1. WEL COME
MASTER SEMINAR

2. Deptt. of Soil Science and Agricultural Chemistry, COA Raipur(C.G.)
“MASS PRODUCTION TECHNOLOGY OF BIOFERTILIZER AND THEIR ROLE IN AGRICULTURE”
PRESENTED BY
Santosh Kumar Chouhan
M.Sc.(Ag.),Previous Year
Deptt. of Soil Science and Agricultural Chemistry, COA Raipur(C.G.)

3. *Source: Singh, R.P. 2012. Microbilogy, Kalyani Publishers.
BIOFERTILIZER
Introduction
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.
*Source: Singh, R.P Microbilogy, Kalyani Publishers.

4. CLASSIFICATION

5. The Important Steps In Relation To Production Of Biofertilizer
ISOLATION
MEDIA
FERMENTER
CARRIER MATERIAL
*Source – www. agritech.tnau.ac.in

6. ISOLATION METHOD
1 SERIAL DILUTION METHOD -
This method is using when bacteria are Isolating from soil.
2 BY ROOT NODULE-
when we isolate the bacteria fro the root nodule we have to collect the root nodule and sterilize with 1%Hgcl2+60%ethnol+crushing. After crush the material is transfer to desire nutrient media for colony growth.
*Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

7. ISOLATION OF DIFFERENT BIOFERTILIZER INOCULANT
ISOLATION PLACE
METHOD
RHIZOBIUM
ROOT NODULE OF LEGUME
ROOT NODULE
PSB
RHIZOSPHERE SOIL
SERIAL DILUTION
VAM
AZOLLA
NATURAL HABITAT .
*Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

8. BIOFERTILIZER ORGANISMS
RHIZOBIUM
AZOTOBACTER
PSB
ALGAE BLUE GREEN E
AZOSPIRILLUM
VA-MYCORRHIZA
*Source – www. agritech.tnau.ac.in

9. Commercial scale production
For large-scale production, fermenter are used for growing bacteria. pH is adjusted to Inoculum should be 5%. Continuous aeration is done by forcing sterile air through sparger. Incubate culture till the bacterial population reaches 108 cells/ml, and added to carrier.
The organism count in final broth cultures shall not be less than
108 to 109 cells / ml. Otherwise, the broth should be rejected
*Source-

10. FERMENTER
The organism count in final broth cultures shall not be less than
108 to 109 cells / ml. Otherwise, the broth should be rejected
*Source-

11. PARTS OF FERMENTER
PHASE LIQUID,GAS ,SOLID.
PROVIDE – 1 AGITATION
2 AREATION
3 STERILIZATION
4 REGULATIO FACTOR (ph, temperature, pressure, etc)
5 WITHDRAWAL OF CELL
Cooling jacket -- Large scalefermentor are almost constructed of stainless steel.
A fermentor is a larger cylinder closed to the top and botom and various pipe and valves are fiitted into it.the frementor is externally fitted with a cooling jacket through which steam or cooling water is run.
Areation system--- One most critical part of the fermentor.in a fermentor with the high population of microbial density, there is tremendous demand of oxygen by the culture however the two aereation system are used in fermentor , Sparger and Impeller.
Baffels----The baffels are normally incorporated into the fermentor of all size to prevent a vortex and to improve aereation efficiency.
*Source- Singh, R.P Microbilogy, Kalyani Publishers.

12. laboratory scale production

13. Laboratory scale production Preparation of the Inoculum culture
prepare specified broth into flasks, and plug with
non­absorbent cotton
Autoclave flasks at 121°C for 20 minutes
Transfer 5-6 days old non contaminant cultures of
bacteria into the flasks with inoculation needle
Incubate flasks at 32°C on shaker for 2-6 days
*Source:Singh, R.P Microbilogy, Kalyani Publishers.

