Simple Autoclave Diagram

Production of Penicillin by Penicillium chrysogenum Through Submerged Fermentation.

Aim: To produce penicillin antibiotic by submerged fermentation using Penicillium chrysogenum and detect its antibacterial activity by agar diffusion method.

Principle: Penicillin is a secondary metabolite produced by the fungus Penicillium chrysogenum. The organism grows first. Then it starts releasing antibiotics into the medium. This usually happens when nutrients begin to reduce and the culture enters the stationary phase. Submerged fermentation works well for this fungus. The mould grows as small mycelial clumps inside the liquid medium. Oxygen is needed. Shaking or aeration helps. Penicillin interferes with bacterial cell wall synthesis. Gram-positive bacteria are sensitive to it. When culture filtrate containing penicillin is placed on a bacterial lawn, a clear inhibition zone appears around the sample. Small circle. No bacterial growth.

Requirements:

• Pure culture of Penicillium chrysogenum
• Nutrient agar plates
• Bacillus subtilis culture (test organism)
• Conical flasks (500 ml or 1000 ml)
• Incubator at 25 to 28°C
• Rotary shaker or manual shaking
• Cotton plugs
• Sterile pipettes
• Whatman filter paper
• Cork borer
• Sterile distilled water

Penicillin Fermentation Medium (Modified Czapek Yeast Extract Medium) (for 1000 ml):

• Lactose: 30 g/L
• Yeast extract: 5 g/L
• Peptone: 5 g/L
• Sodium nitrate: 3 g/L
• Dipotassium phosphate: 1 g/L
• Magnesium sulfate: 0.5 g/L
• Calcium carbonate: 5 g/L
• Distilled water: up to 1000 ml

Procedure:

1. Firstly, prepare the penicillin fermentation medium and adjust the pH to around 6.5.
2. Then, add  250 mL medium into a 500 mL conical flask.
3. Now, sterilize the medium by autoclaving at 121°C for 15-20 minutes.
4. Allow the medium to cool to room temperature.
5. After that, inoculate the flask with a small spore suspension of Penicillium chrysogenum.
6. Plug the flask with a sterile cotton plug.
7. Incubate the flask at 25 to 28°C on a rotary shaker for 5 days.
8. Observe fungal growth daily. White mycelial growth starts appearing inside the broth.
9. After 5 days, filter the culture through sterile filter paper.
10. Collect the clear culture filtrate in a sterile flask.
11. Prepare nutrient agar plates and spread the Bacillus subtilis culture evenly.
12. Use a cork borer to make small wells in an agar plate.
13. Add 0.2 ml culture filtrate into each well.
14. Incubate plates at 37°C for 24 hours.
15. Observe the plates for inhibition zones around the wells.

Observation: A clear circular zone appears around the well containing culture filtrate on the bacterial lawn. The surrounding area shows dense bacterial growth, but the region near the well remains clear.

Result: Penicillin produced by Penicillium chrysogenum during fermentation inhibited the growth of Bacillus subtilis, confirmed by a zone of inhibition in the agar diffusion assay.

Conclusion: Submerged fermentation allows Penicillium chrysogenum to grow and release penicillin into the medium within about five days. The antibiotic activity can be demonstrated easily using the agar diffusion test with sensitive bacteria.