measure oxygen uptake by respirometer

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BIOLOGY LAB REPORT

TITLE : MEASURING THE RATE OF OXYGEN UPTAKE OF YEAST BYUSING RESPIROMETER

PREPARED BY :

I/C NUMBER :

STUDENT ID :

GROUP :

LECTURERS NAME :

PRACTICAL DATE

SUBMISSION DATE :


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Abstract

One of the key ways to gain energy is by aerobic respiration. Aerobic respiration is the process

where organisms consume oxygen gas in order to generate energy in the form of ATP. In this

experiment, we are assigned to measure the rate of oxygen uptake of yeast. The oxygen uptake rate of

the yeast can be measured by a simple respirometer comprises two test tubes containing potassiumhydroxide solution, manometer containing coloured liquid, stoppers, and several other apparatus. The

yeast is placed in one of the test tubes and left to respire. As the yeast consumes oxygen, carbon

dioxide is excreted and absorbed by the potassium hydroxide solution, creating a decrease in pressure

in the respirometer. This pressure will draw the coloured liquid in the manometer towards the test tube

that contains yeast. This movement will be measured in centimeters, and the value should give an idea

of how much oxygen the yeast consume.

Introduction

1. Yeast

Figure 1 : Structure of Yeast (1)

Yeast is a single celled fungus 1020 times bigger than bacteria. Yeast cell is only living organism

that should come into contact with the beer until it is drunk by the customer. It is from plantaekingdom under Mycota division and ordered as Endomycelates. The yeast cell is made up of 34 %

carbohydrates ( which made up the cell wall,gives internal energy reserves), 40.5 % protein ( make up

the enzymes, cell wall, membranes), 10% ribonucleic acid (RNA) for protein synthesis, 5%

phospholipid membranes, 3 % triglycerides 7 % ash, minerals, trace elements and the 0.5 % make up

the DNA, fibre, vitamins.


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Figure 2 : Reproduction of Yeast(1)

Yeast most commonly reproduce Asexually by Mitosis, but the process is slightly different from other

forms of Mitosis, in that it involves Budding. When the cell first begins to reproduce, a Bud is formed

of the surface of the cell. The cell then proceeds through Interphase, duplicating its Chromosomes

and Organelles. Next the Yeast cell undergoes Mitosis, where the new Chromosomes and DNA are

placed in the Bud. After this occurs, the Bud contains nucleus with an identical copy of the parent

cell's DNA. Finally, the Bud separates from the parent cell, producing a new Yeast Cell that

is Genetically Identical to its parent Cell.


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Figure 3 : Suitable condition for Yeast growth(2)

Figure 4 : Fermentation in Yeast (3)


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Figure 5: Both Aerobic and Anaerobic Respiration in Yeast (3)

2. RespirometerA respirometer is a device used to measure the rate of respiration of a living organism by

measuring its rate of exchange ofoxygen and/orcarbon dioxide. They allow investigation into how

factors such as age, chemicals or the effect of light affect the rate of respiration. Respirometers are

designed to measure respiration either on the level of a whole animal (plant) or on the cellular level.These fields are covered by whole animal and cellular (or mitochondrial) respirometry, respectively.

A simple whole animal respirometer designed to measure oxygen uptake or carbon dioxide

release consists of a sealed container with the living specimen together with a substance to absorb the

carbon dioxide given off during respiration, such as soda lime pellets or cotton wads soaked with

potassium hydroxide. The oxygen uptake is detected by displacement of manometric fluid in a thin

glass U-tube connected to the container. When the organism takes in oxygen it gives off an equal

volume of carbon dioxide. As this is absorbed by the soda lime, air is sucked in from the U-tube to

keep the pressure constant, displacing the liquid. The rate of change gives a direct and reasonably

accurate reading for the organism's rate of respiration.
http://en.wikipedia.org/wiki/Respiration_%28physiology%29http://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Carbon_dioxidehttp://en.wikipedia.org/wiki/Respirometryhttp://en.wikipedia.org/wiki/Soda_limehttp://en.wikipedia.org/wiki/Cottonhttp://en.wikipedia.org/wiki/Potassium_hydroxidehttp://en.wikipedia.org/wiki/Manometerhttp://en.wikipedia.org/wiki/Manometerhttp://en.wikipedia.org/wiki/Potassium_hydroxidehttp://en.wikipedia.org/wiki/Cottonhttp://en.wikipedia.org/wiki/Soda_limehttp://en.wikipedia.org/wiki/Respirometryhttp://en.wikipedia.org/wiki/Carbon_dioxidehttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Respiration_%28physiology%29