14. Carriers for Bacterial inoculants
Carrier is the medium in which organisms are allowed to multiply .
Different carrier materials viz., peat lignite , compost, leaf manures, cellulose powder, charcoal powder, coconut shell powder, rice husk powder, press mud etc are extensively used carrier for inoculum preparation.
A: PRESS MUD B:LIGNITE C:CHALCOL D:COCONUT SHEEL
E:RICE HUSK F:CELLUOSE G:LEAF H:PEAT
POWDER
*Source-

15. Mass production of Rhizobium
Isolation –
Rhizobium can isolate from root nodule of leguminous crop.or root nodule of the cross inoculum groups.for isolation the root nodule are collected and than surface sterilize with 1%Hgcl2+60%ethnol+crushing.

16. Cross inoculation group of rhizobium
Rhizobium japonicum
Glycine max(soybean)
Rhizobium meliloti
Medicago sativa(alfa alfa)
Rhizobium leguminocerum
Pisum satium(pea)
Rhizobium lupini
Lupinus albus(lupinus grass)
Rhizobium trifolli
Trifoilium alxandrium(berseem)
Singh, R.P Microbilogy, Kalyani Publishers.

17. Media for Rhizobium Culture
YEM (YEast extract - Manitol)
Distilled water
1 liter
Mannitol
10 g
Yeast extract
0.5 g
MgSO4.7 H20
0.2 g
NaCl
0.1 g
K2HPO4
add water to
1 l
Source- Subba Rao, N.S. Soil Microbiology.

18. Large scale production
Starter culture- selected strain are used in starter culture. the tested strain which is kept in tube is transfer in conical flask. conical flask placed on rotary shaker and contains incubate for 3-7 days.
The contents of these flask usually attain a load of cell called seed(500 – 1000 ml). Broth is prepared by fermenter. .

19. Fermenter are used for large scale production of biofertilizer.
Both production rate is more than times higher then
rotary shaker.
In fermenter aeration is ragulated by forcing sterile air
through sapargers. airation and ph are the two important
facter which regulate the multiplication rate of rhizobium.
For rhizobium growth production small fermenter of
capacity less than 100 lit I batch fermentation sysatem are
usable.
To produce large amount of rhizobium biofertilizer we have
to put the mother culture or starter culture into the
fermenter. Temperature in fermenter is C and ph is 6-
7.5 wihtin 48 hours
Singh, R.P Microbilogy, Kalyani Publishers.

20. Carrier material
Medium matrix on which microbial inoculant grow to resonable higher number with long self life are called carrier material.
Peat most suitable for carrier matrial.bettre growth of rhizobium is obtain in sterlile carrier material .the best method is gamma radiation for sterilization of carrier material.
While using auto clave ,lime mixed lignite or plain charcol powder is filled up to two third capacity of stell trys and is sterlize at 15 PSI for 3-4 hours countionously or 1 hour each for 3 succesive days .
Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

21. Carrier material for Rhizobium
Water holding capacity %
Organic matter %
Charcoal 90 77
Lignite 92 28
FYM
40-50 30
Peat
Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

22. mixing with carrier material

23. Harvest ,Storage and packaging
Harvest – after complete fermentation process the no. of
microbial colony reached to 106 – 109 than it will be
harvested by harvesting drain pipe of fermenter.
Storage – store in cool place away from direct sun and
heat.
Packaging – LDPE packetes (200 g) as then can be
readially sealled allow high gas exchange it relative low
moisture loss .if production technology is best on
carreir material sterilization through autoclave than HDPE bags
Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

24. PSB (phosphate solubilizing bacteria)
Phosphate solubilizing bacteria (PSB) are beneficial bacteria capable of solubilizing inorganic phosphorus from insoluble compounds. P-solubilization ability of rhizosphere microorganisms is considered to be one of the most important traits associated with plant phosphate nutrition. The use of PSB as inoculants increases P uptake by plants. Simple inoculation of seeds with PSB gives crop yield responses equivalent to 30 kg P2O5 /ha or 50 percent of the need for phosphatic fertilizers. Some of the phosphate solubilizing bacteria are pseudomonas, bacillus, azotobacter etc.
Singh, R.P Microbilogy, Kalyani Publishers.

