CELLULAR RESPIRATION AND FERMENTATION

OBJECTIVES ➢ Write the generalized equation of aerobic cellular respiration ➢ Write the generalized equation of alcoholic fermentation ➢ Describe a simple test to measure carbon dioxide production ➢ Identify the major steps of aerobic cellular respiration and where these processes occur in eukaryotic

cells ➢ Relate carbon dioxide production to cellular respiration ➢ Relate oxygen consumption to aerobic cellular respiration Introduction All living organisms carry out some form of cellular respiration - the catabolic process that breaks chemical bonds to release energy. In most cells, the molecule that is being catabolized is glucose and some of the energy released is used to form adenosine triphosphate (ATP), the energy molecule of the cell. Organisms use the energy stored in ATP molecules to perform cellular work such as synthesis, reproduction, waste removal, and active transport.

Aerobic cellular respiration (ACR) involves three major steps: glycolysis, the Krebs (citric acid) cycle, and the electron transport chain. In eukaryotic organisms, glycolysis takes place in the cytoplasm

while the Krebs cycle and the electron transport chain take place in the mitochondria. In prokaryotic organisms, ACR takes place in the cytoplasm and at the cell membrane. The overall equation for ACR is

C6H12O6 + 6O2 → 6CO2 + 6H2O + energy

Glucose + oxygen → carbon dioxide + water + energy

In ACR, oxygen serves as the final electron acceptor. If oxygen is not present, eukaryotic cells may switch to a form of anaerobic cellular respiration (fermentation). Glucose is still catabolized but not as thoroughly as it is in ACR. The products produced as a result of fermentation will vary depending on the organism. Yeasts, plants, and some microorganisms have the ability to produce alcohol by fermentation. Animals and some microorganisms have the ability to produce lactic acid by fermentation.

Alcoholic fermentation

C6H12O6 → 2CO2 + 2C2H5OH + energy

Glucose → carbon dioxide + ethanol + energy

Lactic acid fermentation

C6H12O6 → 2C3H6O3 + Energy

Glucose → Lactic acid + Energy

EXPERIMENT 1: FERMENTATION Introduction In this laboratory exercise you will study fermentation. During fermentation, glucose is incompletely broken down, and much energy remains in the organic molecule that results. During fermentation, a small amount of chemical energy is converted into ATP molecules for use by the cell. ATP molecules are absolutely essential to cellular metabolism because they supply energy whenever a cell carries on activities such as active transport, synthesis, muscle contraction, and nerve conduction. Fermentation is referred to as anaerobic respiration since it occurs without the presence of oxygen. During anaerobic conditions high levels of NADH develop, leaving a shortage of NAD. Low levels of NAD slow the rate of glycolysis. Fermentation restores NAD levels while producing alcohol and CO2. During anaerobic respiration, baker’s yeast breaks glucose down into ethyl alcohol and carbon dioxide. This process only yields 2ATP per glucose molecule. Yeasts (unicellular fungi) can use several types of sugars as an energy source. Glucose and fructose are monosaccharides; sucrose is a disaccharide that contains glucose & fructose. Fructose can be converted to glucose, the usual molecule acted on by yeast. Yeast is used by the wine & beer industries to ferment

the carbohydrates in fruits and grains to alcohol. In baking, the carbon dioxide given off from yeast fermentation causes bread to rise. Fermentation in animals and certain microbes produces lactic acid. This form of fermentation helps produce yogurt & many cheeses, as well as such products as sourdough breads, chocolate, and pickled foods. In the following experimental procedure, you will test the ability of yeast to ferment under specific conditions by measuring the amount of carbon dioxide produced (evolved) over time, which will assess the relative rate of respiration. These measurements will be obtained using a respirometer that you will construct in lab. A respirometer is a device that can be used to assess the rate of respiration by measuring the amount of oxygen consumed or carbon dioxide produced. In this experiment the rate of anaerobic cellular respiration (alcoholic fermentation) will be determined by measuring the quantity of carbon dioxide produced by the yeast. Fermentation is a series of reactions all catalyzed by enzymes. As part of the experiment, you will test different substrates, temperature, pH, or other factors to determine if the rate of fermentation changes. Materials ❏ 5 test tubes and test tube rack ❏ 1-ml graduated pipet ❏ Rubber or aquarium tubing ❏ Binder clips ❏ 10cc syringe ❏ Water bath (30oC) ❏ Yeast solution ❏ Glucose solution

❏ Distilled water ❏ Choice of potential inhibitors and different

substrates (sucrose, sugar substitutes, pH buffers…)

❏ Timer

Procedure Label your 5 test tubes 1-5 and add the following to each tube:

Table 1: Contents of Tubes for Fermentation Experiment

TUBE: DI Water YEAST GLUCOSE 1 4 0 3 2 6 1 0 3 3 1 3 4 1 3 3

5* 3

*Tube 5: this is your experimental tube. You will add or replace an activator, inhibitor, or a different sugar source to determine IF or how the rate of fermentation is influenced by the other substance. (Ex: Will the yeast ferment faster with a different sugar source such as honey or a sugar substitute?). Remember to limit the number of items you change in the experiment to obtain accurate results. For example, if you choose to use sucrose instead of glucose, repeat and add the same contents as you did in Tube #4, but replace the glucose with the sucrose.

