1. Presentation one: Synthesis of PHB
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2. First two solutions are introduced in the fermentor.
Solution A
For 1 liter of solution (grams)
Urea (1,0),
Yeast extract (0,16),
KH2PO4 (1,52),
Na2HPO4 (4,0),
MgSO4∙7H2O (0,52),
CaCl2 (0,02),
Glucose (40)
Solution B
For 1 liter of solution (grams)
ZnSO4∙7H2O (0,13),
FeSO4∙7H2O (0,02),
(NH4)6Mo7O24. 4H2O (0,06)
H3BO3 (0,06)
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3. First two solutions are introduced in the fermenter.
Solution A
Solution B
2 mL of culture
200 mL
20 µL
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4. Put the fermenter in the water bath
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5. Equipment of the water bath
Rotating speed
(adjusted at minimum)
Overhead stirrer
Temperature controller
Low 36°C
High 38°C
Stirrer
Insert the stirrer guide in the
fermenter
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6. The fermenter is stirred and heated at 37°C for 45 hours.
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7. Take the fermenter off the water bath.
After 45 hours:
Take the fermenter off the water bath.
Let it decant 15 minutes.
Extract gently 150 mL of the supernatant with a pipette.
The rest is centrifuged (15 min at rpm)
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8. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Add to pellets 10 mL of sodium hypochlorite.
Incubate the mixture at 60 °C for 45 minutes.
10 mL
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9. The mixture is centrifuged at 5000 rpm for 10 min .
After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Washed with 5 mL of distilled water.
Centrifugate the mixture for 5 minutes
5 mL
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10. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Washed with 5 mL of acetone.
Centrifugate the mixture for 5 minutes
5 mL
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11. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Washed with 5 mL of methanol.
Centrifugate the mixture for 5 minutes
5 mL
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12. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Transfer pellets in a glass petri dish.
Under a filtered fume hood, measure 5 mL of chloroform.
Poor it in the glass petri dish.
5 mL
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13. Stir the mixture with a rotating movement.
Put the glass petri dish below an upsidedown funnel, connected to a venturi vacuum.
Stir the mixture with a rotating movement.
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14. When the chloroform is entirely evaporated:
You can put off the plastic film:
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15. First two solutions are introduced in the fermentor.
Solution A
For 1 liter of solution (grams)
Urea (1,0),
Yeast extract (0,16),
KH2PO4 (1,52),
Na2HPO4 (4,0),
MgSO4∙7H2O (0,52),
CaCl2 (0,02),
Glucose (40)
Solution B
For 1 liter of solution (grams)
ZnSO4∙7H2O (0,13),
FeSO4∙7H2O (0,02),
(NH4)6Mo7O24. 4H2O (0,06)
H3BO3 (0,06)
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16. First two solutions are introduced in the fermenter.
Solution A
Solution B
2 mL of culture
200 mL
20 µL
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17. Put the fermenter in the water bath
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18. Equipment of the water bath
Rotating speed
(adjusted at minimum)
Overhead stirrer
Temperature controller
Low 36°C
High 38°C
Stirrer
Insert the stirrer guide in the
fermenter
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19. The fermenter is stirred and heated at 37°C for 45 hours.
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20. Take the fermenter off the water bath.
After 45 hours:
Take the fermenter off the water bath.
Let it decant 15 minutes.
Extract gently 150 mL of the supernatant with a pipette.
The rest is centrifuged (15 min at rpm)
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21. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Add to pellets 10 mL of sodium hypochlorite.
Incubate the mixture at 60 °C for 45 minutes.
10 mL
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22. The mixture is centrifuged at 5000 rpm for 10 min .
After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Washed with 5 mL of distilled water.
Centrifugate the mixture for 5 minutes
5 mL
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23. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Washed with 5 mL of acetone.
Centrifugate the mixture for 5 minutes
5 mL
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24. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Washed with 5 mL of methanol.
Centrifugate the mixture for 5 minutes
5 mL
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25. After centrifugation:
Keep pellets in the tubes (discard the supernatant)
Transfer pellets in a glass petri dish.
Under a filtered fume hood, measure 5 mL of chloroform.
Poor it in the glass petri dish.
5 mL
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26. Stir the mixture with a rotating movement.
Put the glass petri dish below an upside down funnel, connected to a venturi vacuum.
Stir the mixture with a rotating movement.
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27. When the chloroform is entirely evaporated:
You can put off the plastic film:
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