BIOLOGY LAB REPORT TITLE : MEASURING THE RATE OF OXYGEN UPTAKE OF YEAST BY USING RESPIROMETER PREPARED BY I/C NUMBER

STUDENT ID GROUP LECTURERS NAME PRACTICAL DATE SUBMISSION DATE : : : : : :

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 potassium hydroxide 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 10 20 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 plantae kingdom 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.

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.

Figure 3 : Suitable condition for Yeast growth (2)

Figure 4 : Fermentation in Yeast (3)

Figure 5: Both Aerobic and Anaerobic Respiration in Yeast

(3)

2. Respirometer A respirometer is a device used to measure the rate of respiration of a living organism by measuring its rate of exchange of oxygen and/or carbon 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.

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 oxygen from 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)

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.

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.

Objective To demonstrate the uptake of oxygen in yeast respiration and to measure the rate at yeast respires. To measure the level of difference of liquid in manometer

Problem 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 Manipulated Variable: Presence of yeast Two test tubes were used , one with the presence of yeast and another without the yeast. Ways to control the variables

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.

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 dried and wiped using a paper towel. 2. Using a micropipette, a small volume of Brodies fluid or paraffin deeply coloured with Sudan III 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 carefully assembled, ensuring good seals throughout. 4. 15cm3 of potassium hydroxide solution was transferred into the two test tubes respectively using 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 the manometer), 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 connected to 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.

11. The initial level of manometer fluid was observed and recorded. Then, the stopwatch was started. 12. The position of the coloured fluid in the manometer was recorded at subsequent 1 minute intervals 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.

Results Initial level of monometer: 4.4 cm Initial position of syringe: 0.5cm3 Displace in manometer level: Manometer reading (cm) 4.4 4.9 5.9 6.2 7.2 7.5 7.8 Displacement in manometer level (cm) 0.0 4.9 4.4 = 0.5 5.9 4.4 = 1.5 6.2 4.4 = 1.8 7.2 4.4 = 2.8 7.5 4.4 = 3.1 7.8 4.4 = 3.4

Time (second) 0 60 120 180 240 300 360

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 cm 4.4 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

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 obtained in 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.

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 the experiment. Other evidence that portrait the choices of this yeast is because they are easy to obtain, and easy to keep. In additio n, 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 observe d 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.

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 of gas within the respirometer causes temperature fluctuation. The amount of yeast used in the repeating experiments is not identical so their oxygen uptake rate 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 with the 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.

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 very important 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 light intensity, 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 be differ 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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