Nitrogen-fixing bacteria, microorganisms capable of transforming atmospheric nitrogen into fixed nitrogen (inorganic compounds usable by plants). More than 90 percent of all nitrogen fixation is effected by these organisms, which thus play an important role in the nitrogen cycle. Two kinds of nitrogen-fixing bacteria are recognized. The first kind, the free-living (nonsymbiotic) bacteria, includes the cyanobacteria (or blue-green algae) Anabaena and Nostoc and genera such as Azotobacter, Beijerinckia, and Clostridium. The second kind comprises the mutualistic (symbiotic) bacteria; examples include Rhizobium, associated with leguminous plants (e.g., various members of the pea family); Frankia, associated with certain dicotyledonous species (actinorhizal plants); and certain Azospirillum species, associated with cereal grasses. The symbiotic nitrogen-fixing bacteria invade the root hairs of host plants, where they multiply and stimulate formation of root nodules, enlargements of plant cells and bacteria in intimate association. Within the nodules the bacteria convert free nitrogen to ammonia, which the host plant utilizes for its development. To ensure sufficient nodule formation and optimum growth of legumes (e.g., alfalfa, beans, clovers, peas, soybeans), seeds are usually inoculated with commercial cultures of appropriate Rhizobium species, especially in soils poor or lacking in the required bacterium.

2. Synopsis
 INTRODUCTION
 Free living microorganism
 Symbiotic microorganis

4.  Nitrogen fixation- conversion of Dinitrogen Into ammonia on the
presence of catalyst or enzyme known as a Nitrogen fixation
 Biological Nitrogen fixation- fixation of atmospheric Nitrogen into
plant available form on the presence of microorganism known as a
biological Nitrogen fixation
 Nitrogen fixing microorganism- those- microorganism which is
capable to Nitrogen fixation by nitrogenase enzyme known as a
nitrogen fixing micro organism
 Example – Rhizobium ,anabeana.nostoc etc.

6.  On the basis of nitrogen fixation-
Types of Main nitrogen fixing microorganism
1) free-living or asymbiotic nitogen fixing microorganism
2) Symbiotic nitrogen fixings microorganism

7.  Free living nitrogen fixing microorganism - Soil contain a numbers
of free living nitrogen fixing organism these includes a number of
aerobic and anaerobic photosynthetic and chemosynthetic
 Free-living nitrogen fixing bacteria means that they don’t need to
create symbiotic relationships with plants to survive and replicate.
 Asymbiotic nitrogen fixer classified into following – (a) Aerobic
(b) Anaerobic (c) photosynthetic (d) chemosynthetic

8.  Aerobic-The bacteria that grow in the presence of oxygen are
called aerobic bacteria. They have the ability to detoxify oxygen
with the help of enzymes
 Example -; Azotobacter
 Characteristics of Azotobacter
Azotobacter is gram negative bacteria varying cell size and
shape.
The motility of the bacteria is with the help of peritrichous
flagella. .
 Oval and spherical shaped

9. Example- Azotobacter
 The genus Azotobacter belongs to the γ-subclass of the
Proteobacteri .
 Azotobacter is free-living, motile, bacteria that form cysts.
 They are aerobic and play a large role in nitrogen fixation.
 They are used as model organisms in the study of
diazotrophs, and also for the production of food additives,
biopolymers and some biofertilisers.
 They are mostly found in neutral and alkaline soils, in
association with plant.
 The cells of Azotobacter are resistant to environmental
stresses because they secrete a thick mucus-like layer,
forming a cyst.
 Some of the species produce pigments that range from

10.  Azotobacter are heterotrophic and aerobic bacteria and their main
property is the ability to fix nitrogen non-symbiotically, with a genomic
content of G-C of 63–67.5 % ™ distributed in soils, water and
sediments .
 Anaerobic- An anaerobic organism or anaerobe is any organism
that does not require molecular oxygen for growth. It may react
negatively or even die if free oxygen is present.
 Example clostridium
 Characteristics of clostridium

11. Example-clostridium-
 Clostridium is a genus of gram-positive,
 spore-forming bacteria belonging to the family Clostridiaceae
 . Vegetative cells are rod shaped and arranged in pairs or short chains.
 The majority of species are obligate anaerobes; however, some species can
grow under aerobic conditions or are aerotolerant.
 Several solvent-producing clostridia, including Clostridium
acetobutylicum and C. beijerinckii, were previously shown to be nitrogen-
fixing organisms based on the incorporation of 15N2 into cellular material.
 The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for
nitrogenase component proteins as well as nifE, nifN, nifB and nifV for
synthesis of the iron-molybdenum cofactor (FeMoco) of nitrogenase, have
now been identified in C. acetobutylicum
 And this genes cause a nitrogen fixation in clostridium.

