Online Ex09 Cellular Respiration and Fermentation PDF

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EXERCISE 9

CELLULAR RESPIRATION AND FERMENTATION LEARNING OBJECTIVES • Use a respiration chamber to assess the rate of cellular respiration of germinating peas. • Determine the fermentation rate of yeast with different sugars and under different temperatures

MATERIALS For Aerobic Respiration Lab: Provided by GTC

Provided by Student

• Dry peas

Two small clear disposable plastic cups

• 0.1% Bromothymol Blue solution

Plastic spoon Timer or phone

For Fermentation or Anaerobic Respiration Lab: Provided by GTC

Provided by Student

• 1 Thermometer

4 Clear plastic 12oz or other size water bottles

• 4 Balloons

Measuring spoons, measuring cup

• Glucose

1 small funnel (optional)

• Yeast

A timer or stopwatch Small ruler Sucrose (table sugar) Corn Starch

INTRODUCTION Cellular respiration is the process organisms use to transfer energy in the presence of O2 from food to adenosine triphosphate (ATP), the energy molecule used in cells. The process is complex, requiring

many enzymatic steps. The enzyme reactions are grouped into three pathways: glycolysis, the Krebs cycle (the citric acid cycle), and electron transfer phosphorylation (the electron transport chain and chemiosmosis). While glucose is the most efficient food source for most cells, other types of food may also be converted into intermediates in these pathways. As a result, ATP energy is produced from all our food by these three pathways. See Figure 9.1. Proteins

Carbohydrates

Amino acids

Sugars

Glycolysis Glucose

Glyceraldehyde – P

Pyruvate

NH3

Acetyl CoA

Krebs Cycle

Electron transfer phosphorylation

Figure 9.1: Overview of metabolism.

The overall reaction for cellular respiration is:

C6H12O6 + 6 O2

~

6 CO2 + 6H2O + 36 ATP

Fats

GlycerolFatty acids

Plants produce glucose by photosynthesis. This glucose can then be stored as starch (large polymers of glucose) for later use. When energy is needed, the starch is broken down into individual glucose molecules, which then go through cellular respiration to produce ATP. Animals store excess glucose temporarily as glycogen (polymers of glucose) in liver and skeletal muscle cells. As in plants, when energy is needed, glycogen is broken down into glucose molecules and used to generate ATP. No matter whether you are studying plants or animals, the first step in glucose metabolism is glycolysis, which requires no oxygen (anaerobic). Then the products of glycolysis are further broken down in steps that do require oxygen. Fermentation is an alternative method to generate ATP when oxygen is not available. It is an inefficient process, producing only 2 ATP rather than a possible 36 ATP per molecule of glucose. This is not enough energy for animal metabolism. But some organisms, such as yeast and bacteria, can slow their metabolism enough to survive on the energy produced by fermentation. When oxygen is not +

available, pyruvate, the product of glycolysis, is reduced by NADH, regenerating NAD that can reenter the glycolysis pathway. Fermentation itself does not produce ATP. What it does do is allow glycolysis to continue. Without fermentation, glycolysis would eventually be unable to continue. The products of fermentation vary between organisms, depending on the specific enzymes in the organism. For example, animals produce lactic acid, some bacteria produce acetic acid (vinegar), propionic acid (the flavor in Swiss cheese), or alcohols. Baker’s yeast and brewer’s yeast produce ethyl alcohol (ethanol) and CO2. Glucose

CYTOPLASM

2ATP

GLYCOLYSIS

(net)

2 NAD+2 NADH

If lactate fermentation: 2 lactate

2 Pyruvate

FERMENTATION

If alcoholic fermentation: 2 CO2 and 2 ethanol

Figure 9.2: Overview of fermentation.

In these exercises, you will first learn how to measure the respiration rate of germinated peas using a respiration chamber. Although glucose is the preferred carbohydrate for glycolysis and fermentation, you will also perform an experiment to measure the fermentation rate in yeast for sugars other than glucose. The overall reaction for alcohol fermentation is: C6H12O6

2C2H5OH + 2CO2 + 2ATP

PRE-LAB QUESTIONS 1. List two differences between aerobic and anaerobic cellular respiration. Explain clearly for each process. 1. Cellular respiration utilizes oxygen and creates ATP, anaerobic

cellular respiration does not utilize oxygen and does not create ATP

2. Explain the differences between cellular respiration and breathing. Explain clearly. a.

Cellular respiration is the process through which cells decompose nutrients into ATP. Breathing is the exchange of gases such as oxygen and carbon dioxide in our longs and does not release ATP or any form of energy.

