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Lab report: Cellular respiration and fermentation Title In this experiment we observed the conversion of succinate to fumarate by using DCPIP (dichlorophenol-indophenol) and using fermented yeast to measure what effect different sources of sugar and different temperatures has on the production of carbon dioxide. Introduction There are two types of respirations that occur in the mitochondria that we will be looking at in this experiment known as, aerobic and anaerobic. Cellular respiration is the process of the cell converting sugar into energy, the energy it produces is known as ATP, which our bodies need. In this it goes through two different enzymatic pathways during the break down the chemical bonds such as citric acid, electron transport system and glycolysis. One way that the cell produces ATP is through aerobic conditions, where oxygen is the reactant. It begins with glucose and breaks it down using oxygen to create ATP. In this experiment we will be a lima bean as a suspension of mitochondria. It will allow us to record the conversion of succinate to fumarate. This goes through the Krebs cycle; it goes through an aerobic cycle to create energy through losing electrons from different types of carbohydrates. We also will be using DCPIP dye as an alternate electron acceptor. By using this dye, we can use a spectrophotometer to determine the amount of the percent transmittance and the color change. The process of the conversion of succinate to fumarate. In this reaction it reduces FAD to FADH2, this produces freer electron acceptor. When the free electrons go into the electron transport chain the DCPIP dye changes colorless when the dye “takes” the electrons. We will use different levels of succinate to the mitochondrial suspension and record the amount of transmittance there was. In the anaerobic cycle it has less oxygen present but still breaks down oxygen in a way to produce a smaller amount of ATP. It also produces more NAD+ for the aerobic cycle to pack more glucose in to go through glycolysis and finally make more ATP. Because of the lack of oxygen in this cycle, pyruvic acid is put through fermentation, lactaid and alcoholic fermentation although we humans cannot ferment alcohol. Fermentation is still breaking downing glucose but with minimal to no oxygen. In fermentation it produces waste such as carbon dioxide and takes place in the cytoplasm. In the experiment we will be working with yeast because in baking we see the fermentation of yeast. With the yeast it activates at certain temperatures and at those temperatures can it produce more or less carbon dioxide. We will be measuring the amount of carbon dioxide produced at different temperatures and seeing the amount of carbon dioxide based on the yeast reacting with different types of sugars. By using a test tube to incubate a water and yeast mixture. We incubate the tubes at different temperatures and record the final amount of carbon dioxide. For the alternative sugars, each tube will contain a different sugar mixed with water and yeast, then record the amount of carbon dioxide produced.

Hypothesis 1: If more succinate added, then there will be more reduction of DCPIP causing the color to change clear on spectrophotometer. Hypothesis 2: If the yeast were in temperatures around 45 degree Celsius and 60 degrees Celsius then it will produce the most amount of carbon dioxide because if the yeast is too hot of an environment it will cause the yeast to die.

Hypothesis 3: If the alternative sugar source is fructose, then there will be a greater production of carbon dioxide because yeast can break down monosaccharides easier than disaccharides.

Materials and Methods: I followed the procedure and used the materials as directed by the lab manual (Malinoski et al., p 5-3 – 5-8) Results For the cellular respiration experiment, the results measured the percentage of transmittance and the amount of succinate solution added. The test tubes were measured in increments of 5 minutes starting at 5 minutes reaching 30 minutes, while the spectrophotometer was set at 600nm. Tube 1 was set to be blank for the experiment and the percent transmittance was 100nm during the duration of 30 minutes. Tube 2 results started at 22.3 at 5 minutes and ended at 41 at 30 minutes. Tube 3 started at 22 at 5 minutes and ended 72.2 at 30 minutes. Tube 4 started at 23.3 and ended at 80.7 at 30 minutes. Tube 5 at 38.4 and ended at 34.8 at 30 minutes. The results of the experiment where we measure the production of carbon dioxide using different temperatures in the yeast and water mixtures. In tube one the temperature was 0 degrees Celsius and the final production of carbon dioxide was 0mL. In tube two the temperature was in 22 degrees Celsius and the final production of carbon dioxide was 0.5mL. In tube 3 the temperature was 45 degree Celsius and the final production of carbon dioxide was 5mL. In test tube four the temperature was 60 degree Celsius and the final production of carbon dioxide was 3mL. In tube five the temperature was 100 degrees Celsius and the final production of carbon dioxide was 0mL degrees Celsius. The results of the experiment where we measure the production of carbon dioxide using different types of sugar on fermented yeast. Tube six contained galactose and had an initial carbon dioxide of 1mL and in total had 0.3mL of carbon dioxide produced. Test tube seven contained sucrose, it had an initial of 1mL of carbon dioxide and had a total of 4mL of carbon dioxide produced. Test tube eight contained fructose, it had an initial of 1mL carbon dioxide and had a total of 4.5mL of carbon dioxide produced. Test tube nine contained maltose, it had an initial of 1mL carbon dioxide and had a total of 2.5mL of carbon dioxide. Test tube ten contained lactose, it had an initial of 1.5mL of carbon dioxide and in total produced 0.5mL of carbon dioxide. Test tube eleven contained lactose plus lactaid and had an initial carbon dioxide of 1mL and produced a total of 7.25mL carbon dioxide. Discussion In the aerobic respiration experiment my hypothesis was correct and supported because there was a reduction in DCPIP. In tube four where there was more succinate added after 30 minutes there was the greatest amount of reduction at 80.7. The more succinate added the more reduction was shown in the spectrophotometer and the DCPIP showed there more electrons gained. While tube five had a low amount of succinate added and after 30 minutes resulted with the lowest percent transmittance at 34.8. The process of the conversion of succinate to fumarate. In this reaction it reduces FAD to FADH2, this produces more free electron acceptor. When the free electrons go into the electron transport chain the DCPIP dye changes colorless when the dye “takes” the electrons.

