LAB 7 – CELLULAR RESPIRATION



Lab #7 – Cellular Respiration

OBJECTIVE

T

his laboratory will introduce you to the biochemical processes, reactant, and products involved in aerobic and anaerobic respiration. In this laboratory you will design an experiment to investigate fermentation (anaerobic respiration) in yeasts and determine the presence of aerobic respiration in plants.

INTRODUCTION

T

he energy source for most cellular activity is ATP (adenosine triphosphate). This molecule consists of adenine and ribose sugar onto which are attached three phosphate groups, two being joined by high-energy bonds. Hydrolysis of these high-energy bonds results in the release of energy. Hydrolysis is a reaction in which large molecules are broken apart by the chemical addition of water.

Human beings eat food in order to obtain the energy stored within the chemical bonds of the food molecules. The most common food energy source in humans is carbohydrate. Staple foods high in carbohydrates (or polysaccharide) include bread, rice, and potatoes. The most important carbohydrate food source is glucose, a monosaccharide or simple sugar. Generally animals digest polysaccharides into monosaccharides, then break down the monosaccharide sugars through the various chemical reactions of glucose metabolism to free the energy contained within the chemical bonds of glucose. These reactions provide energy which may be used to form ATP, known as the “energy currency” of the cell.

ATP is formed by the addition of inorganic phosphate to ADP (adenosine diphosphate) by means of high-energy bond. ATP can be formed during cellular respiration, either in the cytoplasm during glycolysis in the absence of oxygen or in the mitochondria by means of the Krebs cycle and electron transport system if oxygen is present. ATP is also formed during photosynthesis in the chloroplasts of green plants, again using an electron transport system. This energy-rich molecule is broken apart to release energy for various chemical reactions and cellular activities, such as active transport, synthesis of new materials, nerve transmission, and muscle contraction. It has been estimated that a single active cell requires more than two million molecules of ATP per second to drive its biochemical reactions. The reversible equation below is known as the ATP reaction.

ATP + H2O ( ADP + Pi + Energy

Glycolysis, occurring in the cytoplasm, is the first stage of cellular respiration and occurs in the presence or absence of oxygen. Glycolysis does not need oxygen to occur; therefore it is an anaerobic reaction. The Krebs cycle and the electron transport system, occurring in the mitochondria, comprise the remaining stages of cellular respiration and occur only in the presence of oxygen (aerobic reactions). Glycolysis converts glucose to ATP and pyruvate. If oxygen is not present (anaerobic conditions) the pyruvate proceeds through fermentation reactions producing carbon dioxide and either alcohol (in yeasts) or lactate (in animals and some bacteria). If oxygen is present (aerobic conditions), the pyruvate proceeds through the Krebs cycle and electron transport chain producing various high-energy molecules (NADH, FADH, and ATP), carbon dioxide and metabolic water. Most ATP needed by the cell is produced during aerobic reactions; glycolysis produces only 2 ATP molecules while the complete aerobic metabolism of glucose may produce up to 38 ATP molecules.

Exercise 7.1 – Anaerobic Metabolism: Fermentation

Every living organism requires energy. Some organisms, like plants, obtain energy from photosynthesis as you will find out in Lab # 8. Other organisms obtain energy by metabolizing molecules synthesized by plants. One such molecule is the sugar glucose which is produced by photosynthesis. Sugar is metabolized by plants and animals to a variety of compounds. The type of compounds and the amount of energy produced depends on the availability of oxygen.

The first stage of sugar metabolism is glycolysis which occurs in the presence OR absence of oxygen. In glycolysis, a 6-carbon sugar is metabolized into two, 3-carbon molecules called pyruvate. In this process, very little ATP (usually 2) is generated.

If oxygen is present, the sugar molecule is further metabolized by going through the Krebs cycle and the electron transport chain and begin ultimately converted to CO2 and H2O. Much more ATP is generated by the Krebs cycle and electron transport chain, in the presence of oxygen, than by glycolysis alone. Since they require oxygen, these stages of respiration are called the aerobic phases.

