UNIVERSITI TEKNOLOGI MARA INTERNATIONAL EDUCATION COLLEGE (INTEC)

NAME GROUP STUDENT ID TITLE OF PRACTICAL

:HANNAH CHEN YEE SZE :12M12 :2011219506 :MEASURING THE RATE OF OXYGEN UPTAKE USING A RESPIROMETER

DATE LECTURER

:3/10/2012 :MS FATHIAH

Introduction:

Living organisms require energy for various activities and living processes. Some of the processes which require energy are excretion of waste products, muscle contraction which enables locomotion, cell division in which new cells are produced for growth and development, transmission of nerve impulses, absorption of digested food through active transport, active transport of biochemical substances, maintaining the body temperature in warm-blooded animals) and synthesis of lipids, hormones, proteins and enzymes. Organic substances like carbohydrates, lipids and proteins contain chemical energy which enters living organisms in the form of food and in order to be converted into a form of energy which can be readily used by cells, respiration is carried out. There are two types of respiration- aerobic respiration and anaerobic respiration. Aerobic respiration takes place in the cells of living organisms to convert chemical energy from food into energy in the form of ATP. It is a redox reaction and its equation is as shown below. C6H12O6 + O2 CO2 + H2O + energy The main substrate for respiration is glucose. When glucose is not available, amino acids and fatty acids can be used as substrates too. There are many complicated steps involved in respiration, which are all controlled by enzymes. The first stage of respiration is glycolysis. In this process, the 6-carbon glucose is phosphorylated by using two molecules of ATP. This activates the glucose and prevents glucose from being transported across the cell membrane. Then, the phosphorylated 6-carbon molecules will then be split into two carbon triose phosphate called glyceraldehyde-3-phosphate. Each molecule of triose phosphate will be oxidised by nicotiamide adenine dinucleotide (NAD). Two molecules of ATP are also produced. This production of ATP is known as substrate level phosphorylation. Each molecule of triose phosphate will result in a pyruvate. The net gain of ATP from glycolysis is two molecules.

Diagram 1: Glycolysis Next, the pyruvate will enter the link reaction and this process requires oxygen. Pyruvate is decarboxylated to form a molecule of carbon dioxide. It is also dehydrogenated to form a molecule of reduced NAD. Pyruvate becomes acetate which combines with coenzyme A to form acetyl coenzyme A (acetyl coA). This will proceed to the Krebs Cycle, which is also known as the citric acid cycle.

Diagram 2: Link Reaction and Krebs Cycle Acetyl coA will combine with a four-carbon molecule called oxaloacetate to form citrate, a six-carbon molecule. Coenzyme A will then be released and recycled in the next link reaction. Citrate is decarboxylated and dehydrogenated, forming one molecule of carbon dioxide and one molecule of reduced NAD. It will form a five-carbon molecule known as alpha-ketoglutarate. dephosphorylated, decarboxylated, and then undergo three times of dehydrogenation to form one molecule of ATP, one molecule of carbon dioxide, two molecules of reduced NAD and one molecule of reduced flavine adenine dinucleotide (FAD). Alpha-ketoglutarate itself becomes oxaloacetate which combines with acetyl coA. This forms a cycle, hence the name Krebs cycle. The hydrogen acceptors (reduced NAD and reduced FAD) will enter the electron transport chain (ETC) to be oxidised. As the electrons pass from carriers to carriers, energy is released in the form of ATP

Diagram 3: Electron Transport Chain In the chemiosmotic theory by Peter Mitchell, he said that protons were actively transported into the intermembrane space using energy provided by the electrons as electrons are passed down the electron transport chain. The protons cannot diffuse the inner membranes as they are impermeable to protons. The pH gradient , concentration gradient and electrochemical gradient that exist between the intermembrane space and the matrix of the mitochondria causes the protons to move back into the matrix via special pores found on stalked particles. The ATP-ase on these pores are used to drive the synthesis of ATP and 38 ATP are gained. Anaerobic respiration is carried out when oxygen is absent. In plants, this is known as alcoholic fermentation, where else in animals, it is known as lactic acid fermentation. Glycolysis is still carried out, but pyruvate will not enter the link reaction but undergo anaerobic respiration. The product is lactic acid and only two molecules of ATP. No carbon dioxide is produced. It is much less efficient compared to aerobic respiration because only two molecules of ATP are formed in contrast to the 38 molecules of ATP formed by aerobic respiration. This shows that oxygen is very important as the final electron acceptor which allows the Krebs cycle and electron transport chain to work.

