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Fermentation
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Fermentation Lab

Do Different pH Levels Affect the Amount of Carbon Dioxide in Fermentation? Abstract In this lab, different levels of pH was used to see if the amount of carbon dioxide, which is the dependent variable, has a significant amount of change in fermentation as the independent variable. The research hypothesis states the rate of fermentation will change based on the pH conditions, indicating that an optimal pH exists at which fermentation will occur at an increasing rate. The null hypothesis states is the surrounding pH is altered, fermentation will occur at the same rate. After the data was obtained, a TTest was performed as well as graphs were constructed. Due to the p-values being greater than 5%, we the data is not significantly different and we fail to reject the null hypothesis stating if the surrounding pH value is altered, fermentation will occur at the same rate. Introduction Fermentation is of any metabolic pathway which occurs in the cytosol that regenerates oxidizing agents, for example NAD+. (Freeman, 2017). Fermentation transfers electrons to an electron acceptor which allows pathways to continue making ATP with little to no oxygen. (Freeman, 2017). Fermentation does not have an electron transport chain which regenerates NAD+ due to the absence of oxygen. (Freeman, 2017). Yeast cells, which was the organism used in the experiment, conduct ethyl alcohol fermentation. (Khan Academy). Ethyl alcohol fermentation involves two chemical reactions: pyruvate (a three-carbon molecule) converts to acetaldehyde (a two-carbon molecule). (Khan Academy). The third carbon is released in the form of carbon dioxide. (Khan Academy). The second reaction has the acetaldehyde reduced to ethanol, and NADH is oxidized to NAD+. (Khan Academy). With alcohol fermentation, yeast cells are able to produce NAD+, an energy carrier. (Khan Academy). In this lab, different levels of pH was used to see if the amount of carbon dioxide, which is the dependent variable, has a significant amount of change in fermentation as the independent variable. The concentration of protons in a solution determining if it’s acidic or basic is what pH stands for. (Freeman, 2017). Solutions that are acidic have a proton concentration that is less than seven. (Freeman, 2017). Acidic molecules tend to release more protons into the solution. (Freeman, 2017). Solutions that are basic have a proton concentration that is more than seven. (Freeman, 2017). A pH level that equals to seven is considered neutral because it is the concentration of pure water. (Freeman, 2017). The purpose of this lab was to reject or accept either the research hypothesis. The research hypothesis states the rate of fermentation will change based on the pH conditions, indicating that an optimal pH exists at which fermentation will occur at an increasing rate. The null hypothesis states is the surrounding pH is altered, fermentation will occur at the same rate.

Methods In the lab, materials were available at the bench station. At the station, the materials available were two beakers labeled “With Glucose” and “Without Glucose”, approximately six one milliliter pipettes, an erlenmeyer flask with yeast, 5% glucose, six syringes, and deionized water. The four different levels of pH (3, 5, 7, and 11) were obtained from the instructor. In this lab, the pH level 7 was used as the control. At first, 1 mL of each pH level was put in four separate 150 mL beakers. After the pH levels were obtained, 10 mL of yeast and 9 mL of 5% glucose were inserted in the beakers containing the pH levels 11 and 7 with a micropipette. After each beaker with the solutions were swirled, a timer for five minutes was set to allow each solution to intubate. As the timer is going, the respirometer was prepared. For the respirometer, a 1 mL pipette was obtained. With the 1 mL pipette, the point was placed into a 50 mL of deionized water. A small amount of water moved into the pipette by capillary action. After the water droplet entered the pipette, the pipette was carefully inverted. The water droplet traveled to approximately at the zero reading on the pipette. Repeat this process for the rest of the pipettes provided at the lab station. After the five minutes, each solution was drawn into three syringes to the 3 mL mark. After the solution were drawn up, the syringe was inverted and was drawn an additional 1 mL of air into it. Then, for pH 11 and 7, each set of syringe was attached to a pipette containing the water therefore at the same time there were three syringes attached to three pipettes for pH 11 and pH 7 were occurring at the same time. A timer was set for 10 minutes. At each minute mark, the pipette was read and recorded in the data tables for each pH level triplicate. After the 10 minutes, the syringes and beakers were cleaned with DI water and pouring the solution out into the waste container. The steps above were repeated for the pH levels 5 and 3. The pipette readings were recorded in the data tables. After the data was obtained, a T-Test was performed as well as graphs were constructed. Results