3. Explain how germinating and dry non-germinating seeds are different. Focus on cell growth, energy requirement and rate of cellular respiration. a. Dried or non-germinating peas are embryos in a state of dormancy, or metabolic inactivity. When environmental conditions change, such as access to water and increased temperatures, they will begin to germinate, or grow. Once germinated, the peas will grow into a plant and develop the structures needed in order to start photosynthesizing. Until they can make their own food through photosynthesis, however, they depend on the stored carbohydrates as fuel for aerobic respiration.

4. Examine the substrates used in the Fermentation lab activity. Determine what type of carbohydrate each represents. Fill in the table below with your answers. (Note: water is NOT a substrate.)

Type of Carbohydrate (monosaccharide, or di...etc.)

Glucose

Disaccharide Disaccharide

Sucrose Polysaccharide

Corn Starch

ACTIVITY #1 – AEROBIC CELLULAR RESPIRATION DURING PEA GERMINATION In this experiment you are going to investigate aerobic cellular respiration rate during pea germination by the amount of carbon dioxide they produce. Refer back to the cellular respiration equation – as glucose is broken down, oxygen is consumed and carbon dioxide and water are produced. Dried or non-germinating peas are embryos in a state of dormancy, or metabolic inactivity. When environmental conditions change, such as access to water and increased temperatures, they will begin to germinate, or grow. Once germinated, the peas will grow into a plant and develop the structures needed in order to start photosynthesizing. Until they can make their own food through photosynthesis, however, they depend on the stored carbohydrates as fuel for aerobic respiration. You will investigate aerobic respiration by peas during the germination process. The experimental setup involves a qualitative estimate of CO 2 amount in the test tubes by the use of a pH indicator, bromothymol blue. When the pH of a solution is less than 6, bromothymol blue will show yellow color; when the pH is between 6-8, it will show green color; when the pH is greater than 8, it will show blue color in solution. pH 8 Lower CO 2 level

Green

Blue

As oxygen is consumed during cellular respiration in the germination process, more and more CO 2 are produced, causing the pH to decrease, which leads to a color change in the solution.

Procedure 1. Obtain all the materials and put on a counter. You will need: dry peas, 1 drop bottle of 0.1% bromothymol blue solution, two clear small disposable plastic cups, a timer or phone or clock, a plastic spoon to stir the solution. 2. Add about 50 peas into one plastic cup. No peas in the other cup. Then add 1/4 cup of lukewarm tap water into both cups. 3. Add 5 drops of bromothymol blue solution into each cup and stir with a spoon to mix the solution. The solution should be blue, if the color is too light, add a few more drops. Leave the cups on the counter and start your timer. 4. Record the color of the solution in the table below (Table 9.1). 5. At the one-hour mark, observe the color of solution in each cup and record your data in the table. 6. Continue with 2, 4, 6, 8 and 24 hours, record data in the table. Take a picture of the two cups side by side at the end of 24 hours. 7. Insert your image in this document, below Table 9.1. Be sure that your result images include your photo ID, for authentication. The image (including ID) must be clear and the details of the ID must be clear and legible. 8. Clean up.

Table 9.1: Solution’s Color Change during Germination in Peas. Solution Color with Peas

Solution Color with No peas

Time (hour)

0

Blue

Blue

1

Aqua

Blue

2

Light Green

Blue

4

Yellowish Green

Blue

6

Yellow ish

Blue

8

Yellow

Blue

24

Yellow

Blue

QUESTIONS: 1.

What’s the purpose of the cup with no peas? Do you expect any color change during the time period? Why?

a.

The cup with no peas is the control group and I do not expect it to change much unless the water reacts with the bromothyl blue due to an unexpected pH change.

2. Describe what happened to the color in the tube with peas? Explain in detail how the color change relates to the rate of cellular respiration during pea germination. a. The cellular respiration occurring consumed the carbon dioxide and increased the pH therefore it turned yellow.

