INOCULATION FOR BIOFERTILIZER

Presented by; W.L.D.P.A.DE ALWIS SC/2007/6518 & SC/2008/6786 DANUSHIKA RATNAYAKA

Biofertilizers are;

Preparations having living cells or latent cells of efficient strains of microbes, that increase crop productivity & soil In oligotrophic conditions in soil, number of beneficial microbes are& Not efficient. Artificially multiplied cultures of efficient strains of selected microbes can accelerate..

Equipments needed in . 1).Auto clave- sterilize growth medium

2).laminar air flow culture transfer & inoculation

3).BOD incubators-provide controlled conditions, for microbes development.

4).rotary shaker: agitate culture fiasks for

aeration.

5).hot air oven: sterilize glassware.

6). pH meter

7).refrigerator: preserve mother cultures

8).fermenter:provides the proper environment for the growth of a desired organism.

Types of biofertilizer (acc. To the physical nature)

1). Carrier based powder form 2). Liquid 3).granules

How to prepare the inoculums

Steps:

Broth is prepared in flasks of 250ml,500ml,3L,5L & inoculumn from mother culture is transferred to flasks of 250ml. Culture is grown under shaking conditions at 30C as submerged cultures. The culture is incubated until maximum cell population level of 1010 to 1011 CFU/ml is prodused. (Rhizobium-4-5days,Azospirillum-5-7days,Phospobacteria-23days,Azotobacter-6-7days) The culture now called as starter culture. For large scale production inoculumn from starter culture is transferred to large flasks/seed tank fermenters. Above media is prepared in large quantities in fermenters & inoculated with the log phase culture grown in 5L flask.

The cells are grown in fermentor by providing aeration & given continous stirring. The cells are harvested with the population load of 109 cells/ml after incubation periods. There should not be any fungal or other bacterial contamination at 10 -6 dilution level.

culturing of microbes
1).media use for mass culture: a). Rhizobium-yeast extract Manitol broth Mannitol K2 HPO4 Mg So4 7H2 O NaCl Yeast extract Agar Distilled water 10.0 g 0.2 g 0.1 g 0.5 g 20.0 g 1000.0 ml Colonies are: White Translucent Glistering Elevated Small Dont take the colour of Congo red stain.

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

Malic acid Potassium hydroxide Dipotassium hydrogen orthophosphate Magnesium sulphate Sodium chloride Calcium chloride Fe-EDTA (1.64% w/v aqueous) Trace element solution BTB (0.5% alcoholic solution) Agar Distilled water pH Trace element solution Sodium molybdate Manganous sulphate Boric acid Copper sulphate Zinc sulphate Distilled water

5.0g 4.0g 0.5g 0.2g 0.1g 0.2g 4.0 ml 2.0 ml 2.0 ml 1.75 g 1000 ml 6.8

Azospirillum : Dobereiner's malic acid broth with NH4Cl (1g per liter) Composition of the N-free semisolid malic acid medium

200 mg 235 mg 280 mg 8 mg 24 mg 200 ml

Waksman medium No.77 (N-free Mannitol Agar Medium for Azotobacter)

Mannitol Ca CO3 K2HPO4 Mg SO4.7H2O NaCl Ferric chloride MnSO4.4H2O N-free washed Agar pH Distilled Water

: : : : : : : : : :

10.0 g 5.0 g 0.5 g 0.2 g 0.2 g Trace Trace 15.0 g 7.0 1000 ml

Phosphobacteria : Pikovskayas Broth

glucose Ca3(PO4)2 (NH4)2SO4 KCl MgSO4. 7H2O MnSO4 FeSO4 Yeast Extract Distilled Water

10.0 g 5.0 g 0.5 g 0.2 g 0.1 g Trace Trace 0.5 g 1000 ml

Carrier based powdered form Processing of carrier materials.

Peat soil, lignite, vermiculite, charcoal, press mud, farm yard manure, soil mixture can be used as carrier material. The neutralized peat soil /lignite are found to be better .

Facts for selecting an ideal carrier material; Cheaper in cost Locally available High organic matter content No toxic chemicals Water holding capacity more than 50% Easy to process, friability & vulnerability

Procedure;

Carrier material is powdered to a fine powder .(so as to pass through 212 micron sieve) The pH of the carrier material is neutralized by CaCO3 (1:10 ratio) Since the peat/lignite are acidic in nature. The neutralized carrier material is sterilized in an autoclave to eliminate contaminants.

Mixing the carrier & the broth culture & packing Procedure;

The neutralized , sterilized carrier materials spread in a clean, dry, sterile metallic or plastic tray. The bacterial culture drawn from the fermentor is added to the sterilized carrier & mixed well by manual or by mechanical mixturer. The inoculants packets of 200g in polythene bags, sealed with electrical sealer & allowed for curing for 2-3 days at room temperature. The polythene bag should be low density grade. Thickness around 50-75 micron.

Granule form of biofertilizer:

Consists of granules (1-2mm) made from tank bed clay. Granules are baked at 200C in a muffle furnace, which help to sterilize the material & gives porosity to granules. The baked granules are soaked in a suspension of N fixing bacteria grown in a suitable medium overnight. The clay granules are dried at room temperature under aseptic conditions. The granules are contain more than a billion of bacteria per gram of granules. suitable for field application along with seeds & fertilizers in furrows.

Liquid bio fertilizer How to make?

References

Thank you