12.  Photosynthetic bacteria- microorganis that carried out photosyntesis
process known as a photosynthetic bacteria
 Example- chlorobium cyanobacteria etc
 Characteristics of cyanobacteria- Blue-Green Algae are a type of
photosynthetic bacteria consisting either of single cells or colonies which is
also known as the Cyanobacteria.
 These bacteria are unicellular, colonial or filamentous, marine or terrestrial
algae.
 They form blooms in polluted water bodies.
 It includes Nostoc and Anabaena

13. Cyanobacteria
 Heterocystous cyanobacteria carry out nitrogen fixation in heterocysts
 and photosynthesis in vegetative cells. These organisms can fix nitrogen in light as well as dark.
 In dark it requires oxygen and ATP for nitrogen fixation generated by respiratory processes,
 and in light ATP is generated in heterocysts which contains only part of photosynthetic apparatus.
 Heterocysts lack both 0, evolution and CO, fixation but retain the ability to generate ATP and
reductant. Heterocysts are connected to vegetative cells by large pores which permit a photosynthate
disaccharide to heterocysts and glutamine out of heterocysts.
 Sugars passing in, help to generate NADPH by pentose phosphate pathway or scavange oxygen via
respiratory processes.
 Heterocyst wall allows sufficient nitrogen in but restrict oxygen diffusion to a level which can be
removed by respiration.


15. Thiobacillus
 Chemosynthetic bacteria- bacteria that synthesize organic
compounds, using energy derived from the oxidation of
organic or inorganic materials without the aid of light.
 Example – Thiobacillus
 Characteristics of Thiobacillus-

16.  Genus Thiobacillus have been subject to reclassification
based on myriad characteristics like molecular phylogenetics,
ecology, and physiology
 Species of the genus Thiobacillus generally occupy extreme
ecological niches especially acidic environment and cling
themselves to sulfur/iron-rich environment with heavy-metal
interactions, which make them survive in nutrient-deficient
conditions.
 Hydrothermal vents, activated sludge disposal sites, and
sources of sulfur gases, such as sediments or anaerobic soils
releasing H2S can become potential sites of occurrence of
ThiobacilluS
 They drive their metabolic activities using sulfur, sulfides,

17.  Thiobacillus plays an integral role and has wide applications
in the field of wastewater treatment, agriculture, , and
maintenance of geomicrobiological cycle.
 . Thiobacillus ferrooxidans has been known to reduce
acetylene to ethylene in the absence of nitrogen sources
indicating the possession of nitrogenase enzyme by this
microbe The role of Thiobacillus and its coexistence with
Leptospirillum spp. In the bioleaching ecosystems is of
utmost significance to the microbiologists
 Strains of Acidithiobacillus (At.) ferrooxidans have been
reported to grow by coupling the oxidation of hydrogen to the
reduction of elemental sulfur .With increase in various
molecular biology techniques, identification of many species
is possible which are responsible for mineral dissolution .

18. Symbiotic microorganism
 Symbiosis was first defined by Marko de Bary in 1869 .
 in which he defined the symbiosis as a term as “namely, the living
together of parasite and host”.
 Symbiotic bacteria are bacteria living in symbiosis with another
organism or each other. For example, rhizobia living in root
nodules of legumes provide nitrogen fixing activity for these plants.

19.  There are three main types of symbiotic relationships:
 commensalism, mutualism, and parasitism.
 Commensalism is when one organism benefits and the other
is neither harmed nor benefits.
 Mutualism is when both organisms benefit.
 Lastly, parasitism is when one organism benefits while the
other organism is harmed.

20. Example-rhizobia
 Example rhizobia
 Rhizobium is a diazotrophic bacteria that fix atmospheric nitrogen.
 It is a gram-negative bacteria that establish in the roots of the plants such as
peas and pulses.
Rhizobium and leguminous plants live in a symbiotic association with each
other.
In this, both the organisms are benefited from each other.
The bacteria fix atmospheric nitrogen and make it available to the plants. On
the other hand, Rhizobium receives nutrition from the plant in the form of
organic acids.

22. Example- Frankia
Frankia strains are N2-fixing actinomycetes whose isolation and cultivation were first
reported in 1978. They induce N2-fixing root nodules on diverse nonleguminous (actinorhizal)
plants that are important in ecological successions and in land reclamation and remediation
. The genus Frankia encompasses a diverse group of soil actinomycetes that have in
common the formation of multilocular sporangia,
filamentous growth, and nitrogenase-containing vesicles enveloped in multilaminated lipid
envelopes.
 Recent studies of the genetics and molecular genetics of these organisms have begun to
provide new insights into higher-plant-bacterium interactions that lead to productive N2-
fixing symbioses.
Sufficient information about the relationship of Frankia strains to other bacteria, and to each
other, is now available to warrant the creation of some species based on phenotypic and
genetic criteria.

23.  Other nitrogen fixing micro organism-
• Gluconacetobacter Diazotrophicus: Gluconacetobacter
diazotrophicus is a nitrogen-fixing, acetic acid bacterium first isolated
from sugarcane plants.
• It belongs to phylum Proteobacteria (comprising Gram negative
bacteria) in section a-Proteobacteria, order Rhodospirillales and
family Acetobacteraceae.
• Currently, this family contains three nitrogen-fixing genera,
comprising of seven species, namely Acetobacter nitrogenifigens,
Gluconacetobacter kombuchar. Gluconacetobacter johannae.
Gluconacetobacter azotocaptans, Gluconacetobacter diazotrophicus.
Swaminathania salitolerans and Acetobacter peroxydans

24.  Azospirillum: Azospirillum species belong to the facultative endophytic
diazotrophs groups .
 which colonize the surface and the interior of roots and this kind of
association is considered as the starting point of most ongoing BNF
(Biological Nitrogen Fixation) programs with non-legume plants world wide.
 Nitrogen fixing organisms such as Azospirillum, directly benefits plants
improving shoot and root development and increasing the rate of water and
mineral uptake by roots [14]