It is a biofertilizer that contains symbiotic Rhizobium bacteria which is the most important nitrogen-fixing organism. These organisms have the ability to drive atmospheric Nitrogen and provide it to plants. It is recommended for crops such as Groundnut, Soybean, Red-gram, Green-gram, Black-gram, Lentil, Cowpea, Bengal-gram and Fodder legumes, etc.

1. RHIZOBIUM
Dr. Rachana Choudhary
Shri Shankaracharya Mahavidyalaya , Junwani Bhilai

2. SYNOPSIS
• Introduction
• Classification
• What is The importance of Rhizobium?
• Morphology
• Nodulation
• Mechanism of Nitrogen Fixation
• Isolation of Rhizobium
• Steps of Mass Production
• Application of Rhizobium
• Biofertilizer Company in India & Chhattisgarh
• Conclusion
• References

3. INTRODUCTION
• Rhizobium is a bacteria that live in symbiotic
association with the root nodules of the leguminous
plants.
• Fixation of nitrogen cannot be done independently.
• Rhizobium is a vital source of nitrogen to agricultural
soils including those in arid regions.
• They convert dinitrogen into ammonia.
• Rhizobium is a diazotrophic bacteria

4. CLASSIFICATION
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Alphaproteobacteria
Order: Rhizobiales
Family: Rhizobiaceae
Species: Rhizobium leguminosarum,R. lentil.

5. CLASSIFICATION
Rhizobium can be classified on the basis of the types of the plant
they are associated with and also the rate of growth.
Few species of Rhizobium bacteria include:
• Rhizobium leguminosarum
• Rhizobium alamii
• Rhizobium lantis
• Rhizobium japonicum
• Rhizobium trifolii
• Rhizobium phaseolii
• Rhizobium smilacinae

6. TAXONOMY OF RHIZOBIA
Genus Species Host plant
Rhizobium leguminosarum bv. trifolii
“ bv. viciae
“ bv. phaseoli
tropici
etli
Trifolium (clovers)
Pisum (peas), Vicia (field beans),
Lens (lentils), Lathyrus
Phaseolus (bean)
Phaseolus (bean), Leucaena
Phaseolus (bean)
Sinorhizobium meliloti
fredii
saheli
teranga
Melilotus (sweetclover), Medicago
(alfalfa), Trigonella
Glycine (soybean)
Sesbania
Sesbania, Acacia
Bradyrhizobium japonicum
elkanii
liaoningense
Glycine (soybean)
Glycine (soybean)
Glycine (soybean)
Azorhizobium caulinodans Sesbania (stem nodule)
‘Meso rhizobium’ loti
huakuii
ciceri
tianshanense
mediterraneum
Lotus (trefoil)
Astragalus (milkvetch)
Cicer (chickpea)
Cicer (chickpea)
[Rhizobium] galegae Galega (goat’s rue), Leucaena
Photorhizobium spp. Aeschynomene (stem nodule)

7. WHAT IS THE IMPORTANCE OF
RHIZOBIUM?
Rhizobium is a bacterium found in soil that helps in
fixing nitrogen in leguminous plants. It attaches to
the roots of the leguminous plant and produces
nodules. These nodules fix atmospheric nitrogen and
convert it into ammonia that can be used by the plant
for its growth and development.

8. MORPHOLOGY
• They appear as elongated rods when viewed under
the microscope
• Like a number of other bacteria, Rhizobium
leguminosarum do not form spores in their life
cycle.
• They posses several flagella on their polar end.
• They are aerobic as such, they need oxygen for
respiratory purposes.
• Various strains of the bacteria have granules
• They can tolerate higher temperatures of about
380 C.

