TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB Introduction Most organisms, including yeasts, use oxygen in a process called cellular respiration. Cellular respiration is the controlled breakdown of carbohydrate to carbon dioxide and water with capture of some of the energy in the form of ATP. The rest of the energy is lost in the form of heat. The first stage of the breakdown is called glycolysis and the second stage is called the Krebs Cycle. During this process, electrons are transferred from the carbohydrates to oxygen in the process called electron transport and water is formed as the final product of electron transport. Electron transport produces a chemosmotic gradient of protons (H+) and positive charges across a membrane and this gradient can drive the formation of ATP. Cellular respiration produces approximately 38 ATP molecules from each molecule of the sugar glucose that is broken down. The carbon that was in the carbohydrate is fully oxidized to form CO2 during respiration. For glucose, the 6 carbons become 6 CO2 molecules. Table 1. yeast.

Comparison of respiration and fermentation of glucose in

PROCESS

CONDITION S

PRODUCTS FROM GLUCOSE

AMOUNT OF ATP

RESPIRATION

AEROBIC

6 CO2 + 6 H2

38

FERMENTATION

ANAEROBIC

2 CO2 + 2 C2H6O

2

Fermentation, a process that can occur in the absence of oxygen, partially breaks down carbohydrate by glycolysis to capture a small amount of energy in the form of ATP. The initial reactions of fermentation and respiration are the same, but fermentation stops after glycolysis whereas respiration continues into the Krebs Cycle. The carbohydrate leftovers are different depending upon the organism that performs the fermentation; usually one product is more oxidized (electron-poor) than the starting molecule and the other is more reduced (electron-rich). In the case of yeast fermentation, the products from one glucose (C6H12O6) molecule are two molecules of ethanol (C2H5OH) and two molecules of CO2. Human anaerobic (oxygen-free) muscle produces two molecules of lactic acid (C3H6O3). Even though the products are different, each fermentation results in a limited, anaerobic breakdown of carbohydrate with energy release. Since the process does not completely break down the carbohydrate, it does not release much energy that can be captured in the form of ATP. In yeast Biology Yeast Lab, Page 7

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB fermentation, there are 2 ATP molecules produced for each glucose molecule that is fermented. This is a low yield compared to that of respiration, but the ability to perform fermentation allows the yeast to survive and grow in environments where no oxygen exists (see Table 1). Gas Chromatography The major technique that is used to determine the type of organic molecules produced during fermentation is gas chromatography. Gas chromatography (GC) is the separation of compounds in the gas phase, depending on their relative ability to adsorb onto the column packing and their volatility into the gas phase at the temperature used. The gas chromatograph is a simple, sensitive instrument which can be used to separate and identify about 60% of all known organic compounds. The compounds to be separated are injected into a gas stream which passes through a column at a preset speed. Under a constant set of conditions in terms of temperature, gas flow rate, and column packing and size, repeated injections of a compound elute from (come out of) the column at a nearly constant time from injection. Different compounds elute at different times. One factor which affects elution time is the molecular weight of the compound; heavier compounds move more slowly through the column. Elution time is also affected by polarity and other factors. The column is first injected with known compounds called standards, and their retention times are determined. Then, unknown mixtures of compounds can be injected, and if the known compounds are in the mixture, their peaks can be recognized by their characteristic retention times. A gas chromatograph detects the presence of a compound in its eluate (exiting stream) by means of some property of the compound. One common method used by GC detectors is to compare the conductivity of a heated filament which is placed within a stream of pure reference gas (helium in our lab) to a heated filament placed in a stream of gas containing our sample molecules. When molecules from our sample pass the detector filament, the changes in conductivity caused by temperature changes are converted into electrical signals which appear as peaks on a computer data screen. Peaks seen in the eluate are plotted on a chart, and the integrated peak area is proportional to the concentration of the compound. Many GCs report the integral area of each of the peaks, following the plotted graph of the peaks. An approximate proportionality between peak height and concentration can also be seen (see sample printouts).

