Massachusetts Institute of Technology

Department of Biology

TW 2014 Biofuel

Effect of Type of Sugar on Yeast Fermentation*

Summary |

During this lesson, students will be investigating the process of fermentation by measuring the amount of gas pressure produced by yeast metabolizing a specific sugar. Students will also measure the pH at the beginning and the end of the experiment. Since fermentation produces carbon dioxide as a byproduct, some will dissolve in solution to produce carbonic acid resulting in a lower pH. The broader concept here is the process of cellular respiration and how it works in living organisms (yeast). Students should understand that almost all living things need to process a form of sugar in order to make energy.

Each group of students will be given a specific sugar to add to their yeast solutions and measure gas pressure over time immediately following the addition of the sugar. In addition, an “unknown” sugar will be given to each lab group for identification.

Skills practiced during this lab include: (adapted from scientific inquiry standards – MA State Frameworks)

·  Observe the world from a scientific perspective.

·  Pose questions and form hypotheses based on personal observations and knowledge.

·  Articulate and explain the major concepts being investigated and the purpose of an investigation (in lab report)

·  Employ appropriate methods for accurately and consistently making observations,

making and recording measurements at appropriate levels of precision,

and collecting data or evidence in an organized way

·  Properly use instruments, equipment, and materials (e.g., probeware, meter sticks, computers)

·  Follow safety guidelines.

·  Represent data and relationships between and among variables in charts and graphs.

·  Use appropriate technology (e.g., graphing software) and other tools.

·  Assess the reliability of data and identify reasons for inconsistent results, such as sources of error or uncontrolled conditions.

·  Use results of an experiment to develop a conclusion to an investigation that addresses the initial questions and supports or refutes the stated hypothesis.

·  State questions raised by an experiment that may require further investigation.

·  Develop descriptions of and explanations for scientific concepts that were a focus of one or more investigations.

·  Review information, explain statistical analysis, and summarize data collected and analyzed as the result of an investigation. (in lab report)

·  Explain diagrams and charts that represent relationships of variables.

·  Construct a reasoned argument and respond appropriately to critical comments and questions.

·  Use language and vocabulary appropriately, speak clearly and logically, and use appropriate technology (e.g., presentation software) and other tools to present findings.

Background Information

All living things need energy to live, grow and reproduce. Most living things produce energy (ATP) through a process called cellular respiration. During this process, sugar is broken down in cells (by many enzymes) to release energy.

An example of an organism that uses cellular respiration is yeast. Yeasts are single cell eukaryotic fungi that can process sugar both anaerobically (without oxygen) and aerobically (with oxygen). You will observe yeast metabolize sugars under anaerobic conditions (fermentation) and monitor the change in air pressure due to carbon dioxide released by the yeast. When yeast burn sugar under anaerobic conditions, ethanol (ethyl alcohol) and carbon dioxide are released as shown by the following reaction:

C6H12O6 2 CH3CH2OH + 2 CO2 + energy

glucose ethanol carbon

dioxide

Thus, the metabolic activity of yeast may be measured by monitoring the pressure of gas in the test tube. If the yeast were to respire aerobically, there would be no change in the pressure of gas in the test tube, because oxygen gas would be consumed at the same rate as carbon dioxide is produced. In addition, you will also be testing different sugars to see if the yeast are able to metabolize them. Also, an initial and final pH measurement will be taken as an additional indication of the rate of fermentation. As the carbon dioxide level increases in the tube, some will be dissolved in the solution. As a result, carbonic acid is produced.

Key Concepts

·  Cellular respiration

·  Enzyme Activity

·  Fermentation

·  Chemical reactions

Objectives

·  Use a Gas Pressure Sensor to measure the change in pressure due to CO2 release during fermentation.

·  Determine the rate of fermentation of yeast using different sugars.

·  Identify an unknown sugar based on class results.

Biology, High School Course- MA State Frameworks

1.3 Explain the role of enzymes as catalysts that lower the activation energy of biochemical reactions. Identify factors, such as pH and temperature that have an effect on enzymes.

2.4 Identify the reactants, products, and basic purposes of photosynthesis and cellular respiration. Explain the interrelated nature of photosynthesis and cellular respiration in the cells of photosynthetic organisms.

Broad Unit Objectives- Hopkinton High School 10th grade Biology (MCAS course)

Enduring Understandings

1. Chemical elements form organic molecules that interact to perform the basic functions of life.

2. Photosynthesis and cellular respiration are interrelated metabolic processes, essential to life on earth.


Essential Questions

1. How do living things make and use energy?

2. How do the structures of a cell allow it to make and use energy?

•  Observe yeast fermentation.

•  Identify an unknown sugar through the fermentation protocol.

•  Record gas pressure over time and measure pH before and after experiment.

•  Demonstrate knowledge of the process of fermentation and enzyme activity.

•  Communicate results of the lab clearly through a well-written lab report.

QUESTION OF THE LAB: WHAT EFFECT DOES THE TYPE OF SUGAR HAVE ON THE RATE OF FERMENTATION OF YEAST?

HYPOTHESIS: Answer the question above in the form of a hypothesis based on your prior knowledge.

______

MATERIALS

LabPro interface / 18 Í 150 mm test tube
Palm handheld / 600 mL beaker
Data Pro program / vegetable oil in a dropper bottle
Vernier Gas Pressure Sensor / pipet bulb or pump
Graphical Analysis or Logger Pro (optional) / ring stand
yeast suspension
5% glucose solution
5% sucrose solution
5% fructose solution
5% lactose solution
5% maltose solution / Thermometer
pH paper
rubber-stopper assembly / basting bulb or Beral pipet
1 liter beaker (for water bath) / utility clamp
10 mL pipet or graduated cylinder / Microscope slide

PROCEDURE

Part 1

1. Connect the plastic tubing to the valve on the Gas Pressure Sensor.

2. Plug the Gas Pressure Sensor into Channel 1 of the LabPro interface. Connect the handheld to the LabPro using the interface cable. Firmly press in the cable ends.

