TITLE: RESPIRATION (SIMPLE RESPIROMETER)
AIM: To determine the rate of respiration and the oxygen uptake per unit mass of a small
organism with the use of a respirometer.
INTRODUCTION:
Respiration is the process by which organic molecules are broken down in a series of stages
to release chemical potential energy which is then utilized to produce adenosine
triphosphate(ATP). Glucose is the main molecule used as a respiratory substrate and the overall
worded and chemical equations for the process are:
Aerobic respiration is the series of chemical reactions by which ATP is synthesized in the
presence of oxygen while anaerobic respiration is the series of chemical reactions by which ATP
is synthesized in the absence of oxygen. Aerobic respiration consists of four stages: glycolysis,
the link reaction, the Krebs/citric acid cycle, and oxidative phosphorylation.
Glycolysis occurs within the cytosol of the cytoplasm and is the lysis of a hexose sugar
glucose into two pyruvate molecules having three carbon atoms each. The first few stages of
glycolysis are referred to as the energy investment phase as ATP is utilized and the other stages
of glycolysis are referred to as the energy payoff phase as ATP is produced. Hence, as ATP is
both used and produced in this metabolic pathway, there is a net production of two ATP
molecules for every glucose molecule that undergoes lysis. In the first stage, phosphorylation,
glucose is phosphorylated using ATP and glucose-6-phosphate is produced. Next, isomerization
occurs and the atoms of glucose-6-phosphate are rearranged to form fructose-6-phosphate.
Glucose-6-phosphate and fructose-6-phosphate are isomers. This fructose-6-phosphate is then
phosphorylated to form fructose-1,6- bisphosphate as ATP is broken down to ADP and inorganic
phosphate(this is used to phosphorylate fructose). The fructose-1,6- bisphosphate undergoes lysis
to form two 3-carbon isomers: glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
The dihydroxyacetone phosphate is converted to glyceraldehyde-3-phosphate and hence there are
two molecules of glyceraldehyde-3-phosphate produced. These two molecules undergo oxidation
as hydrogen is removed from each molecule and is transferred to a hydrogen carrier molecule:
Nicotinamide adenine dinucleotide( NAD+) to form reduced NAD. SImultaneously,
phosphorylation also occurs and a phosphate molecule is added to each molecule of
glyceraldehyde-3-phosphate. This forms two molecules of 1,3-bisphosphoglycerate. Substrate
level phosphorylation of ADP then occurs as each molecule of 1,3-bisphosphoglycerate loses a
phosphate. Phosphorylation of ADP leads to the production of ATP. This forms
3-phosphoglycerate. Isomerisation then occurs and the atoms of both molecules of
3-phosphoglycerate are rearranged to form an isomer: 2-phosphoglycerate. A condensation
reaction occurs and a double bond is formed as water is lost to form two molecules of
phosphoenolpyruvate which has a very high potential energy. Lastly, substrate level
phosphorylation occurs as each molecule of phosphoenolpyruvate transfers a phosphate to ADP
leading to the production of two three-carbon pyruvate molecules and ATP.
The link reaction follows glycolysis given that there is oxygen available and occurs in the
mitochondrial matrix. An overall equation for the link reaction is:
Before the link reaction can take place, the pyruvate molecules are actively transported into the
matrix of the mitochondria. Here pyruvate undergoes a complex series of oxidation-reduction
reactions that are catalyzed by a multienzyme complex known as pyruvate dehydrogenase. The
pyruvate molecules each lose a molecule of carbon dioxide causing the formation of a
two-carbon molecule. The carbon dioxide produced is an excretory product and diffuses out of
the mitochondria and the cell. Hydrogen is also removed and hence, the two carbon molecules
are oxidized and the hydrogen carrier NAD+ is reduced(forms reduced NAD). Finally, a
coenzyme A(CoA) molecule combines with each two-carbon fragment to form two molecules of
acetyl coenzyme A(acetyl CoA). Acetyl CoA has a high potential energy which is used to
transfer the acetyl group to a molecule in the Krebs cycle, a reaction that is therefore highly
exergonic
The Krebs cycle takes place in the matrix of the mitochondria and is a series of eight
enzyme-catalyzed steps in which a six-carbon molecule is gradually changed to a four carbon
compound. For each acetyl group that enters the cycle, 3 NAD+ are reduced to NADH. First, the
acetyl group of the acetyl coenzyme A molecule combines with a four-carbon molecule called
oxaloacetate. This converts oxaloacetate into a six-carbon molecule called citrate. The citrate
molecule is converted to its isomer isocitrate with the removal and addition of a molecule of
water. Isocitrate is oxidized causing the reduction of NAD+ and the resulting molecule is
decarboxylated. This forms α-Ketoglutarate. This α-Ketoglutarate molecule is decarboxylated
and the resulting compound is oxidized causing the reduction of NAD+(forms reduced NAD).
