Non-symbiotic nitrogen fixation refers to nitrogen fixation by microorganisms that live independently in soil and aquatic environments. Key microorganisms that can fix nitrogen this way include species from the genera Azotobacter, Clostridium, Klebsiella, cyanobacteria, and some fungi. Azotobacter is a common soil bacterium that can fix significant amounts of nitrogen aerobically using the nitrogenase enzyme. Cyanobacteria also fix nitrogen, with some genera like Nostoc forming specialized cells called heterocysts that protect the oxygen-sensitive nitrogenase enzyme.

1. Non Symbiotic Nitrogen
Fixation
Name Poonam Kumar Sonkar
Msc IV Microbiology
SoS in Life Science PRSU Raipur

2. • The Fixation of free nitrogen of the soil by all those micro-
organisms living freely or outside the cell is called as non-symbiotic
biological N2 fixation.
•
• heterotrophic bacteria which reside in ground soil and are able for
fixation of significant levels of nitrogen without the direct interaction
with other beings.
• Examples for this type of nitrogen-fixing bacteria include species of
Azotobacter, Bacillus, Clostridium,and Klebsiella.
Non Symbiotic Nitrogen Fixation

3. • Non symbiotic (Asymbiotic) Nitrogen fixation refers to all Biological
Nitrogen Fixation undertaken by microbial cells growing
independently in soil, terrestial, and aquatic wetland environments as
saprophytes

5. The Groups of Nonsymbiotic Nitrogen-Fixing
Microorganisms
A. Organotrophic bacteria, including
(a) the aerobic Azotobacter
(b) the anaerobic Clostridium butyricum and some related species.
B. Photolithotrophic organisms including
(a) many types of blue-green algae
(b) purple sulfur- and nonsulfur-bacteria which are probably of minor
importance under soil conditions

6. C.Free living chemosynthetic bacteria: Thiobacillus
Desulfovibro
D.Cyanobacteria or Blue green algae:
a]Heterocyst bearing: Nostoc, Anabaena, Rivularia,
Calothrix.
b]Non-Heterocyst : Oscillatoria, Gloeocapsa, Lyngbya,
Plectonema.
E. Free living Fungi: Yeasts and Pullularia

7. Azotobacter
• Gram-negative, oval or spherical bacteria
• Aerobic and free-living soil bacteria
• Form thick-walled cysts, multiple species produce large amounts of
capsular slime
• Around six species present, some of which are motile by means of
peritrichous flagella
• Usually found in neutral to alkaline soils, wate, and in close
association with other plants in their rhizosphere

8. • Sensitive to acidic pH, high salts, and temperature
• Model organism for the study of diazotrophs (esp. asymbiotic
itrogen fixers)
• First species of Azotobacter (Azotobacter chroococcum)
discovered by the Dutch scientist Martinus Beijerinchk
• Azotobacter species are mostly free-living nitrogen fixers (some
live in close association with plants)

9. Azotobacter - Nitrogen
Fixation
• Azotobacter species have a full range of enzymes needed to perform
the nitrogen fixation:
• ferredoxin, hydrogenase, and an important enzyme nitrogenase
• The process of nitrogen fixation requires an influx of energy in the form
of ATP
• Nitrogen fixation is highly sensitive to the presence of oxygen, so
Azotobacter developed a special defensive mechanism against oxygen
• Formation of an alginate capsule in the cell surface forms an effective
barrier for 02 transfer into the cell

10. • Azotobacter -
Nitrogenase
• Nitrogenase is the most important enzyme involved in nitrogen fixation
• Azotobacter species have several types of nitrogenase. The basic one is
molybdenum-iron nitrogenase
• An alternative type contains vanadium; it is independent of molybdenum
ions and is more active than the Mo-Fe nitrogenase at low temperatures
• Vanadium is present in V-nitrogenase in a vanadium and iron-
containing protein (the V-fe protein)

