Fermentation Formal Lab Report PDF

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FERMENTATION OF VARIOUS SUGARS IN BAKER’S YEAST Abstract Living organisms are all similar in that they can take energy from their environments as a means to do biological work. Cellular respiration and alcohol fermentation are examples of this characteristic. Yeast, a single celled organism conducts fermentation when sugar is present, to make chemical energy, and in the process produce alcohol and carbon dioxide. Experiments were performed to understand this process better. One experiment focused on yeast and glucose, while another observed how yeast fermented and produced CO 2 with different types of sugars. Respirometers were used to record the amount of CO 2 production. Both experiments proved to be informative, with all expected sugars fermenting the yeast and producing carbon dioxide. However, the exact predictions made were not fully supported by the results and when comparing to the results of a different group, questions are brought to the surface on the performance the experiments. Were there errors? Should the experiment be administered again with minor changes to protocol?

Introduction Cellular respiration is a process that most living organisms undergo to create and obtain chemical energy in the form of adenosine triphosphate (ATP). The energy is synthesized in three separate stages of cellular respiration: glycolysis, citric acid cycle, and the electron transport chain. Glycolysis and the citric acid cycle are both anaerobic pathways because they do not need oxygen to form energy. The electron transport chain however, is aerobic due to its use of oxidative phosphorylation. Oxidative phosphorylation is the process in which ATP molecules are produced with the assistance of oxygen molecules. (Campbell et al 2008) Fermentation is a process adopted, typically, by anaerobic organisms to obtain ATP without the use of oxygen. Saccharomyces cerevisiae, or baker’s yeast, is a unicellular fungus that uses both fermentation and respiration when needed. Organisms that have this ability are called facultative anaerobes. When yeast is in the presence of oxygen it performs cellular respiration, but when oxygen is absent it undergoes alcohol fermentation. In alcohol fermentation, the sugar is converted into two 3-Carbon sugars known as pyruvate. The pyruvate is then converted into ethanol alcohol in two steps. In the first step, it is converted into acetaldehyde with the release of carbon dioxide (CO2), the independent variable of the experiment. Next the acetaldehyde is reduced to ethanol. (Campbell et al 2008) The experiments performed in Dr. Nuss’ Biology classroom were designed to better understand fermentation. Two experiments were performed a week apart. The first experiment, experiment A, studied the fermentation exclusively between yeast and glucose. A respirometer was used to observe the amount of CO 2 produced during fermentation. Based on previous knowledge of fermentation activity, it can be

2 concluded that a test tube containing a greater volume of yeast, rather than glucose, would have the most CO2 production, because fermentation of glucose is dependent on yeast. The second experiment, experiment B, was designed by the students, with each lab group making their own design. The purpose of this group’s research was to see the effects of yeast fermentation with glucose, sucrose, fructose and lactose. The structures of each of the sugars suggests that the test tube containing glucose would ferment the most because it would require less work to break down into pyruvate and acetaldehyde (Fig. 1&2). Sucrose would follow being a bit more complicated to break down, while fructose would produce even less CO2, because of its 5-ring structure differing greatly from glucose’s 6-Carbon structure (Fig. 1). The yeast, because of its lack of appropriate enzymes, would not ferment lactose.

Figure 2 Molecular structure of lactose. Photo courtesy of http://www.rpi.edu/dept/chemeng/Biotech-Environ/FUNDAMNT/lactose.gif

Methods Figure 1 Molecular structures of glucose, fructose and sucrose.

Experiment A. The experiment began by practicing how to properly use and make a respirometer. The resipormeters used in the experiment consisted of a 1 mL serological pipette, aquarium tubing, binder clip, and a test tube. First, the aquarium tubing was attached to the pipette, which was then placed in a beaker filled half way with water. A pipette pump was attached to the tubing and then drew water from the beaker, up to the 0 mL mark. With the pipette pump still attached, the tubing was bent and the clip placed at the bend, as a way to prevent the liquid from falling back into the tube. The clip was slightly removed and immediately replaced, to adjust the liquid level so that it was as close to the O mL mark as possible. Photo courtesy of http://drpinna.com/wpcontent/uploads/2011/04/glucose-fructose-andsucrose.jpg

Next the test tubes for the experiment were prepared. The test tubes were labeled 1, 2, 3, and 4. Necessary amounts of water and glucose were added to all test tubes (Table 1). The yeast was added last to avoid reaction occurring sooner than needed. The test tubes were placed in a test tube rack and then in a 30°C water bath for 5 minutes. As soon as the five minutes had passed, the respirometers were added to

3 each test tube and made ready for obtaining data. Immediately after they were added, the timer started. The liquid level was recorded every two minutes for 20 minutes. Table 1. Test tube set-up for experiment A. Tube # DI H2O Yeast Glucose 1 5 mL 0 mL 3 mL 2 6 mL 2 mL 0 mL 3 3 mL 2 mL 3 mL 4 1 mL 4 mL 3 mL Experiment B. The second experiment was executed similar to part A, with minor changes, and a week later. All test tubes were labeled and then filled with necessary liquids, with yeast being added last (Table 2). The test tubes were then placed in a 30°C water bath for five minutes. After five minutes had passed the newly made respirometers were added to the test tubes and the timer started at 0 minutes. For the next 20 minutes the level of liquid in the pipette was recorded every two minutes. Table 2. Test tube set-up for experiment B Tube # Type of Sugar DI H20 Yeast Sugars 1 Glucose 3 mL 2 mL 3 mL 2 Sucrose 3 mL 2 mL 3 mL 3 Fructose 3 mL 2 mL 3 mL 4 Lactose 3 mL 2 mL 3 mL

Results

Figure 3 CO2 production of experiment A, through the fermentation of yeast with glucose

Experiment A.

