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Internal Assessment: Fermentation Biology Higher Level Examination session: May 2012 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast” I.- Introduction The world glycolysis (sugar splitting) is thought to have been one of the first biochemical pathways to evolve. It uses oxygen and occurs in the cytosol of the cell. The sugar splitting proceeds efficiently in aerobic or anaerobic environments. Glycolysis is the metabolic pathway that is common to all organisms on Earth. (Miller, & Levine, 2006) However, when oxygen is not present, the process of glycolysis is combined with different pathways. When combining these pathways with glycolysis, fermentation takes place. “Some organisms derive their ATP completely without the use of oxygen without the use of oxygen and are referred to as anaerobic. The breakdown of organic molecules for ATP production in anaerobic way is also called fermentation.” (Damon, McGonegal, & Ward, 2009) “During fermentation, cells convert NADH to NAD+ by passing high energy electrons back to pyruvic acid. This action converts NADH back into the electron carrier NAD+, allowing glycolysis to continue producing a steady supply of ATP.” (Miller, & Levine, 2006) There exist two types of fermentation which are alcoholic fermentation and lactic acid fermentation. However, this practice is focus on alcoholic fermentation due to the production of carbon dioxide (CO2) gas. Alcoholic fermentation. Yeast (single-celled fungus) uses this type of fermentation in order to produce ATP molecules. Yeast cells take in glucose from the environment and generate a net of gain of two ATP by way of glycolysis. (Damon, McGonegal, & Ward, 2009) Glycolysis produces organic products known as pyruvate molecules. The next step is taken when yeast converts both of the 3-carbon pyruvate molecules to produce “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 molecules of ethanol. “Ethanol is a 2-carbon molecule, which means that a carbon atom was lost during the conversion. Therefore, the carbon atom is given off in a carbon dioxide molecule. The waste products produced by yeast (ethanol and carbon dioxide) are spread into the environment.” ( D a m o n , M c G o n e g a l , & W a r d , 2 0 0 9 ) II.- Objective The objective of this practice is to study the effect of different concentrations of glucose on the rate of anaerobic respiration in yeast for the production of carbon dioxide gas (CO2). III.- Research Question How will the effect of different concentrations of glucose (5%, 10%, 15%, 20%, 25%) be on the production of carbon dioxide gas (CO2) by yeast fermentation at 37°C? IV.- Hypothesis If increasing the concentration of glucose then the rate of anaerobic respiration in yeast will be faster. Therefore, the carbon dioxide (CO2) produced by the reaction will increased at a higher concentration. This will happen because for a great part of the reactions occurring in nature in our daily life, increasing the concentration of one of the reactants increases the rate of the reaction. For a reaction to occur, the particles of the reactants must collide. Therefore, “at a higher concentration the collisions are greater.” (Clark, 2002) As yeast contains certain types of enzymes, the effect of substrate concentration in enzymes is as follows: “As the concentration of substrate increases, the rate of reaction will increase as well” (Damon, McGonegal, & Ward, 2009). This is because there is an increase in collisions between molecules so if there is a higher quantity of molecules, these will collide in a greater rate. Increasing glucose concentration, there will be more molecules of glucose to break down in order to produce more ethanol and CO2 gas. In breaking down molecules, there are more heat energy released and, as a result more molecules will collide with one another and with the enzymes in yeast. However, we cannot generalize 2 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 the previous statement, because not in all the cases the following statement will have the same effect. Enzymes have a certain rate at which they can work better. V.- Variables For anaerobic respiration in yeast, there is one main factor that affects directly the rate of reaction between glucose and yeast: temperature. In order for the reaction to take place, several factors must be met. This factor will be controlled as it can be appreciate in the following table: Table 1. Identification of Variables Type of Variable Units Control Method variable Independent variable Glucose concentration Percentage The percentage of glucose solution (%) was controlled at the moment of making the solution. Five different concentrations of glucose solution were used (5%, 10%, 15%, 20%, and 25%) Dependent variable Carbon dioxide Milliliters The amount of carbon dioxide gas (CO2) gas (ml) produced was monitored using a 250 production ml graduated cylinder (±5%). It was measured