3. Prepare a water bath for the yeast. A water bath is simply a large beaker of water at a certain temperature. This ensures that the yeast will remain at a constant and controlled temperature. To prepare the water bath, obtain some warm and cool water from your teacher. Combine the warm and cool water into the 1 liter beaker until it reaches the temperature you were assigned. The beaker should be filled with about 600 – 700 mL water. Place the thermometer in the water bath to monitor the temperature during the experiment.

4. Using a 10 mL pipette or graduated cylinder, transfer 2.5 mL of the SUGAR SOLUTION GIVEN TO YOU BY YOUR TEACHER into a clean test tube. Record the type of sugar in data table 1. (If you were assigned the control no sugar will be added). Label this test tube 1.

5. Gently swirl the yeast suspension to mix the yeast that settles to the bottom. Transfer 2.5 mL of yeast into test tube 1. Gently mix the yeast into the sugar solution. Be gentle with the yeast—they are living organisms!

6. Gently pipette a drop of the yeast/sugar solution onto a microscope slide. Test the pH of the solution by dipping the indicator paper into the drop. Record in Data Table 1.

7. In the test tube, place enough vegetable oil to completely cover the surface of the yeast/sugar mixture as shown in Figure 3. Be careful to not get oil on the inside wall of the test tube. Set the test tube in the water bath.

8. Insert the single-holed rubber-stopper into the test tube. Note: Firmly twist the stopper for an airtight fit. Secure the test tube with a utility clamp and ring-stand as shown in Figure 1.

9. Incubate the test tube for 10 minutes in the water bath. Be sure to keep the temperature of the water bath constant. If you need to add more hot or cold water, first remove about as much water as you will be adding, or the beaker may overflow. Use a basting bulb to remove excess water.

Note: Be sure that most of the test tube is completely covered by the water in the water bath. The temperature of the air in the tube must be constant for this experiment to work well.

10. While one team member is performing Step 8, another team member should prepare the handheld and interface for data collection. Press the power button on the handheld to turn it on. To start Data Pro, tap the Data Pro icon on the Applications screen. Choose New from the Data Pro menu or tap to reset the program.

11. Set up the handheld and interface for a Gas Pressure Sensor.

  1. If the handheld displays PRESS(kPa) in CH 1, proceed directly to Step 11. If it does not, continue with this step to set up your sensors manually.
  2. Tap on the Main screen.
  3. Tap to select Channel 1.
  4. Choose PRESSURE-GPS (kPa) from the sensor menu.
  5. Tap to return to the Main screen.

12. When incubation has finished, connect the free-end of the plastic tubing to the connector in the rubber stopper as shown in Figure 4.

13. Tap to begin data collection. Maintain the temperature of the water bath during the course of the experiment. Record the average temperature of the water bath in Table 1.

14. Data collection will end after 15 minutes. Monitor the pressure readings displayed on the handheld screen. If the pressure exceeds 130kilopascals, the pressure inside the tube will be too great and the rubber stopper is likely to pop off. Disconnect the plastic tubing from the Gas Pressure Sensor if the pressure exceeds 130kilopascals.

15. When data collection has finished, an auto-scaled graph of pressure vs. time will be displayed on the handheld screen. To examine the data pairs on the displayed graph, tap or any data point. As you move the examine line, the pressure values of each data point are displayed to the right of the graph.

16. Determine the rate of fermentation for the curve of pressure vs. time. The rate of fermentation can be measured by examining the slope of the pressure vs. time curve:

  1. Tap the Selection button, .
  2. Tap on the data point at the beginning of the sloping portion of the graph to define the left boundary of the selection. An arrow (>) is displayed on this line.
  3. Tap on the data point at the end of the sloping portion to define the right boundary. An arrow (<) is displayed on this line.
  4. Tap , then tap .
  5. From the Fit Equation menu, choose Linear. The linear-regression statistics for these two data columns are displayed for the equation in the form

y = ax + b

where x is time, y is pressure, a is the slope, and b is the y-intercept. Note: The correlation coefficient, r, indicates how closely the data points match up with (or fit) the regression line. A value of 1.00 indicates a nearly perfect fit.

  1. Record the slope of the line (rate of fermentation) in Table 1.
  2. Tap to display the regression curve on the graph of pressure vs. time. Tap to return to the Graph screen.

17. When data collection is complete, disconnect the plastic tubing from the Gas Pressure Sensor. Using a pipette, remove a small drop of the yeast/sugar solution and place on a microscope slide. Test the pH of the solution by placing the indicator strip in the drop. Record the result in Data table 1.

PART 2

Repeat the Part 1 procedure but label a second test tube #2. An unknown sugar will be assigned to you. Record your data in Data Table 2. Use the class data google spreadsheet to determine the unknown sugar.

DATA

Data Table 1

Type of Sugar / pH / Avg. Temp (°C) / Rate of Fermentation (kPa/min)
Initial pH
Final pH

Data Table 2

Type of Sugar / pH / Avg. Temp (°C) / Rate of Fermentation (kPa/min)
UNKNOWN / Initial pH
Final pH

CLASS DATA SHEET

QUESTIONS:

1.  What is the purpose of the control tube?

2.  What is the purpose of the vegetable oil?

3.  According to the class data, what sugar could the yeast metabolize the best?

4.  Hypothesize why some sugars are not utilized by yeast while other sugars are metabolized.

5.  According to the class data, what was the unknown sugar?

6.  How do the pH levels relate to the rate of fermentation?

7.  Explain the sources of error in this lab.