9. LEGUME-RHIZOBIUM
SYMBIOSIS
• The subfamilies of legumes (Caesalpinioideae,
Mimosoideae, Papilionoideae), 700 genera, and 19,700
species of legumes
• Only about 15% of the species have been evaluated for
nodulation
Rhizobium
 Gram -, rod
 Most studied symbiotic N2-fixing bacteria
 Now subdivided into several genera
 Many genes known that are involved in
nodulation (nod, nol, noe genes)

10. NODULATION

11. NODULATION IN LEGUMES

12. MECHENISM OF NITROGEN
FIXATION
• All nitrogen fixing bacteria use highly conserved
enzyme complex called Nitrogenase.
• Nitrogenase is composed of two subunits: an iron-
sulfur protein and a molybdenum-iron-sulfur protein
• Aerobic organisms face special challenges to nitrogen
fixation because nitrogenase is inactivated when
oxygen reacts with the iron component of the proteins

13. NITROGENASE
FeMo Cofactor
N2 + 8H+
2NH3 + H2
8e-
4C2H2 + 8H+ 4C2H2
Dinitrogenase
Dinitrogenase
reductase
Fd(red)
Fd(ox)
nMgATP
nMgADP + nPi
N2 + 8H+ + 8e- + 16 MgATP  2NH3 + H2 + 16MgADP

14. Nitrogenase

15. Genetics of Nitrogenase
Gene Properties and function
nifH
nifDK
nifA
nifB
nifEN
nifS
fixABCX
fixK
fixLJ
fixNOQP
fixGHIS
Dinitrogenase reductase
Dinitrogenase
Regulatory, activator of most nif and fix genes
FeMo cofactor biosynthesis
FeMo cofactor biosynthesis
Unknown
Electron transfer
Regulatory
Regulatory, two-component sensor/effector
Electron transfer
Transmembrane complex

16. ISOLATION OF RHIZOBIUM

17. CULTURE ON YEMA MEDIUM

18. STEPS OF MASS PRODUCTION
1. Inoculum preparation
2. Processing of carrier material
3. Selection of ideal carrier material.
4. Preparation of carrier material
5. Mixing the carrier and the broth culture and
packing
6. Preparation of Inoculants packet

19. 1. Inoculum preparation
• Prepare appropriate media for specific to the bacterial
inoculant in 250 ml, 500 ml, 3 litre and 5 litre conical flasks
and sterilize.
• The media in 250 ml flask is inoculated with efficient
bacterial strain under aseptic condition
• Keep the flask under room temperature in rotary shaker (200
rpm) for 5- 7 days.
• Observe the flask for growth of the culture and estimate the
population, which serves as the starter culture.
• Using the starter culture (at log phase) inoculate the larger
flasks (500 ml, 3 litre and 5 litre) containing the media, after
obtaining growth in each flask

20. 2.Processing of carrier material
The use of ideal carrier material is necessary in the
production of good quality biofertilizer
1.Peat soil
2.Lignite
3.Vermiculite
4.Charcoal
5.Press mud
6.Farmyard manure and soil mixture can be used as
carrier materials.
The neutralized peat soil/lignite are found to be better
carrier materials for biofertilizer production.

21. 3.Selection of ideal carrier material.
1. Cheaper in cost
2. Should be locally available
3. High organic matter content
4. No toxic chemicals
5. Water holding capacity of more than 50%
6. Easy to process, friability and vulnerability

22. 4.Preparation of carrier material
• The carrier material (peat or lignite) is powdered to
a fine powder so as to pass through 212 micron IS
sieve.
• The pH of the carrier material is neutralized with
the help of calcium carbonate, since the peat soil /
lignite are acidic in nature ( pH of 4 - 5).
• The neutralized carrier material is sterilized in an
autoclave to eliminate the microorganisms

23. 5.Mixing the carrier and the
broth culture
Inoculant packets are prepared by mixing the
broth culture obtained from fermenter with sterile
carrier material.

