1.8: Respiration and Fermentation

Introduction

All organisms must break down organic molecules to release chemical energy to synthesize adenosine triphosphate (ATP). The energy stored in ATP can be released to perform cellular work. Organisms break down organic molecules, such as glucose, through the common processes of cellular respiration and fermentation (Figure 1). Cellular respiration is generally described as an aerobic process, requiring oxygen, which yields the most possible ATP generated from one molecule of glucose. But, technically, cellular respiration can occur in an anaerobic environment in some microorganisms. Anaerobic cellular respiration yields variable amounts of ATP, but much less than is generated in aerobic cellular respiration. In this laboratory, our discussion of cellular respiration will focus on aerobic cellular respiration.

Both aerobic cellular respiration and fermentation involve many chemical reactions that release high energy electrons from organic molecules and transfer the electrons to other molecules, often referred to as electron carriers (or coenzymes). These chemical reactions involving the transfer of electrons are called reduction-oxidation reactions, or redox reactions. In a redox reaction, one of the molecules gains electrons and becomes reduced (rig, reduction is gain of electrons) and one of the molecules loses electrons and becomes oxidized (oil, oxidation is loss of electrons). In cellular respiration, electrons are often transferred to the electron carrier nicotinamide adenine dinucleotide (NAD+). When this redox reaction occurs, the organic molecule that loses the electrons has been oxidized. When NAD+ gains the electrons it forms NADH. NADH is the reduced form of NAD.

\[\ce{NAD^{+} + 2e^{-} + H^{+} ->[\text{Redox Reaction}] NADH}\]

Aerobic cellular respiration involves a series of three processes of enzymatic chemical reactions: glycolysis, the citric acid cycle (also known as the Kreb’s cycle), and the electron transport chain. Aerobic cellular respiration begins in the cytoplasm with glycolysis and ends in the mitochondria with the citric acid cycle and the electron transport chain. Aerobic cellular respiration results in fully oxidizing glucose, and can yield a maximum of 32 ATP per glucose molecule. At the culmination of the electron transport chain, the electrons are passed to oxygen, a highly electronegative element, to form water. Therefore, at the end of this process, the high energy electrons that were previously a part of glucose are now at a lower energy state, as they are held very closely by the electronegative oxygen.

Fermentation is an anaerobic process of breaking down organic molecules. It occurs in the absence of oxygen. Fermentation breaks down organic molecules, such as glucose, into smaller organic molecule end products. Fermentation begins with the process of glycolysis to produce pyruvic acid and 2 net ATP. Enzymes then carry out chemical reactions to convert pyruvic acid into various fermentation end products. Two common types of fermentation are named for their end products, alcoholic fermentation and lactic acid fermentation. Fermentation produces organic end products that still contain high-energy electrons. Fermentation does not fully oxidize glucose, and yields only 2 net molecules of ATP, along with organic end products.

Figure 1. Cellular respiration and fermentation phases (original figure)

PART 1: CELLULAR RESPIRATION

Exercise 1 : Investigating Cellular Respiration in Plants

This part of the lab investigates cellular respiration in pea seeds. Seeds of plants are stuffed full of sugars like starch. Cellular respiration involves breaking down sugars to generate ATP. Therefore, this process allows plants to harvest energy necessary to produce roots, shoots, and leaves. The process of cellular respiration also results in the release of carbon dioxide gas. Carbon dioxide will react with water to form carbonic acid. The formation of carbonic acid will affect the pH of an aqueous solution. Since carbon dioxide is colorless, odorless, and very hard to detect, we are going to use a pH indicator to detect the presence of carbonic acid and thus carbon dioxide. pH indicators, like red cabbage juice, bromothymol blue, and phenol red are chemicals that change color when pH is altered. In this experiment, we will observe cellular respiration in germinating pea seeds by detecting the production of carbon dioxide and monitoring the changes in the pH of the solution.

Materials:

• Pea seeds (20 germinating/ lab group and 20 dormant / lab group)
• Large sealable bag
• Test tube rack (that can accommodate wide mouth test tubes)
• Wide mouth test tubes with rubber stoppers (3/ lab group)
• Distilled water or spring water (non-chlorinated water)
• Paper towels
• Nonabsorbent cotton plugs
• Glass beads (20 / group)
• Glass rods
• Gloves and safety goggles
• Sharpie or red wax pencil
• Indicator reagent [Choose 1: red cabbage juice, or bromothymol blue (0.04% solution), or phenol red (0.04% solution)] (need 15 ml of indicator solution / lab group)

Overall Timeline:

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Procedure:

1. Two days before beginning the experiment, pour half of the peas into a glass container and cover with several inches of non-chlorinated water to compensate for the expansion of the seeds as they swell. Allow the seeds to soak overnight.