If oxygen is not present, an anaerobic process, fermentation, takes places. In plants, the end result is ethyl alcohol (ethanol) and carbon dioxide, and in animals and some bacteria the end result is lactic acid (lactate). It is important to realize that fermentation does not generate any ATP for the cell. Why then, would a cell be adapted for anaerobic respiration? The reason is that the cell must have a plentiful supply of the electron acceptor NAD+ if glycolysis is to occur. Fermentation allows the cell to recycle NADH back into NAD+, thus fermentation assures the continuation of glycolysis which produces at least some energy (Something is better than nothing!).

Before beginning this laboratory exercise, review your text for more information on respiration and the organelles of the cell where metabolism occurs.

OBJECTIVE

At the end of this exercise, the student should be able to design an experiment in order to look at the effects of alcohol concentration on the fermentation ability of yeasts.

FERMENTATION

Yeasts are unicellular eukaryotes in the Kingdom Fungi that can live with or without oxygen. They are called facultative anaerobes. In the presence of oxygen, they can metabolize sugar completely to CO2 and H2O. In the absence of oxygen, they produce ethyl alcohol and carbon dioxide by the anaerobic process known as fermentation.

For at least 6000 years, human cultures have used fermentation products. Drinking water was safer because fermentation killed microbes. In early cultures, fermented products were used as medicine and foodstuffs. Alcoholic beverages were not clarified like they are today, and the leftover yeast provided many minerals and vitamins. Today, alcoholic beverages are clarified, and they have no vitamins or minerals. Alcohol cannot be metabolized by most body cells. It must be metabolized by an enzyme (alcohol dehydrogenase) found mostly in the liver. Alcohol has virtually no nutritional value. Since recorded history, people have made a variety of beverages, such as mead, beer, wine and distillate spirits. Beers typically have 4 % alcohol by volume. There are naturally aged wines and fortified wines. Natural wines have a low alcohol content, usually between 12 – 14 %, even though there still may be sugar remaining. Fortified wines have up to 20 % alcohol because alcohol has been added to them. Distillation concentrates the alcohol content of a beverage; alcohol concentrations in distilled spirits may exceed 40 % to 60 % alcohol.

1. Consider the differences between naturally aged and fortified wines. Speculate why the alcohol content of naturally aged wines is between 12 % and 14 % alcohol (even though there may be sugar remaining in the brewing mixture)? Visualize the ingredients of the brew and the end products.

2. Rewrite your speculation into a testable hypothesis? (Consider carefully the materials and equipment available.)

Materials & Equipment:

1. Yeast (1/2 pack, or 3.5 g if measured out)

2. Salt

3. Sugar

4. Ethanol solution (100 %)

5. Warm water bath (40 °C)

6. Test tubes

7. Sealed glass pipettes

Procedure:

1. Activation of yeast: The yeast should be activated before proceeding with the experiment. To a beaker containing 100 mL of warm water, add a pinch of salt, a level teaspoon of sugar, and the yeast. Stir gently and place in warm water bath until yeast is activated (foam is observed; usually takes about 15-20 minutes).

2. While the yeast is activating, design an experiment to test your hypothesis. Don’t forget to include controls. Your design should be based on the equipment you have available and on what you know of the reaction. Briefly describe your design in the space below.

3. Preparation of alcohol (ethanol) solutions: Use the formula C1V1=C2V2 to help you determine the volume of alcohol solution you’ll need. Remember that the final volume will be halved once you add the activated yeast/sugar mixture. Use the table below to help you work out your volumes. For example, if you want to have a final alcohol concentration after adding yeast mixture if 20%, you’ll need a 40% alcohol concentration. Using the above formula to get this 40%:

C1V1 = C2V2

100% x V1 = 40% x 10mL

V1 = (40% x 10mL)/100%

V1 = 4 mL (volume of stock needed)

Record your measurements below:

Table 7.1: Preparation of alcohol solutions

|Final |Initial |Dilution to get Initial [Alc] |

|Concentration |Alcohol |Stock |Distilled |

|Yeast/alcohol mixture |Concentration |(100%) |Water |

| | |Solution |(mL) |

| | |(mL) | |

|20% |40% |4 mL |6 mL |

| | | | |

| | | | |

| | | | |

| | | | |

| | | | |

| | | | |

| | | | |

| | | | |

| | | | |

| | | | |

A. Consider the differences between naturally aged and fortified wines. Can you speculate why the alcohol content of naturally aged wines is between 12 % and 14 % alcohol (even though there may be sugar remaining in the brewing mixture)?

