Cellular Respiration Lab Report PDF

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Relationship Between Succinate Presence and Percent Transmittance of DCPIP in Examining Aerobic Respiration and Relationship Between Temperature, and the Presence of Different Types of Carbohydrates on CO2 Gas Production in Examining Fermentation in Yeast Samples

Hypothesis If yeast samples are combined with varying amounts of succinate in addition to phosphate buffer, mitochondrial suspension and 1 mM DCPIP and allowed to perform aerobic respiration for 30 minutes at room temperature, then the sample with the greatest amount of succinate will display the greatest increase in percent transmittance at 600 nm over time and thus display the highest rate of aerobic respiration due to its participation in the electron transport chain during cellular respiration. If yeast samples are combined with glucose placed in different temperature environments including 0°C, 20°C, 45°C, 60°C, and 100°C and allowed to perform fermentation for 30 minutes, than the increase or change in amount of gas in mL will be the greatest toward the middle of the temperature spectrum around 45°C thus indicating the highest rate of fermentation and the most preferable conditions for fermentation among yeast samples. If yeast samples are combined with carbohydrates including galactose, sucrose, fructose, maltose, lactose, and lactose + lactaid and allowed to perform fermentation for 30 minutes at 45°C then the yeast mixed with the lactose + lactaid carbohydrate would display the greatest change in CO2 volume from beginning to end due to the ability of lactaid to expedite the process of breaking down lactose thus increasing the rate of fermentation. Introduction Percent transmittance is used to measure the percent of light absorbed by a given sample at a specified wavelength. The DCPIP used in samples when testing the effects of succinate starts off blue and as it is reduces; the blue color begins to disappear yielding a clear solution. The DCPIP serves as an electron donor aiding in pumping protons across a membrane and the faster rate of proton movement across the membrane the faster the rate of respiration (Le Laz 2014). It is by means of the change of percent transmittance of DCPIP as it is reduced that the rate of aerobic respiration was observed against differing amounts of succinate. Fermentation, a form of anaerobic respiration, yields CO2. In examining the amount of CO2 produced over a 30 minute time period by identical yeast samples placed in different

temperatures we can roughly determine the rate of fermentation. Temperature is a factor that affects the rate of respiration as the right amount of heat can provide preferable conditions for the execution of necessary redox reaction and through examining the differences in CO2 output across the varying temperature samples an optimal temperature for performing anaerobic respiration can be observed. Fermentation as performed with glucose deviates in performance when enacted using various other carbohydrate compounds. As the structure of these carbohydrates will differ from that of glucose, the testing of these simple sugars aids in the assessment of how the rate of anaerobic respiration differs not only as it relates to these other compounds but how the addition of an enzyme such as in the lactose + lactaid compound, ultimately expedites the respiration process in the presence of simple sugars. Materials and Methods Materials and procedures for all experimental components followed directly as listed within Biology 1107 Lab Manual (Lombard 2017). Results In testing the relation between amount of succinate present and percent transmittance, thus rate of aerobic respiration over time, percent transmittance at 600 nm was tested over the course of 30 minutes and recorded every 5 minutes. The following beginning and ending percent transmittance was recorded for each tube: tube 1; 100-100, tube 2; 30.5-73.4, tube 3; 30.5-98.4, tube 4; 32.6-109.3, tube 5; 67.8-68.4. In testing the relation between temperature and CO2 production, or fermentation, temperatures 0° C, 20°C, 45°C, 60°C, and 100°C were tested as conditions for aerobic respiration using yeast sample and yielded the following volumes of CO2 gas production over a 30 minute time period, 0.75 mL, 1.5 mL, 7.5 mL, 4.5 mL, 0 mL. In testing the relation between the presence of varying carbohydrates and volume of CO2 produced or fermentation, carbohydrates tested included, galactose, sucrose, fructose, maltose, lactose, and lactose + lactaid all of which yielded net CO2 volume increases of the following, 0.3 mL, 5.5 mL, 4.4 mL, 2.5 mL, 0.5 mL, and 8 mL respectively. Conclusions Observing the effects of amount of succinate on percent transmittance over time,. Our hypothesis was ultimately supported by the readings provided by the three experimental tubes, 2,

3, and 4 containing 0.0 mL, 0.1 mL and 0.2 mL of succinate with the largest percent transmittance by the end of the 30 minute period being 109.3 in the tube with 0.2 mL, succinate while only 98.4 and 73.4 were the final reading of tubes 3 and 2 containing 0.1 mL and 0 mL respectively. The increased rate of DCPIP reduction in tube 4 containing the greatest amount of succinate corresponded with the highest rate of aerobic respiration among the samples thus aiding in displaying a positive correlation between succinate present and the speed of electron transport due to increased availability of free electron due to FAD to FADH reduction completed with the assistance of succinate and subsequently the flow of protons across the membrane leading to ATP production (Freeman 2017). Observing the effects of temperature on CO2 production and the rate of fermentation, our hypothesis was again supported by our attained data with 45°C yielding the greatest increase in CO2 volume. The volume of CO2 produced over the course of 30 minutes by the yeast sample in 45°C totaled 7.5 mL while the volumes of CO2 produced by the other samples only ranged from 0-4.5 mL. It could be deduced that 45°C was likely an optimal temperature at which yeast were able to perform fermentation at the highest rate thus producing the largest volume of CO2 over the given time interval. Furthermore, when observing the CO2 production and rate of fermentation of the boiled yeast, or 100°C conditions, there was no change in CO2 levels over the 30 minute time period thus indicating the absence of fermentation which could support the notion that the heat got so high that the yeast were no longer capable of performing fermentation. Observing the effects of various carbohydrates on fermentation, our final hypothesis was supported. All of the single carbohydrates showed changes in CO2 less than that of the regular glucose when allowed to perform fermentation at the optimal 45°C however, when the combination of lactose and lactaid was added to the yeast, the final net change in CO2 over the 30 minute time period was 8 mL, 0.5 mL more than was seen in the fermentation and CO2 production of glucose at the optimal 45°C. Taking into account the results of the combination of lactose and lactaid in comparison to the carbohydrates added alone, it can be further hypothesized that the rate of fermentation was seen to increase as a result of the lactaid’s ability to assist in the break down of lactose thus allowing for expedited anaerobic respiration and hence increasing CO2 production over time.