The remaining molecule is then attached to a coenzyme A molecule by an unstable bond. This
forms succinyl CoA. Coenzyme A is replaced by a phosphate group which is transferred to
Guanosine diphosphate(GDP) via substrate-level phosphorylation to form guanosine
triphosphate(GTP). This GTP can then be used to generate ATP. The succinate formed is then
oxidized to form fumarate and the two hydrogens lost are transferred to flavin adenine
dinucleotide(FAD) forming reduced FAD( or FADH2). A molecule of water is added to fumarate
which rearranges the bonds within its structure forming malate. Malate is oxidized causing the
reduction of NAD+ and the regeneration of oxaloacetate. The significance of the Krebs cycle to
the last stage of respiration(oxidative phosphorylation) is that it produces hydrogen atoms which
are carried by FAD and NAD (hydrogen carriers) to the electron transport chain for oxidative
phosphorylation and the production of ATP by chemiosmosis.
In oxidative phosphorylation, the energy for the phosphorylation of ADP comes from the
activity of the electron transport chain. In this process, reduced FAD and NAD are passed to the
electron transport chain where they are oxidized. The hydrogens lost are split into protons(H+)
and electrons(e-). The energetic electron is transferred to the first of a series of electron carriers.
As an electron moves from a carrier at higher energy level to one of a lower energy level, energy
is released and the higher energy carrier is oxidized and the lower energy carrier is reduced. This
energy is used to actively transport protons from the matrix of the mitochondria to the
intermembrane space. This causes the formation of an electrochemical gradient. As there is then
a higher concentration of protons within the intermembranal space, the protons move down their
electrochemical gradient back into the matrix through chemiosmotic channels of the ATP
synthase complexes by a process known as chemiosmosis. The movement of these protons back
into the matrix leads to the phosphorylation of ADP within the ATP synthase complexes. Finally,
oxygen functions as the final electron acceptor. In the matrix, an electron and a proton are
transferred to oxygen, reducing it to water.
The rate of respiration of an organism can be measured in terms of its oxygen uptake per
unit time. This oxygen uptake can be measured using a respirometer. A respirometer consists of a
capillary tube and graduated scale, syringe, three-way tap, and tubing connecting the container
containing the respiring organism to the syringe and capillary tube. The carbon dioxide produced
during respiration is absorbed by soda lime or a concentrated solution of potassium hydroxide or
sodium hydroxide. As the organism takes up oxygen from its immediate surrounding, the volume
of air around the organism decreases. Oxygen consumption per unit time can be measured by
reading the level of the manometer fluid against the scale.
The chickpea is an annual legume of the family Fabaceae. Chickpea seeds are spherical in
shape, wrinkled, and have a pointed beak. Under optimum conditions of temperature and
moisture content, chickpea seeds undergo germination. Availability of oxygen is an important
factor of seed germination. This oxygen allows for the resumption of respiration and in turn
metabolic activity during seed imbibition. Germinating seeds respire to attain energy needed for
growth and development of structures as these seeds do not yet contain the necessary
components to carry out photosynthesis needed to produce the precursor molecules for their
energy formation and supply.