2. The next day, pour the water off of the seeds. Place the seeds onto a wet paper towel, place in a plastic sealable bag, seal the bag, and store the seeds overnight in the dark.

3. On the day of the experiment carefully remove the rehydrated (germinating) seeds from the paper towel.

4. Label 3 wide mouth tubes #1 - #3.

5. Wear gloves when handling the indicator solution. Place 5 ml of the indicator solution into each test tube.
6. Using the glass rod, push a plug of nonabsrobent cotton into each test tube until it sits right above the indicator solution.
7. Add the following to tubes #1 - #3.
   1. Tube 1: add 20 glass beads
   2. Tube 2: add 20 germinating peas
   3. Tube 3: add 20 dry dormant peas

8. Tightly cap the tubes with rubber stoppers. ( If the rubber stoppers have a hole, cover the stoppers with cling wrap, and then place each stopper into a tube.)
9. Observe the color of the indicator reagent at the beginning of the experiment and record your results in Table 1.
10. Observe the color of the indicator reagent after the 2-hr incubation and record your results in Table 1.

11. Observe the color of the indicator reagent after the 24-hr incubation and record your results in Table 1.

12. Once the experiment has been completed, carefully pour the indicator reagent into the appropriate location as indicated by your instructor, being sure to collect the glass beads by pouring through a wire mesh filter.

13. Rinse and wash the test tubes thoroughly.

Table 1. Cellular Respiration of Germinating Peas

Indicator Used:
Tube contents	Initial color of indicator	Color after 2 hrs	Color after 24 hrs	Acidic or basic?
Control
Germinating peas
Dormant Peas

Questions for Review

1. What is the color of the indicator at at

• Neutral pH?
• Basic pH?
• Acidic pH?

2. What was the purpose of Tube #1?

3. What specifically was produced as a result of cellular respiration that changed the color of the indicator?

4. How is carbon dioxide an indicator that cellular respiration is taking place in these peas?

5. Germination is the process by which a dormant seed begins to sprout and grow into a seedling. What are some possible metabolic processes that are required for seed germination?

6. During respiration, a seed metabolizes sugars. What is the source of the sugar metabolized by the seed?

7. What variables do you think may affect the respiration rate of the seeds?

8. The equation for cellular respiration is:

\[\ce{C6H12O6 + 6O2 → 6CO2 + 6H2O + 32 ATP}\]

The energy released from the complete oxidation of glucose under standard conditions is 686 kcal/mol. The energy released from the hydrolysis of ATP to ADP and inorganic phosphate under standard conditions is 7.3 kcal/mol. Using the equation for cellular respiration above, calculate the efficiency of respiration (i.e. the percentage of chemical energy in glucose that is transferred to ATP). *For help with answering this question, refer to Concept 9.4 (Campbell Textbook).

9. How might the process of photosynthesis affect pH? Form a hypothesis.

PART 2: AEROBIC RESPIRATION IN YEAST

Optional Activity or Demonstration

This part of the lab investigates aerobic cellular respiration by Saccharomyces cerevisiae, also referred to as “baker’s yeast” and “brewer’s yeast.” Yeast is a unicellular fungus that can convert glucose into carbon dioxide and ATP when oxygen is present. Methylene blue dye can be used as an indicator for aerobic respiration in yeast. Aerobic respiration releases hydrogen ions and electrons that are picked up by the methylene blue dye, gradually turning the dye colorless. This redox reaction can be observed when viewing a wet mount of yeast and methylene blue under the compound light microscope. The mitochondria of yeast cells undergoing aerobic respiration will appear as a clear area surrounded by a ring of light blue cytoplasm. If cellular respiration is not taking place, the mitochondria will absorb the blue dye and will not turn colorless.

Materials:

• Yeast (not quick rise)
• Distilled water
• Transfer pipette
• Methylene blue dye (in dropper bottle)
• Compound light microscope
• Microscope slide and cover slip
• Electronic balance, spatula, and weigh paper

Procedure:

1. Prepare yeast suspension: Add 7 grams yeast to 50 ml warm tap water. Stir to mix. Save the yeast suspension for Part 3.

2. Place a drop of yeast suspension on a clean microscope slide with a transfer pipette.

3. Add one drop of methylene blue dye and place a cover slip on the microscope slide over the yeast suspension.

4. Observe the yeast using the scanning objective lens. Use the coarse adjustment knob to focus on the yeast cells. Switch to the low power objective lens and then to the high power objective lens.