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB SAMPLE GAS CHROMATOGRAPH PRINTOUTS

Peak #1

Water / Peak #2 Methanol / Peak #3 Ethanol / Peak #4 Isopropanol Biology Yeast Lab, Page 9

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB Equipment Gas chromatograph Micro syringes (4) for standards, control, aerobic, anaerobic Hot plates Standards for GC: ethanol; mix of ethanol, methanol, and propanol; distilled water Supplies GROWING 3 1 2 1 set 600mL 45 gm 1/2pkg

YEAST CULTURES: (per class) Plastic 1 L Erlenmeyer flasks Aquarium pumps (2 flasks per pump) & gang valve 3' lengths tygon tubing Rubber stoppers: 1 2-hole/1 1-hole/1 solid and bent glass tubing to connect. DI Water Dextrose Active dry Yeast

DEMONSTRATION (to detect carbon dioxide): bottle Bromothymol blue indicator solution 2 100 ml beakers 1 Drinking straw DISTILLATION: 3 600 mL Beakers labeled A/B/C 10 100 mL Graduated Cylinders 10 125 mL Glass Erlenmeyers flasks 10sets distillation tubing and stopper 10 100 mL Glass beakers 10 Thermometers 20 3-finger clamps 10 Ring stands 10 Screw cap vials (labeled A, B, or C) 10 Hot plates Paper towels Ice MASS OF YEAST CELLS: 2 Top loading balances 10 Centrifuge tubes 1 Centrifuge 10 Bamboo skewers to clean centrifuge tubes. GAS CHROMATOGRAPHY: 3 25 µL syringes 1 set alcohol standards 2 Gas Chromatographs

Computer paper Helium gas Extension cords Biology Yeast Lab, Page 10

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB Laboratory procedures Before the laboratory, flasks of yeast culture should be prepared. We have found that five days in advance yields the best results (greatest difference between aerobic and anaerobic conditions.) You should have one aerobic/anaerobic flask set per class. We have 10 flasks which usually go out with the van. This will support 4 classes if you use two flasks for controls. If the teacher supplies flasks for the controls, these 10 flasks will support 5 classes. Additional flasks and pumps may be available if needed. Set up each one-liter flask as follows: 500 ml water (300 ml tap water for minerals and 200 ml distilled or deionized water) + 28 g dextrose (another name for glucose). Label the flasks: A = Aerobic, B = Anaerobic, C = Control. Add NO YEAST to the (C) control flasks. Add 1.5 g of active dry yeast to each of the other flasks. Table 2. Flask A (Aerobic)

Flask B (Anaerobic)

Flask C (Control)

300 mL Tap Water

300 mL Tap Water

300 mL Tap Water

200 mL DI Water

200 mL DI Water

200 mL DI Water

28 g Dextrose

28 g Dextrose

28 g Dextrose

1.5 g Yeast

1.5 g Yeast

NO Yeast

Air pumped in

NO Air added

NO Air added

The flask labeled A (aerobic) is stoppered with a 2-hole rubber stopper with a long aeration tube that becomes submerged below the liquid in the flask and a short escape tube for the air in the flask, which should be bent over or attached to a piece of Tygon (aquarium) tubing to prevent contamination. This flask will be vigorously aerated for 4-5 days. Be careful to provide sufficient aeration to prevent the aerobic flask from fermenting. Either a compressed air line or an aquarium pump can be attached to the long aeration tube in the flask. We have found that each aerobic flask should have a separate air pump, except for the large dual port pump which can support two flasks. Insert a cotton plug into the long aeration tube before connecting the pump. Aeration stones and agitation may help increase aeration efficiency. See Figure 1 for a diagram of the aerobic flask. The flask labeled B (anaerobic) is stoppered with a 1-hole rubber stopper containing a short tube that is attached to a long piece of Tygon tubing. The end of this tubing outside of the Biology Yeast Lab, Page 11

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB flask is placed under water in a 600 mL beaker. The yeast in this flask will use up the oxygen inside the flask and will ferment glucose to produce carbon dioxide. The CO2 will displace the remaining air, which will bubble out through the water. This flask will not contain oxygen after the first hour or two of the experiment. See Figure 2 for a diagram of the anaerobic flask.