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As changes in temperature or pressure can also affect the displacement of the manometric

fluid, a second respirometer identical to the first except with a dead specimen (or something with the

same mass as the specimen in place of the organism) is sometimes set up. Subtracting the

displacement of the second respirometer from the first allows for control of these factors.

Yeast is an organism which respire both aerobic and anaerobic . Yeast must obtain oxygenfrom their environment in order to survive. They use the same metabolic reactions as other animals

(glycolysis, Kreb's cycle, and the electron transport system) to convert nutrients into the chemical

bond energy of ATP. During the final step of this process, oxygen atoms react with hydrogen ions to

produce water, releasing energy that is captured in a phosphate bond of ATP.

Figure 6: A Simple Respirometer(4)


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3. Cellular Respiration(5,6, 7 and 8)Cellular respiration is the process by which cells obtain energy from food through chemical reaction

with an inorganic electron acceptor, usually oxygen. The principal product is adenosine triphosphate

(ATP), a high energy compound used for a wide variety of energy-requiring processes in the cell.

Cellular respiration occurs in three main stages: glycolysis, Krebs cycle, and oxidative

phosphorylation.

Sugars and fatty acids are the primary food sources for cells. Each contains large numbers of C-C and

C-H bonds, which are relatively weak compared to C-O and H-O bonds. During cellular respiration,

these weaker bonds are broken, while the stronger bonds with oxygen are formed, thus releasing

energy. This energy is used to form the weakly bonded ATP molecule from its constituents, adenosine

diphosphate (ADP) and inorganic phosphate (Pi). The formation of ATP absorbs energy, which is thus

stored and available for driving reactions elsewhere in the cell.

Glycolysis, the first stage of cellular respiration, occurs in the cytosol of the cell. Only sugars undergo

glycolysis. During glycolysis, a glucose molecule (C6H12O6) is split to form two molecules of pyruvic

acid (C3H8O3). Hydrogens from glucose are removed by the carrier molecule nicotinamide adenine

dinucleotide (NAD+), forming NADH. The bond joining the H to the NAD

+is weak, meaning the

electrons of the bond are still high-energy electrons. In this way, NADH serves as a transporter of

high-energy electrons from the cytosol into the mitochondria, where the rest of cellular respiration

takes place. Two NADH are formed for each glucose molecule reacted, along with two molecules of

ATP. Most of the energy of the glucose remains in the pyruvates, however.

Pyruvic acid passes into the inner compartment of the mitochondrion, the cell organelle chiefly

responsible for ATP production. In this compartment, called the matrix, the pyruvic acid is

decarboxylated to a two-carbon acetyl group, releasing a CO2 molecule, and is enzymatically joined to

Coenzyme A, a large carrier molecule. This reaction creates another NADH molecule. Fatty acids are

also linked with coenzyme A, two carbons at a time. The product in all cases is acetyl- coA.


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Acetyl-coA then enters the Krebs cycle. In this series of reactions, the two- carbon acetyl group is

linked to a four-carbon compound, forming citric acid. (The Krebs cycle is also called the citric acid

cycle, and, because of the three carboxyl groups in citric acid, it is also known as the tricarboxylic acid

cycle.) In a series of transformations, the four-carbon compound is regenerated, carbon dioxide is

released, and ATP, NADH, and FADH2 are formed. FADH2 is another high-energy electron carrier.

The final stage of cellular respiration occurs in two steps. In the first step, NADH and FADH 2 are

stripped of their electrons, regenerating the original carrier molecules, which are recycled to their

original locations. The electrons are attracted away from their carriers by NADH-Q reductase, the first

in a series of increasingly electronegative proteins that form an electron transport chain in the inner

membrane of the mitochondria. Each protein in turn is first reduced when it accepts the electrons, then

oxidized as they are removed by the next protein in the chain. In succession, these carriers are

ubiquinone, cytochrome reductase, cytochrome c, cytochrome oxidase. The electrons are finally

accepted by molecular oxygen, which together join with H+

ions to form water. The energy released

during this series of redox reactions is used to transport other H+

ions across the inner mitochondrial

membrane, creating an electrochemical gradient.

