Yeast and Molasses - NBHS Biologynbhsbio.weebly.com/uploads/1/8/7/5/18753328/yeast_and_molasses_… · AP Biology: I. Molecules and Cells, C. Cellular Energetics 2. Fermentation and

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Yeast and Molasses 15Yeast and Molasses

Examining the Effect of Food Concentration on Fermentation

OBJECTIVE Students will determine the concentration of molasses that will yield the greatest rate of fermentation by yeast cells. Students will make a hypothesis and then evaluate the validity of that hypothesis based on collected data.

LEVEL Biology I

NATIONAL STANDARDS UCP.1, UCP.2, UCP.3, A.1, A.2, B.2, B.3, C.1, C.5, F.2, G.1, G.2

TEKS 2 (A), 2 (B), 2 (C), 2 (D), 9 (A)

CONNECTIONS TO AP AP Biology:

I. Molecules and Cells, C. Cellular Energetics 2. Fermentation and cellular respiration.

TIME FRAME 30 min day 1 45 min day 2

MATERIALS (For 28 students working in groups of 4)

56 test tubes 25 u 200 mm (70 mL) 100 mL yeast solution 7 test tube racks 1 pkg yeast + 1 L water 28 ea # 4 test tube stoppers 500 mL of 5 % molasses solution 14 graduated cylinders (100 mL) 500 mL of 10 % molasses solution 56 graduated centrifuge tubes 17 mm u 120 mm (15 mL) 0.1 mL graduations

500 mL of 20 % molasses solution 500 mL of 40 % molasses solution 500 mL of 60 % molasses solution

28 scissors 500 mL of 80 % molasses solution 28 glue sticks 500 mL of 100 % molasses solution 500 mL distilled water

448 Laying the Foundation in Biology

©2004 Laying the Foundation, Inc. All rights reserved. Visit: www.layingthefoundation.org

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15 Yeast and Molasses

TEACHER NOTES This lab activity supplements a unit on cellular respiration.

Make the following molasses solutions by mixing:

Percentage Amount of Molasses Amount of Water

0 % 0 mL 500 mL

5 % 25 mL 475 mL

10 % 50 mL 450 mL

20 % 100 mL 400 mL

40 % 200 mL 300 mL

60 % 300 mL 200 mL

80 % 400 mL 100 mL

100 % 500 mL 0 mL

If time permits, you may want each lab group to make their own molasses solution by mixing the following:

Percentage Amount of Molasses Amount of Water

0 % 0 mL 40 mL

5 % 2 mL 38 mL

10 % 4 mL 36 mL

20 % 8 mL 32 mL

40 % 16 mL 24 mL

60 % 24 mL 16 mL

80 % 32 mL 8 mL

100 % 40 mL 0 mL It may take some time to get the molasses and the water thoroughly mixed.

Prepare a stock yeast solution by adding 1 package of brewer’s yeast (7g) to 1 L of warm water about 15 minutes before class. When the yeast is needed, dilute 30 mL of the stock solution with 70 mL of warm water.

Laying the Foundation in Biology 449


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Yeast and Molasses 15Part I of this lab investigates the relationship between fermentation and increasing concentration of the food source, molasses. The rate of fermentation is indicated by the amount of carbon dioxide produced. Typically, students predict that increasing concentrations of molasses will result in an increased rate of fermentation. However, the results of this activity will show that when the concentration of molasses is above 80%, there is a decrease in the amount of energy produced. This decline is attributed to resultant low levels of water within the cells.

Prepare a transparency of the data table to facilitate the sharing of data.

Part II is a cut and paste exercise which examines the specific events of glycolysis and fermentation in a manner engaging to tactile/kinesthetic learners. If you have no need to differentiate your instructional activities you may simply have the students write in the missing information.