ACTIVITY #2a – YEAST FERMENTATION WITH DIFFERENT CARBOHYDRATES During fermentation, yeast produces ethanol and CO 2. While glucose is the preferred substrate, other sugars can also be fermented by yeast. In this experiment you will determine yeast’s relative fermentation rates of different carbohydrates. The rate of fermentation can be measured by capturing and measuring the CO2 as it is produced by the yeast.

Procedure 1. Obtain all the materials and put them on the kitchen counter or a table. You will need: 4 empty clear plastic water bottles with caps, 1 thermometer (optional), 1 small funnel (optional), 1 timer, 4 balloons, dry yeast, 1 measuring teaspoon, ½ measuring cup, marker or sharpie, 1 small ruler and substrates: dextrose (glucose), sucrose(table sugar), corn starch. 2. Use your sharpie to label the water bottles: W for water, G for glucose, S for sucrose, C for corn starch. Arrange them in the order on the kitchen counter. 3. Add 1 tsp of dry yeast into each of the bottles. Using a small funnel to avoid spillage. You can make a funnel by cutting out the top of a small water bottle 4. Then, add 1 tsp of glucose into the G bottle; 1 tsp of sucrose into the S bottles; 1 tsp of corn starch into the C bottle. No substrate into the W bottle (think Why?) 5. Turn on your kitchen faucet for some warm water. If you have a thermometer, try to use the water with a temperature of about 37oC which is 97oF. If you don’t have a thermometer, put your hand under the water until your hand feels warm but not hot. 6. Using your measuring cup, add 1/2 cup of warm water into each of the four bottles. Use a small funnel to aid the step if possible. 7. Cap the bottles and mix the contents by gently shaking the bottles. Each of the 4 bottles should have exactly the same volume of solution. 8. Now, remove the caps. Put balloons on the bottles by stretching the mouth of a balloon over the mouth of each bottle. This creates an anaerobic environment for the yeast. 9. Leave the bottles on the counter and take a picture of the four bottles side by side then start the time for 20 minutes. 10. After 20 minutes, observe what happens in the bottles and what happens to the balloons. Using a small ruler, roughly measure the diameter of each of the balloons and record the data in Table 9.2. 11. Continue the observation, data collection, and picture taking at 40 minutes and 60 minutes. 12. Insert your image in this document, below Table 9.2. Be sure that your result images include your photo ID, for authentication. The image (including ID) must be clear and the details of the ID must be clear and legible. 13. Once you are done, clean up your counter and wash the bottles in the sink for next activity. 14. Calculate the CO2 production from yeast fermentation by following the formula in Table 9.2. 15. Complete the pre-lab, post-lab questions.

Table 9.2: Yeast Fermentation with Various Carbohydrates

Substrate

Volume of CO2 production (cm3) Vol = 4/3 π(D/2)3 0 min 20 min 40 min 60 min

Diameter of balloon (cm) 0 min

20 min

40 min

60 min

Water

0

0

0

0

Glucose

0

5

5.5

Sucrose

0

6

6.5

7.5

Cornstarch

0

0

0

0

6.5

0 0

0

0

0

0

523.598 696.909 77559 9703213 4

1150.34 650998 95

904.778 1150.34 684239 650998 95

1767.14 5867644 3

0

0

QUESTIONS 1. What was the control for this experiment? Why was the control necessary? •

The control was the water and the control is necessary to gage the change in the rest of the variables.

2. What gas inflated some of the balloons? How is it produced? •

Carbon dioxide was produced

3. Which substrates are monosaccharides? Disaccharides? Polysaccharides? 4. – Glucose and sucrose are disaccharides. Corn starch is a polysaccharide.

4. Which substrate was fermented the most? Was this consistent with your prediction? Explain any discrepancy. - Sucrose fermented the most because it is the molecule closest to glucose.

5. Water is not a substrate, explain why. Did any substrates fail to ferment at all? If so, explain why this happened and why there was no major fermentation activity. - Water failed to react with a anything because it is not a substrate.

6. Create a graph using the data in Table 9.2. Plot the substrate vs. total volume of CO2 produce. Determine which type of graph is most appropriate for the data and use the proper format.

Yeast Fermentation with Various Carbohydrates 1400 1200 1000 800 600 400 200 0

0 min

20 min

40 min Water

Figure 9.3

60 min Glucose

0 min Sucrose

20 min

Cornstarch

40 min

60 min

ACTIVITY #2b – YEAST FERMENTATION UNDER DIFFERENT TEMPERATURES Fermentation is also affected by temperature. You will compare yeast fermentation rates at cold temperature (~ 4-8oC), room temperature (~ 25oC), warm temperature (~ 37oC) and boiling temperature (~ 100oC) using sucrose (table sugar) as the substrate.