APPARATUS: potometer, stopwatch, retort stand, cotton balls, 15% potassium hydroxide,
chickpeas, plasticine, wire mesh, balance
DIAGRAM:
METHOD: The capillary tube was submerged in coloured water and a bubble was introduced at
the zero mark. The three-way tap was adjusted to prevent backflow of the manometer fluid and a
piece of plasticine was used to seal the end of the capillary tube while adjusting the tap. The
syringe was carefully removed from the apparatus setup. The plunger was removed from the
barrel of the syringe and a dry cotton ball was introduced and was pushed all the way to the end
of the syringe. A few drops of potassium hydroxide were added to partially saturate the cotton
ball without touching the sides of the syringe. The wire mesh was inserted into the syringe barrel.
The weight of the chickpea was measured and the chickpea was placed inside the barrel and the
plunger was gently reinserted half-way into the syringe. The position of the meniscus was noted
and the distance moved by the coloured water was recorded every two minutes up to a maximum
of ten minutes.
RESULTS:
TABLE 1. SHOWING THE INITIAL VOLUME, CHANGES IN VOLUME, AND FINAL
VOLUME OF COLOURED WATER OVER A TEN-MINUTE TIME PERIOD
Time
(mins)
Initial
Volume
(ml)
Trial 1:Change
in Volume (ml)
Trial 2:Change Trial 3 :Change
in Volume
in Volume (ml)
(ml)
Average
Volume (ml)
Final Volume
(ml)
0
10.0
0.0
0.0
0.0
0.0
10.0
2
10.0
0.7
0.8
0.7
0.7
9.3
4
9.3
0.7
0.6
0.7
0.7
8.6
6
8.6
0.7
0.6
0.6
0.6
8.0
8
8.0
0.8
0.7
0.6
0.7
7.3
10
7.4
0.7
0.6
0.6
0.6
6.7
TRENDS: The mass of the chickpea was 0.78g. As time increased over a 10-minute period, the
final volume of the coloured liquid within the capillary tube decreased.
GRAPH:
CALCULATIONS:
1. Average change in volume(ml):
Using values for the two-minute period from Table 1
T1= 0.7ml
T2= 0.8ml
T3= 0.7ml
Average change in volume = (T1 + T2 + T3)/3
= (0.7 + 0.8 + 0.7)/ 3
= 0.7 ml
2. Final volume (ml):
Using values for the two-minute period from Table 1:
Initial Volume = 10 ml
Average change in volume = 0.7 ml
Final volume = Initial volume - average change in volume
= 10 - 0.7
= 0.3 ml
3. Gradient of graph of final volume against time ( rate of respiration in ml per min):
(x1, y1): (1.2, 9.6)
(x2, y2): (17.6, 4.1)
Gradient = (y2 - y1)/ (x2 - x1)
= ( 4.1 - 9.6)ml / 17.6 - 1.2) min
= -0.34 ml/min
4. Metabolic rate of the chickpea:
Mass of the chickpea = 0.78g
Rate of respiration of the chickpea = -0.34 ml/min
Metabolic rate = respiration rate / mass of organism
= -0.34 ml/min / 0.78g
= -0.44 ml min-1g-1
DISCUSSION:
A chickpea may undergo aerobic respiration and this rate of respiration may be measured by
determining the volume of oxygen taken up by the chickpea over a period of time. Aerobic
respiration has four stages: glycolysis, link reaction, krebs cycle, and oxidative phosphorylation.
This process occurs in the presence of oxygen and the presence of oxygen is important as oxygen
acts as the final electron acceptor in the final stage of respiration. Glycolysis occurs in the
cytosol of the cytoplasm and is the process/metabolic pathway by which a hexose sugar(glucose)
is converted into two molecules of pyruvate(triose sugar). The link reaction takes place in the
mitochondrial matrix and is the process by which pyruvate molecules are decarboxylated,
oxidized and combined with coenzyme A molecules to form molecules of acetyl coenzyme A.
This coenzyme A molecule acts as an acetyl carrier for the Krebs cycle. The krebs cycle is the
series of enzyme controlled reactions by which ATP is produced and most importantly, the
process by which hydrogen atoms are produced and taken up by hydrogen carriers(NAD and
FAD) for use in oxidative phosphorylation. Oxidative phosphorylation produces relatively large
amounts of ATP via the movement of electrons produced from the hydrogen atoms carried by
NAD and FAD down the electron transport chain.
A graph illustrating the final volume of coloured water versus time was plotted and showed
that final volume and time had an inversely proportional relation. This type of relationship
indicates that as time increased the volume of coloured water within the capillary tube decreased.