POSSIBLE ANSWERS TO THE CONCLUSION QUESTIONS AND SAMPLE DATA

DATA AND OBSERVATIONS

Data Table 1

Amount of CO2 Collected (mL)

Test Tube # 1 2 3 4 5 6 7 8

Percent of Molasses 0 % 5 % 10 % 20 % 40 % 60 % 80 % 100 %

Individual Team Data

0 5.0 11.0 14.0 15.0 15.0 7.5 0

Team # 1 0 5.5 10.5 15.0 15.0 15.0 7.0 0

Team # 2 0 5.0 10.0 13.5 15.0 150. 7.0 1

Team # 3 0 6.0 10.0 12.5 15.0 15.0 8.0 0

Team # 4 0 4.0 10.5 15.0 15.0 15.0 7.5 0

Team # 5 0 5.5 12.0 15.0 15.0 15.0 7.0 0

Team # 6 0 4.0 10.5 15.0 14.5 15.0 8.0 0

Team # 7 0 5.5 11.0 14.0 15.0 13.0 7.5 0

Class Average 0.0 5.1 10.6 14.4 14.9 14.7 7.4 0.1

CONCLUSION QUESTIONS

1. What happened to the amount of carbon dioxide gas produced as the concentration of molasses increased? x In the beginning, an increase in the concentration of molasses resulted in an increase in the

production of carbon dioxide, however, after a certain point, an increase in the concentration of



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molasses resulted in a decrease of carbon dioxide production. This is because there is insufficient water to support life for the yeast cells.

2. What percentage of molasses resulted in the greatest amount of fermentation? Does this result support your hypothesis? x The individual data indicated that the greatest amount of fermentation occurred when the

molasses was concentrated between twenty and sixty percent. The data supported the hypothesis up to sixty percent and then after that, an increase in the concentration of molasses resulted in a decrease in the amount of fermentation produced. This is because the molasses solution becomes hypertonic to the yeast cells, and the yeast cells are dehydrating.

3. Design an experiment based on this protocol that would investigate the effect of temperature on fermentation. x This is an open-ended question that will result in a variety of answers but the following is a

possible answer: The data indicated that a molasses concentration of 20í60% resulted in the greatest amount of energy production. This lab could use the same protocol with 40% molasses for three sets of test tubes. One set could be put into the refrigerator, the second in an incubator, and the third at room temperature for 24 hours. Require the students to include control in their experimental design.



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Yeast and Molasses 15PART II Diagram Key — Biochemical Pathway for Glycolysis and Fermentation.

CH OH2

C OHHH

C

HO OH HCOH

C CH OH ATP

ADPC O

HHH

C

HO OH HCOH

C CH OH

O

C C

H H HO OH

C CHO H

CH OH2

Glucose

Glucose 6-phosphate

Fructose16- bisphosphate

1. A phosphate is added to glucose. Itcomes from ATP. This phosphateincreases the amount of energy ofglucose.

2. Atoms are rearranged andglucose 6-phosphate is turned intofructose 6-phosphate.

CH O– P2

CH O– P2



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O

C C

H H HO OH

C CHO H

CH OH2

Fructose 6-phosphate

CH O– P2

O

C C

H H HO OH

C CHO HFructose1-6 bisphosphate

CH O– P2

ATP

ADP

3. Another phosphate is added tofructose 6-phosphate. It comes fromATP. This phosphate increases theamount of energy of fructose.

4. Fructose 1-6 bisphosphate has somuch energy and is so unstable that thishexose is cleaved into two trioses,phosphoglyceralaldehyde or PGAL anddihydroxacteone phosphate.

5. Atoms are rearranged and dihydroxy-acetone phosphate is turned intophosphoglyceraldehyde, PGAL. FromThis point in time, everything is multipliedby a factor of two because there are twotrioses.

H

CHOH

CH OH2

C=O

CH O– P2

Dihydroxy-acetonephosphate

Phosphoglycer-aldehyde

CH O– P2

C=O

CH O– P2



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Yeast and Molasses 15



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REFERENCES Biological Science, Interaction of Experiments and Ideas. Englewood Cliffs: Prentice-Hall, Inc., 1983. pp. 16í22




Yeast and Molasses Examining the Effect of Food Concentration on

Fermentation All cells need energy, and the most useful energy molecule for cells is ATP. The full name given to ATP by chemists is adenosine triphosphate. ATP is composed of the sugar, ribose, and the nitrogenous base, adenine, and three phosphate groups. Two of the bonds connecting the phosphates are considered high-energy bonds and, when broken, useful energy is released. Cells use this energy to power a variety of cellular activities. Cells produce ATP through the process of respiration. During respiration, energy is moved from the bonds of organic compounds into the phosphate bonds of ATP. If a phosphate group is removed from ATP, a molecule called adenosine diphosphate (ADP) will be formed. ADP has one less phosphate group than ATP and thus less energy. If two phosphate groups are removed from ATP, a molecule called adenosine monophosphate (AMP) is formed.

