Biofertilizers, Nitrogen fixation

1. BIOFERTILIZERS
 It involves inoculation of beneficial microorganisms that help nutrient
acquisition by plants through fixation of nitrogen, solubilization and
mobilization of other nutrients.
 Multifarious advantages of biofertilizers leads to its wide applicability in
sustainable agriculture.
 The term “biofertilizer” refers to preparation containing live microbes which
helps in enhancing the soil fertility either by fixing atmospheric nitrogen,
solubilization of phosphorus or decomposing organic wastes or by augmenting
plant growth by producing growth hormones with their biological activities.
ADVANTAGES OF BIOFERTILIZERS
• Renewable source of nutrients.
• Sustain soil health.
• Supplement chemical fertilizers.
• Replace 25-30% chemical fertilizers.
• Increase the grain yields by 10-40%.
• Decompose plant residues, and stabilize C:N ratio of soil.
• Improve texture, structure and water holding capacity of soil.
• No adverse effect on plant growth and soil fertility.
• Stimulates plant growth by secreting growth hormones.
• Secrete fungistatic and antibiotic like substances.
• Solubilize and mobilize nutrients.
• Eco-friendly, non-pollutants and cost-effective method.
TYPES OF BIOFERTILIZERS

2. N2 fixing
Bacterial Fixers
❖ The live cells of bacteria used as a biofertilizers.
❖ These microbes contain unique gene called as Nif-Gene which make them
capable of fixing nitrogen.
❖ The nitrogen fixing bacteria work under two conditions,
Symbiotically
 The symbiotic bacteria make an association with crop plants through forming
nodules in their roots.
Free living bacteria (non-symbiotic)
 The free-living bacteria do not form any association but live freely and fix
atmospheric nitrogen.
SYMBIOTIC NITROGEN FIXERS
 Most important symbiotic Nitrogen fixing bacteria is Rhizobium and
Azospirillum.
Rhizobium
• The name Rhizobium was established by Frank in 1889.
• Rhizobium is a gram negative anaerobic microorganisms which fix atmospheric
nitrogen symbiotically with leguminous plant.
• In addition to fixing the atmospheric nitrogen through nodulation, it shares
many characteristics with other PGPRs including hormones production and
solubilization of organic and inorganic phosphate.
• This genus has seven distinct species based on "Cross Inoculation Group
Concept".
• More than twenty cross-inoculations groups have been established.
• A new classification has been established for Rhizobium.
• That is 'slow growing rhizobia' known as Bradyrhizobium and the other group
is 'fast growing rhizobia' called Rhizobium.
• Rhizobium can fix 50-300 kg/ha.

3. Azospirillum
• It mainly presents in cereal plants.
• Inhabits both root cells as well as surrounding of roots forming symbiotic
relation and increasing nitrogen fixing potential of the cereal plant.
• Azospirillum is recognized as a dominant gram negative bacteria.
• Fixes nitrogen in the range of 20- 40 kg/ha in the rhizosphere in non-
leguminous plants such as cereals, millets, Oilseeds, cotton etc.
• These species have been commercially exploited for the use as nitrogen
supplying Bio-Fertilizers.
Azotobacter
• Azotobacter is a heterotrophic free-living nitrogen fixing bacteria present in
alkaline and neutral soils.
• Azotobacter is the most commonly occurring species in arable soils of India.
• Apart from its ability to fix atmospheric nitrogen in soils, it can also synthesize
growth promoting substances such as auxins and gibberellins and also to some
extent the vitamins.
• Many strains of Azotobacter also exhibit fungicidal properties against certain
species of fungus.
• Response of Azotobacter has been seen in rice, maize, cotton, sugarcane, pearl
millet, vegetable and some plantation crops.
• It improves seed germination and plant growth.
• Azotobacter is heaviest breathing organism and requires a large amount of
organic carbon for its growth.
Cyanobacteria
• Another group of free-living nitrogen fixers are cyanobacteria.
• Commonly called as Blue green algae.
• More than 100 species of BGA can fix nitrogen.
• Nitrogen fixation takes place in specialized cells called ‘Heterocyst’
• BGA very common in rice field.
• Unlike Azotobacter BGA are not inhibited by the presence of chemical
fertilizers.
• No chemical fertilizers added, inoculation of the algae can result in 10-14%
increase in crop yields.

