Strain collection

A strain of Penicillium chrysogenum has been preserved at the Doctoral School at the Lebanese University in Tripoli.

Preparation of the liquid medium (Broth):

 0.5g peptone, 0.1g tryptone, 0.1g glucose, 0.1g yeast extract, 0.25g NaCl, 50ml distilled water were mixed and heated in a beaker. The pH was measured and adjusted to 7 by adding a few ml of H2SO4.

The medium was then placed in tubes, autoclaved for 1h and then cooled. 

An inoculum is taken from the strain Penicillium chrysogenum using a sterile loop and then transferred and mixed in the liquid medium. The tube is incubated at room temperature for 4 days.

Constitutes of penicillin G fermentation broth:

1. Prepare the nutrient media in 100ml distilled water.
2. Measure the PH and then adjust it to 7 by adding a few ml of H2SO4
3. Heat and mix for about 15min 
4. Autoclave the medium for 1h then cool it down for 30min
5. Add the inoculum to the medium
6. Incubate the mix at 28˚C for 10 days with shaking (120 RPM)

Filtration and purification:

• After 10 days of medium incubation with shaking we proceed to Filtration using a filter whose pores have a diameter of 0.2 µm. The microorganisms are too large and are therefore retained by the filter.
•  After filtration, the pH of the filtrate is adjusted to 1.5-2.2 by adding H2SO4 (penicillin ionized in the solution) in order to stop any reaction that could degrade penicillin.
•  The filtrate was then kept cold (5-10˚C).
• Add the butyl acetate (1V solvent/2V filtrate) which serves as solvent for separating the organic phase and the aqueous phase(F1) using a separating funnel.
• Add 2% (W/V) phosphate buffer(PB) to the organic phase(F2).
• Adjust the PH to 7.5 by adding NaOH.
• Add 2% (W/V) NaHCO3 in order to carry out the crystallization. 
• Cool the solution (F3) at 4˚C about 7 days .
• Filtrate and harvest penicillinG sodium salt.

Results:

Trial 1:

We obtain clear white powder of crude PenG that will be used later for quantification so the crystallization has succeeded .

Note that we realize a sensitivity test for:

_F1(Aqueous phase after the  organic solvent extraction)

_F2(Organic phase After PB extraction)

_F3(the final solution obtained after organic solvent extraction)

The results show : 

A clear zone of inhibition in F1 which means that : 

• There is PenG In F1 so we can say that the experiment has succeeded and the PAA produced in the lab is effective.

• Not all the PenG was extracted into the used organic solvent(there is a percentage of PenG loss).

A clear zone of inhibition in F3 which means that : 

• There is crude PenG in the final solution so the solvent used is effective.
• The purification has succeeded with a percentage of PenG loss in aqueous phaseF1.

No zone of inhibition in F2 which means that there is no loss of produced PenG in F2.

Trial 2:

according to the sensitivity test :

_F1(Aqueous phase after the  organic solvent extraction)

_F2(Organic phase After PB extraction)

_F3(the final solution obtained after organic solvent extraction)

Penicillin Quantification (May 2023)

The procedure for quantification is the same as that mentioned before using disc diffusion method leading to a calibration curve helping to calculate the concentration of our crude PenG obtained from F1 and F3 solution:

Quantification of Crude PenG obtained from F1 crystallization:

Table1 : Measurements of diameters of bacterial growth inhibition zones for different concentrations of standard dilute commercial penicillinG and F1.

Graph 1: Graph showing the variation of penicillin concentration (LogC) as function of the diameter of the inhibition zone

According to the calibration curve y=1.15x+(-2.43)

So the concentration of PenG in F1 is 0.04mg/ml

Quantification for Crude PenG obtained from F3 crystallization

Table 2 : Measurements of diameters of bacterial growth inhibition zones for different concentrations of standard dilute commercial penicillinG and F3 solution.

Graph 2 : Graph showing the variation of penicillin concentration (LogC) as function of the diameter of the inhibition zone.

According to the calibration curve y=0.623x+(-0.847)

So the concentration of PenG in F3 is 0.388mg/ml ~ 0.4 mg/ml.

 Table showing every step with its duration and working date

Trial 3:

Same protocol reused in this trial (except: adding PAA : Phenylacetic acid every 24h at 30˚C for the first 48h then decrease the temprature to 25˚C for the rest of the fermentation).

PS : due to disfunction of the incubator we couldn't maintain the temprature ( between 25-30˚C) and solubility of the PAA

RESULTS:

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                                                                                                                       trial 4

Same protocol reused in this trial (except:aeorbie respiration and medium: without glucose , lactose 3g ).

                                                                                                                    results 

Graph showing the variation of penicillin concentration (LogC) as function of the diameter of the inhibition zone.