25. Isolation mass production and technique for PSB
Isoaltion – from rhizosphere soil through serial dilution technique and growth in plating .
Mass production -
Mass production technique is similar with rhizobium inoculants only the growth media is different.

26. Pikovsakya media composition
For PSB
Pikovsakya media composition
Ingredients
g/l
Yeast extract
0.5
Dextrose 10
Calcium phosphate 5
Ammonium phosphate
Pottasium chloride
0.2
Magnesium sulphate
0.1
Manganese sulphate
0.0001
Ferrous sulphate
0.001
Agar 15 Ph
6.8
Source- Subba Rao, N.S. Soil Microbiology.

27. Kings B broth (for pseudomonas spp.)
Peptone
20 g
Di potassium phosphate
1.5 g
Magnesium di chloride
Glycerol
10 g ph
Source- Subba Rao, N.S. Soil Microbiology.

28. Mass production of azospirillum
Isolation – screening method
Half cm root piece are surface sterilize into 70 % alcohol for 3-5 sec and washed in phosphate buffer ph 7.0. these root pieces then placed in semisolid nitrogen free sodium mallet medium. The tube re incubated in 35 C for 48 hours.
Tilak and subbarao 1987 have reported highest increase grain yield(33.67%) and plant biomass yield 32.71% by application of inoculants azospirillum brasilance and gomus faciculatum.
Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

29. Media for Azospirillum
Malic acid 
5.0g
Potassium hydroxide 
4.0g
Dipotassium hydrogen orthophosphate
0.5g
Magnesium sulphate 
0.2g
Sodium chloride 
0.1g
Calcium chloride 
Fe-EDTA (1.64% w/v aqueous) 
4.0 ml
Trace element solution 
2.0 ml
BTB (0.5% alcoholic solution) 
Agar
1.75 g
Distilled water 
1000 ml pH
6.8
Source- Subba Rao, N.S. Soil Microbiology

30. Mass production of Azolla
Azolla (mosquito fern, duckweed fern, fairy moss, water fern) is a genus of seven species of aquatic fernsin the family Salviniaceae. They are extremely reduced in form and specialized, looking nothing like other typical ferns but more resembling duckweed or some mosses. In azolla there are calothrix, anabena, nostock etc.
Blue green algae (BGA) when cultured in small pit and used as an inoculum in paddy kg /ha resulted in nitrogen economy to rice crop of the order of kg N with yield increase of percent.
Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

31. Isoaltion of Azolla
Isolation of azolla involves the transfer of an azolla from its natural habitat to medium where it can be grown without any contamination.
Serial dilution of soil and algal sample from the field location are prepared .
Aliquotes of appropriate dilution are then inoculated into liquid media, in flask and incubated for several weeks in illuminated growth room, at room temperature oC.
Source- biofertilizers.html

32. Mass production of Azolla
Cement cistern method –
Azolla can be multiplied I cement cistern of 2×1×3 m. a layer of cm soil is provided at the bottom of the cisterns. About cm depth of water is maintained in the cistrens. Super 8-12 g and 2.0 g are applied. About 200 g of the fresh azolla can be harvseted . After each harvest, super phosphate is applied to facilited fast multiplication. Every time, 200 g of azolla is inoculated for further multiplication.
Source-

33. Mycorrhiza
A mycorrhiza is a symbiotic association composed of a fungus  and roots of a vascular plant .In a mycorrhizal association, the fungus colonizes the host plant's roots, either intracellularly as in arbuscular mycorrhizal fungi (AMF or AM), or extracellularly as inectomycorrhizal fungi.
These fungi cannot be grown in pure culture but must be grown in association with plant roots. They form branched structures called arbuscules within the host’s root cells, and thus they are known as arbuscular mycorrhizal fungi. The arbuscules are sites of nutrient exchange between the fungus and the host. This manual focuses on arbuscular mycorrhizal fungi
.source, Venkatashwarlu B. Role of bio-fertilizers in organic farming: Organic farming in rain fed agriculture: Central institute for dry land agriculture, Hyderabad, 85-95