The flask labeled C (control) contains no yeast and no air circulation is needed. A color coded system has been developed to reduce confusion when distributing yeast cultures. Aerobic is red, anaerobic is blue, control is green. The color coded system is used on the graduated cylnders, dispensing beakers (400ml-plastic), distillation vials, and culturing flasks and tubing. Also it may be desirable to color code syringes and GC used in analysis of products. NOTE: Please note that there are no student directions in the lab for Part I (demonstration) or for Part III (mass of yeast cells). This is because many teachers opted not to have students do these parts individually. PART I: Demonstration The purpose of the following demonstration is to verify that the gas being produced in both yeast and human respiration is carbon dioxide. If a gas sample is mixed with bromothymol blue indicator solution, the color of the solution will change from blue to green and finally to yellow as the carbon dioxide Biology Yeast Lab, Page 12

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB produces carbonic acid and drops the pH of the solution. After 24 hours, the production of CO2 can be demonstrated in the anaerobic yeast culture as follows: 1.

Place 50 mL of Bromothymol blue solution into a 125 mL Erlenmeyer flask.

2.

Place the exit tubing from the anaerobic culture flask into the solution and observe the results.

3.

Using a straw, exhale into a second beaker with bromothymol blue solution and again observe the results. Several students may demonstrate this as time permits. Caution the students not to blow so hard as to splatter the solution out of the flask (bromothymol blue will stain).

PART II: Distillation The products of the respiration or fermentation should be collected by distillation. For each culture, including the control, transfer the contents of the flask to a labeled 600 mL beaker for convenience in dispensing. Each lab group should obtain 50 ml of one of the cultures, noting which one they have, and place it into a 125 mL Erlenmeyer flask and assemble the distillation apparatus (see Figure 1 in student handout). Ethanol can be separated by distillation because it boils at 78°C, while water boils at 100°C. The hot plates for distillation should be turned on and the flask should be placed on the hot plate. Be sure the thermometer does not extend into the liquid. You want to measure the temperature of the vapors as the product distills. Use the thermometer to maintain the temperature of the vapors at 80-90° C. The flask should boil vigorously (boiling point of 95% ethanol azeotrope is 78.15°C). Do not overheat. Maintaining the temperature at the lower end of this range will give a slower distillation and better (i.e.purer) results. Wrap the glass tube with cool, wet paper towels to help condense the alcohol. Students should not add the cool wet paper towel to their distillation flask until the temp is above 70°C so that the wet paper does not heat up excessively before boiling of the culture begins. Be careful that students do not let excess water from their paper towel drip into the collection vial. When distillate begins to collect in the vial, turn off the hot plate and continue collecting several more drops. Students Biology Yeast Lab, Page 13

