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MEASURING FERMENTATION OF YEAST UNDER VARIOUS CONDITIONS AND

CELLULAR RESPIRATION OF MITOCHONDIRA IN LIMA BEANS

April 14, 2015

BSC 2010L Section: 904


Lab Parteners: Angelid Pabon, Taylor Wright, Riley Blake
Abstract

The problem addressed in the first part of the experiment is how do the metabolic rates of yeast
compare under varying conditions which include food source and temperature. This problem was
approached setting up nine different combinations of environments for the yeast; sucrose,
glucose and saturated starch solution along with 37C, 25C and 4C. The results supported the
hypothesis with glucose having on average the fastest fermentation rate for food source and 37C
for the temperature.
Introduction
Cellular Respiration refers to the biological pathway by which cells release energy from
chemical bonds of food molecules and provide that energy for essential processes of life (Nave,
2012). All living cells must carry out cellular respiration wether it be aerobic respiration (in the
presence of oxygen) or anaerobic respiration (without oxygen). Prokaryotic cells carry out
cellular respiration within the cytoplasm or on the inner surfaces, whereas in eukaryotic cells it
occurs in the mitochondria. The outcome of cellular respiration is the production of ATP. Figure
1 shows the equation for cellular respiration.

Figure 1:
Cellular respiration involves the oxidizing of glucose to yield carbon dioxide water and 36 ATP

Under anaerobic conditions, fermentation occurs. Fermentation makes it possible for ATP
to be continually produced in the absence of oxygen. as shown in figure 2 the equation for
fermentation yields ethanol and carbon dioxide . There are two types of fermentation, alcohol
fermentation and lactic acid fermentation. Lactic acid fermentation occurs in the muscles of
animals when they need energy faster than the blood is can supply oxygen. Alcohol fermentation
is performed by yeast used in brewing, wine making and baking.

Figure 2:
Summarized equation of fermentation

The importance of cellular respiration is it provides the energy for living organisms to
perform all of the other necessary functions to maintain life and most single-celled organisms,
such as bacteria, do not require much energy and are able to survive on glycolysis and
fermentation.

In terms of food sources monosaccharides and disaccharides represent only a small fraction of
the total amount of carbohydrates in the natural world. The great bulk of the carbohydrates in
nature are present as polysaccharides, which have relatively large molecular weights. The
polysaccharides serve two principal functions. They are used by both plants and animals to store
glucose as a source of future food energy and they provide some of the mechanical structure of
cells. Plants store food energy as polysaccharides known as starch.
Cellular Respiration is a redox reaction oxidizing food molecules, like glucose to carbon
dioxide and water. A redox reaction is a chemical reaction in which a substance loses electrons
and another gains electrons, there is a transfer of electrons between reactants. Overall it involves
four main subdivisions, shown in figure 3 ; glycolysis is when glucose is broken down releasing
hydrogen molecules to form pyruvate; the Krebs cycle the carriers NAD+ and FAD pick up
electrons and hydrogen ions to form NADH and FADH; the electron transport chain and
chemiosmosis also occur.

Figure 3:
Inputs and Outputs of Cellular Respiration

In the citric acid cycle, also known as krebs cycle, a critical steps occurs, enzymecatalyzed conversion of succinate to fumarate through redox reaction occurs. FAD depletes
hydrogen ions from succinate and in the process fumarate is formed, this is shown in figure 4. In
this reaction DPIP acts as a electron acceptor that intercepts the hydrogen ions released from
succinate. The oxidation of succinate is required for the Citric Acid cycle to proceed.

Figure 4:
the oxidation of succinate occurs through the transfer of hydrogen ions to FAD.

In part one this experiment cellular respiration and alcohol fermentation will be observed.
The goals is to first compare the fermentation rates of yeast under various conditions. These
conditions included growing the yeast in one of the three carbohydrate solutions as a food source
(0.2 M sucrose, 0.2 M glucose or a saturated starch solution), at one of the three different
temperatures (37C, ~25C, or 4C). And secondly measure cellular respiration in mitochondria
that have been isolated from lima beans.
My hypothesis for how the metabolic rates would compare is, the metabolic rate would
increase with temperature. Yeast is known for breaking down complex carbohydrates to imply
sugars, so the glucose would have a faster rate, following sucrose then the saturated starch
solution.
Materials and Methods

My lab parter and I were assigned 0.2 M sucrose and 25C. With the use a graduated
cylinder, 15 ml of 0.2 M sucrose was measured out into a 50 ml beaker and 0.5 g of yeast was
added to it. With a transfer pipette, a drop of yeast was placed onto a slide and set aside for
viewing. Following with the experiment the yeast suspension was immediately transferred to a
fermentation tube and inverted properly. As shown in figure 5 it shows how a pocket of CO2
collected during fermentation. Every five minutes for forty minutes the observation of CO2 gas
forming in tube was recorded in a table in units of millimeters.