The second, final step of this stage uses the energy stored in the electrochemical gradient to produce

ATP. H+

ions flow through a membrane protein called ATP synthase. The energy released by this flow

drives the synthesis of ATP from ADP and Pi. This process is known as chemiosmosis. The

combination of electron transport and chemiosmostic ATP synthesis is known as oxidative

phosphorylation, sometimes abbreviated as OXPHOS.

The overall ATP harvest from one glucose molecule is either 36 or 38 ATP, depending on the cell type

involved. Of these, all but four are formed by ATP synthase.


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Objective

To demonstrate the uptake of oxygen in yeast respiration and to measure the rate at yeastrespires.

To measure the level of difference of liquid in manometerProblem Statement

There are oxygen uptakes by yeast and thus cause the changes in the level of the colouring fluid in the

manometer.

Hypothesis

The respiration rate can be measured by the means of a respirometer by calculating the uptake of

oxygen per unit time.

Variable

Types of Variables Ways to control the variables

Manipulated Variable:

Presence of yeastTwo test tubes were used , one with the presence

of yeast and another without the yeast.

Responding Variables:

Rate of oxygen uptake by yeast The change in the initial and final coloured liquid

level divided with time taken for the experiment.

Repeat the experiment.

Fixed Variables:

Volume of coloured liquid Use 0.5l of coloured solution, using

micropipette.


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Apparatus

2 test tubes, capillary manometer with calibrated scale, coloured liquid (Brodies fluid or paraffin

deeply coloured with Sudan III), micropipette, three-ways taps, 1cm3

syringe, two small mesh basket,

potassium hydroxide solution, stopwatch, 1000cm3

beakers, rubber stoppers and connection tubes,

plastic sucker, forceps and scissors.

Materials

Yeast, paper towel

Procedure :

1. A manometer was cleaned. Water found on the inner wall of the U-tube manometer was driedand wiped using a paper towel.

2. Using a micropipette, a small volume of Brodies fluid or paraffin deeply coloured with SudanIII was drawn carefully into the capillary manometer, ensuring that it was free from bubbles.

The fluid was drawn up to about half-way up each arm of the manometer.

3. The capillary manometer with calibrated scale, three-way taps and stoppers were carefullyassembled, ensuring good seals throughout.

4. 15cm3 of potassium hydroxide solution was transferred into the two test tubes respectivelyusing a plastic dropper.

5. Filter paper with a lot of yeast was chosen.6. This filter paper inserted in a small basket into a test tube filled with potassium hydroxide

solution. Meanwhile, another test tube was filled with only potassium hydroxide.

7. Both the three-ways taps were turned to the atmosphere position (pointing away from themanometer), and both the test tubes were connected to the manometer by ensuring the stoppers

seal the test tube mouths properly.

8. The piston of the syringe was adjusted so that it was at the 0.5cm3 mark. It was then connectedto the respirometer at the side where there was test tube with living crickets.

9. The three-ways taps were turned to connect with the respirometer.10.The position of the manometer fluid was adjusted using the syringe so that the levels were the

same on both sides.


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11.The initial level of manometer fluid was observed and recorded. Then, the stopwatch wasstarted.

12.The position of the coloured fluid in the manometer was recorded at subsequent 1 minuteintervals until there was no further change. Observed change to the position of the syringe was

recorded if there was any.

13.The distance travelled by the liquid during each minute is measured and recorded in a table.14.From the data collected, the mean rate of oxygen uptake during the period is calculated.

Safety precaution and Risk Assessment

In order to avoid any accident or injury during the experiment in laboratory, the precautionary

steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when

conducting an experiment in the lab at all times to protect the skin and clothing from spillage of any

chemical substance or blood. Hands need to be thoroughly washed before and the experiment. This is

to avoid ourselves from getting infections. Furthermore, the glassware such as test tubes should be

handled with full care because they are fragile. Next, potassium hydroxide is caustic. Caustic simply

means that the solution able to corrode, burn and destroying living tissue. Goggles need to be worn all

the time when using KOH solution to avoid any eye irritations. After using all samples and apparatus

at the end of experiment, they should be discarded properly and returned back to their places to avoid

injuries and unnecessary accidents that may result fatal results. Liquid waste must be considered

infections and discarded according to local safety regulations.


