Mass production and how to process of Bacterial Biofertilizer


Mass production of   Bacterial Biofertilizer

Biofertilizers are carrier based preparations containing efficient strain of nitrogen fixing or phosphate solubilizing microorganisms. Biofertilizers are formulated usually as carrier based inoculants. The organic carrier materials are more effective for the preparation of bacterial inoculants. The solid inoculants carry more number of bacterial cells and support the survival of cells for longer periods of time.
   The mass production of carrier based bacterial biofertilizers involves three stages.
   Culturing of microorganisms
   Processing of carrier material
   Mixing the carrier and the broth culture and packing

 Culturing of Microorganisms
Although many bacteria can be used beneficially as a biofertilizer the technique of mass production is standardizedfor Rhizobium, Azospirillum, Azotobacter and phosphobacteria.
The media used for mass culturing are as follows:
Rhizobium : Yeast extract mannitol broth.
Growth on Congo red yeast extract mannitol agar medium
Mannitol
-
10.0 g
K2 HPO4 
-
0.5 g
Mg So4 7H2 O 
-
0.2 g
NaCl
-
0.1 g
Yeast extrac

-
0.5 g
Agar

20.0 g
Distilled water 

1000.0 ml
Add 10 ml of Congo red stock solution (dissolve 250 mg of Congo  red  in 100ml water) to 1 liter after adjusting the PH to 6.8 and before adding agar.
Rhizobium forms white, translucent, glistening, elevated and comparatively small colonies on this medium. Moreover, Rhizobium colonies do not take up the colour of congo red dye added in the medium. Those colonies which readily take up the congo red stain are not rhizobia but presumably Agrobacterium, a soil bacterium closely related to Rhizobium.
Azospirillum : Dobereiner's malic acid broth with NH4Cl (1g per liter)
Composition of the N-free semisolid malic acid medium
Malic acid 
-
5.0g
Potassium hydroxide 
-
4.0g
Dipotassium hydrogen orthophosphate
-
0.5g
Magnesium sulphate 
-
0.2g
Sodium chloride 
-
0.1g
Calcium chloride 
-
0.2g
Fe-EDTA (1.64% w/v aqueous) 
-
4.0 ml
Trace element solution 
-
2.0 ml
BTB (0.5% alcoholic solution) 
-
2.0 ml
Agar
-
1.75 g
Distilled water 
-
1000 ml
pH
-
6.8
Trace element solution
Sodium molybdate
-
200 mg
Manganous sulphate 
-
235 mg
Boric acid 
-
280 mg
Copper sulphate 
-
8 mg
Zinc sulphate 
-
24 mg
Distilled water 




Waksman medium No.77 (N-free Mannitol Agar Medium for Azotobacter)
Mannitol
:
10.0 g
Ca CO3           
:
5.0 g
K2HPO4
:
0.5 g
Mg SO4.7H2O
:
0.2 g
NaCl
:
0.2 g
Ferric chloride
:
Trace
MnSO4.4H2O
:
Trace
N-free washed Agar
:
15.0 g
pH
:
7.0
Distilled Water
:
1000 ml
Phosphobacteria : Pikovskaya’s Broth
Glucose
10.0 g
Ca3(PO4)2
5.0 g
(NH4)2SO4
0.5 g
KCl
0.2 g
MgSO4. 7H2O
0.1 g
MnSO4
Trace
FeSO4
Trace
Yeast Extract
0.5 g
Distilled Water
1000 ml
The broth is prepared in flasks and inoculum from mother culture is transferred to flasks. The culture is grown under shaking conditions at 30±2°C as submerged culture. The culture is incubated until maximum cell population of 1010 to 1011 cfu/ml is produced. Under optimum conditions this population level could be attained with in 4 to 5 days for Rhizobium; 5 to 7 days for Azospirillum; 2 to 3 days for phosphobacteria and 6-7 days for Azotobacter. The culture obtained in the flask is called starter culture. For large scale production of inoculant, inoculum from starter culture is transferred to large flasks/seed tank fermentor and grown until required level of cell count is reached.
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.
   The above media is prepared in large quantities in fermentor, sterilized well, cooled and kept it ready.
   The media in the fermentor is inoculated with the log phase culture grown in 5 litre flask. Usually 1 -2 % inoculum is sufficient, however inoculation is done up to 5% depending on the growth of the culture in the larger flasks.
   The cells are grown in fermentor by providing aeration (passing sterile air through compressor and sterilizing agents like glass wool, cotton wool, acid etc.) and given continuous stirring.
   The broth is checked for the population of inoculated organism and contamination if any at the growth period.
   The cells are harvested with the population load of 109 cells ml-1 after incubation period.
   There should not be any fungal or any other bacterial contamination at 10-6 dilution level
   It is not advisable to store the broth after fermentation for periods longer than 24 hours. Even at 4o C number of viable cells begins to decrease.



