Purpose

The purpose of the activity is to explore the rate of fermentation. Measure the production of carbon dioxide gas in a sampling bottle containing yeast and grape juice and then measure the gas production when a chemical inhibitor is added to the grape juice/yeast mixture.

Background

All organisms require a source of energy to maintain cell physiology and growth. Cellular respiration is the process utilized to oxidize food molecules and release the energy to fuel life processes.

There are two types of cellular respiration – aerobic and anaerobic – and both begin with glycolysis. Glycolysis is a biochemical process utilized by most microorganisms (yeast, bacteria) and “higher” animals to convert glucose to pyruvate and adenosine triphosphate (ATP). Prior to glycolysis, enzymes break down starch into complex sugars (such as sucrose) and then simple sugars (such as fructose and glucose). During glycolysis, the glucose breaks down into pyruvate.

Animal cells and some unicellular organisms convert the pyruvate to lactic acid (lactic acid fermentation). Some plant cells and unicellular organisms convert the pyruvate to ethanol and carbon dioxide gas (alcoholic fermentation).

Yeasts are versatile organisms. Unlike most other organisms that obtain their cellular energy either through aerobic respiration (requiring gaseous oxygen) or through anaerobic respiration – fermentation – (requiring the absence of oxygen), yeast cells respire in either condition, depending upon the availability of gaseous oxygen.

During fermentation, enzymes break down complex carbohydrates into simpler ones. The loss of structural integrity, which can result from changes in pH or high temperatures, usually leads to a loss of enzyme activity.

In this activity, the yeast cells use fermentation(also known as anaerobic respiration) to transform the sugars in grape juice into carbon dioxide gas, ATP molecules, and ethanol.

Pre-lab Questions

Measure carbon dioxide gas production during the metabolism of yeast in grape juice.

1. How would a chemical inhibitor that stops enzyme actions affect the carbon dioxide gas production?
2. How would a change in temperature (either very cold or very hot) affect the carbon dioxide gas production?
3. How will the gas production change over time?

Safety Precautions

• Follow all directions for using the equipment.
• Wear protective gear (e.g., safety goggles, gloves, apron).

Procedure

Yeast and Grape Juice Preparation   

1. Connect a Fast-Response Temperature Probe (included with the GLX) into Port 1 on the left side of the Xplorer GLX. The Graph Screen will automatically open with Temperature (ËšC) versus Time (s).
2. Press the Home key () to go to the Home Screen. Select ‘Digits’ and press the Activate key ().
3. Pour 450 mL of grape juice into a beaker. Put the end of the temperature probe into the juice. Press the Start key () on the GLX so you can watch the temperature of the grape juice.

4. Place the beaker on a hot plate and slowly warm the juice to a temperature of 30 to 35ï‚°C (yeast will die above 40°C). When the temperature is between 30 and 35°C, adjust the hot plate so the grape juice remains warm, but does not get any hotter.
5. While the juice is warming, add 100 mL of warm tap water to another beaker. [Remember to keep the temperature below 40ï‚°C – use the temperature probe to make sure.] Add a package of dry yeast to the beaker and stir well. The yeast will become active in 15 to 20 min.

GLX Setup

1. Stop recording temperature data. Remove the Fast-Response Temperature Probe from the GLX.
2. Connect a PASPORT Extension Cable into Port 1 on the top of the Xplorer GLX. Connect the other end of the Extension Cable to the PASPORT CO₂ Sensor.
   • The Graph Screen will automatically open with CO2 Concentration (ppm) versus Time (s).
3. Open the GLX setup file labeled grape juice.glx (check the appendix at the end of this activity). The file is set to record data once per second.

Sensor Calibration (Optional)

• See the appendix at the end of this activity.

Equipment Setup

1. Transfer 150 ml of warmed grape juice to the sampling bottle. Add a stir bar.
2. Mix the yeast suspension well and add 10 mL to the juice.
3. Use a dropper to add a layer of mineral oil to the surface of the grape juice/yeast mixture so the yeast will have anaerobic conditions.
4. Put the end of the CO₂ Gas Sensor into the sampling bottle loosely. (You do not want gas pressure to build up too high in the sampling bottle.) Do not push the rubber stopper down into the end of the sampling bottle.
   • Note: Avoid bumping the CO₂ Gas Sensor during data collection because it may record erratically.
5. Put the sampling bottle on the magnetic stirrer. Turn on the stirrer.