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Results

Initial level of monometer: 4.4 cm Initial position of syringe: 0.5cm3 Displace in manometer level:

Time (second) Manometer reading (cm) Displacement in manometer level (cm)

0 4.4 0.0

60 4.9 4.94.4 = 0.5

120 5.9 5.94.4 = 1.5

180 6.2 6.24.4 = 1.8

240 7.2 7.24.4 = 2.8

300 7.5 7.54.4 = 3.1

360 7.8 7.84.4 = 3.4

Table 1: Displacement in Manometer Level

Displacement is obtained from [Displacement = Final reading (cm) Initial reading (cm)]

Final level of manometer fluid: 7.8cm Final position of syringe piston: 0.5cm3 Total increment in manometer reading:

7.8 cm4.4 cm = 3.4cm

Calculated rate of oxygen uptake (cm s-1):Total Amount of Oxygen Consumed/Total Time Taken

= 3.4 cm / 360 seconds

= 9.44 x 10-3

cm s-1


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DISCUSSION

This discussion will look at the overall procedure and the result obtained. This experiment was carried

out to investigate the rate of oxygen uptake of yeast using a respirometer. Yeast are easily found and

relatively easy to handle. Yeast respire both aerobic and anaerobically, but they prefer aeobic

respiration in the presence of oxygen. Thus, any outcome produced by them respiring can be obtainedin a shorter amount of time.In order to determine the rate of respiration, we must measure the amount

of oxygen inspired by the yeast over various periods of time. The amount of oxygen inspiration is

indicated by the change in the level of fluid in the manometer. Any carbon dioxide produced by the

yeast (as carbon dioxide is a by-product of respiration) is absorbed by the potassium hydroxide

solution, so it can be assumed that the change in level of fluid is due only to the inhalation of oxygen

by the yeast.

At 1 mol dm-3

, the potassium hydroxide solution used is quite highly concentrated. This is so that it

can absorb large amounts of carbon dioxide before becoming saturated. Thus, when air is inhaled and

carbon dioxide is absorbed by the solution, the pressure within the test tube drops as the volume of

gases decrease causing it to move towards the tube containing the yeast. If carbon dioxide is not

removed, the level of the fluid would remain relatively static as it is assumed that the amount of

oxygen inhaled is equal to the amount of carbon dioxide exhaled.

In order to ensure reliability, some factors must be kept constant throughout the experiment. For

instance, the surrounding temperature should be kept at a constant temperature as changes in the

external temperature affects the rate of metabolism of the yeast. To do this, the experiment set up was

left in a beaker without its location being changed. A control set with no yeast in the test tube could

also be introduced so that comparisons can be made between the two, ensuring that any changes are

due solely to the yeast breathing.

From the results obtained, it is shown that the manometer reading in the capillary tube nearer to the

side of the test tube with yeast in it increases. The initial reading was 4.4cm. When the stopwatch

initiated, the level of coloured fluid increased constantly up to the point 7.8cm after a total time of 6

minutes. The reading was constant after that point and no further increment was observed. Overall, the

rate of oxygen uptake increases.


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EVALUATION

In this experiment, yeast is used as a subject of living organisms. We used this to give a clear

picture that only living organism can ever carry out cellular respiration. One tube consist of this yeast

act as the manipulated variable where as the empty tube (contain nothing) act as control throughout theexperiment. Other evidence that portrait the choices of this yeast is because they are easy to obtain,

and easy to keep. In addition, this consume only small spaces, so the apparatus doesnt need much

alteration to fit in the samples.

A small capillary U-tube is used because of its small area and diameter which makes the

capillary more sensitive. Thus, any slight change can be observed via the tube. Brodies fluid or

paraffin oil deeply coloured with Sudan III is used as indicators to indicate the relative volume

changes between the two test tubes. Other coloured indicators can be also used in this experiment; as

long as it can be seen clearly. Paraffin oil is naturally less dense than water, so it is more sensitive and

that is why water is not used in this experiment.