24. 6.Preparation of Inoculants packet
• The neutralized, sterilized carrier material is spread
in a clean, dry, sterile metallic or plastic tray.
• The bacterial culture drawn from the fermenter is
added to the sterilized carrier and mixed well by
manual (by wearing sterile gloves) or by
mechanical mixer.
• The culture suspension is to be added to a level of
40- 50% water holding capacity depending upon
the quantity of carrier material

25. • The inoculant packet of 200 g quantities in
polythene bags, sealed with electric sealer and
allowed for curing for 2-3 days at room
temperature.
• Curing can be done by spreading the inoculant on
a clean floor/polythene sheet/ by keeping in open
shallow tubs/ trays with polythene covering for 2-
3days at room temperature before packaging.

26. Specification of the polythene bags
• The polythene bags should be of low density grade.
• The thickness of the bag should be around 50 - 75
micron.
• Each packet should be marked with the
name of the manufacturer
name of the product, strain number
 the crop to which recommended ,method of inoculation
 date of manufacture, batch number, date of expiry, price
 full address of the manufacturer and storage
 instructions to farmers

27. Storage of biofertilizer packet
• The packet should be stored in a cool place away
from the heat or direct sunlight.
• The packets may be stored at room temperature or
in cold storage conditions in lots in plastic crates or
polythene / gunny bags.
• The population of inoculant in the carrier inoculant
packet may be determined at 15 days interval.
• There should be more than 109 cells / g of inoculant
at the time of preparation and 107 cells/ g on dry
weight basis before expiry date

29. MASS PRODUCTION

30. Application of Rhizobium
Seed treatment or seed
inoculation
Seedling root dip
Main field application
1
.
2
.
3

31. 1.Seed treatment
• One packet of the inoculant is mixed with 200 ml of
rice to make a slurry.
• The seeds required for an acre are mixed in the slurry
so as to have a uniform coating of the inoculant over
the seeds and then shade dried for 30 minutes.
• The shade dried seeds should be sown within 24
hours.
• One packet of the inoculant (200 g) is sufficient to
treat 10 kg of seeds

32. 2.Seedling root dip
• This method is used for transplanted crops.
• Two packets of the inoculant is mixed in 40 litres
of water.
• The root portion of the seedlings required for an
acre is dipped in the mixture for 5 to 10 minutes
and then transplanted

33. 3.Main field application
• Four packets of the inoculant is mixed with 20 kg of
dried and powdered farm yard manure and then
broadcasted in one acre of main field just before
transplanting.

34. BIOFERTILIZER COMPANY IN INDIA
 Ampada Overseas Enterprises. Pune-India.
 Agri Science India. Solan- 173205 ,India.
 Kan Biosys Pvt. Ltd. Surya Bio Products
Hyderabad, India.
 Phms Technocare Private Limited,
Gujarat
 Aquatic Agro. Thane- 421201 ,India.
 Criyagen Agri And Bio Tech Ltd. Crop
Chemicals India Ltd.
 Shree Samarth Agro Tech.

35. BIOFERTILIZER COMPANY IN
CHHATTISHGARH
 Maa Danteshwari Herbal Products
 SRT Agro Science Pvt. Ltd Patan
 Shri Krishna Agro - Organic (Bio) Fertilizers
Manufacturer
 GK Bio Fertilizer Company Raipur
 Rajshree agro and farming nogwa baloda dist
janjgir chappa,
 Rals agrocare pvt. Ltd Ambikapur Chhattisgarh.

36. CONCLUSION
Rhizobium is a biological fertilizer based on a
selected strain of naturally-occurring beneficial
bacteria - Rhizobium japonicum They actively fix
atmospheric nitrogen through a symbiotic relation
with the leguminous plants. Successful nodulation of
leguminous crop by rhizobium largely depends on
the availability of a compatible stain for a particular
legume. Its population in the soil is dependent on the
presence of legumes crops in field.

37. REFERENCES
• Textbook of Microbiology by R. P. Singh
• Textbook of Microbiology by Dubey &
Maheshwari.
• Soil Microbiology by N.S. Subba Rao.
• Agricultural Microbiology G. Rangaswami
• Google Search

38. THANK YOU