BIOL1408 Introductory Biology Name Lab Unit 9

16 Jun BIOL1408 Introductory Biology Name Lab Unit 9

Question
BIOL1408 Introductory Biology Name

Lab Unit 9: Respiration date

Dr. Flo Oxley

In this lab unit, you will follow your eSciences ACC Lab Manual (posted in Blackboard: “Lab Manual”) to learn about the processes of energy transformation that work inside mitochondria of your cells to support life.

This document will serve as your guide, sending you to the relevant lab activities and introductory information found in the ACC custom eSciences lab manual (pages for this unit are 97-106) uploaded to Blackboard.

NOTE: I recommend that you read from this lab guide & report document first, going to the eSciences manual materials only as directed. The additional background information provided in this document and the step-by-step guidance through the eSciences lab materials is for your benefit.

In this lab, you will study different aspects of cellular respiration: anaerobic fermentation by yeast and aerobic respiration by germinating seeds.

BACKGROUND INFORMATION: CELLULAR RESPIRATION

Plants obtain the energy to meet their metabolic needs through the harvest of solar energy to transform carbon dioxide into sugar molecules. These sugar molecules are then used by the plant cell to fuel the necessary metabolic reactions for their growth and for the maintenance of their cells. They are sometimes referred to as“autotrophs”, meaning literally“self-feeders” since they do not consume other organisms in order to survive.

In contrast, animals and other organisms that feed off of others are referred to as“heterotrophs”, meaning literally“other-feeders”. They can be animal-eaters (carnivores), or plant eaters (herbivores), or rely on both plants and animals for their nutritional needs (omnivores). Herbivores also indirectly benefit from the energy harvested by plants from the sun, using the sugars and other biomolecules synthesized by the plants for their own needs. And carnivores indirectly benefit, as the carbon molecules go up the food chain.

We have a caloric requirement in our daily diet to meet our own energy needs, derived from the foods that we eat. The primary sources of calories used in our cellular metabolism are sugars and fats. In this lab unit, we will focus our attention on how we derive energy from sugars in the cell.

Cellular RespirationBackground Questions

Go to: http://www.sumanasinc.com/webcontent/animations/content/cellularrespiration.html to watch the“The Big Picture”animation on cellular respiration, and answer the questions below.

1. Photosynthesis and cellular respiration are in many ways opposites. Answer the following questions as you watch the animation.

a. What are the reactants of the overall photosynthesis reactions, and what are the products of the overall cellular respiration reactions? (Include the energy sources and products in your answer.)

b. What are the products of the overall photosynthesis reactions, and what are the reactants of the overall cellular respiration reactions?

c. What are the 3 major steps of cellular respiration?

BACKGROUND INFORMATION: FERMENTATION

The first step of cellular respiration is glycolysis, where glucose is broken down to smaller-sized molecules (called“pyruvate, or pyruvic acid”) with the formation of some energy-harvesting molecules (ATP and NADH). If oxygen is available, a considerably larger amount of energy can still be extracted from the products of glycolysis by shuttling these pyruvate molecules into the mitochondria to undergo further digestion.

If oxygen is not available, the harvest energy from sugars ends with glycolysis. If this happens in your muscles during a heavy workout, your muscles will accumulate lactic acid (formed from the pyruvic acid products of glycolysis). The lactic acid buildup in your muscles can cause soreness over time if you don’t do some cool-down exercises to clear out the lactic acid.

When yeast cells are grown anaerobically (without oxygen), the glycolytic products (pyruvate) are turned into alcohol instead of lactic acid. (How fortunuate for us!) The process of growing yeast anaerobically is called“fermentation”.

If any of you make your own homebrews of beer, this activity should be very familiar to you because in the absence of oxygen, yeast cells will ferment glucose into ethanol and carbon dioxide, both of which make the product delightful to the taste!

C6H12O6  CO2 + 2 C2H5OH + 2 ATP

Glucose ferments to carbon dioxide and alcohol, releasing 2 ATP

Carbon dioxide is a by-product of anaerobic respiration, and is the gas that will inflate the balloons in your fermentation experiment. The release of carbon dioxide in water leads to the formation of carbonic acid, which can be detected by a pH indicator such as Phenol Red or Bromothymol Blue. Carbon dioxide also can be measured by its absorbance of infrared radiation (a‘greenhouse effect’).

There is evidence for the production of beer by fermentation of barley in prehistoric civilizations from thousands of years ago. Today, beer production is a fine art, and breweries ferment lagers slowly at lowered temperatures, while the ales ferment much faster at higher temperatures. The lower temperatures slow the yeast’s metabolism, resulting in longer periods of brewing before the sugars are converted to ethanol and carbon dioxide.

Anaerobic fermentations have been used to leaven bread for nearly as many years. The CO2produced when yeast metabolize carbohydrates in bread provides thousands of small bubbles in the dough. This is what causes the bread to“rise”.