Literature Cited Freeman, Scott et al., 2017, Biological Science, Third Custom Edition for University of Connecticut-Storrs, 3rd edition, New York, NY, USA, p. 1027 Sébastien Le Laz, Arlette Kpebe, Marielle Bauzan, Sabrina Lignon, Marc Rousset, Myriam BrugnaPLoS One. 2014; 9(1): e86343. Published online 2014 Jan 22. doi: 10.1371/journal.pone.0086343 PMCID:PMC3899249 Lombard, Karen et al., 2018, Principles of Biology, Biology 1107 Laboratory Manual, United States of America, p. 310

Table 1. Sample preparation for aerobic respiration in a mitochondrial extract Tub Phosphate Mitochondrial e Buffer Suspension 1 mM DCPIP Succinate 1 4.4 mL 0.5 mL 0 mL 0.1 mL 2 4.2 mL 0.5 mL 0.3 mL 0 mL 3 4.1 mL 0.5 mL 0.3 mL 0.1 mL 4 4.0 mL 0.5 mL 0.3 mL 0.2 mL 5 4.6 mL 0 mL 0.3 mL 0.1 mL Table 2. Percent transmittance (%T) at 600 nm Tub e 1 5 10 1 100 100 100 2 30.5 44.1 51.4 3 30.5 47.7 60.7 4 5

32.6 67.8

52.8 66.8

69.3 67.5

15

20 100 57.9 72

100 64.1 82.7

82.3 66.8

87.6 67.6

25 30 100 100 68.6 73.4 92.5 98.4 109. 99.9 3 67.8 68.4

Table 3. Effect of incubation temperature over 30 minutes Tub Initial Gas Final Gas Volume of CO2 Produced, e Temperature Volume, mL Volume, mL in mL 1 0°C (ice) 1 1.75 0.75 20°C (room 2 temp.) 1 2.5 1.5 3 45°C 1 8.5 7.5 4 60°C 1 5.5 4.5 100°C (boiled 5 yeast) 1 1 0 Table 4. Effect of carbohydrate sources on fermentation over 30 minutes Tub Carbohydra Initial Volume of Final Volume of Volume of CO2 e te CO2, mL CO2, mL Produced, mL 6 Galactose 1.2 1.5 7 Sucrose 1.2 6.7 8 Fructose 1.5 6 Table 5.Effect of carbohydrate sources on fermentation over 30 minutes

0.3 5.5 4.4

Tub e Carbohydrate 9 Maltose 10 Lactose Lactose + 11 Lactaid

Initial Volume of CO2, mL

Final Volume of CO2, mL

Volume of CO2 Produced, mL

1 1

3.5 1.5

2.5 0.5

1

9

8

120

Percent Absorbance (nm)

100

Tube 1; 4.4 mL Phosphate Buffer, 0.5 mL Mitochondrial Suspension, 0 mL 1 mM DCPIP, 0.1 mL Succinate

80

Tube 2; 4.2 mL Phosphate Buffer, 0.5 mL Mitochondiral Suspension, 0.3 mL 1 mM DCPIP, 0 mL Succinate

60

Tube 3; 4.1 mL Phosphate Buffer, 0.5 mL Mitochondrial Suspension, 0.3 mL 1 mM DCPIP, 0.1 mL Succinate

40

Tube 4; 4.0 mL Phosphate Buffer, 0.5 mL Mitochondrial Suspension, 0.3 mL 1 mM DCPIP, 0.2 mL Succinate

20

0 1

5

10

15 Time (Minutes)

Tube 5; 4.6 mL Phosphate BufSus-30 20 fer, 0 mL Mitochondiral 25 pension, 0.3 mL 1 mM DCPIP, 0.1 mL Succinate

Figure 1. Affect of succinate on percent transmittance over the course of 30 minutes when combined with DCPIP, mitochondrial suspension, and phosphate buffer

9 8 7

Gas Volume, mL

6 5

1 0°C (ice) 2 20°C (room temp.) 3 45°C 4 60°C 5 100°C (boiled yeast)

4 3 2 1 0 0

30 Time (minutes)

Figure 2. The affect of incubation temperature on CO2 gas volume of a yeast and glucose sample when performing fermentation over a 30 minute time period

10 9 8

Volume CO2, mL

7 6

Glucose Galactose Sucrose Fructose Maltose Lactose Lactose + Lactaid

5 4 3 2 1 0 0 mins

30 mins Time (minutes)

Figure 3. The affect of varying carbohydrates on CO2 gas volume over a 30 minute time period when combined with yeast and allowed to perform fermentation at 45°C...