Yeasts are eukaryotic microorganisms from the kingdom Fungi. They are unicellular and their size varies depending on the species, usually ranging between 3-4m in diameter. They usually reproduce asexually by a process called budding. Yeasts are chemoorganotrophs, as they use organic compounds as a source of energy and do not require sunlight to grow. They are also facultative anaerobes, which means they can respire both aerobically and anaerobically.

Diagram 4: Yeast

Objective: To investigate the rate of oxygen uptake of yeast using a respirometer. Problem Statement: What is the rate of oxygen uptake of yeast? Hypothesis: The respiration rate can be measured by the means of a respirometer by calculating the uptake of oxygen per unit time. Null hypothesis: The respiration rate cannot be measured by the means of a respirometer by calculating the uptake of oxygen per unit time. Apparatus: Boiling tubes, 1cm3 syringe, three-way tap, manometer tube, gauze, measuring cylinder, stopper, delivery tubes, micropipette Materials: Yeast, red dye, filter paper, potassium hydroxide solution, cling film

Procedure:

Diagram 5: Apparatus Set-up 1. The apparatus was set up as shown above

2. Both of the boiling tubes were filled with 7cm3 of potassium hydroxide solution.

3. One of the boiling tubes was stoppered, and a syringe was inserted on top of the stopper, as shown in Figure . 4. A gauze was placed in the other boiling tube. 5. Two filter papers soaked in yeast were placed in the experimental tube, on the gauze and not touching the potassium hydroxide solution.
6. The stoppers were replaced.

7. The manometer tube was filled with approximately 200 l of red dye using a micropipette.

8. The manometer tube was connected to both of the boiling tubes simultaneously.
9. The liquid in the manometer tube was adjusted using the syringe attached to the non-

experimental tube so that the level of the liquid in the manometer tube became equal 10. The starting position of the fluid was recorded 11. The three way tap was turned to ensure air flows between the manometer and the boiling tubes 12. The readings of the level of the coloured dye is recorded every minute for 6 minutes. The increment of red dye level was assumed to be the volume of oxygen absorbed by the yeast. Results: Initial level of manometer fluid = 2.5cm Initial position of syringe piston = 0.5cm3 Displacement in manometer level (cm) = Final reading (cm) Initial reading (cm) *Displacement in manometer level corresponds to the total amount of oxygen consumed. Time/Minutes Readings on Manometer/cm Displacement in

Manometer Level/cm 0 2.5 0.00 1 2.6 0.10 2 3.1 0.60 3 3.4 0.90 4 3.8 1.30 5 4.4 1.90 6 4.5 2.00 7 4.7 2.20 8 5.0 2.50 9 5.3 2.80 10 5.6 3.10 11 5.8 3.30 12 6.1 3.60 Table 1: Table showing Displacement of manometer Level in a Period of 12 Minutes Final level of manometer fluid = 6.1cm

Final position of syringe piston = 0.5cm3 Total increment in manometer reading = Total amount of oxygen consumed (cm) = 6.1 cm 2.5 cm = 3.6 cm Calculated rate of oxygen uptake (cm s-1) = = 3.6 cm 720 seconds = 5.00 x 10-3 cm s-1