N

CN

HCCH

N

NC

C

H

C C

O

CH

OH OH

H HH

CC O P O

H O

H O-

O P

O

OHP

O

O- O-

Energy-rich bonds

3 Phosphates

Ribose

Adenine

Many biochemical reactions require the addition of phosphates to one or more reactants in order to proceed. The addition of these phosphates increases the energy content of the molecule. For example, in the biochemical reaction called glycolysis, the process of breaking down glucose begins by adding a phosphate to the molecule producing glucose-phosphate. Glucose-phosphate has more energy than plain glucose because of the addition of a phosphate group.

Cells need a plentiful supply of ATP. Once the ATP is used and converted into ADP, the cells need to regenerate ATP by adding a phosphate to ADP. Since energy was released during the breaking of the phosphate bond, the reverse process of forming a phosphate bond requires an energy input.



Yeast and Molasses 15The energy needed to regenerate ATP is transferred by cells from the organic compounds in food sources such as glucose, sucrose, or other such organic compounds. Glucose has considerably more energy than ATP. One molecule of ATP has approximately 16 kcal/mole whereas glucose has approximately 680 kcal/mole. You may ask, why not use glucose instead of ATP as the cell energy currency? Cells cannot directly use glucose as an energy source because glucose has too much energy. Releasing all of glucose’s energy at once would be like putting a match into a gas can. The cell cannot handle the release of such large amounts energy at once. The resulting increase in temperature would denature the enzymes and destroy the cell. Instead, cell respiration releases the energy from organic compounds in small amounts at a time through a series of steps. Another analogy that illustrates using glucose as a direct energy source in the cell would be like you trying to buy a candy bar with a one thousand dollar bill. You have money but it is in an unusable form. The bill has to be taken to the bank and exchanged for useable money such as ten-dollar bills. In the cell’s energy economy, glucose is like the one thousand dollar bill and ATP is like ten-dollar bills. The cell can use ATP directly and easily. The process by which cells retrieve the energy from molecules such as glucose is called cellular respiration. The chemical equation below summarizes the process of cellular respiration.

There are three major parts to cell respiration. The three parts to cellular respiration are glycolysis, the Krebs cycle, and oxidative phosphorylation. Glycolysis occurs in the cytoplasm of the cell. The major events in the process of glycolysis are the following:

During the process of glycolysis no oxygen is required.

The other 34 molecules of ATP are made in the Krebs cycle and oxidative phosphorylation. These processes occur in the inner compartment of the mitochondria. It is at the very end of these processes that oxygen is needed. Respiration is often referred to as aerobic respiration because oxygen is required. If there is no oxygen present, then the Krebs cycle and oxidative phosphorylation will not occur.

If oxygen is not present, then glycolysis can continue to make pyruvic acid and two molecules of ATP. After all, the synthesis of two ATP molecules is better than making none. The limiting factor in this process is having enough NAD to make the NADH. In order to regenerate NAD, several additional steps are needed. These additional steps complete the process known as fermentation. There are several types of fermentation. The most common types of fermentation are lactic acid fermentation and alcohol fermentation. Muscle cells perform lactic acid fermentation when muscles are vigorously contracting and are unable to obtain enough oxygen. Yeast cells, plant cells and certain bacteria perform alcoholic



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Yeast and Molasses

fermentation in the absence of oxygen. The alcohol produced is ethanol and during this reaction carbon dioxide is released.

This lab exercise uses yeast cells to investigate the relationship between fermentation and food concentrations. If yeast cells have more food available, will they generate more ATP? Yeast cells use food sources like molasses through alcoholic fermentation to regenerate ATP. During alcoholic fermentation, carbon dioxide is released as a by-product. This lab measures the amount of carbon dioxide released as an indicator of the amount of fermentation occurring. Below are the final steps added to glycolysis to complete the process of fermentation.

PURPOSE In this activity you will investigate the process of fermentation and its relationship to the availability of food for fermentation.