4 Figure 3 shows the amount of CO 2 produced over a 20 minute time period. Test tubes containing either only yeast, or only glucose, produced almost no CO 2, if any at all. Test tubes 3 and 4, which contained both yeast and glucose, produced a significant amount of CO2 (Table 1). The test tube containing more yeast than glucose, produced the most CO2 and in a faster amount of time than the tube containing a larger amount of glucose over the yeast. Figure 4 The production of CO2, through fermentation of yeast and various sugars, over a period of time.

Experiment B. Figure 4 demonstrates the amount of CO2 produced when yeast fermented with various sugars (Table 2). CO2 was produced in all tubes ranging from 0.06-0.9 mL. In the time period of the experiment, the test tube containing sucrose had reached the maximum amount of production before the 20-minute mark, while the test tube containing glucose had reached the maximum at the very end of the experiment. Fructose had produced more CO2 than expected, while lactose produced a negligible amount of CO2 as predicted.

Discussion The purpose of these experiments was to better understand fermentation by yeast. Both experiments worked with yeast and a sugar. The first experiment had two controls, tubes 1 and 2 (Table 1), which each contained either yeast or glucose. The remaining tubes both contained yeast and glucose. Tube 4 had more yeast than glucose while tube 3 had more glucose than yeast. We had predicted that the test tube containing more yeast would produce a higher volume of CO2. Figure 2 supports our prediction. Tube 4 had produced the most CO2, while tubes 1 and 2 showed almost no production. Tube 3 produced carbon dioxide, but not as much as tube 4. This was because there was much less yeast to ferment the amount of glucose present.

5 In experiment B, we used various sugars to ferment the yeast (Table 2). Upon examining the structures we determined that glucose would show more production of CO2 than the other sugars, because of its structure and the results obtained from experiment A. Sucrose would evolve the next highest amount of CO 2, because it’s structure was a bit more complex, being a disaccharide (two sugars) made up of glucose and fructose (Fig. 1.1). We felt that it may take more energy to break down the sugar. We also considered the fact that a 6-Carbon sugar attached to a 5-Carbon sugar would limit production of CO2. Fructose, a 5-Carbon sugar, was not taken into much consideration in the amount of carbon dioxide production; however we did believe that some CO2 would evolve from the combination. We predicted that lactose would produce almost no carbon dioxide. The disaccharide was complex and we assumed there were no enzymes in the yeast to break down galactose. Our results both refuted and supported our predictions. While glucose did produce the maximum amount of CO 2, sucrose also produced the same and in a shorter amount of time (Fig. 3). The results showed that sucrose produced more CO2 and at a faster rate than glucose. Fructose, as we predicted, produced a small amount of CO2 and lactose produced almost no CO2. It is possible that yeast has enough enzymes to break down the sucrose, allowing for rapid fermentation of the sugar. When looking at the results from Alhasan et al (2011), who tested all the same sugars as us, their glucose mixture produced the maximum amount of CO2 in the 20-minute time frame while fructose produced approximately 0.79 mL and sucrose produced 0.77 mL. These results contradict ours completely. Assuming that they had executed their experiment perfectly, the only conclusion that can be made about the varying results is that when the respirometers were being read for liquid levels, they were pulled out completely from the sugar-yeast mixture. However, this was consistent throughout both experiments. There are other possible experiments that can be conducted to determine what sugar best ferments with yeast. The experiment can be repeated again, twice with the respirometers remaining in the mixtures when liquid levels are being read and once more with them being pulled out. It is also possible and would be interesting to work with compressed yeast. Compressed yeast, often used for baking breads, is a dry solid form of yeast packed into blocks. Would placing simple syrup (sugar dissolved in water), cause an immediate reaction? Are other things, such as flour, necessary for a reaction to occur? Would the reaction be similar to putting salt on an ice cube, where the ice begins to “melt”?

Acknowledgement Many thanks to the NEIU Biology Department, for providing equipment and substances necessary and to Dr. Nuss for reviewing and providing comments on the draft. Also, many thanks to my lab mates, Mohammed Hadiyousufi and Amanda Hartjes, for the collaboration, effort and work put into the experiment. Also to Alhasan et al. for making their results available for comparison.

References

6 Campbell, N., Reece, J., Urry, L., Cain, M., Wasserman, S., Minorsky, P., Jackson, R., (2008) Biology. 8th ed. Pearson Education, San Francisco Alhasan, Haider, Salamah, Velasquez, (2011) Biology 201 Fermentation Lab. Northeastern Illinois University, Chicago Illinois....