In this article we will discuss about the fermentation of penicillin.
1. Starter cultures on M2 Agar.
2. M2 agar (pH 7.0):
3. Seed medium:
4. Fermentation medium:
Dissolve 1.5 g of phenyl acetic acid in warm distilled water. Adjust pH to 7 and make up the volume to 100 ml. Add 1 ml of this to the medium.
1. Grow P. chrysogenum on M2 agar for 120—144 hours at room temperature (28°-30°C).
2. Make a suspension of spores and inoculate 10 ml of it on seed medium in Erlenmeyer flasks with 500 ml capacity.
3. Incubate at room temperature (28°— 30°C) on a shaker (240 rpm) for 48- 72 hours seed.
4. Inoculate this seed at the rate of 10% (V/V) to 45 ml of fermentation medium in 500 ml Erlenmeyer flasks (45 and medium + 5 ml seed).
5. Incubate at room temperature (28°- 30°C) for 120 hours on a rotary shaker (240 rpm).
6. Check the pH of the medium first before inoculation (pH will be around 7.0- 7.5). Adjust it to 5.2 using 20% H3PO4.
7. After fermentation filter the broth and use the filtrate for chemical and biological assays.
Chemical Assay— Iodimetric Method:
Active penicillin molecule does not absorb iodine. Alkali inactivation product, Na penicillate does absorb I2. Penicillin G inactivated with alkali absorbs nine atoms of I2 per penicillin molecule. Residual I2 is estimated with sodium thiosulphate.
1. Standard curve.
2. 1(N) NaOH (40 g NaOH in 1000 ml distilled water.).
3. I2 solution 0.1 (N1).
Grind I2 and KI in a mortar and pestle. Add little water in it and remove the solution. Add, little more water and grind. Repeat the process till all the I2 and KI are brought into solution. Make up the volume to 1000 ml and keep in brown bottle. Take 10 ml of this and make it upto 100 ml = N/58 I2.
4. Sodium thiosulphate 0.1N-24.3g. Na2S203 in 1000 ml distilled water + 1 ml chloroform as preservative.
5. Starch solution: lg starch powder—boil in 100 ml water and keep it at low temperature.
6. Methyl orange 0.04% in 20% ethanol.
7. Buffers for penicillin.
P04 buffer pH 6.0
KH2P04-5.81g + lN NaOH-6.7ml made upto 1000 ml. Check pH on pH meter.
Acetate buffer pH 4.5
Na acetate-5.44 g + Glacial acetic acid – 2.4 ml made upto 1000 ml.
8. 1.2N HCl-100 ml conc. HCl 12(N) made upto 1000 ml.
9. Titration apparatus.
10. Test tubes.
1. For standard curve, dissolve the contents of Penicillin G from a vial (5,00,000 units) in 50 ml phosphate buffer (pH 6.0). One ml of this solution has 100,00 units of penicillin.
2. Prepare dilutions of penicillin G. as follows:
1) Total units in the vial=500,000 units
2) Solution of this in 50 ml PO4 buffer = 500,000/50 = 10,000 units/ml
3) 1 ml of dilution (2) + 9ml PO4 buffer 10000/10 = 1000 units/ml (stock)
4) 1 ml of dilution (3) + 99 ml PO4 buffer = 1000/100 = 10 units/ml (working stock)
5) 0.5 ml of dilution (4) + 9.5 ml PO4 buffer = 5/10 = 0.5 units/ml (working stock)
6) 0.7 ml of dilution (4) + 9.3 ml PO4 buffer = 7/10 = 0.7 units/ml
7) 1.0 ml of dilution (4) + 9.0 ml PO4 buffer = 10/10 = 1.0 units/ml
8) 1.2 ml of dilution (4) + 8.8 ml PO4 buffer = 12/10= 1.2 units/ml
9) 1.5 ml of dilution (4) + 8.5 ml PO4 buffer = 15/10 =1-5 units/ml
10) 2.0 ml of dilution (4) + 8.0 ml PO4 buffer = 20/10 = 2.0 units/ml
BS = Blank standard.
ES = Experimental standard.
BT = Blank test (fermented broth).
ET = Experimental test (fermented broth).
Label these dilutions and use for chemical and biological assay.
1. Test organism-Bacillus subtilis.
2. Fermented broth.
3. Standard graph of penicillin with diameter of zone of inhibition against units of penicillin.
4. Petri plates.
5. Bioassay medium butts.
6. Cork borer.
7. Glass marking pencil.
9. Base agar (any fungal medium).
1. Pour base agar into sterile Petri plates and allow it to solidify.
2. Inoculate B. subtilis into sterilised cooled (to 45°C), 10 ml medium in tubes, mix well by rolling it gently between the palms.
3. Pour it to the surface of the base agar, allow it to solidify.
4. With a sterile cork borer punch holes in the agar medium (3—4) in such a way that the zones of inhibition should not overlap.
5. Add different dilutions of standard penicillin along with fermented broth into the wells using a micropipette and label respective concentrations.
6. Keep the plates in a refrigerator (4°C) for an hour to allow even diffusion of the test solutions into the surrounding agar medium.
7. Incubate at 37°C for 24-48 hours and measure the diameter of the zones of inhibition.
8. Prepare a graph with concentration of penicillin against the diameter of the zone of inhibition. From this find out the concentration of penicillin in the fermented broth.