5. In the circle below, draw several yeast cells undergoing aerobic respiration and several yeast cells not undergoing aerobic respiration. Label the cytoplasm and nucleus if visible.

PART 3: ALCOHOLIC FERMENTATION IN YEAST

This part of the lab investigates alcoholic fermentation by Saccharomyces cerevisiae, also referred to as “baker’s yeast” and “brewer’s yeast.” Yeast converts pyruvate from glycolysis into acetaldehyde, releasing carbon dioxide gas. Acetaldehyde is then enzymatically converted by the enzyme alcohol dehydrogenase into ethanol (Figure 2). In this lab, we will measure the accumulation of carbon dioxide released in the first enzymatic reaction as an indicator of the progression of fermentation.

Figure 2. Alcoholic Fermentation (original figure)

Exercise 1: Investigating Different Concentrations of Yeast

Materials:

• 4 identical saccharometers (glass fermentation hydrometer with either a 10-cm or a 15-cm vertical tube, Figure 3) / lab group
• Yeast (not quick rise)
• Wax pencil or Sharpie
• Distilled water
• 10% glucose solution
• Transfer pipettes
• Test tube rack
• 4 large (20 ml) test tubes or small Erlenmeyer flasks for larger volumes
• Large plastic tray
• Masking tape or lab tape
• Large weigh boat (4/group)
• Metric ruler
• Electronic balance
• Spatula
• Weigh paper
• Red food coloring (optional)

Figure 3. Saccharometer

Table 2. Contents of Saccharometers when testing fermentation with various yeast concentrations.

Saccharometer	DI Water	Glucose Solution	Yeast Suspension
1	*8 ml	*6 ml	0 ml
2	*12 ml	0 ml	*2 ml
3	*6 ml	*6 ml	*2 ml
4	*2 ml	*6 ml	*6 ml

*Double these amounts if using saccharometers that have a 15-cm vertical tube. See table below

Saccharometer	DI Water	Glucose Solution	Yeast Suspension
1	16 ml	12 ml	0 ml
2	24 ml	0 ml	4 ml
3	12 ml	12 ml	4 ml
4	4 ml	12 ml	12 ml

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Procedure:

1. Prepare yeast suspension: Add 7 grams yeast to 50 ml warm tap water. Stir to mix. Alternatively, you can use the yeast suspension from Part 2. Optional: Add a few drops of red food coloring to the yeast to increase contrast, allowing easier measuring of the height of yeast in saccharometers.
2. Label 4 test tubes and 4 saccharometers # 1- 4. Use a transfer pipette to add the appropriate amount of glucose and distilled water listed in Table 2 to the corresponding labeled test tubes.
3. Use a transfer pipette to add the appropriate amount of yeast solution listed in Table 1 to the corresponding labeled test tubes. It is important to work carefully and quickly after adding the yeast solution to the glucose and water.

4. Carefully pour the contents of the test tubes into the correspondingly labeled saccharometer, ensuring that the solutions are well mixed.

5. Carefully tilt the saccharometers to allow any air bubbles that are trapped in the arms of the vertical tube to escape.

6. Begin the timer for the experiment and measure the size of any bubbles (in mm) that are trapped in the vertical arms of the saccharometers. Record this measurement as the 0 time point.

7. Position the saccharometers on the large plastic tray, positioning them around a plastic weigh boat to catch any fermentation overflow that may occur.

8. Carefully tape the saccharometers to the large plastic tray to prevent them from falling and breaking.

9. Every 2 minutes measure and record the total amount of bubbles that accumulate in the top of the vertical arm of the saccharometer. Record the mm of carbon dioxide (bubble) measurements in Table 3.

10. Continue recording the total amount of carbon dioxide released every 2 minutes for 20 minutes.

11. After completing the experiment carefully carry the saccharometers to a sink for washing. Carry only one saccharometer at a time. Spill the yeast mixture into the sink and wash the saccharometer carefully and thoroughly. Return the saccharometer to the plastic tray, laying it down on its side when not in use.

Table 3. Carbon dioxide evolved during fermentation by different concentrations of yeast. Carbon dioxide levels measured in millimeters (mm).

