of N
fixation from which ammonia is formed-
through reduction.

44. Biochemistry of nitrogen fixation
• N₂ fixers utilize atm. N₂ to synthesize NH3
• In this process , N₂ is first split up into free N₂
atoms by breaking the triple bond , with help
of enzyme nitrogenase.
• This reaction is endergonic (energy
consuming), it requires an input of nearly
160kcal energy.

45. • Free nitrogen combines with hydrogen
forming NH3
• This reaction is exergonic (energy releasing)
• Mediated by enzyme hydrogenase & it
releases nearly 13 kcal energy.
• BNF requires a net input of 147 kcal energy &
an expenditure of nearly 16 mols of ATP per
each molecule of nitrogen.

46. N₂ + 160Kcal 2N
2N + 3H₂ 2NH3 + 13 Kcal
NITROGENASE

47. Nitrogenase Complex
Two protein components: nitrogenase
reductase and nitrogenase
• Nitrogenase reductase is a 60 kD homodimer
with a single 4Fe-4S cluster
• Very oxygen-sensitive
• Binds Mg ATP
• 4 ATP required per pair of electrons
transferred
• Reduction of N2 to 2NH3 + H2 requires 4 pairs
of electrons, so 16 ATP are consumed per N2

49. Nitrogenase
A 220 kD heterotetramer
• Each molecule of enzyme contains 2 Mo, 32
Fe, 30 equivalents of acid-labile sulfide
(Fe8S 7-8clusters, etc)
• Four 4Fe-4S clusters plus two Fe, Mo, Co, an
iron-molybdenum cofactor (fe 7 S9 Mo
homocitrate)
• Nitrogenase is slow - 12 e- pairs per second,
i.e., only three molecules of N2 per second

51. Pathway of nitrogen fixation in root
nodules
• Glucose-6-phosphate acts as a electron donor
• Glucose-6-phosphate is converted to
phosphogluconic acid
Glucose-6-phosphate + NADP+ + H2O 6-phosphogluconic acid + NADPH + H+
• NADPH donates electrons to ferrodoxin. Protons
released and ferrodoxin is reduced
• Reduced ferrodoxin acts as electron carrier. Donate
electron to Fe-protein to reduce it. Electrons
released from ferrodoxin thus oxidized

52. • Reduced Fe-protein combines with ATP in the
presence of Mg +2
• Second sub unit is activated and reduced
• It donates electrons to N2 to NH3
• Enzyme set free after complete reduction of
N2 to NH3
Mo – N=NH
Mo=N-NH2
MoΞN+NH3
Mo + NH3
NΞN
Mo-NΞN

53. N2+16ATP+8H++8 e− → 2 NH3+H2+16ADP+16Pi
4 ATP
4 ATP
4 ATP
4 ATP

54. • Nitrogenase protection mechansims
• 1. Leghaemoglobin scavenges O2 to protect
nitrogenase in legume rhizobium symbiosis
• 2. Confirmatory protection in Azotobacter as
well as the higher respiratory rate.
• 3. Thick walls of Heterocyst protect O2 in BGA,
since Nitrogenase are present in the
heterocyst.
• 4. Microaerophilic nature in Azospirillum

55. Genes involved in Nitrogen fixation
• Genes involved in root nodule formation - called
nodulin genes ( nod genes)
• Nodulin genes essential for infection of plant root and
nodule formation by symbiotic N₂ fixing bacteria -
divided into 2 classes
• 1) include genes that specify biochemical composition
of bacterial cell surface.
• such as gene determining the synthesis of
exopolysaccharides ( exo genes)
• Lipopolysaccharides (lps gene)
• Capsular polysaccharides or K antigen & β 1,2 glucans
(ndu genes)

56. • exo & lps genes - play a role in determining
host specificity
• 2) consist of nodulation genes (nod or nol)
• nod genes are involved in nodulation of
particular host -called host specific nod(hsn)
genes.
• Fast growing rhizobium sps - nod genes are
located on large sym plasmids
• Slow growing bradyrhizobium sps – carry late
nod gene on the bacterial chromosome.

58. • nif and fix genes - structural genes for
nitrogenase enzyme
• Sym plasmid carry hup genes coding for
hydrogenase activity
• Nif genes - 22 genes are involved , arranged in
7/8clusters
• Nif Q,B,A,L,F,M,Z,W,V,S,U,X,N,E,Y,T,K,D,H,J

59. REFERENCES
• Microbial ecology-fundamentals & application
- Atlas & Bartha. pp- 108-115
• Ramesh ,vijaya. ENVIRONMENTAL
MICROBIOLOGY .pp -309-315,291-295
• Bhaskaran ,kk ENVIRONMENTAL BIOLOGY
WILDLIFE CONSERVATION .3rd ed.(2013) . pp -
76-78