4. ❖ They are easy to produce
❖ Usually they are mass produced in cement tanks filled with fresh water.
❖ Not require any processing
❖ Quite and cheap
❖ Cost of 10kg may be Rs. 30-40 only
❖ Beneficial in certain crops like vegetables, cotton, sugarcane.
❖ E. g. of some algal biofertilizers are
Anabena
Nostoc
Oscillatoria
AZOLLA AS A BIOFERTILIZER
❖ Azolla is a tiny fresh water fern common in ponds, ditches and rice fields.
❖ It has been used as a biofertilizer for a rice in all major rice growing countries
including India, Thailand, Korea, Philippines, Brazil and West Africa.
❖ The nitrogen fixing work is accomplished by the symbiotic relationship
between the fern and BGA, Anabena azollae.
❖ In addition to nitrogen the decomposed Azolla also provides K, P, Zn and Fe to
the crop Good manure for flooded rice.

5. ❖ Increase of crop yield up to 15-20% has been observed while fertilizing the rice
with Azolla
❖ Hybrids are growing faster
❖ Tolerant to heat and cold
❖ Fix 4-5% more nitrogen
COMMERCIAL PRODUCTION OF RHIZOBIUM
1) Isolation and identification of efficient strain of Rhizobium from rhizosphere
soil.
2) To identify suitable medium for production Rhizobium.
3) To standardize the procedure for the mass production of Rhizobium.
4) Use of Rhizobium Biofertilizer
Isolation and identification of efficient strain of Rhizobium from rhizosphere soil

6. Isolation and identification of efficient strain of Rhizobium from rhizosphere soil
❖ Bacterial colonies grown on YEMA are streaked over CRYEMA after incubation
of these plates at 28-30 °C for 7 days.
❖ It has been observed that utilizes Congo red slowly and form white, circular,
translucent, glistening, elevated and raised colonies
To identify suitable medium for production Rhizobium
The selective and optimized mediums used for mass culturing of biofertilizers are as
follows:
SELECTIVE YEAST EXTRACT MANNITOL BROTH
Components g/L
Mannitol 10.0
K2HPO4 0.5
MgSO4.7H2O 0.2
NaCl 0.1
Yeast extract 0.5
Distilled water 1 L
OPTIMIZED BROTH
Mannitol 10.0
K2HPO4 0.5
MgSO4.7H2O 0.2
NaCl 0.1
Yeast extract 0.1
FeCl3 .6H O 0.02
Cacl2 .7H O 0.04
Thymidine Trace
Congo red 0.001
Distilled water 1 L

7. To standardize the procedure for the mass production of Rhizobium
Use of Biofertilizer
❖ In soil at root system of plant
❖ Mixing with soil
❖ By dissolving into water

8. Plant Growth Promoting Rhizobacteria (PGPRs)
 The term “Plant growth promoting rhizobacteria (PGPR)” for beneficial
microbes was introduced by Kloepper JW, Schroth MN (1981).
 The term “plant growth promoting bacteria” refers to bacteria that colonize
the roots of plants (rhizosphere) that enhance plant growth.
 Rhizosphere is the soil environment where the plant root is available and is a
zone of maximum microbial activity resulting in a confined nutrient pool in
which essential macro and micronutrients are extracted.
INTERACTION OF PGPRs -Root

9. Classification of PGPRs
MECHANISM OF ACTION

10. DIRECT MECHANISMS
NITROGEN FIXATION
 The plant growth promoting rhizobacteria widely presented as symbionts are
Rhizobium, Bradyrhizobium, Sinorhizobium, and Mesorhizobium with
leguminous plants, Frankia with non-leguminous trees and shrubs.
 Non-symbiotic Nitrogen fixing rhizospheric bacteria belonging to genera
including Azoarcus, Azotobacter, Acetobacter, Azospirillum, Burkholderia,
Diazotrophicus, Enterobacter, Gluconacetobacter, Pseudomonas and
cyanobacteria (Anabaena, Nostoc)
PHOSPHATE SOLUBULIZATION
 The main phosphate solubilization mechanisms employed by plant growth
promoting rhizobacteria include:
(1) release of complexing or mineral dissolving compounds e.g. organic acid anions,
protons, hydroxyl ions, CO2,
(2) liberation of extracellular enzymes (biochemical phosphate mineralization) and
(3) the release of phosphate during substrate degradation (biological phosphate
mineralization)
 Phosphate solubilizing PGPR included in the genera Arthrobacter, Bacillus,
Beijerinckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium,
Microbacterium Pseudomonas, Rhizobium, Rhodococcus, and Serratia
PHYTOHORMONE PRODUCTION
 A wide range of microorganisms found in the rhizosphere are able to produce
substances that regulate plant growth and development.
 Plant growth promoting rhizobacteria produce phytohormones such as auxins,
cytokinins, gibberellins and Ethylene can affect cell proliferation in the root
architecture by overproduction of lateral roots and root hairs with a
subsequent increase of nutrient and water uptake

