PRODUCTION OF BIOFERTILIZER-mass cultivation of AZOSPIRILLUM

PRODUCTION OF BIOFERTILIZER-AZOSPIRILLUM

AIM:

To isolate and mass cultivate the biofertilizer Azospirillum.

INTRODUCTION:

Azospirillum was first described as Spirillum lipoferum by beijerinck in 1925 as a nitrogen fixing bacterium. Tarrand et al., (1978) renamed this organism as Azospirillum (N-fixing Spirillum). Azospirillum have been found to be associated with the roots of rhizosphere of many members of the Gramineae particularly in the tropics. Digitaria, maize, sorghum, rice, sugarcane, wheat and forage grasses are most frequently cited as hosts. Azospirillum species are characterized as micro aerobically nitrogen fixing bacteria. Diverse nitrogen fixing organisms contribute to the soil nitrogen pools. The major biological nitrogen fixation systems include cyanobacteria and photosynthetic bacteria that inhibit flood waters and soil surface and heterotrophic bacteria present in the rhizosphere and bulk soil. Azospirillum is recognized as a ubiquitous soil organism capable of colonizing effectively not only the roots of a wide variety of plants but also their above ground portions forming apparently an associative symbiosis

MATERIALS REQUIRED: 

Crystal violet, Safranin, Malate Semisolid Medium, BMS Agar, Lignite, CaCO_3, Polythene bags, Nfb medium (Nitrogen Free Bromothymol Blue (Nfb) Medium)

PROCEDURE:

Collection of soil samples:

  1. The soil samples were collected at 0.30 cm soil depth.

 2. Collected samples were mixed and placed in sterile polythene bags that was sealed and then brought to the laboratory and kept at 5-10⁰c.

  3. To 9 ml sterilized distilled water in a test tube 1 g of soils were added and mixed thoroughly from this 1 mL of diluted sample was transferred into another tube.

4. This process was continued upto 10^-9 dilution from the above diluted samples 1mL of 10^-5, 10^-6, 10^-7, 10^-8, were inoculated into NFb semisolid medium and incubated into 48 hours of 28± 2⁰c in the incubate.

 Isolation of Azospirillum:

  5. Isolation from soils 25 mL test tubes with 5 mL of NFb semi-solid medium were inoculated with one gram rhozophere soil were inoculated with malate semisolid medium.

  Bacterial smears preparation:

  6. Flame heat fixed smears prepared from light suspension of cells, flooded with crystal violet solution for 1 minute and washed for 30 seconds in the running water.

7. Rinse off excess water and flooded in iodine solution for 1 min and again washed in running water for 30 seconds.

8. Slides were passed through iodinated alcohol solution for removing excess stain and washed with tap water for five seconds.

9. The excess water removed and flooded with counter stain (safranin) for 1 minute and again washed in tap water, air dried and examined under microscope.

 Mass inoculums production:

 10. The isolated strains were used for large scale multiplication. The isolated strain selected for the preparation of Azospirillum inoculums.

11. These strains were inoculated into BMS agar slants. They are called starter culture.

12. These starter cultures were transferred into 100mL NFb liquid medium containing 250mL conical flask.

13. These cultures were incubated at 28±2⁰c for 4 days with occasional shaking of the conical flasks, for proper aeration.

14. After 4 days, the cultures were mixed with carrier materials. Lignite was used as carrier for Biofertilizer production, 4kg lignite used per liter of broth culture for mixing.

15. After mixing 2% CaCO3 was added and the mixed inoculums covered by polythene sheets for curing for 24 hours. After 24 hours the carrier based inoculums were pocketed into polythene bags.

Media Preparation:

Nfb medium (Nitrogen Free Bromothymol Blue (Nfb) Medium)

DL-malic acid: 5.0 g
K₂HPO₄: 0.5 g
MgSO₄ • 7H₂O: 0.2 g
NaCl: 0.1 g
CaCl₂ • 2H₂O: 0.02 g
Micronutrient solution: 2 ml
Bromthymol blue solution (0.5% in 0.2N KOH): 2 ml
Fe(III) EDTA (1.64%): 4.0 ml
Vitamin solution: 1.0 ml
Distilled water: 1.0 L

Adjust pH to 6.8
For semisolid medium, add 0.5 g of agar; for solid medium, add 15 g of agar. Autoclave at 121°C for 15 min.

Micronutrient solution:
CuSO₄ • 5H₂O: 0.4 g
ZnSO₄ • 7H₂O: 0.12 g
H₃BO₃: 1.4 g
Na₂MoO₄ • 2H₂O: 1.0 g
MnSO₄ • H₂O: 1.5 g
Distilled water: 1.0 L
Vitamin solution:
Biotin: 10 mg
Pyridoxol HCL: 20 mg
Distilled water: 0.1 L

BMS AGAR (POTATO INFUSION AGAR MEDIUM)

Components gms/litre

Potatoes 200 g

Mallic acid 2.5

KOH 2.0

Cane sugar 2.5

Biotine 2.1

 Cook washed potatoes and filter through cotton. Prepare potassium malate by dissolving 2.5g malate in 50ml water adding 2 drops of Bromothymol blue (0.5% ethanol) and 2 g of KOH. Adjust pH to green colour. Add malate, sugar, biotine to potato filtrate and dilute to 1000ml. Solid media is obtained by adding 2% agaragar.