34. Mass production technology of VAM
A trench – 3×3×1 m and line with black polythene 500kg vermiculite ,50 kg sterilize soil up to 20 cm , spread 10 kg VAM inoculums 2- 5 cm below the surface of vermiculite .
Maize seed surface sterilize 5% sodium hypochloride than sown than applied 20 g urea 10g MOP than further 10 g urea applied for 30 and 40 days.
Source- Singh, Purohit, Parihar. Soil Microbiology, AGROBIOS,INDIA

35. PACKING AND STORAGE After curing, the inoculant is ready to be packed
Select micron polythene bags (6 x 10 in.)
Dispense 200 g of inoculant in each bag
Seal the polythene bags leaving 2/3 vacant spaces
Pin bags on few places for aeration
Keep inoculants for a week at room temperature
Store in a cold room and dispatch
Source-

38. Source - National Centre of Organic Farming, Annual Report 2013 - 2014

43. Applications of Biofertilizers to crop
Seedling root dip:-  This method is applied to the rice crop or the ransplanted crop.2 packet of inoculant is mixed with 40 liter of water for one acr area plant root are dipped into this mixture to 5-10 minute and than trnsplanted.
Seed treatment:-  1 packet of inoculum mixed with 200ml rice gruel to make aslury the sed require for sowing mixed with slury uniform coating of inoculant oer the seed raised I shed for 30 minute within 24 hours it should be sown
200gm---100k g seed.
Soil treatment:-  All the biofertilizers along with the compost fertilizers are mixed together. They are kept for one night. Then the next day this mixture is spread on the soil where seeds have to be sown. 4 packet inoculant made with 20kg fym.
Source-

44. Role of Biofertilizers
They help to get high yield of crops by making the soil rich with nutrients and useful microorganisms necessary for the growth of the plants. 
Biofertilizers have replaced the chemical fertilizers as chemical fertilizers are not beneficial for the plants. They decrease the growth of the plants and make the environment polluted by releasing harmful chemicals. 
Plant growth can be increased if biofertilizers are used, because they contain natural components which do not harm the plants but do the vice versa.
Promotes the development of mycorrhizal associations, which increases the availability of phosphorus (P) on the soil.
Biofertilizers destroy those harmful components from the soil which cause diseases in the plants. Plants can also be protected against drought and other strict conditions by using biofertilizers. 
Biofertilizers are not costly and even poor farmers can make use of them. 
They are environment friendly and protect the environment against pollutants.
Arun K.S., Bio-fertilizers for sustainable agriculture. Mechanism of P-solubilization Sixth edition, Agribios publishers, Jodhpur, India, (2007)

45. Conclusion
Bio-fertilizers being essential components of organic farming play vital role in maintaining long term soil fertility and sustainability by fixing atmospheric di-nitrogen (N=N), mobilizing fixed macro and micro nutrients or convert insoluble P in the soil into forms available to plants, there by increases their efficiency and availability. Currently there is a gap of ten million tones of plant nutrients between removal of crops and supply through chemical fertilizers. In context of both the cost and environmental impact of chemical fertilizers, excessive reliance on the chemical fertilizers is not viable strategy in long run because of the cost, both in domestic resources and foreign exchange, involved in setting up of fertilizer plants and sustaining the production. In this context, organic manures (biofertilizers) would be the viable option for farmers to increase productivity per unit area
Arun K.S., Bio-fertilizers for sustainable agriculture. Mechanism of P-solubilization Sixth edition, Agribios publishers, Jodhpur, India, (2007)

46. ?

47. THANK YOU