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB need to get only the first ten drops of distillate which should be the most concentrated alcohol mixture. If students collect several mL of fluid, chances are that they are collecting a lot of excess water. Now the distillate will be tested by means of gas chromatography. Please refer to the Gas Chromatograph instructions beginning on page 19. First, as a teacher demonstration, inject about one microliter of the ethanol standard into the gas chromatograph. Observe the time that it takes for the peak to appear. This should be done on both gas chromatographs. Then, a standard containing a mixture of water, methanol, ethanol, and propanol will be injected. Observe the time it takes for each of the four peaks to appear. When running standards, run a few samples through GC before you use the printouts for instruction. The first few samples may not give clear results. Have a print out for each GC available and labeled for student reference. Make sure that students use the standards for the same machine that they will be using for the analysis of their sample (A for aerobic, B for anaerobic, C can be on either-your choice) Have the students record the retention times and names of the standards onto their data tables. Notice that the retention times will vary slightly with each machine due to the condition of its columm and rate of gas flow. Remind students that they must run their samples through the same machine for which they are recording standard retention times. For convenience, the machines will be labeled A and B. Have students make predictions (Question #4) before injecting their sample in GC. Explain that even the aerobic culture will have some alcohol production due to incomplete aeration of the flask. Next, have students inject about one microliter of their distillate in turn into the gas chromatograph. This will take 810 minutes per group. Have students record their culture code letter (A, B, or C), the machine code number (A or B), and then identify the peaks by name and the percentage yield of each compound in their sample (see pages 7-8). This information will be written on their computer printout first, and then transfered to their data table. The students will need to refer to the data from the standards to identify the components of their sample. Doing examples on paper prior to the lab day will greatly increase the students' understanding of these procedures. They should see two peaks, one for water and one for ethanol. Any other peaks are contaminants and can be ignored for this lab. Have students average results from all the student samples Biology Yeast Lab, Page 14

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB before making conclusions about the results. Sloppy technique in distillation will produce results which do not follow the theory of the lab.

Note: In case no gas chromatograph is available, 2.5 ml of each distillate can be mixed with 1 ml of concentrated I2/KI and 1.5 ml of 1.5 M NaOH. If ethanol is present, an iodoform reaction occurs producing a yellow precipitate in about 10 minutes.

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB SAMPLE AEROBIC CULTURE GAS CHROMATOGRAPH RESULTS Yeast culture A

Gas Chromatograph B

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB SAMPLE ANAEROBIC CULTURE GAS CHROMATOGRAPH RESULTS Yeast Culture B

Gas Chromatograph B

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TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB PART III: Mass of yeast cells (Optional, if time permits. Can be done while waiting for distillation to occur or for opportunity to inject sample) The purpose of this experiment is to see how much growth was possible for the aerobic versus the anaerobic yeast culture. For this experiment, have students label and mass a centrifuge tube on the top loading balance to .01 gms. Then have students tranfer aproximately 10 ml of a yeast culture to their tube from the culture remaining in the beakers from Part II. This volume will fill up the tube to its white label. Be sure that the students stir up the culture in the beaker before transferring it to their tubes, so as to get the yeast cells mixed evenly. This is important if you want to get consistent results. Have students centrifuge their sample for 5 minutes. Have them pour off the supernatant (the liquid portion) into the sink. The mass of the remaining pellet in the bottom of the centrifuge tube can be estimated by massing the tube again on the top loading balance. (If necessary, try to dry the inside of the tube with a kimwipe before massing it). The tube mass should be subtracted from the combined mass of the (tube + pellet) to get the wet mass of the cells. This is a crude approximation but if means of the masses for the class are compared, there should be a difference between aerobic and anaerobic cultures. Aerobic cultures should have larger masses due to increased energy utilization and reproduction. When performing this portion of the lab it may be better to change the sequence of weighing the tubes to help you with clean up. Have the students weigh their tubes with a yeast cell pellet first and then again after they have rinsed out the pellet (using a bamboo skewer or a bent paper clip helps remove the cells from the tip of the centrifuge tube). The water which is inside the tube can be ignored because it is present at both weighings. This is a crude technique, but if you average all the data from the entire set of classes or completely centrifuge the contents of the distribution beakers (400ml plastic) you should find a difference in the weight of the yeast cells between the two cultures. Again making conclusions from a single sample does not yield good conclusions. References Warren D. Dolphin, Biology Laboratory Manual, Third Edition. Wm C Brown Publisher, 1992. Campbell, Neil. Biology, Second Edition. Benjamin Cummings, Menlo Park, 1991. Pietrzyk, D and Frank, C. Analytical Chemistry, An Introduction. Academic Press, New York and London, 1974. Biology Yeast Lab, Page 18

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