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Date: 19/10/2023 Lab#3
TITLE: ANAEROBIC RESPIRATION IN YEAST.
AIM: To use a differential respirometer to determine the rate of respiration in yeast.
APPARATUS: Respirometer, (1) 1000 mL beaker, laboratory thermometer, paper towels,
timer,
2.5 mL baker’s yeast suspension, 8 mL of 5% glucose solution, 2 measuring
cylinders (for glucose and for yeast), paraffin, dropper, water bath at 37-40°C
METHOD:
A 1000 mL beaker was filled to 900 mL with tap water and allowed it to
reach room temperature. The temperature was monitored with a laboratory
thermometer. 2.5 mL of yeast suspension and 2.5 mL of 5% glucose solution
was added to the experimental test tube in bracket ‘E’. A dropper was used
to add enough paraffin to cover the surface of the yeast/glucose solution.
The paraffin was not allowed to touch the walls of the test tube.The
yeast/glucose solution was incubated in the water bath for 10 minutes at 37-
40°C to ‘jump start’ the fermentation reaction. The control test tube in
bracket ‘C’ was filled with 5 mL of 5% glucose solution, then the same
amount of paraffin was added in the experimental test tube. Before inserting
the stoppers into the test tubes, the plunger of the control-chamber syringe
(on left) was pulled all the way to the top. The plunger of the experimental-
chamber syringe (on right) was pushed all the way down. The stoppers were
inserted into the mouth of the test tubes. By pushing the control plunger
down and/or pulling the experimental plunger up, the initial levels of blue-
fluid in the control and experimental pipette was adjusted so that the control
pipette (on left) reads 0.700 mL and the experimental (on right) reads 0.100
mL. The entire assembly was placed over the edge of the room temperature
1000 mL beaker water bath that was filled in step 1. The submerged test
tubes were allowed to equilibrate in the water bath for a few minutes and
then the temperature was recorded. A final adjustment of the experimental
tube was made to read 0.100 mL and this was recorded as the initial volume
at time 0. As the fermentation of the sugar progressed, the pressure from
the carbon dioxide gas forced the blue liquid to move down the tube. When
the volume changed at 0.5 minutes (30 seconds) for a minimum of 10-15
minutes it was recorded.The carbon dioxide produced per interval was
calculated by taking the difference between the initial reading at 0.100 mL
and the final reading. The volume of carbon dioxide (mL) versus time
(minutes) was plotted. The graph should be linear. The slope (gradient) is
calculated by ∆Y/∆X = (Y2-Y1)/(X2-X1).
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RESULTS:
A TABLE CREATED FOR THE RESULTS RECORDED OF THE CHANGE IN
VOLUME (ml) FOR THE ANAEROBIC RESPIRATION IN YEAST WITH GIVEN
TIME.
Time (min) Change in Volume (mL)
Group 1 Group 2 Group 3 Group 4
0.00 0.00 0.00 0.00 0.00
0.50 0.04 0.08 0.05 0.05
1.00 0.08 0.16 0.10 0.13
1.50 0.12 0.23 0.14 0.19
2.00 0.16 0.29 0.18 0.24
2.50 0.20 0.35 0.20 0.29
3.00 0.24 0.42 0.27 0.39
3.50 0.28 0.48 0.32 0.39
4.00 0.32 0.54 0.36 0.44
4.50 0.36 0.60 0.40 0.49
5.00 0.40 0.60 0.44 0.54
5.50 0.44 0.60 0.48 0.60
6.00 0.48 0.60 0.52 0.64
6.50 0.51 0.60 0.56 0.66
7.00 0.56 0.60 0.60 0.66
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Calculations:
Average rate of CO2 production with regards to time (min.)=Average gradient of the 4 graphs
Group 1=
M1= (0.48-0.24)/(6-3)
= 0.08ml CO2 /min.
Group2=
M2= (0.54,0.42)/(4-3)
=0.12ml CO2 /min.
Group 3=
M3= (0.44-0.18)/(5-2)
= 0.09ml CO2 /min.
Group 4
M4= (0.44-0.29)/(3.50-2.50)
= 0.15ml CO2 /min.