11. INDIRECT MECHANISMS
ANTIBIOTICS
 The production of antibiotics is considered to be one of the most powerful and
studied biocontrol mechanisms of plant growth promoting rhizobacteria
against phytopathogens.
 A variety of antibiotics have been identified, including compounds such as
phenazine, tropolone.
 Some rhizobacteria are also capable of producing volatile compound known as
hydrogen cyanide (HCN) for bio-control of black root rot of tobacco.
LYTIC ENZYMES
 Plant growth promoting rhizobacterial strains can produce certain enzymes
such as chitinases, dehydrogenase, β-glucanase, lipases, phosphatases,
proteases etc.
 Through the activity of these enzymes, plant growth promoting rhizobacteria
play a very significant role in plant growth promotion particularly to protect
them from biotic and abiotic stresses by suppression of pathogenic fungi.
 Pseudomonas fluorescens has been suggested as potential biological control
agent due to its ability to colonize rhizosphere and protect plants against a
wide range of important agronomic fungal diseases such as black root-rot of
tobacco, root-rot of mustard and damping-off of sugar beet in field condition
SIDEROPHORE PRODUCTION
 Siderophores may be defined as low molecular mass compounds (< 1000 Da)
with a great affinity for Fe+3 chelation, followed by the shift and
accumulation of Fe within the cells of bacteria.
 Siderophores are secreted to solubilize iron from their surrounding
environments, forming a complex ferric-siderophore that can move by
diffusion and be returned to the cell surface.
 Microbial siderophores enhance iron uptake by plants that are able to
recognize the bacterial ferric-siderophore complex.

12. Examples of Siderophores
 Pseudobactin -Pseudomonas sp.
 Schizokein -Bacillus subtilis
 Ferribactin - Pseudomonas fluorescens
 Cepabactin - Pseudomonas cepacia
 Pyoverdin - Pseudomonas aeruginosa
INDUCED SYSTEMIC RESISTANCE (ISR)
 ISR may be defined as a physiological state of enhanced defensive capacity
elicited in response to specific environmental stimuli and consequently the
plant’s innate defenses are potentiated against subsequent biotic challenges.
 PGPR induced resistance is a state of enhanced defensive capacity developed
by a plant reacting to specific biotic or chemical stimuli

13. EXO POLYSACCHARIDES PRODUCTION OR BIOFILM FORMATION
 Certain bacteria synthesize a wide spectrum of multifunctional polysaccharides
including intracellular polysaccharides, structural polysaccharides, and
extracellular polysaccharides.
 Production of exo polysaccharides is generally important in biofilm formation;
root colonization can affect the interaction of microbes with roots
appendages. Effective colonization of plant roots by EPS-producing microbes
helps to hold the free phosphorous from the insoluble one in soils and
circulating essential nutrient to the plant for proper growth and development
and protecting it from the attack of foreign pathogens.
 Other innumerable functions performed by EPS producing microbes
constitute shielding from desiccation, protection against stress, attachment
to surfaces plant invasion, and plant defence response in plant–microbe
interactions

14. ARBUSCULAR MYCORRHIZAL FUNGI
 The word Mycorrhizae was first used by German researcher A.B Frank in 1885
and originates from the Greek mycos, meaning “fungus” and “rhiza” meaning
“root”.
 Mycorrhizae is a symbiotic mutualistic relationship between special soil fungi
and fine plant roots: it is neither the fungus nor the root but rather the
structures from these two partners.
 Mycorrhizal associations involve 3-way interactions between host plants,
mutualistic fungi and soil factors.
Mutualistic relation
 Since the association is mutualistic, both organisms benefit from the
associations.
 The fungus receives carbohydrates (sugars) and growth factors from the plant,
which in turn receives many benefits, including increased nutrient absorption.
MYCORRHIZA- CATEGORIES
Inside root
• Intercellular mycelium
• Intracellular arbuscule
• tree-like haustorium
• Vesicle with reserves
Outside root
• Spores (multinucleate)
• Hyphae
• thick runners
• filamentous hyphae
Form extensive network of hyphae even connecting different plants
Host
plant
Soil
factors
Fungi

15. ENDOMYCORRHIZA
 Arbuscular mycorrhizas, or AM (formerly known as vesicular-arbuscular
mycorrhizas, or VAM), are mycorrhizas whose hyphae enter into the plant
cells, producing structures that are either balloon-like (vesicles) or
dichotomously branching invaginations (arbuscules).