Processing of carrier material
The use of ideal carrier material is necessary in the production of good quality biofertilizer. Peat soil, lignite, vermiculite, charcoal, press mud, farmyard manure and soil mixture can used as carrier. The neutralized peat soil/lignite are found to be a good carrier for biofertilizer production
   Cheaper in cost
   locally available
   High organic matter contain
   No toxic
   Water holding capacity of more than 50%
   Easy to process




Preparation of carrier material






   The carrier material (peat or lignite) made it into a powder so as to pass through 212 micron IS sieve.
   The pH of the carrier material is neutralized with the help of calcium carbonate (1:10 ratio) , 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 contaminants.



Mixing the carrier and the broth culture and packing
Inoculant packets are prepared by mixing the broth culture obtained from fermentor with sterile carrier material
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 fermentor 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 population.
   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


 Schematic representation of mass production of bacterial biofertilizers
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 etc.,
Storage of biofertilizerpacket
   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 and107 cells/ g on dry weight basis before expiry date.


Mass production of Mycorrhizal biofertilizer

The commercial utilization of mycorrhizal fungi has become difficult because of the obligate symbiotic nature and difficulty in culturing on laboratory media. Production of AM inoculum has evolved from the original use of infested field soils to the current practice of using pot culture inoculum derived from the surface disinfected spores of single AM fungus on a host plant grown in sterilized culture medium. Several researches in different parts of the world resulted in different methods of production of AM fungal inoculum as soil based culture as well as carrier based inoculum. Root organ culture and nutrient film technique provide scope for the production of soil less culture.
As a carrier based inoculum, pot culture is widely adopted method for production. The AM inoculum was prepared by using sterilized soil and wide array of host crops were used as host. The sterilization process is a cumbersome one and scientists started using inert materials for production of AM fungi. The researchers tried use of perlite, montmorillonite clay etc., In TNAU vermiculite was tried as substrate for the replacement of soil sterilization, which resulted in the best method of inoculum production. 
Method of production
 Vermiculite contained raised AM infected maize plants
   A trench (1m x 1m x 0.3m) is formed and lined with black polythene sheet to be used as a plant growth tub.

  
Mixed 50 kg of vermiculite and 5 kg of sterilized soil and packed in the trench up to a height of 20 cm
   Spread 1 kg of AM inoculum (mother culture) 2-5 cm below the surface of vermiculite
   Maize seeds surface sterilized with 5% sodium hypochlorite for 2 minutes are sown
   Applied 2 g urea, 2 g super phosphate and 1 g muriate of potash for each trench at the time of sowing seeds.  Further 10 g of urea is applied twice on 30 and 45 days after sowing for each trench
   Quality test on AM colonization in root samples is carried out on 30th and 45th day
   Stock plants are grown for 60 days (8 weeks).  The inoculum is obtained by cutting all the roots of stock plants.  The inoculum produced consists of a mixture of vermiculite, spores, pieces of hyphae and infected root pieces.
   Thus within 60 days 55 kg of AM inoculum could be produced from 1 sq meter area.  This inoculum will be sufficient to treat 550 m2 nursery area having 11,000 seedlings.                        



AM fungi
Nursery application: 100 g bulk inoculum is sufficient for one metre square. The inoculum should be applied at 2-3 cm below the soil at the time of sowing. The seeds/cutting should be sown/planted above the VAM inoculum to cause infection.
For polythene bag raised crops: 5 to 10 g bulk inoculum is sufficient for each packet. Mix 10 kg of inoculum with 1000 kg of sand potting mixture and pack the potting mixture in polythene bag before sowing.
For out –planting: Twenty grams of VAM inoculum is required per seedling. Apply inoculum at the time of planting.
For existing trees: Two hundred gram of VAM inoculum is required for inoculating one tree. Apply inoculum near the root surface at the time of fertilizer application.