Figure 5:
Fermentation Tube
The arrow shows how and where CO2 gas collected in
the tube

In part two of the experiment the spectrophotometer was turned on and set to read the
percentage of transmittance of 600 nm of light. Then six cuvettes were obtained labeling the tops
B-1, B-2, 1 ,2 ,3 , and 4. Six squares of parafilm paper was then cut to properly cover the tops of
the tubes. The tubes were prepared according to a table similar to figure 5. B-1 and B-2 were
used as blanks. The blanks were prepared first then the cuvettes were prepared. The succinate
was added last due to its fast reaction time to the sample. Once the succinate was added each
tube was covered with a piece of Parafilm and was then vigorously shaken for two seconds to
mix the components.

Sample ID

Buffer (ml)

Mitochondrial
Suspension (l)

DPIP (l)

Succinate (l)

B-1

2.15

150

200

B-2

2.3

200

2.2

150

150

2.1

150

150

100

2.0

150

150

200

2.15

150

200

Figure 6:
Scheme for DPIP Reduction Reactions
(the mitochondrial suspension was previously prepared and kept on ice)

Before placing the cuvette in the spectrophotometer, each tube was wiped down with a Kimwipe
to clear any smudges from interfering with data.
The Parafilm from B-1 was removed and placed into the spectrophotometer to blank the
machine. It was then removed and sample id 1 was inserted. Readings at the zero time point was
taken. Then sample id 2 was inserted and zero time point was recorded, and this was repeated for
sample id 3 as well. The spectrophotometer was then blacked using B-2, and the zero time point
for sample id 4 was taken. For every five minutes these steps were repeated for a total of thirty
minutes.

Results
Fermentation

In the fermentation experiment, the amount of gas produced varied among the different
carbohydrates and the different temperatures, Figure 7 shows the results of the experiment.
Time
(mins)
37C

~25C

4C

Food Source

Control

0.2 M
sucrose

0.2 M
glucose

saturated
starch

H2O

10

2.7

0.2

15

0.3

20

2.4

0.5

25

2.6

3.5

0.7

30

4.5

0.7

35

6.1

9 (est)

0.8

40

7.0

11 (est)

0.8

0.1

0.1

10

0.3

0.1

15

0.1

0.7

0.2

20

0.4

0.9

0.4

25

0.9

1.0

0.4

30

1.5

1.1

0.4

35

1.8

1.8

0.5

40

1.9

2.5

0.5

1.0

0.3

10

1.8

0.9

15

2.5

1.5

0.2

20

3.1

1.8

0.3

25

3.5

2.3

1.4

30

4.0

2.6

2.5

35

4.4

3.1

2.7

40

4.7

3.6

3.1

Table 1-1
Time (mins)

Sample ID

10

15

20

25

30

14.0

14.9

14.6

14.9

14.5

13.9

15.0

16.7

17.1

18.6

19.5

20.7

21.6

22.1

16.9

18.9

20.4

21.1

23.2

24.0

23.9

9.5

9.1

9.2

9.7

10.1

8.9

9.2

% Transmittance Readings of Reduced DPIP


30.0
24.0

% Transmittance1

18.0
12.0

6.0
0.0

Time (mins)

Discussion
The data highly supported the data. The difference in metabolic rates for carbohydrates
and temperature difference could be accounted for the rate of alcohol production increasing with
temperature up to a range of 40C. The cold temperature would slow down the reaction. Glucose
is a monosaccharide, sucrose is a disaccharide, and starch a polysaccharide, this accounted for
the difficulty in yeast breaking down the sugars, the more complex the sugar the slower the rate
of metabolic reaction.

The reason for sample ID showing the small % of transmittance was due to the lack of
mitochondrial suspension for the DPIP to react on.

References
Nave, Carl R. (2012). Cellular Respiration. Respiration. Department of Physics and Astronomy,
Georgia State University. [Online]. Available: http://hyperphysics.phyastr.gsu.edu/hbase/biology/celres.html. [April 6, 2015].

Carbohydrates. (2013). Retrieved April 14, 2015, from


http://chemed.chem.purdue.edu/genchem/topicreview/bp/1biochem/carbo5.html