Simple Autoclave Diagram

Production of Ethanol by fermentation using Saccharomyces cerevisiae

Aim: To produce ethanol from grape juice by fermentation using Saccharomyces cerevisiae and confirm ethanol formation by chemical test.

Principle: Fermentation is one of the oldest biochemical processes humans learned to use. Yeast converts sugars into ethanol and carbon dioxide when oxygen is limited. Grapes naturally contain glucose and fructose, both easy sugars for yeast metabolism. When yeast cells are added to grape juice, they start breaking down these sugars through the glycolysis and alcoholic fermentation pathways. The final products are ethanol and CO₂ gas. Bubbles appear. The smell changes. Inside the yeast cell, pyruvate formed from glucose is converted to ethanol with the help of enzymes like pyruvate decarboxylase and alcohol dehydrogenase. A temperature of around 28 to 30°C works well. If conditions are right, fermentation begins within 24 hours only.

Requirements:

• Fresh grapes (approximately 500 g)
• Pure culture of Saccharomyces cerevisiae
• Distilled water
• Conical flask (500 ml or 1000 ml)
• Cotton plugs
• Muslin cloth or filter cloth
• Mortar and pestle or blender
• Incubator set at 28 to 30°C
• Fermentation lock or a simple rubber tube arrangement
• Test tubes
• Spirit lamp
• Potassium dichromate reagent
• Concentrated sulfuric acid

Yeast Fermentation Medium (for 1000 ml):

• Glucose: 50 g/L
• Yeast extract: 3 g/L
• Peptone : 5 g/
• Distilled water: up to 1000 ml

Procedure:

1. Take about 500 g of ripe grapes. Wash them gently with distilled water.
2. Crush the grapes using a mortar and pestle or a blender to extract juice.
3. Filter the crushed mixture through muslin cloth to obtain clear grape juice.
4. Transfer around 400 ml of grape juice into a 500 ml conical flask.
5. Add 50 ml of yeast fermentation medium to provide nutrients.
6. Sterilise the mixture by heating at 70°C water bath for about 10 minutes. Do not boil. High heat will destroy natural sugars.
7. Allow the flask to cool to room temperature.
8. Inoculate the flask with 5 mL actively growing culture of Saccharomyces cerevisiae.
9. Plug the flask with cotton and attach a fermentation lock or rubber tube to allow CO₂ to escape.
10. Incubate the flask at 28 to 30°C for 3 to 5 days.
11. Observe the flask daily for bubble formation and smell changes.
12. After the fermentation period, 
13. Filter the fermented broth through muslin cloth to remove yeast biomass and grape solids. Collect the clear filtrate.
14. Transfer the filtrate to a round-bottom flask and set up a simple distillation apparatus.
15. Heat slowly. Ethanol distils between 78 and 82 °C. Collect the distillate in a receiver flask kept in ice.
16. Collect the first 20 to 30 ml fraction of ethanol-enriched distillate.
17. Take 1 ml of distillate in a clean test tube.
18. Add 1 ml of potassium dichromate reagent in a test tube (5 g K2Cr2O7 dissolved in 4 N H2SO4).
19. Warm gently in a water bath at 60°C for 2 to 3 minutes.
20. Observe colour change. Orange-yellow dichromate turning green confirms ethanol is present, as Cr6+ gets reduced to Cr3+.

Observation: During fermentation, bubbles of carbon dioxide appear in the flask, and the liquid develops a mild alcoholic smell. When the potassium dichromate test is performed, the orange colour slowly turns green, showing ethanol oxidation.

Result: Ethanol was successfully produced from grape sugars by yeast fermentation and confirmed by the potassium dichromate chemical test.

Conclusion: Grapes contain natural fermentable sugars which yeast easily convert into ethanol under anaerobic conditions. In about 4 to 5 days, fermentation becomes clearly visible through gas bubbles and an alcoholic smell.