Biological nitrogen fixation (BNF) can be defined as the conversion of atmospheric dinitrogen (N2) to ammonia (NH3) under the combined action of biological and chemical activities

1. Biological Nitrogen fixation
Dr. Rachana Choudhary
(H.O.D.)Department Of Microbiology
Shri Shankaracharya Mahavidyalya,Junwani Bhilai

2. SYNOPSIS
1. INTRODUCTION
2. MODES OF NITROGEN FIXATION
3. ROOT NODULE FORMATION IN RHIZOBIUM-LEGUME
ASSOCIATION
4. ENZYMES OF NITROGEN FIXATION
5. MECHANISM OF NITROGEN FIXATION
6. BIOCHEMISTRY OF NITROGEN FIXATION IN NODULES
7. MOLECULAR BASIS OF NITROGEN FIXATION
8. CONCLUSION
9. REFERENCE

3. INTRODUCTION
The biological availability of N,P & K are
considerable an economic importance because
they are the major plant nutrients derived from th
e soil.
Of the three nitrogen is the most important
one this element is a key building block of protei
n molecule upon which life is based.
So the conversion of molecular nitrogen
into ammonia is known as Nitrogen Fixation.

4. Nitrogen is major
nutrient for plants
Atomospheric
N2
1.INTRODUCTION
Plant incapable to
Use this N2 gas
Triple bond makes
Highly inert
◆ Utilization of atmospheric nitrogen gas as a source of
cell nitrogen by way of its reduction to amonia is
called nitrogen fixation.

5. 2. MODES OF NITROGEN FIXATION
• ( (a) Spontaneously nitrogen Fixation
(b) Industrial Process
(c) Biological Nitrogen Fixation

6. 2. MODES OF NITROGEN FIXATION
(a) Spontaneously nitrogen Fixation
N2 H2
H2O
NH3
NO2
NO3

7. (b) Industrial process
Fig: Production of ammonia by
HABER PROCESS

8. A large number of micro-organism are known to
have the ability to reduce atmospheric nitrogen
into nitrogenous compounds. This conversion of
molecular nitrogen into nitrogenous compounds
by micro-organism is called Biological Nitrogen
fixation
1.Non-symbiotic nitrogen fixation
2.Symbiotic nitrogen fixation
(c) Biological nitrogen fixation

9. 1. Non-symbiotic nitrogen fixation
◆ Some microorganisms live freely and independently in
the soil.
◆ Including bacteria and some cyanobacteria.
FREE LIVING BACTERIA
Azotobactor,
Azomonas,
Azotococcus,
Thiobacillus ferooxidant,
Rhodospirillum rubrum
CYANOBACTERIA
Anabaena,
Nostoc

10. 2. Symbiotic nitrogen fixation
■ Through nodules formation in legume plant:
■ Through Nodule formation in non-legume
plants
■ Without Nodulation

11. Legume
Plant
Association
Rhizobium
spp.
● Nodulating bacteria Rhizobium is gram negative, Motile rods.
● This association is fix larg quantity of nitrogen and have
considerable agricultural significance
■ Through nodules formation in legume plant:

12. Legume plant Nodulating bacteria
Alfalfa Sinorhizobium meliloti
Beans Rhizobium biovar
Clover R. trifolii
Lotus Mesorhizobium loti
Pae R.viciae
Soybean Bradyrhizobium japonicum

13. Through Nodule formation in non-legume plants
◆ Some non-legume plants involve in nodule formation.
◆ The best known plant is Alder associated with
actinomycete, Frankiya sp.
◆ Other plants are Casuarina and Hippophae.
Alder nodule Hippophae nodule

14. Without Nodulation
Example – Nostoc+Gunnera macrophylla.
Spirillum notatum+Maize

15. 3. NODULES FORMATION IN RHIZOBIUM- LEGUME
ASSOCIATION
NODULES FORMATION TAKES PLACE BY
FOLLOWING STAGES :
(a) Recognition
(b) Attachment
(c) penetration
(d) Travel
(e) Bacteroid formation
(f) Development of mature nodule

16. (a) Recognition
◆ Legume plant secret
Metabolite including
Lectin protein.
◆ Which attracte Rhizobia
◆ Rhizobia comes in the
Rhizospheric region and
Increase population.