Mass production and field application of cyanobacteria


 Blue green algal inoculation with composite cultures was found to be more effective than single culture inoculation. A technology for mass scale production of composite culture of blue green algae under rice field condition was developed at TNAU and the soil based BGA inoculum could survive for more than 2 years. At many sites where algal inoculation was used for three to four consecutive cropping seasons, the inoculated algae establish well and the effect persisted over subsequent rice crop. Technologies for utilizing nitrogen fixing organisms in low land rice were the beneficial role of blue green algal inoculation in rice soils of Tamil Nadu.  
The blue green algal inoculum may be produced by several methods viz., in tubs, galvanized trays, small pits and also in field conditions.  However the large-scale production is advisable under field condition which is easily adopted by farmers.
I. Multiplication in trays
   Big metallic trays (6’x 3’x 6”lbh) can be used for small scale production
   Take 10 kg of paddy field soil, dry powder well and spread
   Fill water to a height of 3”
   Add 250 g of dried algal flakes (soil based) as inoculum
   Add 150 g of super phosphate and 30 g of lime and mix well with the soil
   Sprinkle 25 g carbofuran to control the insects
   Maintain water level in trays
   After 10 to 15 days, the blooms of BGA will start floating on the water sources
   At this stage stop watering and drain. Let the soil to dry completely
   Collect the dry soil based inoculum as flakes
   Store in a dry place.  By this method 5 to 7 kg of soil based inoculum can be obtained.
II. Multiplication under field condition
Materials
   Rice field
   Super phosphate
   Carbofuran
   Composite BGA starter culture
Procedure
Select an area of 40 m2 (20m x 2m) near a water source which is directly exposed to sunlight.
Make a bund all around the plot to a height of 15 cm and give it a coating with mud to prevent loss of water due to percolation.
   Plot is well prepared and levelled uniformly and water is allowed to a depth of 5-7.5 cm and left to settle for 12 hrs.
   Apply 2 kg of super phosphate and 200 g lime to each plot uniformly over the area.
   The soil based composite starter culture of BGA containing 8-10 species @ 5 kg / plot is powdered well and broadcasted.
   Carbofuran @ 200 g is also applied to control soil insects occurring in BGA.
   Water is let in at periodic intervals so that the height of water level is always maintained at 5 cm.
   After 15 days of inoculation, the plots are allowed to dry up in the sun and the algal flakes are collected and stored.
Observations
The floating algal flasks are green or blue green in colour.  From each harvest, 30 to 40 kg of dry algal flakes are obtained from the plot.

Method of inoculation of BGA in rice field
Blue green algae may be applied as soil based inoculum to the rice field following the method described below.
   Powder the soil based algal flakes very well.
   Mix it with 10 kg soil or sand (10kg powdered algal flakes with 10 kg soil / sand).
   BGA is to be inoculated on 7-10 days after rice transplanting.
   Water level at 3-4” is to be maintained at the time of BGA inoculation and then for a month so as to have maximum BGA development.
Observation
A week after BGA inoculation, algal growth can be seen and algal mat will float on the water after 2-3 weeks. The algal mat colour will be green or brown or yellowish green. 
Mass production and field application of Azolla

Azolla is a free-floating water fern that floats in water and fixes atmospheric nitrogen in association with nitrogen fixing blue green alga Anabaena azollae. Azolla fronds consist of sporophyte with a floating rhizome and small overlapping bi-lobed leaves and roots. Rice growing areas in South East Asia and other third World countries have recently been evincing increased interest in the use of the symbiotic N2 fixing water fern Azolla either as an alternate nitrogen sources or as a supplement to commercial nitrogen fertilizers.  Azolla is used as biofertilizer for wetland rice and it is known to contribute 40-60 kg N ha-1 per rice crop. The agronomic potential of Azolla is quite significant particularly for rice crop and it is widely used as biofertilizer for increasing rice yields. Rice crop response studies with Azolla biofertilizer in the People’s Republic in China and in Vietnam have provided good evidence that Azolla incorporation into the soil as a green manure crop is one of the most effective ways of providing nitrogen source for rice.
The utilization of Azolla as dual crop with wetland rice is gaining importance in Philippines, Thailand, Srilanka and India. The important factor in using Azolla as a biofertilizer for rice crop is its quick decomposition in soil and efficient availability of its nitrogen to rice. In tropical rice soils the applied Azolla mineralizes rapidly and its nitrogen is available to the rice crop in very short period. The common species of Azolla are A. microphylla, A. filiculoides, A. pinnata, A. caroliniana, A. nilotica, A. rubra and A. mexicana.
I.  Mass multiplication of Azolla under field conditions
A simple Azolla nursery method for large scale multiplication of Azolla in the field has been evolved for easy adoption by the farmers.
Materials
   One cent (40 sq.m) area plot
   Cattle dung
   Super phosphate
   Furadan
   Fresh Azolla inoculum
Procedure
   Select a wetland field and prepare thoroughly and level uniformly.
   Mark the field into one cent plots (20 x 2m) by providing suitable bunds and irrigation channels.
   Maintain water level to a height of 10 cm.
   Mix 10 kg of cattle dung in 20 litres of water and sprinkle in the field.
   Apply 100 g super phosphate as basal dose.
   Inoculate fresh Azolla biomass @ 8 kg to each pot.
   Apply super phosphate @ 100 g as top dressing fertilizer on 4th and 8th day after Azolla inoculation.
   Apply carbofuran (furadan) granules @ 100 g/plot on 7th day after Azolla inoculation.
   Maintain the water level at 10 cm height throughout the growth period of two or three weeks.
   Observations
   Note the Azolla mat floating on the plot. Harvest the Azolla, drain the water and record the biomass.



