Presentation one: Synthesis of PHB Evreux meeting

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) Evreux meeting

First two solutions are introduced in the fermenter. Solution A Solution B 2 mL of culture 200 mL 20 µL Evreux meeting

Put the fermenter in the water bath Evreux meeting

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 Evreux meeting

The fermenter is stirred and heated at 37°C for 45 hours. Evreux meeting

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 10000 rpm) Evreux meeting

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 Evreux meeting

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 Evreux meeting

After centrifugation: Keep pellets in the tubes (discard the supernatant) Washed with 5 mL of acetone. Centrifugate the mixture for 5 minutes 5 mL Evreux meeting

After centrifugation: Keep pellets in the tubes (discard the supernatant) Washed with 5 mL of methanol. Centrifugate the mixture for 5 minutes 5 mL Evreux meeting

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 Evreux meeting

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. Evreux meeting

When the chloroform is entirely evaporated: You can put off the plastic film: Evreux meeting

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) Evreux meeting

First two solutions are introduced in the fermenter. Solution A Solution B 2 mL of culture 200 mL 20 µL Evreux meeting

Put the fermenter in the water bath Evreux meeting

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 Evreux meeting

The fermenter is stirred and heated at 37°C for 45 hours. Evreux meeting

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 10000 rpm) Evreux meeting

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 Evreux meeting

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 Evreux meeting

After centrifugation: Keep pellets in the tubes (discard the supernatant) Washed with 5 mL of acetone. Centrifugate the mixture for 5 minutes 5 mL Evreux meeting

After centrifugation: Keep pellets in the tubes (discard the supernatant) Washed with 5 mL of methanol. Centrifugate the mixture for 5 minutes 5 mL Evreux meeting

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 Evreux meeting

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. Evreux meeting

When the chloroform is entirely evaporated: You can put off the plastic film: Evreux meeting