Preparation of Penicillin
IV. MATERIALS AND METHODS
Process Description
The classical penicillin production process is an aerobic fermentation in fed batch fermenters made with some Penicillium strains, usually Penicillium chrysogenum (Nielson, 1997) that transforms substrates rich in carbohydrates into penicillin. As with other antibiotic production processes (Cunha et al., 2002), the penicillin process operated at antibiotics involves four stages. The incubation of the culture strain provides the seed that grows in seed fermenters until a stage of maturity is reached. Then, the seed is transferred to a final-stage fermenter. These fermenters are operated in fed-batch mode under standard conditions in order to optimize the synthesis of penicillin. After that, the product is withdrawn by solvent extraction in the downstream. Penicillium chrysogenum ATCC 16520 (Obtained from American Type Culture Collection) is used for the production of Penicillin.
Preparation of Different Salts of Phenyl acetic acid.
Sodium phenyl acetate
For 100 ml preparation, few grams of PAA (of known percentage) are dissolved in certain volume of De-Mineralized water. The pH of the solution is maintained between 6.6-7 by adding NaOH in drops. Molecular Weight-159.
Potassium phenyl acetate
For 100 ml preparation, few grams of PAA (of known percentage) are dissolved in certain volume of De-Mineralized water. The pH of the solution is maintained between 6.6-7 by adding KOH in drops. Molecular Weight-175.
Ammonium phenyl acetate
For 100 ml preparation, few grams of PAA (of known percentage) are added in certain volume of De-Mineralized water. The pH of the solution is maintained between 6.6-7 by adding NH4OH in drops. Molecular Weight-154.
Industrial penicillin production from Penicillin chrysogenum involves two stages:
a. Lab stage and
b. Plant stage.
The plant stage was further divided into Pilot plant stage and Production plant stage. This project work was carried out in both Lab and Pilot plant stage.
LAB SCALE
The production of Penicillin is carried out in two consecutive cycles that is G1, G2 cycle
G1 Cycle
Colony selection
Ampoules containing strain are broken and serially diluted in De-Mineralized water. 0.1 ml sample from each dilution is taken and incubated in lactose corn steep agar (LCSA) plate, at 250 c with relative humidity of 60±5% that is optimum for the growth of fungi.
After 13 days, green colored colony of 5 – 10 mm size that will be raised and containing 3 - 4 craters will be formed and one such type of colony is picked out naturally which is considered the best. The colony is macerated to 2 ml and taken in 10 tubes and it is inoculated to 100 gm of rice flask.
Rice flask preparation
Good quality, partially boiled, unbroken Ponni rice with slendor, long grains is preferable for good and consistent results.
Required quality of rice is weighed and taken in a flask. In 1:3ratio DM (De Mineralized) water is added and the rice is soaked for 10-30 minutes to attain a moisture level at 21-24%. The wet rice is then carefully transferred into a stainless steel sieve based and kept for 10-15 minutes, till the water gets drained. The rice is then shade dried on a coarse filter paper for 10-30 minutes. The moisture content in the rice is calculated as follows
Moisture content % = Weight of wet rice –weight of dry rice (100
Weight of wet rice
= X%
The optimum moisture content should be 22-23%
Weigh and distribute 100 gm of rice into 1-liter wide mouth Erlenmeyer flask (Figure: 02). Plug the flask with loose flexible bacteriological plug made of non-absorbent cotton. Sterilize the rice flask at 121ºc ±1ºc for 25 min.
Moisture loss during sterilization
Weigh each flask separately along with rice and plug before sterilization. Weigh each flask after sterilization to calculate and compensate the water...
Process Description
The classical penicillin production process is an aerobic fermentation in fed batch fermenters made with some Penicillium strains, usually Penicillium chrysogenum (Nielson, 1997) that transforms substrates rich in carbohydrates into penicillin. As with other antibiotic production processes (Cunha et al., 2002), the penicillin process operated at antibiotics involves four stages. The incubation of the culture strain provides the seed that grows in seed fermenters until a stage of maturity is reached. Then, the seed is transferred to a final-stage fermenter. These fermenters are operated in fed-batch mode under standard conditions in order to optimize the synthesis of penicillin. After that, the product is withdrawn by solvent extraction in the downstream. Penicillium chrysogenum ATCC 16520 (Obtained from American Type Culture Collection) is used for the production of Penicillin.
Preparation of Different Salts of Phenyl acetic acid.
Sodium phenyl acetate
For 100 ml preparation, few grams of PAA (of known percentage) are dissolved in certain volume of De-Mineralized water. The pH of the solution is maintained between 6.6-7 by adding NaOH in drops. Molecular Weight-159.
Potassium phenyl acetate
For 100 ml preparation, few grams of PAA (of known percentage) are dissolved in certain volume of De-Mineralized water. The pH of the solution is maintained between 6.6-7 by adding KOH in drops. Molecular Weight-175.
Ammonium phenyl acetate
For 100 ml preparation, few grams of PAA (of known percentage) are added in certain volume of De-Mineralized water. The pH of the solution is maintained between 6.6-7 by adding NH4OH in drops. Molecular Weight-154.
Industrial penicillin production from Penicillin chrysogenum involves two stages:
a. Lab stage and
b. Plant stage.
The plant stage was further divided into Pilot plant stage and Production plant stage. This project work was carried out in both Lab and Pilot plant stage.
LAB SCALE
The production of Penicillin is carried out in two consecutive cycles that is G1, G2 cycle
G1 Cycle
Colony selection
Ampoules containing strain are broken and serially diluted in De-Mineralized water. 0.1 ml sample from each dilution is taken and incubated in lactose corn steep agar (LCSA) plate, at 250 c with relative humidity of 60±5% that is optimum for the growth of fungi.
After 13 days, green colored colony of 5 – 10 mm size that will be raised and containing 3 - 4 craters will be formed and one such type of colony is picked out naturally which is considered the best. The colony is macerated to 2 ml and taken in 10 tubes and it is inoculated to 100 gm of rice flask.
Rice flask preparation
Good quality, partially boiled, unbroken Ponni rice with slendor, long grains is preferable for good and consistent results.
Required quality of rice is weighed and taken in a flask. In 1:3ratio DM (De Mineralized) water is added and the rice is soaked for 10-30 minutes to attain a moisture level at 21-24%. The wet rice is then carefully transferred into a stainless steel sieve based and kept for 10-15 minutes, till the water gets drained. The rice is then shade dried on a coarse filter paper for 10-30 minutes. The moisture content in the rice is calculated as follows
Moisture content % = Weight of wet rice –weight of dry rice (100
Weight of wet rice
= X%
The optimum moisture content should be 22-23%
Weigh and distribute 100 gm of rice into 1-liter wide mouth Erlenmeyer flask (Figure: 02). Plug the flask with loose flexible bacteriological plug made of non-absorbent cotton. Sterilize the rice flask at 121ºc ±1ºc for 25 min.
Moisture loss during sterilization
Weigh each flask separately along with rice and plug before sterilization. Weigh each flask after sterilization to calculate and compensate the water...
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