B. What is the hypothesis being tested in this experiment? Use some of the space to draw a sample of your experimental setup!!!

3. Complete the following table:

Table 7.2: Results of your experiment

|Time (min) |Alcohol Concentrations |

| | | | | | | |

|0 | | | | | | |

|5 | | | | | | |

|10 | | | | | | |

|15 | | | | | | |

A. In which direction did the dye move? _______________________________________

Why?

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B. What was the purpose of adding the heat-killed peas to one tube?

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C. What does this experiment tell you about the influence of temperature on oxygen consumption during cellular respiration?

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Exercise 7.2 (B) – Carbon Dioxide Production During Germination in Peas.

( This is a computer based lab exercise adapted from “Biology with Vernier” – computer 11B. Vernier Software and Technology, )

Cell respiration refers to the process of converting the chemical energy of organic molecules into a form immediately usable by organisms. Glucose may be oxidized completely if sufficient oxygen is available by the following equation:

C6H12O6 +6O2 (g) 6H2O + 6CO2 (g) + energy

All organisms, including plants and animals, oxidize glucose for energy. Often, this energy is used to convert ADP and phosphate into ATP. It is known that peas undergo cell respiration during germination. Do peas undergo cell respiration before germination? The results of this experiment will verify that germinating peas do respire. Using your collected data, you will be able to answer the question concerning respiration and non-germinating peas.

Using the CO2 Gas Sensor, you will monitor the carbon dioxide produced by peas during cell respiration. Both germinating and non-germinating peas will be tested. Additionally, cell respiration of germinating peas at two different temperatures will be tested.

Objectives

In this experiment, you will

• Use a CO2 Gas Sensor to measure concentrations of carbon dioxide.

• Determine whether germinated and non-germinated peas respire.

• Compare the rates of cell respiration in germinated and non-germinated peas.

Materials

Computer 25 germinating peas

Vernier computer interface 25 non-germinating peas

Logger Pro Vernier CO2 Gas Sensor

250 ml respiration chamber

Procedure

1. Connect the CO2 Gas Sensor to Channel 1 of the Vernier computer interface.

2. Prepare the computer for data collection (if not already done by the instructor).

3. Obtain 25 germinating peas and blot them dry between two pieces of paper.

4. Place the germinating peas into the respiration chamber.

5. Place the shaft of the CO2 Gas Sensor in the opening of the respiration chamber.

6. Wait one minute, then begin measuring carbon dioxide concentration by clicking

Data will be collected for 5 minutes.

7. Remove the CO2 Gas Sensor from the respiration chamber.

8. Use a notebook or a notepad to fan air across the openings in the probe shaft of the CO2 Gas Sensor for 1 minute.

9. Determine the rate of respiration:

a. Move the mouse pointer to the point where the data values begin to increase. Hold down the left mouse button. Drag the mouse pointer to the end of the data and release the mouse button.

b. Click the Linear Fit button, to perform a liner regression. A floating box will appear with the formula for a best fit line.

c. Record the slope of the line, m, as the rate of respiration for germinating peas at room temperature in Table 2.

d. Close the linear regression floating box.

10. Move your data to a stored run. To do this, choose Store Latest Run from the Experiment menu.

11. Wash out the respiration chamber and dry it completely. Obtain 25 non-germinating peas and place them in the respiration chamber.

12. Repeat steps 5 – 10 for the non-germinating peas.

13. To print a graph showing all data runs:

a. Label all curves by choosing Text Annotation from the Insert menu, and typing “Room Temp Germinated” (or Room Temp Non-Germinated) in the EDIT box. Then drag each box to a position near its respective curve. Adjust the position of the arrowhead.

b. Print a copy fo the graph, with all three data sets and the regression lines displayed. Enter your name(s) and the number of copies of the graph you want.

DATA

|Table 1 |

|Peas |Rate of Respiration (ppm/min) |

|Germinating, room temperature | |

|Non-germinating, room temperature | |

|Germinating, cool termparature | |

QUESTIONS

1. Do you have evidence that cell respiration occurred in peas? Explain.

2. What is the effect of germination on the rate of cell respiration in peas?

3. Why do germinating peas undergo cell respiration?

EXTENSIONS

1. Compare the respiration rate among various types of seeds.

2. Compare the respiration rate among seeds that have germinated for different time periods, such as 1, 3, and 5 days.

3. Compare the respiration rates of various small animal types, such as insects or earthworms.

Exercise 7.3 – Carbon Dioxide Production during Respiration by Humans

This exercise is designed to demonstrate the production of CO2 in humans during respiration. This exercise will be done as a demonstration by a volunteer or by the instructor.