In the temperature and fermentation experiment my hypothesis was correct and supported because at 45 and 60 degrees Celsius it had the highest amount of CO2 produced at 5mL and 3mL. These temperatures were found to work best for the yeast and water mixture to create more CO2, while at 1 and 100 degrees Celsius the amount of CO2 was both 0mL. This was because there wasn’t enough heat for the yeast to activate and too much heat caused it to die. When it’s at a temperature such as 40 degrees Celsius it causes there to be enough energy to react. At too high of temperature, it can lose this energy and die. This is because there is more collision between the reactants.

In the alternative sugar’s fermentation experiment my hypothesis was incorrect because lactose + lactaid produced the most amount of CO2 at 7.25 mL compared to the other sugars such as fructose. “Lactase insufficiency means that the concentration of the lactose- cleaving -galactosidase, also called lactase, in the brush border membrane of the mucosa of the small intestine is too small. This hypolactasia causes insuffient lactose maldigestion” (de Vrese et al. page 421) Yeast uses oxygen to release ATP from carbohydrates and the more carbs it has the more it grows so having a disaccharide the yeast releases creates more energy causing it to grow more.

References Malinsoki, chris. Biology 1107: Principle of Biology-aLaboratory Manual. Dep. Biological sciences at the University of Connecticut, 2021

Russell, Peter J., et al. Biology: the Dynamic Science. Cengage, 2021

Michael de Vrese, Anna Stegelmann, Bernd Richter, Susanne Fenselau, Christiane Laue, Jürgen Schrezenmeir, Probiotics— compensation for lactase

insufficiency, The American Journal of Clinical Nutrition, Volume 73, Issue 2, February 2001, Pages 421s–429 Michael de Vrese, Anna Stegelmann, Bernd Richter, Susanne Fenselau, Christiane Laue, Jürgen Schrezenmeir, Probiotics— compensation for lactase insufficiency, The American Journal of Clinical Nutrition, Volume 73, Issue 2, February 2001, Pages 421s–429

Micheal de Vrese, Anna Stegelmann, Bernd Richter, Susanne Fenselau, Christiane Laue, Jurgen Schrezenmeir, Probiotics – compensation for lactase insufficiency,The American Journal of Clincial Nutrition, Volume 73, Issue 2, February 2001, Pages 421 .

Tables/Figures Figure 1

Percent Transmittance effects on Succinate 120

Percent transimittance

100 80 60 40 20 0

1

5

10

15

20

25

30

Time in minutes tube 1

tube 2

tube 3

tube 4

tube 5

Legend for figure 1: Percent transmittance effects on succinate This measured the percentage of transmittance and amount of succinate of solution added. The test tubes were measured of increments of 5 minutes going up to 30 minutes and beginning at 5 minutes. Tube 1 was used as a blank/control and remained the same during the 30-minute test timed

Figure 2

Effect of different temperatures on the production of CO2 6 5

Producded carbon dioxide (mL)

4 3 2 1 0

be Tu

1

(0

g de

re

e

el sc

s) s iu

be Tu

2

2 (2

gr de

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el sc

si u

s)

be Tu

3

5 (4

g de

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) us lsi e sc

be Tu

4

0 (6

gr de

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el sc

si u

Temperature of incubated tubes ( in celsius)

s)

be Tu

5

00 (1

g de

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Legend for Figure 2: Effect of different temperatures on the production of CO2 This measured the production of carbon dioxide at different temperatures. The test tubes were incubated in different temperatures, the temperatures were recorded in Celsius.

Figure 3

Effect of alternatives sugars on the production of CO2 8

Produced carbon dioxide (mL)

7 6 5 4 3 2 1 0

Galactose

Sucrose

Fructose

Maltose

Lactose

Lactose + Lactaid

DIfferent types of suagr

Legend for figure 3: Effect of alternatives sugars on the production of CO2 This was measured in a 30 minute window with six different sugars : galactose, sucrose, fructose, maltose, lactose, lactose + lactaid....