Record Data: Grape Juice and Yeast

1. Press the Start key on the GLX.
2. Record data for 30 minutes and then stop.
3. Carefully remove the CO₂ Gas Sensor from the sampling bottle. Dispose of the contents as directed and rinse the inside of the bottle.

Record Data: Grape Juice, Yeast, and Inhibitor

1. Transfer another 150 mL of warm grape juice to the sampling bottle and add 1.0 g of sodium fluoride.
2. Stir the yeast suspension again and add 10 mL to the grape juice. Add a layer of mineral oil on top of the grape juice as before.
3. Return the CO₂ Gas Sensor to the sampling bottle so that the rubber stopper rests loosely in the end of the bottle.
4. Press the Start key on the GLX, record data for 30 minutes and then stop.
5. Carefully remove the CO₂ Gas Sensor from the sampling bottle. Dispose of the contents as directed and rinse the inside of the bottle.

Record Data: Warm Grape Juice and Yeast

1. Disconnect the CO₂ Gas Sensor and reconnect the Fast-Response Temperature Probe. Select ‘Digits’ as before and put the end of the probe in the remaining grape juice.
2. Use the hot plate to warm the grape juice to between 45 and 50ËšC. Transfer the warmed grape juice to the sampling bottle.
3. Stir the yeast suspension again and add 10 mL to the warmed grape juice. Add a layer of mineral oil on top of the grape juice as before.
4. Disconnect the temperature probe and re-connect the CO₂ Gas Sensor. Return the CO₂ Gas Sensor to the sampling bottle so that the rubber stopper rests loosely in the end of the bottle.
5. Press the Start key on the GLX, record data for 30 minutes and then stop.
6. Carefully remove the CO₂ Gas Sensor from the sampling bottle. Dispose of the contents as directed and rinse the inside of the bottle.

How do your results compare with others in your class?

Analysis

1. Draw a sketch of your CO₂ concentration versus time graph as requested in the Lab Report section.
2. Use your recorded data to find the change in CO₂ concentration for the grape juice and yeast, the grape juice, yeast, and inhibitor, and the warmed grape juice (optional).
   • In the Graph Screen, press F3 to open the ‘Tools’ menu. Select ‘Statistics’ and press ‘Activate’. The Statistics show the minimum and maximum values.
3. Calculate the rate of change of CO₂ concentration versus time, or the ratio of CO₂ concentration (in ppm) divided by the time (in minutes), for each run of data.

Record your results in the Lab Report.

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Appendix:

To open a specific GLX file, go to the Home Screen (press ). In the Home Screen, select Data Files and press the Activate () key. Use the cursor keys to navigate to the file you want. Press F1 () to open the file.

Optional: To calibrate the PS-2110 CO₂ Gas Sensor, see the instructions provided by the instructor.

Name ________________________________ Date ___________

Pre-Lab Questions

Measure carbon dioxide gas production during the metabolism of yeast in grape juice.

1. How would a chemical inhibitor that stops enzyme actions affect the carbon dioxide gas production?
2. How would a change in temperature (either very cold or very hot) affect the carbon dioxide gas production?
3. How will the gas production change over time?

Data

Make a sketch of one run of CO₂ concentration versus time, including labels for the y- and x-axes.

Data Table

Questions

1. What is the overall rate of CO₂ production for grape juice and yeast and how does it change over time?
2. How does the rate of CO₂ production for grape juice, yeast, and the chemical inhibitor compare to the rate for the grape juice and yeast alone?
3. How does the rate of CO₂ production for the heated grape juice and yeast compare to the rate for the grape juice and yeast?
4. What can you conclude about the affect of the chemical inhibitor on the yeast suspension?
5. What can you conclude about the affect high temperature on the yeast suspension?