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Penicillin Production

1-     Experiment 2: Penicillin production

 

 

  • The same steps already mentioned in a last report are followed(10).

                                                                                                     

Penicillin purification:

To purify the produced penicillin we had pursued different three protocol.

The first (P1) without using charcoal (just: filtration-ethyl acetate-centrifugation- sodium bicarbonate)

The second by using charcoal and V/V from the ethyl acetate

The third by using charcoal and V[14:2] from the ethyl acetate( for each 14 ml of the filtrate use 2 ml of ethyl acetate)

 

Then we have tested the filtrate when we had obtain a lot of contaminant.

For this reason the protocol was retried with the autoclaving of the liquid medium before adding the penicillium colony. Then the quantification will be tried.

2- Corrected protocol for Penicillin production :

     2.1     Purification of the isolate:

We took a colony from the cultivated dish and we recultivate into another tryptone medium to obtain a pure strain that will be used below.

2.2     Preparation of liquid medium

  • We weigh 2 g of glucose powder, 0.8 g of lactose, 0.8g of peptone, 1g yeast extract, 0.15g of MgCl2, 0.15g of KCl, and 1g of KH2PO4, 0.2g CaCO3, 0.1g corn oil
  • It is important to add penicillin precursor which called phenyl acetate at proportion 0.5%
  • We add 100ml of distilled water
  • We put the mixture in an erlenmeyer flasks
  • We heat them with mixing for 15min by using a magnetic hot plate stirrer
  • We autoclave the liquid medium, then let it cool down for about 30 min
  • We inoculate it with the cultivated petri dish already prepared (about two or three colony)
  • We incubate them at room temperature for 7 to 10 days with shaking

2.3   Purification:

  • Harvest liquid medium which contain penicillin by filtration
  • Chill to 5-10oC (because penicillin is highly reactive and destroyed by alkali and enzyme)
  • Acidify filtrate to PH 2.0-2.5 with H2SO4 (to convert penicillin to its anionic form)
  • Extract penicillin from aqueous filtrate by adding ethyl acetate (at this very low ph as soon as possible)
  • Discard aqueous fraction
  • Allow the organic fraction to pass through charcoal to remove impurities and extract penicillin (this step is not important)
  • Extract penicillin from ethyl acetate by adding 2% aqueous phosphate buffer ( here the PB can be replaced by distilled wter) at PH 7.5
  • Acidify the aqueous fraction to PH 2-2.5 with mineral acid and re-extract penicillin into fresh ethyl acetate
  • Add sodium bicarbonate to the solvent to christallise the antibiotic as sodium salt
  • Recover crystal in filter centrifuge or by filtration.

140122-Pilot_Plant_Penicillin_Production.docx

110422-MEGBI-App_Report2022

 

140322-Bioreactor and antibiotic production pilot plant.docx