Analysis of results: In this experiment, yeast was used as it is cheap, easily available and also easy and safe to handle. Before the experiment, the level of the red dye in the manometer was adjusted to be at the same level at both sides and the initial level on the experimental tube side was recorded. When the three way tap is turned to the direction of the manometer and the stopwatch was started, it can be seen that the level of red dye at the side of the manometer connected to the experimental tube started increasing, while the level of dye on the control side decreased. This shows that the volume of air in the experimental tube is decreasing as time passes. As the volume of air decreases, the pressure also decreases. Thus, the level of red dye increases in response to the decreased pressure. The volume of air decreased as carbon dioxide released during respiration has been absorbed by the potassium hydroxide solution, while the oxygen in the experimental tube is being used up in respiration. As there is no respiration taking place in the control tube, the volume of gas remains constant and the pressure is constant as well. The control tube is very important as it helps to compensate for the change in temperature and pressure of the atmosphere. This will help to ensure that the change in level of red dye is solely due to the respiration of yeast and not by other external factors. The rate of respiration is calculated by measuring the amount of oxygen consumed by yeast over a period of time. The oxygen consumed is indicated by the level of fluid in manometer. The equation for respiration is as shown below:

Discussion: The main limitation in this experiment is that yeast is a facultative anaerobe and can respire both aerobically and anaerobically. Thus, the rate of respiration calculated may not be accurate as when yeast respires anaerobically, they do not take in oxygen and the carbon dioxide released is absorbed by potassium hydroxide solution. Thus, there is no change in volume of gas in the experimental tube and hence, no change in pressure. The level of red dye does not change. Fluctuations in surrounding temperature also affect the experimental results as gas expands when temperature increases and thus have a bigger volume. Although this may be compensated slightly by the control set, it may still affect the displacement of the red dye over the period of time when it is recorded. The connections to the tubes may not have been completely tight and secure, for example the connection between the delivery tubes to the manometer tube, the stopper for the boiling tubes and the connection between the syringe and the non-experimental tube. This may allow the gas inside the tubes to escape through the unsealed gaps. Besides, parallax error are likely to occur when the measurement of the level of the coloured liquid in respirometer. This will result in wrong readings being taken and the results of the experiment being less accurate. Some ways to overcome these limitations and sources of errors are to use other organisms such as crickets that only undergo aerobic respiration. Fluctuations in the surrounding temperature can be overcome by using a water bath set at a constant temperature. This will ensure temperature is kept constant and does not affect the displacement of red dye, thus making the results of the experiment more reliable. All the connections should be sealed tightly with cling film to ensure the connections are airtight and air is unable to escape via the connections. This will ensure that the air pressure does not change due to gas escaping from the tubes.

When readings are taken, the eye level must be perpendicular to the scale of the respirometer. This will prevent parallax error and wrong readings to be taken and make sure the results are more accurate and reliable.

Safety precautions: 1. Care must be taken when handling the apparatus to avoid the apparatus from breaking. If the apparatus breaks, the broken apparatus should be swept away using a broom, not using a hand as the broken glass may cut the fingers. 2. Potassium hydroxide solution has to be handled with care as it is strong base and corrosive. 3. If the KOH comes into contact with skin accidentally, wash under running tap water immediately.

Conclusion: During aerobic respiration where oxygen is available, living organisms take in oxygen from the surrounding and release carbon dioxide. The respiration rate can be measured by means of a respirometer by calculating the uptake of oxygen per unit time. The displacement of the coloured fluid in the capillary tube is an indication to the uptake of oxygen. In this experiment, we can conclude that the rates of respiration of yeast is 5.00 x 10-3 cm s-1 Further work: For further investigation, a manipulated variable can be included to this experiment, for instance, the surrounding temperature. Water bath with different temperature can be used and their effects on the respiration rate can be investigated. Theoretically, the rate of respiration increases with temperature as the enzyme activity speeds up at higher temperature. However, if the temperature is too high, enzymes may denature, and finally respiration ceases and organisms die. Word count: 2240 words

Bibliography: Non web based:

1. Salters-Nuffield Advanced Biology for Edexcel A2 Biology 2. Clegg C.J, Edexcel Biology, Hodder Education, London, UK, 2008, A Pearson Company. 3. Edexcel A2 Biology, A Pearson Company 2008.

Web based:
1. http://www.purchon.com/biology/respire.htm 2. http://biology.about.com/od/cellularprocesses/a/cellrespiration.html 3. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellularRespiration.html 4. http://www.essortment.com/all/cellularrespira_rmpr.htm 5. http://www.wisegeek.com/what-is-aerobic-respiration.htm 6. http://idv.sinica.edu.tw/ckao/About%20the%20Lab.html