❖ Complete your “experimental” tube #5. We have several different sugar sources you may use OR you may choose to replace the DI water with a different pH or salt solution.

❖ Assemble the respirometers ➢ You will need to assemble 5 respirometers (one for each test tube) ➢ Attach the rubber tubing to a serological pipet (1 ml). You may need to use a lubricant (such as

glycerol) to attach the tubing to the end of the pipet. ➢ Insert the pipet into the test tube. ➢ Using a 10cc syringe (without a needle), siphon the contents of your tube into the pipet. ➢ Try to draw the liquid to the top of the pipet (near the “0” mark). ➢ Clamp the tubing off with a binder clip.

❖ Place all 5 test tubes / respirometers and test tube rack in a water bath set at 30oC. ❖ Record your initial reading in Table 2 for all 5 pipettes. This will be your “INITIAL” reading. ❖ After 2 minutes, record the reading from the pipette in Table 2. This will be your “ACTUAL”

reading. ❖ Subtract your ACTUAL reading from your INITIAL reading to determine the amount of CO2

evolved during the fermentation process. ❖ Continue taking reading every 2 minutes for 20 minutes. Record your results in Table 2. ❖ To calculate the ACTUAL results, always subtract your reading from the initial (time 0) reading for

accurate results.

Results 1. Complete Table 2. 2. After your results are completed and the actual and initial readings have been calculated, graph your

results to illustrate your data. Illustrate your results using five lines (one line for each tube).

Table 2 Total CO2 Evolved (mL) by Different Concentrations of Yeast. Actual values are the graduated pipette readings. For CO2 evolved values – SUBTRACT the initial reading from the

actual reading. This is the amount of CO2 accumulated over time. Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

Time (min)

Actual (A)

CO2 Evolved

(A-I)

Actual (A)

CO2 Evolved

(A-I)

Actual (A)

CO2 Evolved

(A-I)

Actual (A)

CO2 Evolved

(A-I)

Actual (A)

CO2 Evolved

(A-I) Initial Reading (I)

2

4

6

8

10

12

14

16

18

20

Discussion Questions 1. Explain the effect of different concentrations of yeast on the rate of fermentation.

2. What is (are) your independent variable(s)? This will be graphed on the x-axis

3. What is the dependent variable? This will be your y- axis

4. What is the purpose for each tube? What was (are) the control tube(s)?

5. What is the substrate in this reaction? What is the enzyme in this reaction?

6. What tube had the highest rate of fermentation? Explain why.

7. What tube had the lowest rate of fermentation? Explain why. 8. What are your conclusions based on your experimental tube #5?

EXPERIMENT 2: CELLULAR RESPIRATION Introduction While 2 ATP produced per glucose molecule is clearly better than nothing, it is not nearly enough to meet the energy needs of complex multicellular organisms such as plants and animals. To get the maximum ATP yield from molecules of glucose requires cellular respiration, which can produce up to 36 ATP per glucose molecule. In aerobic organisms, cellular respiration requires O2 (which is why we breathe!), hence the term aerobic respiration. In eukaryotic cells, cellular respiration begins with glycolysis in the cytoplasm and continues in the mitochondria as outlined below:

The Citric Acid Cycle – This is a biochemical pathway involved in breaking pyruvate down to CO2. In the process, energy rich electrons in hydrogen atoms are transferred to NAD+ and FAD producing NADH and FADH2. In addition, 2 ATP per original glucose are also produced.

Oxidative Phosphorylation – This is the process by which the remaining 32 ATP molecules are produced involving two distinct stages: ● Electron Transport - electrons gathered by NADH and FADH2 during glycolysis and the citric acid

cycle are used to produce an H+ gradient within mitochondria in a process that requires O2 ● Chemiosmosis – the H+ gradient produced by electron transport provides energy for ATP synthase

to make 32 ATP per original glucose. The importance of O2 for cellular respiration cannot be overemphasized. O2 is the final electron acceptor in the electron transport chain. Without O2 electron transport does not occur, bringing cellular respiration to a halt, and the only option for ATP production is fermentation. This means 2 ATP per glucose instead of 36. In the next exercise you will detect the oxidation of succinate, a metabolic intermediate in the Citric Acid Cycle, as evidence of cellular respiration. Succinate dehydrogenase (SDH) is an enzyme in the Citric Acid Cycle which catalyzes the removal of 2 hydrogens from succinate (i.e., the oxidation of succinate) which are transferred to the electron carrier FAD. This yields the products fumarate and FADH2 as shown below:

FAD + succinate FADH2 + fumarate FADH2 in turn will donate the electrons from these 2 hydrogens to coenzyme Q in the electron transport chain. The compound DCPIP (di-chlorophenol-indophenol) is not normally found in cells, however when added to mitochondria it will substitute for coenzyme Q and receive electrons from FADH2. Before receiving the electrons (in its oxidized state) DCPIP is a blue color. After receiving the electrons (being reduced by FADH2) DCPIP is colorless. Because of this color change, DCPIP is a good indicator of respiration as illustrated below. In this experiment, you will study cellular respiration with mitochondria obtained from pulverized lima beans. We will use the color from DPIP and a spectrophotometer to measure cell respiration (DPIP will convert from a blue color to colorless as cell respiration occurs).

