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Biological nitrogen fixation and Biofertilizers-Types and advantages

“Biofertilizers are carrier based preparations containing active strains of specific

microorganisms, which help in enhancing the soil fertility either by fixing atmospheric
N2, solubilization/ mineralization of P & K or decomposing organic wastes or by
augmenting plant growth through producing growth promoting substances with their
biological activities.

Biofertilizer Agents are classified as

1. N-fixer
2. P-Solubilizer/Mobilizer
3. Compost Accelerator

Biological N-Fixation:
What is Biological N2 fixation?
“Reduction of Dinitrogen (N2) to NH3 through biological means is termed as Biological
Nitrogen Fixation”
Why reduction of N2 is necessary?
Though atmosphere contains 79% of N, eukaryotes cannot utilize the N directly,
unless it is reduced to NH3. Dinitrogen having triple bond and cannot be broken by higher
plants.
Broadly the BNF is divided into two groups
 Symbiotic
 Non Symbiotic
Symbiotic Nitrogen fixation may be of three different types
 Legume Rhizobium
 Cyanobacteria with plant/fungi
 Frankia with trees
Non Symbiotic Nitrogen Fixation can be divided into another three groups based on the
oxygen requirements
 Aerobic free-living nitrogen fixation. example-Azotobacter,Bejeirinkia
 Microaerophillic , for example Azospirillum
 Anerobic free living , for example Clostridium, Desulfovibrio etc.
In all the BNF system it is the nitogenase enzyme play crucial role in reducing
dinitrogen to ammonia

Symbiotic Nitrogen Fixation

What is Symbiosis?
“An intimate association between two different organisms for which the
coexistence is deemed to be mutually beneficial”
Rhizobium is symbiotic bacterium, occurs in the roots of different legumes &
pulses, produce nodules and fix atmospheric dinitrogen inside the nodules. This
Rhizobium present inside the nodule can be isolated and multiplied in laboratory .After
appropriate multiplication the Rhizobium is mixed with carrier material in aseptic
condition and used as biofertilizers for different pulse crops.
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Necessity of Rhizobium Biofertilizers

Though Rhizobium occurs naturally in soil, but in most of the cases, either their
population is highly insufficient or ineffective for proper nodulation traits. The low pH in
the North Eastern Region is not conducive for proper nodulation that limits the
Rhizobium cells to survive in adequate numbers in free living state. Under such situations
the legume crops need to be inoculated with specific strains of Rhizobium inoculants each
and every time.

The advantages of Rhizobium inoculation.

Large numbers of field trials conducted over the years in India and abroad have
established that the crops inoculated with specific strains of Rhizobium benefits the crop
in many ways.
• Increases crop yield by 10-30%
• Reduce Chemical fertilizer by 50% Leave considerable residual N (3-8kg of per
bigha) after the harvest of the legume crop.
• Benefiting the succeeding crop
• Benefit the companion crop if grown along with legume as inter crop

Based on the ability of Rhizobium to infect diverse leguminous crop –in

1932,Fred proposed the concept of “Cross Inoculation Group”. The cross inoculation
group refers a collection of leguminous species that develop nodules when exposed to
rhizobium, isolated from the nodules of any member of that particular group .
In 1981 Allen and Allen reported that the family leguminaceae comprise 748
genera and 19000 species of which 49% genera and 16% species were examined for
nodulation that resulted 41% genera 14% species could produce nodule.Jordan in 1984
classified the Rhizobia into two groups based on
 the growth pattern and
 nif and nod genes location
If growth is fast and nif & nod genes locate in plasmid ,it is termed as Rhizobium
where as if slow growth and nif & nod genes locate in chromosomes ,termed as
Bradyrhizobiumi

Non-Symbiotic Azospirillum and Azotobacter

Out of sixteen known elements, N play vital role in crop production. Due to
intensive agriculture and injudicious use of N through Chemical fertilizers jeopardize the
soil ecosystem. To sustain the fertility of soils, maintenance of appropriate N2 fixing
microflora is very essential .In this context, Azospirillum & Azotobacter play significant
role as renewable nutrient source especially nitrogen in sustainable agricultural
production.
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Azospirillum biofertilizers is made of live cells of bacteria with microaerophillic

and free-living habitat. .Azospirillum biofertilizers can be used in crops. Being
microaerophillic in nature these bacteria perform better in medium heavy to heavy
textured soils. Azotobacter biofertilizers is a product containing high number of live
bacteria, which is aerobic, free living nitrogen fixer..Azotobacter biofertilizers can be
used in non-legume crops mostly grown under upland condition.