Anaerobic RespirationBackground Questions

Go to: http://www.sumanasinc.com/webcontent/animations/content/cellularrespiration.html to watch the“Glycolysis”animation on cellular respiration, and answer the questions below.

1. Where in the cell does glycolysis occur?

2. Energy is harvested from glycolysis. What is the source of the energy that is extracted?

3. How many ATP are required for the preparatory phase, and how many ATP are harvested in the payoff phase of glycolysis?

4. What is the net yield of ATP and NADH molecules from the glycolysis on one molecule of glucose?

5. What is the glucose molecule split into at the end of glycolysis?

EXPERIMENT 1. FERMENTATION BY YEAST
CAUTION: DO NOT OVERHEAT YOUR YEAST AS YOU DISSOLVE IT. TO BE SAFE, KEEP THE TEMPERATURE JUST LUKEWARM, OR BELOW 37C. HIGHER TEMPERATURES WILL KILL THE YEAST CELLS.
NOTE: IF YOU DO NOT OBSERVE SIGNIFICANT DIFFERENCES IN GAS HEIGHTS IN YOUR RESPIROMETER TUBES AFTER TWO HOURS, LET THE FERMENTATIONS CONTINUE FOR ANOTHER HOUR.
RESULTS:
Table 1: Gas Production from Yeast Fermentation

Tube

Initial Gas Height (mm)

Final Gas Height (mm)

Net Change (mm)

1

(1% glucose)

7 mm

8.5 mm

1.5 mm

2

(1% sucrose)

7 mm

9 mm

2 mm

3

(1% Equal)

5 mm

5 mm

0 mm

4

(1% Splenda)

4 mm

4 mm

0 mm

5

(1% sugar)

5 mm

6 mm

1 mm

Experiment 1 Questions

1. If you have evidence of anaerobic respiration, identify the gas that was produced. Suggest two methods for positively identifying this gas.

2. Which of your respirometers showed the largest amount of fermentation? Provide a plausible explanation for these results.

3. Which of your respirometers showed the least amount of fermentation? Provide a plausible explanation for these results.

4. Hypothesize why some of the sweetener solutions were not metabolized by the yeast, while others were. Research the formulation of Equal and Spenda to explain how it would affect yeast respiration. Theobromine is an enzyme which the body cannot process. But people ingest it all the time because it is found in chocolate. But the body cannot process it, so it comes out in your urine. The chemical formula effects of Equal™for respiration was not metabolized because it’s missing sugar. Splenda™is derived from sugar so the respiration results were different.

5. How do the results of this experiment relate to the role yeast plays in baking? The experiment shows a relation between yeast and size in materials. This is what makes bread so light and fluffy after it has been baked. The yeast makes it rise separating the dough and when it’s baked it comes out fluffy.

6. What would you expect to see if the yeasts’metabolism was slowed down? How could you slow down the yeasts’metabolism to check this out? If you slow down the yeast’s metabolism you would have slow or non-rising bread. This can and is accomplished by using the one thing that is sure to slow down all organic reactions, cold temperatures. Such as storage in a refrigerator.

7. Indicate plausible sources of error in your experimental results and suggest some strategies for improvement. There was limited activity in the time frame and heat sources I used. I used a portable heater but there wasn’t much activity after just one hour. Also, the tubes that I worked with weren’t made to fit exactly into each other so this may have affected the levels measured. 1. In some cases the human body cannot digest the substance in question. If a child swallows a button, for example, it will just pass through the body and come out with the feces.

BACKGROUND INFORMATION: AEROBIC RESPIRATION

Here are some ways in which aerobic respiration is similar to the processes found in photosynthesis, and some ways in which it differs.

SIMILARITIES

1. Both take place in an organelle (mitochondria for respiration vs chloroplast for photosynthesis).

2. ATP is produced as a consequence of an electron transport system in an internal membrane system.

3. Electron transport in an internal membrane system powers pumps in the membranes which set up a concentration gradient of hydrogen ions (H+) that in turn powers ATP productions by ATP Synthase enzymes.

DIFFERENCES

1. Oxygen is the terminal electron transport acceptor in mitochondria, generating water, while water is split into oxygen and hydrogen ions in chloroplasts to supply the electron transport system with electrons.

2. NADP is the terminal electron transport acceptor in chloroplasts, generating NADPH, while NADH is the source of high-energy electrons for the electrons transport system in mitochondria.

3. Chemical energy (from glucose) is harvested in mitochondria for ATP production, while solar energy powers ATP production in chloroplasts.

Aerobic Respiration Background Questions

1. Go to: http://www.sumanasinc.com/webcontent/animations/content/cellularrespiration.html to watch the“Krebs Cycle”animation on cellular respiration, and answer the questions below.

a. What are the carbon compounds that begin the Krebs cycle?

b. How many carbon atoms does Acetyl-CoA contribute to the Krebs cycle? What becomes of the carbon atoms during the oxidation reactions of the Krebs cycle?

c. As the carbon atoms become oxidized, what becomes of the freed hydrogen atoms?

d. How do NADH and FADH2 molecules transfer their energy to ATP molecules?

e. Sports training results in the increase of mitochondria in muscle tissue. Why is the increase in mitochondria of benefit?