Time (min.)	Sacch. 1	Sacch. 2	Sacch. 3	Sacch. 4
0 (initial)
2
4
6
8
10
12
14
16
18
20

Extension Activity: (Optional)

The results of this experiment can be presented graphically. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the final step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

1. Graph your results using the data from Table 3.

2. What is the dependent variable? Which axis is used to graph this data? ___________________________________________________________________

3. What is your independent variable? Which axis is used to graph this data? ___________________________________________________________________

Exercise 2: Investigating the fermentation of different carbohydrates

Materials:

• 4 identical saccharometers (glass fermentation hydrometer with either a 10-cm or a 15-cm vertical tube) / lab group
• Yeast (not quick rise)
• Wax pencil or Sharpie
• Distilled water
• 10% glucose solution
• 1% starch solution
• 10% sucrose solution
• Metric ruler
• Transfer pipettes
• Test tube rack
• 4 large (20 ml) test tubes or small Erlenmeyer flasks for larger volumes
• Large plastic tray
• Masking or lab tape
• Large weigh boat
• Electronic balance
• Spatula
• Weigh paper
• Red food coloring (optional)

Table 4. Contents of Saccharometers when testing fermentation of different carbohydrates.

Saccharometer	DI Water	Yeast Suspension	Carbohydrate
1	*2 ml	*6 ml	*6 ml glucose
2	*2 ml	*6 ml	*6 ml sucrose
3	*2 ml	*6 ml	*6 ml starch

*Double these amounts if using saccharometers that have a 15-cm vertical tube. See table below

Saccharometer	DI Water	Yeast Suspension	Carbohydrate
1	4 ml	12 ml	12 ml glucose
2	4 ml	12 ml	12 ml sucrose
3	4 ml	12 ml	12 ml starch

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Procedure:

1. Prepare yeast suspension: Add 7 grams yeast to 50 ml warm tap water. Stir to mix. Optional: Add a few drops of red food coloring to the yeast to increase contrast, allowing easier measuring of the height of yeast in saccharometers.

2. Label 3 test tubes and 3 saccharometers # 1- 3. Use a transfer pipette to add the appropriate amounts of carbohydrates and distilled water listed in Table 4 to the corresponding labeled test tubes.

3. Use a transfer pipette to add 6 ml yeast solution to each of the test tubes. It is important to work carefully and quickly after adding the yeast solution to the carbohydrate.

4. Carefully pour the contents of the test tubes into the correspondingly labeled saccharometer, ensuring that the solutions are well mixed.

5. Carefully tilt the saccharometers to allow any air bubbles that are trapped in the arms of the vertical tube to escape.

6. Begin the timer for the experiment and measure the size of any bubbles (in mm) that are trapped in the vertical arms of the saccharometers. Record this measurement as the 0 time point.

7. Position the saccharometers on the large plastic tray, positioning them around a plastic weigh boat to catch any fermentation overflow that may occur.

8. Carefully tape the saccharometers to the large plastic tray to prevent them from falling and breaking.

9. Every 2 minutes measure and record the total amount of bubbles that accumulate in the top of the vertical arm of the saccharometer. Record the mm of carbon dioxide (bubble) measurements in Table 5.

10. Continue recording the total amount of carbon dioxide released every 2 minutes for 20 minutes.

11. After completing the experiment carefully carry the saccharometers to a sink for washing. Carry only one saccharometer at a time. Spill the yeast mixture into the sink and wash the saccharometer carefully and thoroughly. Return the saccharometer to the plastic tray, laying it down on its side when not in use.

Table 5. Carbon dioxide evolved during the fermentation of various carbohydrates. Carbon dioxide levels measured in millimeters (mm).

Time (min.)	Sacch. 1	Sacch. 2	Sacch. 3
0 (initial)
2
4
6
8
10
12
14
16
18
20

Extension Activity: (Optional)

The results of this experiment can be presented graphically. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the final step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

1. Graph your results using the data from Table 5.

2. What is the dependent variable? Which axis is used to graph this data? ___________________________________________________________________

3. What is your independent variable? Which axis is used to graph this data? ___________________________________________________________________

Questions for Review

1. Fermentation involves redox reactions. Explain what happens to electrons during a redox reaction and how this changes a molecule’s potential energy.

2. Why did we add the Saccharomyces cerevisiae (baker's yeast) to the fermentation tubes? Specifically, what did the yeast provide to the fermentation mixture?

3. What is the purpose of Saccharomyces cerevisiae (“baker’s yeast) in the bread-making process?

4. We measured the formation of what end product to determine the fermentation rate? Name the end product that we measured.

5. List two specific factors (as they relate to the experiment performed in our lab) that affect the rate of fermentation.

Practical Challenge Questions:

1. What other variables could be investigated that might affect the rate of alcoholic fermentation by yeast?