Data Table 1 pH

1

2

3

4

5

6

7

8

9

10

3

0

0.01

0.039

0.092

0.165

0.261

0.375

0.500

0.639

0.791

5

0

0.037

0.051

0.082

0.130

0.184

0.260

0.350

0.452

0.575

11

0

0.020

0.035

0.065

0.120

0.175

0.280

0.400

0.471

0.545

Control

0.01

0.018

0.031

0.057

0.09

0.14

0.207

0.284

0.385

0.504

(pH 7)

Data Table 2

pH

1

2

3

4

5

6

7

8

9

10

3

0.01

0.024

0.068

0.135

0.198

0.297

0.412

0.541

0.690

0.851

5

0.01

0.025

0.042

0.071

0.114

0.172

0.248

0.332

0.449

0.572

11

0.010

0.020

0.030

0.060

0.110

0.170

0.290

0.421

0.501

0.585

Control

0.01

0.02

0.036

0.060

0.094

0.152

0.220

0.331

0.441

0.561

(pH 7)

Data Table 3 pH

1

2

3

4

5

6

7

8

9

10

3

0.025

0.071

0.100

0.132

0.175

0.238

0.321

0.410

0.529

0.691

5

0.040

0.049

0.061

0.085

0.112

0.150

0.210

0.274

0.357

0.460

11

0.025

0.040

0.060

0.090

0.135

0.190

0.290

0.419

0.530

0.673

Control

0.01

0.019

0.034

0.068

0.110

0.180

0.254

0.370

0.490

0.620

(pH 7)

Average from Triplicates (Data 1, 2, and 3) pH level

Average

3

0.293

5

0.198466667

11

0.2253333

Control (pH 7)

0.19353333

T-Test Results pH 3 vs Control

pH 5 vs Control

pH 11 vs Control

0.09252218

0.91583391

0.53539831

Graphs

(*Note: Series 1: pH 3 Series 2: pH 5 Series 3: pH 11 Series 4: pH 7)

Discussion The research hypothesis states the rate of fermentation will change based on the pH conditions, indicating that an optimal pH exists at which fermentation will occur at an increasing rate. The null hypothesis states is the surrounding pH is altered, fermentation will occur at the same rate. According to the t-tests above, if the p-value is greater than 0.05, then the null hypothesis is not rejected. The t-test values that were obtained were 0.09252218 between pH 3 and pH 7 which was the control, 0.91583391 between pH 5 and the control, and 0.53539831 between pH 11 and the control. The values that were used to obtain the p-value from the t-test were the whole data set from each pH value (3, 5, and 11) compared to the whole data set from the control (pH 7) from each data set or triplicate. Due to the p-values being greater than 5%, we the data is not significantly different and we fail to reject the null hypothesis stating if the surrounding pH value is altered, fermentation will occur at the same rate. According to the graphs above in each data table, the pH value that had the most carbon dioxide released was the one with the lowest pH value which in this experiment was pH 3. The pH 3 value can be used as the optimal pH for fermentation of yeast or that yeast fermentation is aided by a more acidic environment. ( Adachi, E.). The pH value 7, which was the control, actually slows down the fermentation process. In low pH value solutions, the concentration of hydrogen ions are greater thus increasing the rate of fermentation. But

according to the graphs above, the data that does not support the statement of “lower pH value increases the rate of fermentation” is the pH 11. The value pH 11 is considered a basic state, but in Data Table 3, there’s a point where pH 3 and pH 11 intersect. This data shows the inconsistency of the pH values therefore more usage of different pH values may provide more clarification. Though cross contamination and other factors were tried to be limited, it is possible that the experimenters mixed pH solutions incorrectly. There also might have been a problem with accurate measurements as well. For future experiments, more usage of a more variety of pH values would show more differences.

Literature Cited

Adachi, E., Torigoe, M., Sugiyama, M., Nikawa, J. I., & Shimizu, K. (1998). Modification of metabolic pathways of Saccharomyces cerevisiae by the expression of lactate dehydrogenase and deletion of pyruvate decarboxylase genes for the lactic acid fermentation at low pH value. Journal of fermentation and bioengineering, 86(3), 284-289. Fermentation and anaerobic respiration | Cellular respiration (article). Khan Academy. [accessed

2017 Nov 7]. https://www.khanacademy.org/science/biology/cellular-respiration-andfermentation/variations-on-cellular-respiration/a/fermentation-and-anaerobic-respiration Freeman S, Quillin K, Allison LA, Black M, Podgorski G, Taylor E, Carmichael J. Biological science. Boston: Pearson; 2017....