II.  Method of  inoculation of Azolla to rice crop
The Azolla biofertilizer may be applied in two ways for the wetland paddy.  In the first method, fresh Azolla biomass is inoculated in the paddy field before transplanting and incorporated as green manure.  This method requires huge quantity of fresh Azolla. In the other method, Azolla may be inoculated after transplanting rice and grown as dual culture with rice and incorporated  subsequently.
A.  Azolla  biomass incorporation as green manure for rice crop
   Collect the fresh Azolla biomass from the Azolla nursery plot.
   Prepare the wetland well and maintain water just enough  for easy incorporation.
   Apply fresh Azolla biomass (15 t ha-1) to the main field and incorporate the Azolla by using implements or tractor.
B.  Azolla inoculation as dual crop for rice
   Select a transplanted rice field.
   Collect fresh Azolla  inoculum from Azolla nursery.
   Broadcast the fresh Azolla in the transplanted rice field on 7th day after planting (500 kg / ha).
   Maintain water level at 5-7.5cm.
   Note the growth of Azolla mat four weeks after transplanting and incorporate the  Azolla biomass by using implements or tranctor or during inter-cultivation practices.
   A second bloom of Azolla will develop 8 weeks after transplanting which may be incorporated again.
   By the two incorporations, 20-25 tonnes of Azolla can be incorporated in one hectare rice field.




4. Application of Biofertilizers
1. Seed treatment or seed inoculation 
2. Seedling root dip 
3. Main field application
Seed treatment
One packet of the inoculant is mixed with 200 ml of rice kanji 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.
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.
Main field application
Four packets of the inoculant is mixed with 20 kgs of dried and powdered farm yard manure and then broadcasted in one acre of main field just before transplanting.
Rhizobium
For all legumes Rhizobium is applied as seed inoculant.
Azospirillum/Azotobacter
In the transplanted crops, Azospirillum is inoculated through seed, seedling root dip and soil application methods. For direct sown crops, Azospirillum is applied through seed treatment and soil application.
Phosphobacteria
Inoculated through seed, seedling root dip and soil application methods as in the case of Azospirillum.
Combined application of bacterial biofertilizers.
Phosphobacteria can be mixed with Azospirillum and Rhizobium. The inoculants should be mixed in equal quantities and applied as mentioned above.


Points to remember
   Bacterial inoculants should not be mixed with insecticide, fungicide, herbicide and fertilizers.
   Seed treatment with bacterial inoculant is to be done at last when seeds are treated with fungicides.
Biofertilizers recommendation (one packet - 200 g)
S. No.
Crop
Seed
Nursery
Seedling dip
Main field
Total requirement of packets per ha
1.
Rice
5
10
5
10
30
2.
Sorghum
3
-
-
10
13
3.
Pearl millet
3
-
-
10
13
4.
Ragi
3
-
5
10
18
5.
Maize
3
-
-
10
13
6.
Cotton
3
-
-
10
13
7.
Sunflower
3
-
-
10
13
8.
Castor
3
-
-
10
13
9.
Sugarcane
10
-
-
36 
(3 split)
46
10.
Turmeric
-
-
-
24 
(2 split)
24
11.
Tobacco
1
3
-
10 g/pit
14
12.
Papaya
2
-
-
10
-
13.
Mandarin
Orange
2
-
-
10 g/pit
-
14.
Tomato
1
-
-
10
14
15.
Banana
-
-
5
10 g/pit
-
Rhizobium (only seed application is recommended)
S. No.
Crop
Total requirement of packets per ha
1.
Soybean
5
2.
Groundnut
5
.
Bengalgram
5
4.
Blackgram
3
5.
Green
ram
3
6.
Redgram
3
7.
Cowpea
3
Phosphobacteria
The recommended dosage of Azospirillum is adopted for phosphobacteria inoculation; for combined inoculation, both biofertilizers as per recommendations are to be mixed uniformly before using.


