Procedure 1. Obtain all the materials and put them on the kitchen counter or a table. You will need: 4 empty clear plastic water bottles with caps, 1 thermometer (optional), 1 small funnel (optional), 1 timer, 4 balloons, dry yeast, 1 tsp and 1 TBS measurer, 1 measuring cup, marker or sharpie, 1 small ruler and sucrose(table sugar) as the substrate. 2. Use your sharpie to label the water bottles: C for cold temperature, R for room temperature, W for warm temperature and B for boiling temperature. Arrange them in the order on the counter. 3. Add 1 tsp of dry yeast into each of the bottles. Using a small funnel to avoid spillage. You can make a funnel by cutting out the top of a small water bottle. 4. Then, add 1 tablespoon of sucrose (table sugar) into all the bottles. 5. Prepare water at different temperatures as follows (use the thermometer if provided): a. Turn on faucet and collect 1 cup of water in a small pan and heat on stove till it’s boiling. Using a funnel, add this boiling water to the bottle labelled B for boiling temperature water. b. Collect another 1 cup of tap water (~ 25oC/77oF) and add this to the room temperature bottle labelled R. c.

For cold temperature of water, collect 1 cup of water from your fridge water dispenser or add a few ice cubes to tap water and add to the bottle labelled with C for cold temperature water.

d. For warm water, turn on your faucet to the warm side and let it run for maybe 30 seconds or so until the water feels warm but not hot to the touch, add 1 cup (~ 37-40oC/ 97102oF) into the bottle labelled with W for warm water temperature. 6. Cap the bottles and mix the contents by gently shaking the bottles. Each of the 4 bottles should have exactly the same volume of solution. 7. Now, remove the caps. Put balloons on the bottles by stretching the mouth of a balloon over the mouth of each bottle. This creates an anaerobic environment for the yeast. 8. Leave the bottles on the counter and take a picture of the four bottles side by side then start the timer for 30 minutes. 9. After 30 minutes, put all the bottles on the counter in order. Observe what happens in the bottles and what happens to the balloons. Using a small ruler, measure the diameter of each of the balloons and record the data in table 9.3. 10. Take pictures of the completed results, after the 30-minute incubation.

11. Insert your image in this document, below Table 9.3. Be sure that your result images include your photo ID, for authentication. The image (including ID) must be clear and the details of the ID must be clear and legible. 12. Once you are done, clean up. 13. Calculate the CO2 production from yeast fermentation and record the data in table 9.3 14. Complete the pre-lab, post-lab questions.

Table 9.3: Yeast Fermentation under Various Temperatures

Temperature (oC) Cold Temp. (~4-8 oC)

1.2

Volume of CO2 production (cm3) Vol = 4/3 π(D/2)3 5.7122888990

Room Temp. (~22-27 oC)

6.3

142.683447

Warm Temp. (~37-40 oC)

1.2

5.7122888990

Boiling Temp. (~100 oC)

1

4.73728900008

Diameter of balloon (cm)

QUESTIONS: 1. Among the four temperatures tested, which one showed the highest rate of fermentation? a. 37-40 degrees Celsius (warm) fermented at a higher rate than the rest of the temperature.

2. What happened to the boiling temperature? Why? a. In order for fermentation to occur the enzymes must be active and the boiling temperature denatured the enzymes therefore it did not ferment.

3. From this experiment and your knowledge, describe how temperature affects the rate of fermentation?

-

Fermentation occurs through enzymes and enzymes can become denatured in extreme temperatures.

4. Make a graph with the data in Table 9.3. Determine the independent and dependent variables. Also determine whether you should use a bar graph or a line graph.

: Yeast Fermentation under Various Temperatures 160 140 120 100 80 60 40 20 0

ld Co

m Te

p

-8 ~4 .(

) oC

om Ro

m Te

p.

27 22 (~

) oC

m ar W

m Te

p.

40 73 (~

) oC

ng il i Bo

m Te

Diameter of balloon (cm) Volume of CO2 production (cm3) Vol = 4/3 π(D/2)3

p

00 ~1 .(

) oC