CH 150: Introduction to Biochemistry

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Fermentation

Fermentation is the biochemical process by which a carbohydrate, usually glucose, is reduced to ethanol by yeast.  The carbohydrate enters the glycolytic pathway at distinct points, depending on its identity, and is oxidized to pyruvate.  Under anaerobic conditions, pyruvate is metabolized to ethanol by way of acetaldehyde, in order to regenerate NAD⁺ utilized in glycolysis.  If NAD⁺ is not regenerated, glycolysis stops and ATP production ceases.

                                    O       pyruvate decarboxylase                                                                  H₃C-C-C-O^- ---------------------------------->    CO₂ +    H₃C-C-H

                        Pyruvate                                                               acetaldehyde

               O                                Alcohol dehydrogenase

H₃C-C-H  +   NADH  -------------------------------------®  H₃C-CH₂-OH   + NAD⁺

    Acetaldehyde                                                                    ethanol   

The products of the reactions above are CO₂, ethanol, and NAD⁺. In this experiment, you will measure the generation of CO₂ as an indicator of fermentation using different carbohydrates as substrates. Additional molecules may be added as well to inhibit the process.   The carbohydrate substrates you will investigate are glucose, fructose, sucrose, glycerol, lactose, maltose, and xylose.    Inhibitors are N-ethylmaleimide and sodium phosphate.

 Procedure:

A.     Calibration of Fermentation Tubes:

Using 5 mL increments of water, fill your fermentation tube.  Record the volume of water needed to fill the tube.

 B.    Fermentation:

You will be assigned two of the following carbohydrate solutions to use for your fermentation substrate:

            2 % glucose

            2% fructose

            2% sucrose

            2% glycerol

            2% lactose

            2% maltose

            2 % xylose

            2% glucose + 5 mM N-ethylmaleimide

            2% glucose + 50 mM sodium phosphate

 Mix 15 mL of your assigned carbohydrate solution with 15 mL of 1 % yeast suspension.  You may need to add more of both solutions such that the vertical tube in the fermentation tube is filled.  Save at least 1 mL of the sugar solutions for later use. Place your tube in a warm incubator (about 43 ^oC).  Occasionally examine your tube for the production of a gas.  After 1.5 hrs, remove your tube and estimate the volume of gas produced. Mark the level of the liquid in the glass collection tube with a Sharpie. Decant the fermentation broth into a clean beaker and rinse the fermentation tube with water. Invert the fermentation tube and fill the gas collection tube with water to the line. Decant into a graduated cylinder to measure the volume.

 C.    Analysis of sugar levels (DNSA procedrue)

NOTE: ALWAYS run a standard curve EACH time you perform the DNSA assay, using the same sugar for your standards as in your fermentation / unknowns!!

Standard curve: 

1.      For each sugar your are assigned, prepare 11 tubes containing 0, 2,4,6,8 or 10 mg per mL of the specific sugar. See table 1.

  Table 1: Sugar standard curve samples

2.      Place 0.25 mL of the sugar sample in a test tube using the plastic pipets.

 Unknown samples:

3.   Pipet 1 mL of the fermentation mixture into a labeled microfuge tube. Pellet the cells by spinning the sample for 3 minutes at 1000 rpm.

4. Prepare three replicates of each sample by placing 0.25 mL of the sample (filtrate) in a test tube.

5. Prepare three replicates of the zero-time samples (from sample saved above, before yeast was added) by pipetting 0.125 mL of the sugar solution and 0.125 mL of distilled water into a test tube.

For all samples:

5. Add 8 drops of the DNSA solution to each test tube.

6. Heat in a boiling water bath for 10 minutes.

7. Allow the sample to cool to room temperature.

8. Add 4 mL of water to each tube. 

9. Read and record the absorbance of the sample at 540 nm. 

   standards:

sugar_________________

sugar_________________

sugar samples:

sugar_________________

sugar_________________

fermentation samples:

sugar_________________

sugar_________________

7.      Using Excel, plot mg/mL sugar on the X-axis and absorbance on the y-axis from the "standards" data.   Generate a linear trend line; be sure to print the equation of the line and the regression (r2) value on the plot.

8.      Using unknown sample absorbance data for y-values, calculate the mg/mL of sugar  (x) for each unknown sample.

Results and Discussion:

1.      Draw the structured of all the carbohydrates and inhibitors used in this experiment.

2.      Which carbohydrates to you expect to support glycolysis?  Describe the reactions through which each sugar enters glycolysis.

3.      Which sugars do you expect to not enter glycolysis?  Why?

4.      Tabulate the classes results.  Are these as you would expect?

5.      Why are n-ethylmaleimide and phosphate listed as inhibitors for the process?  What enzyme does each effect?

6.     If galactose were to ferment, would there be any additional cost in terms of energy yield? If so, describe it.

7.      How would glycerol enter glycolysis? Explain why it could not be fermented.