1ml syringe is used by connecting it to the experimental test tube. The syringe acts as a

compensator or more like calibrator. The level of pressure inside the tube can be control as to maintain

the level of coloured indicators; balancing it for both sides in the U-tube.


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Limitations

There are several limitations that have been identified throughout this experiment.

It is hard to maintain a strictly constant surrounding temperature as the pressure and volume ofgas within the respirometer causes temperature fluctuation.

The amount of yeast used in the repeating experiments is not identical so their oxygen uptakerate is not the same, resulting in different metabolic rates.

Air bubbles may have been trapped inside the capillary manometer. The leakage of the connection. The leakage can make the pressure inside the test tube equal to

the surrounding condition. As a result, when the carbon dioxide is absorbed by the potassium

hydroxide, no effect can be seen on the pressure changes. Thus, alter the final result of the

experiment.

The heat source from the surroundings. One of the main sources is from the hand contact withthe test tube. The heat can increase the pressure inside the test tube and alter the real result .

The rate of respiration may also increased by the heat from surrounding. So, in order to

minimize this problem, try to avoid touching the closed apparatus, respirometer.


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Sources of errors

Several sources of error in this experiment were identified and steps were taken to minimize these

errors to make the result more accurate.

Make sure that there are no air bubbles when inserting the coloured liquid. This is veryimportant because if a tiny air bubble present, it might and could significantly change the

accuracy when taking the reading, and so about to reduce the reliability on the data collected.

Hence, we need to really make sure when injecting the coloured indicator using the

micropipette.

Changes in external conditions are also affecting the reliability of the experiment thus lightintensity, pressure, and also surrounding temperature need to be constant and fixed throughout

the experiment. To minimize the errors in the experiment, the lights were still on during and

after the experiment.

Although the experiment is carried out in 1 atm pressure, the pressure in the test tube may bediffer due to external force such as the increase in volume of gases in the experiment tubes as

this changes lead to raise in pressure inside the tube. As the result, if the stopper is not well

sealed, pressure will push the stopper out and gases may escape via opened tube. Hence, it is

also important to make sure that every connection are firmly inserted either the three-way

taps, the manometer and also with the tubes for an air-tight seal.

Conclusion

From this experiment it is true that the level of coloured indicator changes and these changes in the

level of coloured indicator is directly proportional to the amount of oxygen consumed by the yeast as

they respire. Hence, this give clear picture that living organism does consume oxygen when cellular

respiration takes place. Thus, the hypothesis is accepted.

Further Investigation

Another experiment can be carried out using same methods of rate of oxygen uptake of yeast but

altering the other external conditions such as light intensity, temperature and pressure.


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References

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2. http://www.pc.maricopa.edu/Biology/rcotter/Title%205%20Files/EukaryoticCellStructure/EukaryoticCellStructure6.html. Accessed on 4

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October 2012.3. http://www.google.com.my/imgres?q=yeast+fermentation&hl=en&sa=X&biw=1280&bih=886

&tbm=isch&prmd=imvnsb&tbnid=tzv5DH_orY6dUM:&imgrefurl. Accessed on 4th

October

2012

4. http://www.sparknotes.com/biology/cellrespiration/glycolysis/section3.rhtml&docid=YSIWiokx3Hoj4M&imgurl=http://img.sparknotes.com/figures/1/18b9012870c85fba3a8046a767b52ddf/

anaerobicaerobic.gif&w=420&h=345&ei=3GVtUKHcM8LrrQeEioCACw&zoom=1&iact=rc

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5. Pearson International edition Biology, 7th edition, Campbell, Reece 20056. Clegg, C.G. 2009.Edexcel Biology for A.2. 209p. London: Hodder Education.7. Fullick, A. 2009. Edexcel A.2. Biology. 272p. United Kingdom: Pearson Education Limited.8. Reece, J.B. and N.A. Campbell. 2005. Biology. Seventh edition. 1231p. United States of

America: Pearson EducationLimited.

9. Gan, W.Y. 2006. Exploring Biology. 348 p. Selangor : Fajar Bakti Sdn. Bhd.
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measure oxygen uptake by respirometer