Yeast and Molasses 15MATERIALS

Part I 8 test tubes 25 u 200 mm (70 mL) yeast solution test tube rack 5 % molasses solution 4 ea # 4 test tube stoppers 10 % molasses solution

graduated cylinders (100 mL)

8 graduated centrifuge tubes 17 mm u 120 mm (0.1 mL graduations)

20 % molasses solution 40 % molasses solution 60 % molasses solution

80 % molasses solution 100 % molasses solution distilled water Part II scissors glue stick

PROCEDURE PART I 1. Formulate a hypothesis that predicts the relationship between the amount of fermentation and an

increasing amount of available food. Record your hypothesis on your student answer page. Your teacher will divide the class into groups of 3í4 students. Every student should have the opportunity to participate in the experiment.

2. Obtain the above materials and label the large test tubes 1í8.

3. Add 40 mL of the indicated molasses solution to each of the following test tubes: # 1 — 0 % molasses/ only distilled water # 2 — 5 % molasses # 3 — 10 % molasses # 4 — 20 % molasses # 5 — 40 % molasses # 6 — 60 % molasses # 7 — 80 % molasses # 8 — 100 % molasses

4. Add 10 mL of yeast solution to each test tube.

5. Stopper each tube and shake to mix thoroughly.

6. Remove the stopper and rinse the stopper with water.



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Yeast and Molasses

7. Obtain a small centrifuge tube and notice that it has markings on the sides of the tube in mL. The small tube needs to be filled with the yeast-molasses solution. Do this by inverting the small centrifuge tube and sliding it into the large test tube. Then, put the large stopper into the large tube and hold it on its side. When the small tube is completely filled with the suspension, slowly move the large tube back to its upright position. If there is any air bubble in the small tube, repeat the procedure until no air is present.

8. Allow the tubes to sit for 24 hours. After 24 hours, measure the gas in the centrifuge tube for each tube by observing the amount of gas in the tube using the gradations on the side of the centrifuge tube. Record your data in Data Table 1 on your student answer page. Collect data for each group in the classroom and average the data.

9. Graph your data and the class-averaged data.

Optional — These calculations can be done on a TI-83 calculator by doing the following:

1. To make a data table press , the select EDIT (Figure 1) and press . Notice that there are columns or lists to record data.




Figure 1

Figure 2

2. To clear a list that might have data in it, put the cursor at the very top of the list so that the name of

the column is highlighted. Press followed by the .

3. Now enter the percent of molasses in L1 starting at 0 for the first entry and ending at 100 percent for the last entry. In L2, record the amount of carbon dioxide collected. In L3, record the class average

for the amount of carbon dioxide collected. (Figure 2). To view this graphically, press ,

, . At this time make sure to put your cursor on PLOT 1 and press . Then

move the cursor down to ON and press . Both PLOT 1 and ON should be highlighted. All

other plots should be inactivated. Highlight the line graph (Figure 3) and press .

4. Highlight the Xlist and press , [L1].

5. Highlight the Ylist and press , [L2].

6. Highlight the box symbol for the Mark and press (Figure 3).

Figure 3

Figure 4



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Yeast and Molasses

7. To put a second line on the graph, position the cursor on Plot 2 and press . Move the cursor

down to ON and press . Both PLOT 2 and ON should be highlighted. Highlight the line

graphs (Figure 4) and press .

8. Highlight the Xlist and press , [L1].

9. Highlight the Ylist and press , [L3].

10. Highlight the cross hairs symbol for the Mark and press (Figure 4).

11. Press, then press . A graph should appear that will have automatically adjusted the axes so that they fit the window. (Figures 5 and 6).

Figure 5

Figure 6

It may appear that there is only one line on the graph if your data is close to the class average. To

demonstrate that both lines are present press and then use the and to differentiate between the points. Look at the figures above. In Figure 5, the data point, Y=14.9, is class averaged data and in figure 6, the data point Y=15, is the student’s data.

PART II In this portion of the activity you will practice sequencing the events of glycolysis and fermentation. As you read about the steps of these processes, look at the accompanying diagram on your student answer page. You will notice that there are names of products and descriptions of the reaction missing. The empty boxes indicate missing items. At the end of the diagram is a list of missing parts. Using scissors and a glue stick, fill in the missing items to make the biochemical pathway complete. Be sure to place either the missing step or molecule in the correct sequence.

1. A phosphate is added to glucose. It comes from ATP. This phosphate increases the amount of energy of glucose.



Yeast and Molasses 152. Atoms are rearranged and glucose 6-phosphate is turned into fructose 6-phosphate.