16. Benefits of AMF
• Increases yield
• Increases water uptake
• Maximizes nutrient uptake (P)
• Reduces transplantation stress
• Reduces fertilizer inputs
• Reduces heavy metal toxicity
• Maintains soil fertility
• Disease resistance
➢ They help the plant to feed itself, but they are no fertilizer.
➢ They contribute to stress and disease tolerance, but they are no crop
protection product.
➢ They destroy nothing, they are no pesticide
Enhance phosphorus uptake
 Improve physical exploration of the soil pore
 Formation of polyphosphates in the hyphae
 Production of extracellular phosphatases
 Production of organic acids
 Occupy sites of active decomposition

17. ECTOMYCORRHIZA
• Ascomycetes and Basidiomycetes-form large fruiting bodies
• 5000 species interact with 2000 plant species
• Interaction with trees: angiosperms and all Pinaceae
Inside root
• Intercellular hyphae
• Does not enter cells
Outside root
• Thick layer of hyphae around root
• Fungal sheath
• Lateral roots become stunted
• Hyphae
• Mass about equal to root mass
Forms extensive network of hyphae even connecting different plants

18. COMPOSTING
Composting
• process of decomposition of organic waste by micro-organism
• natural process (be made faster and more effective by mixing various
types of waste and adjusting moisture, temperature and aeration)
• contains NPK and other plant nutrients including micro-organisms
 steps of composting:
• preparation (converting waste into raw material)
• production of compost
• marketing
VERMICOMPOSTING
❖ The raising and production of earthworms and harvesting worm castings.
❖ Using worms to decompose organic food waste, turning the waste into a
nutrient-rich material capable of supplying necessary nutrients to help sustain
plant growth.
❖ Vermicompost is similar to regular compost, except that worms take part in
the composting process.
SPECIES OF EARTHWORMS USED IN VERMICOMPOSTING:

19. MATERIALS REQUIRED FOR PREPARATION OF VERMICOMPOST
➢ Bedding material/organic residue
➢ Housing or shed facility
➢ Worm food
➢ Cow dung/biogas slurry
➢ Watering the vermi-bed
STEPS IN PREPARATION OF VERMICOMPOST
❖ Collection of wastes and processing including shredding and separation of
non-degradable material.
❖ Preparation of earthworm bed. A concrete base is required to put the waste
for vermicompost preparation. Loose soil will allow the worms to go into soil
and also while watering; all the dissolvable nutrients go into the soil along with
water.
❖ Collection of earthworms after vermicompost collection. Sieving the
composted material to separate fully composted material. The partially
composted material will be again put into vermicompost bed.
STEPS IN PREPARATION OF VERMICOMPOST
 Storing the vermicompost in proper place to maintain moisture and allow the
beneficial microorganisms to grow.
VARIOUS STEPS OF WASTE DEGRADATION BY EARTHWORMS
 Ingestion of organic waste material.
 Softening of organic waste material by the saliva in the mouth of the
earthworms.
 Softening of organic waste and neutralization by calcium (excreted by the
inner walls of the esophagus) and passed on to the gizzard for further action in
the esophagus region of the worm body.

20.  Grinding of waste into small particles in the muscular gizzard.
 Digestion of organic waste by a proteolytic enzyme in stomach.
 Decomposition of pulped waste material components by various enzymes
including proteases, lipases, amylases, cellulases, and chitinases secreted in
intestine and then absorbing the digested material in the epithelium of
intestine.
 Excretion of undigested food material from worm castings.
BENEFITS OF VERMICOMPOSTING
❖ Vermicompost is an important source of organic manure. It has the following
useful attribution.
❖ helpful in recycling any organic wastes into a useful biofertilizer and leaves no
chance of environmental pollution.
❖ an eco-friendly, non-toxic product, consumes low energy input while
processing.
❖ a preferred balanced nutrient source.
❖ Improves physical, chemical and biological properties of soil without any
residual toxicity.
❖ Reduces the incidences of pests and diseases in crop production.
❖ Improves quality of agricultural produce.