2. Go to: http://www.sumanasinc.com/webcontent/animations/content/cellularrespiration.html to watch the“Electron Transport” animation on cellular respiration, and answer the questions below.

a. Where in mitochondria does the electron transport chain occur?

b. What are the electron donors for the electron transport chain? Where does this released energy end up?

c. Where in the process of electron transport is oxygen required? What happens if oxygen is not available?

d. What powers the proton pump in the inner membrane of mitochondria? What is purpose of this c oncentration gradient?

e. Which of the 3 stages of aerobic respiration results in the highest yield of ATP?

Experiment 2.

In this experiment, you will observe the metabolism of sugars aerobically (with oxygen) which includes the action of mitochondria. Carbon dioxide is by-product of aerobic cellular respiration, the final breakdown product of glucose molecules. In the process, ATP harvests the energy:

C6H12O6 + 6 O2  6 CO2 + 6 H2O + enough energy to make 38 ATP

The CO2 generated will acidify water, causing the pH to drop, as measured by a pH indicator such as Phenol Red or Bromothomol Blue. Notice how this overall chemical reaction is very similar to that of photosynthesis which we studied in the last lab–except the reaction is in opposite direction! In photosynthesis, we observed the rise of pH as carbonic acid was consumed by photosynthesis, and in this lab, we observe the drop in pH as carbonic acid is generated by cellular respiration.

Pay attention to the controls in this experiment, and what variables they eliminate in any conclusions that you might draw. Having one beaker with no beans eliminates the variable of the air itself. Any effect from the air will be present in all three beakers. Having one beaker with dry beans eliminates the variable of the bean itself affecting the results. The controls are necessary to show that it is respiration, and no other variable, that is affecting the results. Without ruling out these factors, you might draw the wrong conclusions from your experimental results.

RESULTS:
Table 2: Bromothymol Blue Color Change Over Time for Pinto Bean Experiment

Time

Beaker with Pre-Soaked Beans

Beaker with Un-Soaked, Dry Beans

Beaker with No Beans

0 min

Blue

Blue

Blue

30 min

Light Green

Blue

Blue

60 min

Green

Blue

Blue

90 min

Dark Green

Blue

Blue

120 min

Green

Dark Green

Blue

150 min

Light Green

Dark Green

Blue

180 min

Light Yellow

Green

Blue

24 hours

Yellow

Green

Blue

Table 3: Bromothymol Blue Color Change Over Time for Kidney Bean Experiment

Time

Beaker with Pre-Soaked Beans

Beaker with Un-Soaked, Dry Beans

Beaker with No Beans

0 min

Blue

Blue

Blue

30 min

Green

Blue

Blue

60 min

Green

Blue

Blue

90 min

Yellowish Green

Blue

Blue

120 min

Green

Light Green

Blue

150 min

Green

Light Green

Blue

180 min

Yellow

Green

Blue

24 hours

Dark Yellow

Dark Green

Blue

Experiment 2 Questions

1. What are the controls in this experiment, and what factors did they rule out? Why is it important to have a control for this experiment? The control in this experiment is the beaker of Bromothymol by itself. This is because it has no variables, beans in other words, in it. This proves that it is the beans themselves that is doing any changes to the fluids in the beakers. It is important to have a control because you cannot tell difference if the CO2 gas is released.

2. What is the chemistry that causes the bromothymol blue solution color change? Bromothymol blue solution is an indicator that changes color in the presence of a weak acid or base. So the mechanism would be the release of carbon dioxide into water which turns into a weak acid. Therefore the cellular respiration is the mechanism that is ultimately driving

the color change.

3. What evidence do you have to prove cellular respiration occurred in beans? Explain your reasoning. Cellular respiration had to occur in the beans because the bromothymol changed color. This

proves that the beans had to release either CO2, which would make a weak acidic

compound or blue when it is in a basic solution.

4. Were there differences in the rates of respiration in pinto beans vs. kidney beans? If so, why?
I know that during germination, the kidney beans need more space as they are producing more oxygen vs the pinto beans which can be placed closer together.

5. If this experiment were conducted at 0°C, what difference would you see in the rate of respiration? Why? The rate would be much slower at 0C because cold temperatures inhibit both the release of gas and most metabolic and cellular functions in organic substances. This means that the color change and release of CO2 would be much slower at room temperature.

6. What effect would extremely high temperature (e.g.,greater than 55°C) have on respiration in beans? Design an experiment to test your hypothesis, complete with controls.

7. What would you predict your results to look like if you added cyanide to the environment of germinating beans.

8. Would you expect to find CO2in your breath? Why? Carbon Dioxide is the results of