3. Another phosphate is added to fructose 6-phosphate. It comes from ATP. This phosphate increases the amount of energy of fructose.

4. The increased energy and phosphate makes fructose 1,6-biphosphate causing it to split into two molecules, phosphoglyceraldehyde (PGAL) and dihydroxyacetone phosphate.

5. Atoms are rearranged to convert dihydroxyacetone phosphate into PGAL. Each PGAL will continue through the process. For this reason there is a “2” in front of the product names in the remaining steps.

6. Hydrogens are stripped from each phosphoglyceraldehyde and transferred to NAD+. In addition, an inorganic phosphate group is added to the molecule. (The phosphate group comes from the cytoplasm.) This step produces 1, 3-biphosphoglyceric acid.

7. Each 1, 3- biphosphoglyceric acid molecule gives up a phosphate to ADP forming ATP. This step produces 3-phosphoglyceric acid.

8. The phosphate group found on carbon number three is transferred to carbon number two producing 2-phosphoglyceric acid.

9. A dehydration reaction occurs as a water molecule is removed to form phosphoenolpyruvic acid or PEP.

10. Phosphoenolpyruvic acid transfers its phosphate to ADP to form ATP. This produces pyruvic acid. If oxygen is present then the pyruvic acid is used in the Krebs cycle. If NO oxygen is present, then fermentation occurs.

11. Lactic acid fermentation- Hydrogens are transferred to pyruvic acid from NADH to form lactic acid and NAD+. Alcoholic fermentation- Hydrogens are transferred to pyruvic acid from NADH to form lactic acid and NAD+ and a molecule of carbon dioxide is produced.



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Yeast and Molasses

Name _____________________________________

Period _____________________________________

Yeast and Molasses Examining the Effect of Food Concentration on

Fermentation

HYPOTHESIS

DATA AND OBSERVATIONS

Data Table 1

Amount of CO2 Collected (mL)

Test Tube # 1 2 3 4 5 6 7 8

Percent of Molasses 0 % 5 % 10 % 20 % 40 % 60 % 80 % 100 %

Individual Team Data

Team # 1

Team # 2

Team # 3

Team # 4

Team # 5

Team # 6

Team # 7

Class Average



Yeast and Molasses 15CONCLUSION QUESTIONS

1. What happened to the amount of carbon dioxide gas produced as the amount of molasses increased?

2. What percentage of molasses resulted in the greatest amount of fermentation? Does this result support your hypothesis? Give a possible explanation for any tube(s) that did not support your hypothesis.

3. Design an experiment based on this protocol that would investigate the effect of temperature on fermentation.



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Yeast and Molasses

PART II

DIAGRAM OF GLYCOLYSIS & FERMENTATION

CH OH2

C OHHH

C

HO OH HCOH

C CH OH

C OHHH

C

HO OH HC

OH

C CH OH

O

C C

H H HO OHC C

HO H

CH OH2

Glucose

Glucose 6-phosphate

1. A phosphate is added to glucose. Itcomes from ATP. This phosphateincreases the amount of energy ofglucose.

2. Atoms are rearranged andglucose 6-phosphate is turned intofructose 6-phosphate.

CH O– P2

CH O– P2




O

C C

H H HO OH

C CHO H

CH OH2

Fructose 6-phosphate

CH O– P2

O

C C

H H HO OH

C CHO HFructose1-6 bisphosphate

CH O– P2

3. Another phosphate is added tofructose 6-phosphate. It comes fromATP. This phosphate increases theamount of energy of fructose.

5. Atoms are rearranged and dihydroxy-acetone phosphate is turned intophosphoglyceraldehyde, PGAL. FromThis point in time, everything is multipliedby a factor of two because there are twotrioses.

H

CHOH

CH OH2

C=O

CH O– P2

Dihydroxy-acetonephosphate

Phosphoglycer-aldehyde

CH O– P2

C=O

CH O– P2



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Yeast and Molasses






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Yeast and Molasses



Yeast and Molasses 15Here are the missing items to make glycolysis and fermentation complete:



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Yeast and Molasses



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Yeast and Molasses - NBHS Biologynbhsbio.weebly.com/uploads/1/8/7/5/18753328/yeast_and_molasses_… · AP Biology: I. Molecules and Cells, C. Cellular Energetics 2. Fermentation and