5. Azolla – The best feed for cattle and poultry
Azolla is a free floating water fern that floats in water and fixes nitrogen in association with the nitrogen fixing blue green algae, Anabaena azollae. Azolla is considered to be a potential biofertilizer in terms of nitrogen contribution to rice. Long before its cultivation as a green manure, Azolla has been used as a fodder for domesticated animals such as pigs and ducks. In recent days, Azolla is very much used as a sustainable feed substitute for livestock especially dairy cattle, poultry, piggery and fish. 
Azolla contains 25 – 35 per cent protein on dry weight basis and rich in essential amino acids, minerals, vitamins and carotenoids including the antioxidant b carotene. Cholorophyll a, chlorophyll b and carotenoids are also present in Azolla, while the cyanobiont Anabaena azollae contains cholorophyll a, phycobiliproteins and carotenoids. The rare combination of high nutritive value and rapid biomass production make Azolla a potential and effective feed substitute for live stocks.
Inputs required
Azolla fronds, Polythene sheet, Super phosphate and Cow dung.










Methodology
The area selected for Azolla nursery should be partially shaded. The convenient size for Azolla is 10 feet length, 2 feet breadth and 1 feet depth. The nursery plot is spread with a polythene sheet at the bottom to prevent water loss. Soil is applied to a depth of 2 cm and a gram of super phosphate is applied along with 2 kg of vermicompost or cow dung in the nursery for quick growth. Azolla mother inoculum is introduced @ 5 kg/plot.
The contents in the plot are stirred daily so that the nutrients in the soil dissolve in water for easy uptake by Azolla. Azolla is harvested fifteen days after inoculation at the rate of 50-80 kg / plot. One third of Azolla should be left in the plot for further multiplication. Five kg cow dung slurry should be sprinkled in the Azolla nursery at  ten days intervals. Neem oil can be sprayed over the Azolla at 0.5 5 level to avoid pest incidence.
Animal
Dosage / day
Adult cow , Buffalo, Bullock
1.5-2  kg
Layer, Broiler birds
20 – 30 grams
Goat
300 – 500 grams
Pig
1.5 – 2.0 kg
Rabbit
100 gram
Value of the technology
The egg yield is increased in layer birds due to Azolla feeding. The Azolla fed birds register an overall egg productivity of 89.0 per cent as against 83.7 per cent recorded by the birds fed with only concentrated feed. The average daily intake  of concentrated feed is considerably low (106.0 g) for birds due to Azolla substitution as against 122.0 g in the control birds. More impotantly Azolla feeding shows considerable amount of savings in the consumption of concentrated feed (13.0 %) leading to reduced operational cost. By considering the average cost of the concentrated feed  as Rs. 17/ Kg, a 13.0 % saving in the consumption ultimately leads to a feed cost savings of 10.0 paise /day/ bird and hence a layer unit maintaining 10,000 birds could cut down its expense towards feed to a tune of rs.1000/day.



Benefits
The Azolla feeding to layer birds increase egg weight, albumin, globulin and carotene contents. The total protein content of the eggs laid by the Azolla fed birds is high and the total carotene content of Azolla eggs(440 g 100 g-1 of edible portion)is also higher than the control. The rapid biomass production due to the high relative growth rate, increased protein and carotene contents and good digestability of the Azolla hybrid Rong ping favour its use as an effective feed supplement to poultry birds.
Effect of Azolla hybrid Rong Ping on the nutritional value of egg
Parameters
Azolla egg
Control
percentage increase over control
Egg weight (g)
61.20
57.40
6.62
Albumin (g /100 g of edible portion)
3.9
3.4
14.70
Globulin (g /100 g of edible portion)
10.1
9.5
6.31
Total protein (g/ 100 g of edible portion)
14.0
12.9
8.52
Carotenes (µg / 100 g of edible portion)
440
405
8.64
Application
In Indian conditions, agriculture is very much coupled with poultry farming. Azolla is an important low cost input, which plays a vital role in improving soil quantity in sustainable rice farming. The twin potentials as biofertilizer and animal feed make the water fern Azolla as an effective input to both the vital components of integrated farming, agricultural and animalo husbandry.
Limitation
Azolla is a water fern and requires a growth temperature of 35-38º C. The multiplication of Azolla is affected under elevated temperature. Hence adopting this technology in dry zones where the temperature exceeds 40ºc is difficult.





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