17. (b) Attachement
The bacterial
rhicadhesin
Attached with lactins.

18. (c) penetration
After attachement Rhizobia
Excrete nod factor which
Induce secretion of indol
acetic acid results
Curling of root hair.

19. (d) Travel
◆ After the root hair and
induce the Plant to develop
a cellulotic tube.
◆ Called infection thread.

20. (e) Bacteroid formation

21. ◆ After releasing in cytoplasm
They became irregular shaped
Called Bacteroids.
◆ Surrounded by plant
Derived membrane called
Peribacteroid membrane.

22. (f) Development of mature nodule
◆ Symbiosome developed as
Nodule.
◆ The Symbiosome secret
Harmon called leghaemoglobin.
◆ which makes pink to nodule.
Vascular supply
To nodule
Rhizobia containing
Area in nodule

23. Formation of a Root Nodule

24. 4. ENZYMES OF NITROGEN FIXATION
Nitrogenase
Dintrogenase
(Mo-Fe protein)
Dintrogenase reductase
(Fe-protein)
Mw= 220,000 Mw= 64, 000
Present in
Two copies
Present in
One copies

26. Alternative
Nitrogenase
◆ Nitrogenase that contain Vanadium in the place of
Molybdenum called alternative Nitrogenase.
◆ Synthesized if molybdenum is not present in medium.

27. Nitrogen fixation is catalysed by nitrogenase which is sensitive to
oxygen. It gets inactivated when the oxygen level exceeds 0.5 atm.
In plant cells, oxygen level is some what high, so the nitrogenase
may fail to reduced the nitrogen.
Leghaemoglobin is a red, myoglobin-like protein present only in
healthy root nodules of legumes. It is found outside the bacteria,
but in close contact with it.
Leghaemoglobin combines with oxygen to form
oxyleghaemoglobin and provides the oxygen to plant cells for
respiration. So the oxygen level around the bacteria is low & this
low content of oxygen does not affect nitrogenase activity for
nitrogen fixation.

28. In non legumin0us plant, where Leghaemo-
globin is absent hydrogenase enzyme is presen
t.
hydrogenase combines H2 and O2 to form wat
er (H2O). Hydrogenase removes oxygen from
the vicinity of nitrogenase in the bacteria.
Thus hydrogenase makes a suitable micro-envi
ronment for nitrogenase activity.
Azotobacter chroococcum &Anabaena cylindrica
contain the enzyme hydrogenase.

29. Hydrogenase

30. Factors Affecting Nitrogenase Activity
Excessive soil moisture,
Soil acidity,
P deficiency,
Excess mineral N, and
Deficiency of Ca, MO, CO and B.

31. Mechanism of biological nitrogen
fixation

32. 5. MECHANISM OF NITROGEN FIXATION

33. 6. BIOCHEMISTRY OF NITROGEN FIXATION IN ROOT
NODULES
Schematic presentation
Of biochemical
Pathway in nodule

34. Molecular Basis of Nitrogen Fixation
Nif Genes
The nif genes are genes encoding enzymes involved in
the fixation of atmospheric nitrogen in to a form of
nitrogen available to living Organisms.
Nif genes also encode a number of regulatory proteins in
volved in nitrogen fixation.
The nif genes are found in both free-living & sym-biotic
bacteria.

35. Conclusion
Biological Nitrogen Fixation is important
Process For the Increasing of soil Fertility .
It is necessary for the growth of the plant.
Agriculture in developed countries is definitely
base of the economy.
"eco- friendly“.

36. 8. REFERENCE
◆ MICROBIOLOGY (Prescott)
◆ TEXT BOOK OF MICROBIOLOGY (R.P.Singh)
◆ INTRODUCTION TO EVS.MICROBIOLOGY (Michel.R.)
◆ CHEMISTRY (M.M.Tandan)