Azospirillum

In 1925, one microaerophillic organisms having spiral shaped was isolated by

Bejeirinck and named as “Spirillum lipoferum” from grasses. Becking demonstrated their
N-fixing ability in 1963. But its importance in agriculture was not realized till 1976,
when Dobereiner and Day reisolated from different grasses. Based on morphology
,physiological and biochemical tests some of the strains were separated from the genus
Spirillum by Tarrand et al in 1978 and renamed as Azospirillum with two species
Azospirillum lipoferum and Azospirillum brasilense . The distinguishing characters in
between these two species are
 Azospirillum lipoferum having denitrification ability and requirements of
biotin for its multiplication
 Azospirillum brasilense does not have these two characters
Azotobacter

Bejerinck ,1901 first time isolated one aerobic N-fixing soil microorganisms and
termed as “Azotobacter”.This Azotobacter placed under the family “Bacteriaceae”by
Krasilnikov in 1956.In 1960 , Norris , replaced the family under the new name
Azotobacteriaceae. In recent classification based on 16S rRNA ,the members of the
family marged with Pseudomonadales under the family Pseudomonadaceae Till now
there are six different species of the genus Azotobacter

What are the differentiating characters among the species of Azotobacter?

 Homopolysaccharide Production
o Azotobacter chroccum,Azotobacter bejeirinkii and Azotobacter nigricans
produce diffusible homopolyssacharides from sucrose and raffinose
o Azotobacter armenicus produce polyssacharide from sucrose only
 Pigmentation
o Azotobacter chrococcum produce black brown pigments
o Azotobacter armenicus produce yellow pigments
o Azotobacter bejeirinkii produce light yellow pigments
o Azotobacter paspali produce red to violet
Benefits of Azospirillum and Azotobacter inoculants

 Benefits the crops by 15-25 kg of N/ha/Season

 Grain yield is increased by 10-20%
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 Vegetative growth and leaf yield increased by 10-30%

 Secretes growth promoting and antibiotic like substances
 Can supplement 50% of nitrogenous fertilizer requirement by the crop

Gluconacetobacter:
Gluconacetobacter diazotrophicus is an endophyte bacterium first isolated from
the sugarcane growing regions of Brazil. It was widely studied and used as a model
system to assess the bacterial endophyte– plant interactions. After its first discovery, it
was reported from variety of crops viz, coffee, ragi , pineapple and a latest report states
Gluconacetobacter sp. As a natural colonizer of the wild rice and a salt tolerant Pokali
rice variety . These reports clearly indicated the wide occurrence of G. diazotrophicus in
different plants .Besides N fixation this bacteria also producing different plant growth
promoting substances.By application of this organisms , the chemical fertilizer can be
reduced to about 50%.

Phosphate Solubilizing Microorganisms

What are the Importance of P-Solubilizing Microorganisms
 Phosphorus (P) is one of the major essential macronutrients limiting plant
growth owing to its low bioavailability in soils.
 Fertilizer P tends to be fixed soon after application and becomes mostly
unavailable, resulting in low recovery by crops and a considerable P
Fixation in soils
 Microorganisms able to solubilize and mineralize P pools in soils are
considered to be vital.
 Bacteria are the predominant microorganisms that solubilize mineral P in
soils, as well as mineralize the Organic P.
 Bacillus & Pseudomonas are two major genera of Phosphate Solubilizing
Bacteria. Aspergillus and Penicillium are belongs to fungi.
Mechanisms of Inorganic P-Solubilizition
 The main mechanism of inorganic P solubilization is through a decrease in pH
 Another mechanism is by secretion of Organic acids such as Oxalic/Propionic/
Fumeric /Gluconic/Acetic/Citric/α-Ketogluconic acid.
 Other compounds such as CO2,H2S, Humic Substances etc.
Organic acid due to its dissociation, the Organic ‘Anions’ make strong complex with
Ca/Al/Fe and release phosphate into solution. Organic Cation such as H+ make complex
with phosphate and may release into solution

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