This occurred because over time the chickpea took up oxygen from its surroundings and this in
turn decreased the level of coloured water. It is assumed that the rate of oxygen uptake is equal to
the rate of respiration of the chickpea. Hence, the gradient of the graph represents the rate of
respiration of the chickpea and this was calculated to be -0.34 ml/min. This negative gradient
also indicates that final volume and time had an inversely proportional relation. This respiratory
rate may also be used to determine the metabolic rate of the chickpea by considering the
respiratory rate and mass of the chickpea. The respiratory rate was determined to be -0.44 ml
min-1g-1.
This type of graph was observed due to the use of oxygen by the chickpea to carry out
respiration. All stages of aerobic respiration with the exception of glycolysis require oxygen.
This is because oxygen acts as the final electron acceptor of the electron transport chain(in
oxidative phosphorylation) by accepting hydrogen ions and electrons to form water. When
oxygen is present, the electron carriers are able to pass on electrons obtained from hydrogen
carriers down the electron transport chain. This movement of electrons down the electron
transport chain allows the synthesis of ATP as this energy is used to produce an electrochemical
gradient for the passive movement of protons. Hence, as hydrogens are transferred from the
electron carriers NAD and FAD to the primary electron carrier, these carriers may be used in the
Krebs cycle and link reaction. Thus, in the presence of oxygen, the chickpea is able to carry out
all the necessary processes within aerobic respiration and hence, over a ten-minute period, the
chickpea continuously took up oxygen from its surroundings thereby decreasing the final volume
of the coloured water in the capillary tube. Two points from the graph that illustrate this are: (,)
and (, ). These points indicate that over a period of …minutes, there was a decrease in the
volume of water in the capillary tube by…..ml.
As the chickpea carried out respiration, carbon dioxide is produced. This carbon dioxide may
be removed from the air by reaction with potassium hydroxide. As carbon dioxide is acidic in
nature and potassium hydroxide is strongly alkaline in nature, these compounds will react to
form potassium carbonate(K2CO3) and water. Three trials of the experiment were conducted
under identical conditions to ensure that the values obtained were reliable and utilizing an
average of a measurement of the same quantity under identical conditions improves the overall
reliability of the data. Coloured fluid was placed in the capillary tube to allow ease of
observation of the movement of the water and the meniscus. This ensured that the final volume
of the coloured water could be accurately recorded.
Respiration rate may be defined as the volume of oxygen taken up by an organism per unit
time in order to carry out the necessary processes of respiration. The theoretical respiration rate
of a chickpea at 25 degrees celsius is -0.01 ml/min (“Lab 5 Ap Sample 3 - BIOLOGY
JUNCTION” 2017). The calculated value for the rate of respiration of the chickpea was
-0.34ml/min. The value calculated was -0.24ml/min lower than the theoretical value. Metabolic
rate may be defined as the volume of oxygen taken up in a given time by an organism to carry
out respiration per unit mass of that organism. The theoretical metabolic rate could not be found.
(2) Sources of error- minimal amounts of soda lime may have touched the chickpea and this may
have affected the accuracy of the results and the setup may not have been completely airtight
which may have affected the results recorded.
(2) Precautions- allowing the apparatus for equilibrate for an adequate period of time before
taking measurements and taking measurements of the final volume of the coloured water at eye
level
(2) limitations- there may have been temperature fluctuations and this could not be controlled,
there may have been minute air bubbles within the coloured water that were too small to be
detected and this may have affected the results
Assumptions- assume the total change in volume is due to the respiration if the chickpea and no
other factors influenced the decrease in the volume of the coloured water
Improvements and recommendations- setting up a control/reference respirometer with inert
material to account for changes in atmospheric conditions
CONCLUSION: The rate of respiration of a chickpea was determined to be -0.34ml/min and the
metabolic rate(rate of oxygen uptake per unit mass) of the chickpea was determined to be
-0.44ml min-1g-1.
REFERENCES:
“Lab 5 Ap Sample 3 - BIOLOGY JUNCTION.” 2017. BIOLOGY JUNCTION. April 21,
2017.
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