To obtain quantitative measurements, you will use a spectrophotometer to measure the light transmitted, or conversely absorbed, by the DPIP.

Materials ❏ Mitochondrial extract from lima beans ❏ Succinate ❏ DPIP solution ❏ Phosphate buffer ❏ 1 ml graduated pipette & pipette pump ❏ Spectrophotometer ❏ Kimwipes ❏ 5 cuvettes ❏ Timer Procedure ❖ Prepare the spectrophotometer

➢ Turn on the power and allow the instrument to warm up for at least 20 minutes ➢ Using the “Wavelength” control knob, set the spectrophotometer at 600 nm (this is the

wavelength of light that will be absorbed by DPIP) ➢ Make sure the spectrophotometer is set to read “Transmittance” (0 A/T button – should be set to

“T”) ➢ Do not change any of the settings once they are set

❖ You will be preparing a cuvette, called a “blank,” for use in zeroing the spectrophotometer. ❖ Set up your blank and experimental cuvettes (Tubes 1-3) as indicated in the Table 3 below. Add the

succinate last and just prior to starting the experiment. The reaction will come to completion prior to measuring the data if the succinate is added too soon or the cuvettes are allowed to sit too long prior to beginning your measurements.

❖ Cuvette 4 will be a student-designed investigation. Choose only 1 variable to change. You may choose items available to you to determine if the rate of cellular respiration will change. ➢ Spices, pH buffers, temperature changes ➢ Different substrates (change the succinate): glucose, sucrose, sugar substitutes, etc. ➢ Use the same other materials as you used in Tube or Cuvette # 3

■ For example, to determine if cell respiration is increased or decreased when a different substrate is used replace the 0.2 ml of succinate with glucose or another substrate

■ If you use spices or another material as an activator or inhibitor: weigh a small amount and dissolve in phosphate buffer (replace the 4.2 ml of phosphate buffer with your new solution)

Table 3: Contents of Experimental Tubes (volumes in ml)

Tube Phosphate Buffer DPIP Mitochondrial Suspension

Succinate (**Add last)

B (blank)

4.6 0 0.3 0.1

1 4.4 0.3 0.3 0 2 4.3 0.3 0.3 0.1 3 4.2 0.3 0.3 0.2

4 0.3 0.3

Tube 4: Student design; adjusted accordingly ❖ Once all of your samples are prepared, place a small piece of parafilm over the tube and gently mix

the contents. ❖ Immediately start your measurements with the spectrophotometer

➢ Gently clean the cuvettes with a Kimwipe to remove any residue or fingerprints from the tube. ➢ Place the Blank cuvette in the spectrophotometer and close the lid. ➢ Zero the spectrophotometer by pushing the 0 A/T button. Press and release the button. The

digital display will read “BLA” and then “0”. Once the spectrophotometer reads “0” you are ready to begin measuring your experimental cuvettes.

➢ Place cuvette #1 in the spectrophotometer and close the lid. Record the measurement in Table 4. Repeat the procedure for cuvettes #2-4. This measurement is for Time “0”.

➢ Repeat the procedure every 5 minutes. You will continue to take measurements every 5 minutes for 30 minutes.

➢ You MUST blank or zero the spectrophotometer prior to taking each set of new measurements every 5 minutes!!!

➢ Record all of your measurements and data in Table 4. ❖ Graph your results to determine the rate of cellular respiration.

Results

Table 4: Results for Cellular Respiration of Mitochondrial Extract and DPIP Time (min)

Tube 0 5 10 15 20 25 30

1

2

3

4

Discussion Questions

1. Using your data, construct a graph to illustrate your results.

a. What is (are) you independent variable(s)?

b. What is the dependent variable?

2. Explain the experimental design. What was the role for each of the components in the test tube?

3. Which experiment tube was the control?

4. In which experimental tube did the transmittance from the spectrophotometer increase more rapidly? Why?

5. Why did you have to add the succinate to the test tube last?

CELL RESPIRATION AND FERMENTATION REVIEW QUESTIONS:

1. Which organisms produce ATP in cellular respiration or fermentation and then use ATP in their

metabolism?

2. What are the three metabolic stages of cellular respiration and where do they occur in the cell?

3. Which processes in cellular respiration and fermentation are anaerobic?

4. Which are aerobic?

5. Which processes takes place in the mitochondria?

6. Which processes take place in the cytoplasm?

7. What is the initials reactant of cellular respiration?

8. What is (are) the product(s) of cellular respiration?

9. What are the two most common types of fermentation?

10. How much ATP is produced in cellular respiration compared to fermentation?

11. Why would an organism choose to undergo fermentation instead of cellular respiration?

12. What is the overall equation for aerobic cellular respiration?