11. • VFe proteins contains vanadium in an environment with iron, suphur
and oxygen (or nitrogen or carbon) as nearest neighbour atoms, very
similar to the environment of Molybdenum in the iron and
molybdenum cofactor (FeMoco) centre of the MoFe proteins
• Synthesis of nitrogenase is controlled by the nif genes

12. Azospirillum
• Gram negative, rod-shaped bacteria
• highly motile
• Utilize glucose, lactate, succinate, fructose, malate, pyruvate, fumarate as
• carbon source
• N sources utilized by Azospirillum:
• Ammonium, Nitrate, Amino acids ,Elemental N (N2)
• 1925, one microaerophillic organisms having spiral shaped was
isolated byBejeirinck and named as “Spirillum lipoferum” from
grasses

13. Azospirillum - Nitrogen Fixation
• Azospirillum can convert atmospheric nitrogen into ammonium under
microaerobic conditions at
• low nitrogen levels, through the action of the nitrogenase complex.
• This enzyme is built from two components:
• 1 dinitrogenase protein (MoFe protein, NifDK),which contains a
molybdenum-iron cofactor,it is the site of N2 reduction;
• 2.dinitrogenase reductase protein (Fe protein, NifH) transfers
electrons from an electron donor to the nitrogenase protein
• Azospirillum only fixes nitrogen in microaerobic N-limiting conditions

14. • nitrogenase enzyme complex is very sensitive to oxygen, biological N2
fixation is tightly regulated

15. Cyanobacteria are often called "blue-green algae"
• They are Prokaryotic organisms, multicelullar organisms have more
types of cells
• They live in water and therefore we call them aquatic organisms.
• Specific blue green In colour
• They produce OXYGEN becouse Obtain energy through Photo
synthesis
• Have chlorophyll a, and photosystems I and II that allow them to
perform oxygenic photosynthesis
• Use the pentose phosphate pathway for carbohydrate metabolism
•
Cyanobacteri
a

16. A. Cyanobacteria that can fix N2 aerobically
1.Cyanobacteria that separate N2 fixation from oxygenic
photosynthesis in space.
*Includes heterocystous genera, for example, Anabaena
2.Cyanobacteria that separate N2fixation from oxygenic
photosynthesis in time.>*Includes non-heterocystous genera, such as
Gloeothece, Cyanothece and Lyngbya
3.Cyanobacteria that separate N2 fixation from oxygenic
photosynthesis both in space and in time.
*Includes non-heterocystous genera, such as Trichodesmium and
Katagnymen
Nitrogen fixer Cyanobacteria

17. B Cyanobacteria that can fix only N2 either anaerobically or
microaerobically
Many non-heterocystous cyanobacteria,for example
Plectonemaboryanum

18. Cynanobacterial Nitrogen fixation by
Heterocyst
• The Heterocyst is the site for cyanobacterial nitrogen fixation which is
an enlarged cell.
• In the process of cyanobacterial nitrogen fixation, hydrogen gas is
also evolved as a by product
• Heterocyst is made up of 3 different cell wall layers- the outer fibrous
and middle homogenous layers are made up of non-cellulose
polysaccharide. the inner laminated laver is made up of glycolipids.
•

19. • Heterocysts protect nitrogenase from inactivation by O2 by
• several mechanisms, including a high rate of respiration and
decreased
• permeability to O2. Heterocysts do
• not evolve O2 and cannot fix CO2,

20. • https://doi.org/10.1111/j.1574-6976.2000.tb00552.x
Reference
• Ohyama T, editor. Advances in biology and ecology of nitrogen fixation.
BoD–Books on Demand; 2014 Jan 29.
• https://www.sciencedirect.com/topics/biochemistry-genetics-and-
molecular-biology/diazotroph
• Steenhoudt, Oda, and Jos Vanderleyden. "Azospirillum, a free-living
nitrogen-fixing bacterium closely associated with grasses: genetic,
biochemical and ecological aspects." FEMS microbiology reviews 24.4
(2000): 487-506.

21. Thank you