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Bioconversion Practical

The document outlines a practical procedure for producing bioethanol from glucose using the microorganism Saccharomyces cerevisiae. It details the requirements, including materials and apparatus, as well as the step-by-step process for inoculum preparation, fermentation, and ethanol estimation using the Potassium Dichromate Method. Additionally, it emphasizes performance indicators for evaluating bioconversion efficiency.

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Rahul Jaiswal
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6 views2 pages

Bioconversion Practical

The document outlines a practical procedure for producing bioethanol from glucose using the microorganism Saccharomyces cerevisiae. It details the requirements, including materials and apparatus, as well as the step-by-step process for inoculum preparation, fermentation, and ethanol estimation using the Potassium Dichromate Method. Additionally, it emphasizes performance indicators for evaluating bioconversion efficiency.

Uploaded by

Rahul Jaiswal
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

Bioconversion Practical: Production of Bioethanol from Glucose

1. Requirements

 Microorganism: Saccharomyces cerevisiae (Active Dry Yeast or slant culture).

 Substrate/Media: Yeast Extract Peptone Dextrose (YEPD) broth or a simplified medium

 Apparatus: 500 mL Erlenmeyer flasks, Cotton plugs, Incubator Shaker, Centrifuge,


Distillation setup.

 Reagents for Estimation: Potassium Dichromate, Concentrated, Standard Ethanol


solutions.

2. Procedure

1. Inoculum Preparation: Culture the yeast in 50 mL of YEPD broth for 18–24 hours at 30°C
in a shaker to reach the log phase.

2. Media Preparation: Prepare 250 mL of fermentation medium in a 500 mL flask. Sterilize


via autoclaving at 121°C (15 psi) for 15 minutes.

3. Inoculation: Aseptically transfer 5–10% (v/v) of the yeast inoculum into the production
medium.

4. Fermentation: * Initially, shake the flask for 6–12 hours to allow for aerobic cell growth
(biomass buildup).

o Seal the flask with a rubber stopper equipped with a gas outlet (to maintain
anaerobic conditions) and incubate statically at 30°C for 48–72 hours.

5. Harvesting: Centrifuge the fermented broth at 5,000 rpm for 10 minutes to remove cell
biomass. The supernatant (the "wash") contains the ethanol.

3. Estimation of Ethanol Produced (Dichromate Oxidation Method)

In a lab setting, ethanol is typically estimated using the Potassium Dichromate Method, which
relies on the oxidation of alcohol to acetic acid.

Principle

Ethanol is oxidized by an acidic solution of potassium dichromate. During this process, the
orange Cr (VI) is reduced to the green Cr (III). The intensity of the green color is proportional to
the ethanol concentration.

Protocol for Estimation


1. Steam Distillation: Distill a known volume of the fermented supernatant to separate
ethanol from interfering sugars and solids.

2. Standard Curve: Prepare a series of ethanol standards (e.g., 2% to 10%).

3. Reaction: Add 1 mL of the sample (distillate) to 5 mL of Acid Dichromate Reagent

4. Heating: Heat the mixture in a water bath at 60°C for 15 minutes.

5. Spectrophotometry: Measure the absorbance at 600 nm (or 540 nm depending on the


protocol) using a blank.

6. Calculation: Determine the ethanol concentration of the unknown sample by comparing


its absorbance against the standard curve.

4. Performance Indicators

To evaluate the efficiency of your bioconversion, calculate the following:

 Ethanol Yield (Bioconversion yield):

(Theoretical maximum is 0.51 g/g).

 Fermentation Efficiency:

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