BIOLOGY Volume 1 - A Guide to General Biology - 2004

9. UTILIZATION OF ENERGY

9.3. Cellular Respiration

9.3.9. Industrial Applications of Fermentation Processes

In various Fermentation industries, which play a highly significant role in the modern economy, fermentation processes are carried out by microorganisms. For this purpose, they are cultivated in special containers known as fermenters or bioreactors. We will discuss these processes in Chapter 12.

Practical 9.1. Investigation of The oxidation of an intermediate in the Krebs cycle

The most efficient way to extract energy from a substrate and store it for subsequent use is to break down the process into a series of simpler, reversible, enzyme-catalysed reactions. One such intermediate reaction is the oxidation of succinic acid to fumaric acid via the removal of hydrogen.

Certain substances are capable of accepting the removed hydrogen atoms while undergoing a colour change. One such substance is 2,6-dichlorophenolindophenol (DCPIP): its oxidised form is blue, while its reduced form is colourless.

If the oxidised form of DCPIP is decolorised when mixed with a tissue extract, it can be hypothesised that this is due to the acceptance of hydrogen atoms from succinic acid. Let us add succinic acid to the mixture. If The rate of decolorization now increases, this will reinforce our hypothesis that DCPIP acts as an acceptor for the hydrogen atoms removed from succinic acid.

Since most reactions in Living organisms are enzyme-catalysed, it is evident that oxidation will only occur if the appropriate enzyme is present in the medium. The oxidation of succinic acid is catalysed by the enzyme succinate dehydrogenase, and further investigations allow us to detect the presence of this enzyme. In our experiment, the source of the enzyme will be a suspension of Cell/35.html">Mitochondria extracted from germinating mung bean seeds (Phaseolus aureus). It is essential to isolate the mitochondria as quickly as possible, since METABOLISM continues for only a very short time after Cell Disruption.

The experiment is divided into two parts: 1) Isolation of the required enzyme, and 2) its use in the oxidation of succinic acid. DCPIP serves as an indicator to determine whether the reaction is taking place.

Everything required for the first part of the experiment (i.e. the preparation of the enzyme extract) should ideally be kept in a refrigerator for at least an hour before starting the work.

Materials and Equipment

4 centrifuge tubes (15 ml capacity)

2 Glass stirring rods

2 10 ml graduated pipettes

2 1 l beakers (preferably plastic)

Ice

Salt

Mung bean seeds

Test tubes with a rack

1 1 ml graduated pipette

Timer

Solutions (instructions for preparing the solutions are given after the Description of the experiment)

Buffered sucrose solution

Buffered sucrose solution + succinic acid solution

0.1% DCPIP solution (prepared in buffered sucrose solution)

Distilled Water

Procedure

1. Germinate mung bean seeds (24 pcs) by placing them on moist cotton wool and leaving them in the dark for 3–4 days.

2. Prepare an ice bath: place ice in a 1 L plastic beaker and add salt to lower the Temperature.

3. Place the flask with the buffered sucrose solution and two centrifuge tubes into the ice bath.

4. Remove the seed coats and roots from 12 germinated seeds.

5. Place 6 seeds into each centrifuge tube.

6. Add 1 ml of the buffered sucrose solution (free of succinic acid) to each tube.

7. Thoroughly grind the seeds using a pre-chilled glass rod; the tubes must remain in the ice beaker at all times.

8. Add another 10 ml of buffered sucrose solution to each centrifuge tube.

9. Place the centrifuge tubes in the centrifuge (opposite each other) and spin at maximum speed for 3 min.

10. Return the centrifuge tubes to the ice beaker.

11. Pipette 15 ml of distilled water into one of the laboratory tubes and mark the meniscus level.

12. Pour out the distilled water and fill the tube to the mark with the supernatant from the centrifuge tubes.

13. This step must be performed very quickly: add 0.5 ml of DCPIP solution to the same laboratory tube and mix the contents by covering the tube with your thumb and inverting it.

14. Start a stopwatch as you mix the solutions.

15. Note the color of the solution after 20 min.

16. Repeat the entire experiment, this time using the buffered sucrose solution containing succinic acid.

The course of the experiment can also be monitored using a colorimeter.

1. Insert the red filter, turn on the colorimeter, and allow it to warm up for 5 min.

2. Add 0.5 ml of DCPIP solution to 15 ml of the supernatant, as described above.

3. Mix the solutions and start the timer.

4. Place the tube into the colorimeter and set the instrument to 0% transmittance.

5. Take colorimeter readings after 1, 2, 5, 10, and 20 min.

6. Repeat the experiment using a buffered sucrose solution containing succinic acid.

7. Plot a graph of transmittance (y-axis) versus time.

8. Formulate Conclusions based on the obtained results.

Preparation of Solutions

Buffered Sucrose Solution (100 mL)

Disodium hydrogen phosphate (Na₂HPО₄) 0.76 g

Potassium dihydrogen phosphate (КН₂РО₄) 0.18 g

Sucrose 13.60 g

Magnesium sulfate 0.10 g

Buffered Sucrose Solution + Succinic Acid (100 mL)

Buffered sucrose solution, prepared as described above

Succinic acid 1.36 g

Sodium bicarbonate 1.68 g

It is best to prepare enough buffered sucrose solution at the start for both PARTS OF THE experiment (using distilled water). Then, divide the solution into two equal parts and add succinic acid and sodium bicarbonate (at the appropriate concentration) to one of them.

When succinic acid and sodium bicarbonate are added to the buffered sucrose solution, the liquid will effervesce. The solution should be thoroughly swirled to release all carbon dioxide, as its presence may interfere with the experimental results.

DCPIP Solution

Dissolve 0.1 g of dichlorophenolindophenol in 10 mL of buffered sucrose solution (without succinic acid for both parts of the experiment). Since this substance dissolves rather poorly, the suspension should be filtered after thorough mixing.