Simple Autoclave Diagram

Production and study of Azotobacter / Rhizobium as biofertilizer using carrier based inoculum method

Aim: To prepare and study Azotobacter or Rhizobium biofertilizer using carrier based inoculum technique under laboratory conditions.

Principle: Biofertilizers are living microbial preparations which improve soil fertility. Two important nitrogen fixing bacteria used in agriculture are Azotobacter and Rhizobium. Azotobacter lives freely in soil and fixes atmospheric nitrogen without needing a plant host. Rhizobium is different. It forms nodules on roots of leguminous plants and fixes nitrogen inside those nodules. In both cases nitrogen gas from air is converted into ammonia which plants can absorb. In laboratory we grow these bacteria in a nutrient medium first. Later the culture is mixed with a sterile carrier material such as peat soil or lignite. This carrier helps the bacteria survive longer and makes the biofertilizer easy to apply in field.

Requirements:

• Pure culture of Azotobacter or Rhizobium
• Laminar airflow cabinet
• Conical flasks (250 mL and 500 mL)
• Autoclave
• Incubator (28–30°C)
• Sterile distilled water
• Sterile carrier material (peat soil or lignite powder)
• Calcium carbonate powder
• Polythene packets for storage
• Weighing balance
• Glass rod or spatula
• Cotton plugs and aluminium foil

Ashby's medium for Azotobacter (1000 mL preparation):

• Mannitol : 20 g/L
• Dipotassium phosphate : 0.2 g/L
• Magnesium sulphate : 0.2 g/L
• Sodium chloride : 0.2 g/L
• Potassium sulphate : 0.1 g/L
• Calcium carbonate : 5 g/L
• Distilled water : 1000 mL

Yeast Extract Mannitol Broth for Rhizobium (1000 mL preparation):

• Mannitol : 10 g/L
• Yeast extract : 1 g/L
• Dipotassium phosphate : 0.5 g/L
• Magnesium sulphate : 0.2 g/L
• Sodium chloride : 0.1 g/L
• Distilled water : 1000 mL

Procedure:

1. Prepare the required culture medium depending on the organism used, Azotobacter or Rhizobium.
2. Dissolve all medium components in distilled water and adjust the volume to 1000 mL.
3. Dispense around 100 mL medium into sterile conical flasks.
4. Plug the flasks with cotton and cover them with aluminium foil.
5. Sterilize the medium in autoclave at 121°C for 15 minutes.
6. Allow the medium to cool to room temperature.
7. Transfer the flasks into laminar airflow cabinet.
8. Inoculate each flask with a loopful of pure bacterial culture.
9. Incubate the inoculated flasks at 28–30°C for 48 hours.
10. During incubation the broth becomes slightly turbid as bacterial cells grow in the medium.
11. Take sterile carrier material such as peat soil or lignite powder and dry it properly.
12. Sterilize the carrier material in autoclave to remove unwanted microorganisms.
13. After cooling, transfer the carrier material into sterile tray or container.
14. Mix the grown bacterial broth culture with the sterile carrier material slowly.
15. Use a sterile spatula to mix until the carrier becomes uniformly moist.
16. Fill the prepared biofertilizer mixture into small sterile polythene packets.
17. Seal the packets and label them with organism name and date.
18. Store the packets at cool temperature until use.
19. Students usually observe this step carefully because proper mixing is important only.

Observation: After incubation the broth medium becomes turbid indicating growth of Azotobacter or Rhizobium cells. The carrier material becomes uniformly moist after mixing with the culture.

Result: Azotobacter or Rhizobium biofertilizer was successfully prepared using carrier based inoculum method.

Conclusion: Biofertilizers are useful in agriculture because they naturally increase nitrogen availability in soil. When Azotobacter or Rhizobium cultures are mixed with a carrier material, the bacteria survive longer and can be applied easily to seeds or soil.