As a result the average rate of CO2 production with regards to time
= average gradient of the 4 graphs= (m1 + m2 + m3+ m4)/4
= 0.11 ml CO2 /min (to 2dp)
Gram of glucose in the test tube:
5% = 5g:100ml
So there is (5 x 2.5/100)= 0.125g (mass of glucose in the test tube)
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Rate per gram = Slope/ total grams of glucose= 0.11/0.125=0.88 ml CO2 /min./g)
DISCUSSION:
Anaerobic respiration is a respiratory process that occurs in both prokaryotes and eukaryotes in
which cells break down the sugar molecules to produce energy without the presence of oxygen.
However, fermentation, a form of anaerobic respiration in yeast cells, was investigated. During
the process, pyruvate is first decarboxylated to acetaldehyde by an enzyme known as pyruvate
dehydrogenase, forming CO2 as a by-product. The acetaldehyde is then reduced by NADH to
ethanol utilizing the enzyme dehydrogenase to form NAD + and ethanol. This NAD+ is then used
in a process known as glycolysis to form a net gain of 2 ATP molecules per molecule of glucose.
The equation for the reaction taking place in this experiment is as follows:
C6H12O6 (aq) →2C2H5OH(aq) + 2CO2(g) . The fermentation of glucose produces an alcohol known
as ethanol and carbon dioxide.
In this lab a differential respirometer was used to determine the rate of respiration in yeast.A
respirometer is a scientific tool used to estimate the rate of respiration due to the changes in
volume and pressure caused by gas absorption.
For the results obtained, it is observed that as time increased the volume of CO 2 increased for
all 4 groups. This indicates that the period of time is directly proportional to the change in
volume. This is proved by the straight- line graphs that were drawn and passed through the
origin. On the other hand, as the period of time extended the more pyruvates were
decarboxylated leading to an increase in the volume of CO 2 and ethanol produced. In addition,
the constant volume of the glucose started to decrease as the occurrence of glycolysis reaction
increased. As a result, the amount of pyruvate made available for the reaction depleted as proven
in group 2 from 4.50-7.00 minutes.
As the graph was plotted the rate per minute was calculated to be (0.11) rate per minute/ (s)
whereas the average rate at which CO 2 was produced per minute was calculated to be 0.11 ml
CO2 /min (to 2dp) average rate. However, since 2.5ml of the 5% glucose was used in the
experiment the gram of glucose would be (5/100 x 2.5/1)= 0.125g of glucose which was used in
the experiment. In addition, the rate per gram was calculated taking the gradient of the slope
(0.11 ml CO2 /min (to 2dp) and dividing it by the gram of glucose (0.125g) which equaled
(0.88ml CO2 /min./g).
The use of the paraffin in the experiment is to act as a barrier between oxygen and the glucose
solution with yeast so that no anaerobic respiration could occur. This is so because there are
factors that affect fermentation and this includes temperature, the optimum temperature for the
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fermentation of yeast cells ranges from 320c-340c because at this temperature the collisions of the
molecules of pyruvate dehydrogenase, pyruvates and acetaldehyde and its enzyme acetaldehyde
dehydrogenase are more likely to work efficiently as they are able to meet the criterias of the
collision theory. Therefore, temperatures out of the range can denature enzymes responsible for
the fermentation process. Also, ph level can have a big effect on the fermentation reaction.
Enzymes in the reaction best operate in a weak acidic range of 4-6. Therefore, if the ph level is
not in this range the enzyme's tertiary structure can be disrupted causing it to be denatured.
LIMITATIONS:
1. Over a period of time the concentration of glucose decreased which affects the
availability of the pyruvates available for the process of glycolysis.
2. Depletion of yeast cells as they are overly exposed to excess ethanol as they undergo
anaerobic respiration affects the availability of yeast cells to carry out anaerobic
respiration which in turn affects the process glycolysis by slowing it down.
PRECAUTION:
1. Ensure that oil did not spatter on the walls of the test tube.
2. Wash apparatus before use.
SOURCE OF ERRORS:
1. Mislabelling of test tube
2. Not reading measurements at eye level
3. Using wrong unit of measurements
CONCLUSION:
A differential respirometer was used to determine the rate of respiration in yeast. As a result, the
average rate of respiration of the yeast cells in the 5% of glucose solution was calculated to be
0.11 ml CO2/min and average rate of respiration per minute was found to be 0.88 ml CO2/ min/g.