Procedure:

1. Your instructor will ask for a volunteer for this demonstration.

2. Fill an Erlenmeyer flask with 1-2 inches of Barium Hydroxide [Ba(OH)2] solution and then place the rubber stopper in the mouth of the flask. Barium hydroxide reacts with carbon dioxide to form an insoluble precipitate according to the formula below:

CO2 + H2O ( H2CO3

H2CO3 + Ba(OH)2 ( BaCO3 + 2H2O

3. Make sure you inhale and exhale on the correct tubes. Only inhale from the short tube and only exhale into the long tube. The solution, although not poisonous, does not taste very good so be careful if you are the one chosen for this demonstration. Test for the presence of CO2 by first applying suction to the short tube and then exhaling several times if necessary, into the longer tube. Record any changes in the color of the solution.

A. Enter your results for the Ba(OH)2 demonstration and explain your results:

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Review questions

Cellular Respiration

1. What is the energy source for most cellular activity?

2. What is hydrolysis?

3. Where does the energy for ATP production come from?

4. When is ATP produced during cellular respiration?

5. What are the steps involved in cellular respiration and where does each step take place?

6. Does glycolysis require energy?

7. To which kingdom does yeast belong?

8. Which steps of aerobic cellular respiration require oxygen?

9. What is produced at the end of glycolysis?

10. In the absence of oxygen (anaerobic conditions) which molecules are produced in animals/organisms like yeast?

11. How many ATP molecules are produced in the absence of oxygen?

12. How many ATP molecules are produced by the complete aerobic metabolism of a single glucose molecule?

13. Do all living organisms require energy?

14. What is a facultative anaerobe? Give an example of one?

15. What was the precipitate produced when CO2 reacted with Ba(OH)2 ?

16. Why was potassium hydroxide used in the oxygen consumption in the peas experiment? Does soda lime perform the same function?

17. Write out the balanced equation for cellular respiration.

LAB REPORT #7

Date: ___________________

Name(s) :____________________________________________________

Respiration

Exercise 7.1 – Fermentation

1. Consider the differences between naturally aged and fortified wines. Can you speculate why the alcohol content of naturally aged wines is between 12 % and 14 % alcohol (even though there may be sugar remaining in the brewing mixture)?

2. What is the hypothesis being tested in this experiment? Use some of the space to draw a sample of your experimental setup!!!

3. Complete the following table:

Table 7.2: Results of your experiment

|Time (min) | | | | | | |

|0 | | | | | | |

|5 | | | | | | |

|10 | | | | | | |

|15 | | | | | | |

2. In which direction did the dye move? ______ Why?

____________________________________________________

____________________________________________________

3. What was the purpose of adding the heat-killed peas to one tube?

____________________________________________________

____________________________________________________

4. What does this experiment tell you about the influence of temperature on oxygen consumption during cellular respiration?

____________________________________________________

____________________________________________________

Exercise 7.2 (B) – Carbon Dioxide Production During Germination in Peas.

( This is a computer based lab exercise adapted from “Biology with Vernier” – computer 11B. Vernier Software and Technology, )

DATA

|Table 1 |

|Peas |Rate of Respiration (ppm/min) |

|Germinating, room temperature | |

|Non-germinating, room temperature | |

|Germinating, cool termparature | |

QUESTIONS

1. Do you have evidence that cell respiration occurred in peas? Explain.

2. What is the effect of germination on the rate of cell respiration in peas?

3. Why do germinating peas undergo cell respiration?

EXTENSIONS

1. Compare the respiration rate among various types of seeds.

2. Compare the respiration rate among seeds that have germinated for different time periods, such as 1, 3, and 5 days.

3. Compare the respiration rates of various small animal types, such as insects or earthworms.

Exercise 7.3 – Carbon Dioxide Production during Respiration by Humans

1. Enter your results for the Ba(OH)2 demonstration and explain your results:

____________________________________________________

____________________________________________________

____________________________________________________

____________________________________________________

____________________________________________________

____________________________________________________

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Collect

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