by means of volume of water displaced. (Continuation) Table 1. Identification of Variables Type of Variable Units Control Method Time Minutes Data will be collected at intervals of 5 variable Controlled 3 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 variables (min) min. during a period of 25 min. (5, 10, 15, 20 and 25 min) by means of a chronometer (± 0.5 s.). Temperature Celsius An optimum temperature of 37°C was degrees used to carry out the reaction. The (°C) temperature of the water bath was monitored by means of a thermometer (± 0.5 ˚C) Yeast Percentage Concentration of yeast was controlled concentration (%) at the moment of making the solution. Yeast was used at a 10% concentration. Yeast volume Milliliters (ml) 50 ml of yeast solution were required for each trial throughout the experiment. Glucose volume Milliliters In each trial 50 ml of glucose solution (ml) were used at five different concentrations (5%, 10%, 15%, 20%, and 25%). VI.- Materials: 250 ml Graduated cylinder (±5 %) 100 ml Graduated cylinder (±5 %) 125 ml Flask (±5 %) 500 ml Beaker (±10 %) Thermometer (±0.5 °C) Chronometer (± 0.5 s) Rubber stopper Water bath Support stand rod (base, support and clamp/holder) Container Incubator 4 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 Test tube rack Reagents 250 ml of 5% Glucose solution 250 ml of 10% Glucose solution 250 ml of 15% Glucose solution 250 ml of 20% Glucose solution 250 ml of 25% Glucose solution 1. 25 L of 10% Yeast solution Tap water VII.- Procedure Focusing on glucose and yeast reaction, the following method was designed with the objective to measure the production of carbon dioxide (CO 2) gas. It must be pointed that the amount of CO2 was measured by means of the volume of water displaced in the 250 ml graduated cylinder (±5%). Five repetitions were carried out for each of the different concentrations of glucose solution at intervals of 5 min (5, 10, 15, 20 and 25 min). The following procedure was used for the development of the experiment: 1. Each flask and beaker used was labelled in order to avoid any confusion. 2. Then the 10% yeast solution was incubated at 37 °C inside the incubator, for a period of one hour. In addition, each of the five glucose concentrations was incubated for a period of one hour (at the same temperature), in order to fast the experiment. 3. While yeast and glucose were being incubated; the electric water bath was filled with 2 liters1 of tap water and it was turned on. 4. Then the temperature of the water bath was monitored by means of a thermometer (±0.5 °C) until it reached an optimum temperature of 37 °C. 5. After setting the water bath, a container was filled with approximately 2 liters2 of tap water. 1 2 The amount of water tap does affect neither the experiment nor the results. The amount of water tap does affect neither the experiment nor the results. 5 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 6. A support stand rod was set up besides the container with tap water in order to hold the 250 ml graduated cylinder (±5%) by means of a clamp/holder attached to the rod. 7. Before holding the 250 ml graduated cylinder (±5%) by means of the support stand rod, the graduated cylinder was submerged inside the container in order to fill the graduated cylinder with tap water. 8. Once the 250 ml graduated cylinder (±5%) was filled with tap water, it was placed in the support stand. 9. The initial volume of the 250 ml graduated cylinder (±5%) was collected before initiating the fermentation process. 10. Then the hose of the rod stopper was placed underneath the 250 ml graduated cylinder (±5%). 11. Once the one hour period was completed, the yeast solution and each of the five concentrations of glucose solution were taken out from the incubator. 12. Then 50 ml of 5% glucose solution were measured by means of the 100 ml graduated cylinder (±5 %). 13. After that, the 50 ml of 5% glucose solution were placed into a 125 ml flask (±5 %) in order to introduce it into the electric water bath. 14. In addition, 50 ml of 10% yeast solution were measured by means of the 100 ml graduated cylinder (±5 %). 15. Then the 50 ml 10% yeast solution were placed into another 125 ml flask (±5 %) labelled in order to introduce it into the electric water bath together with the flask containing the 50 ml of 5% glucose solution. 16. The temperature of both solutions was regulated by means of a thermometer (±0.5 °C), until they reach the optimum temperature of 37 °C. 17. Once the temperature was reached, the reaction was carried out. The flask containing the 5% glucose solution was mixed with the flask containing 10% yeast solution. 18. The flask was closed as fast as possible using the rod stopper. This step was taken into action as fast as possible to prevent a loss of CO2. 19. Then the chronometer (±0.5 s) was activated once the reaction had begun. 6 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 20. In order to measure the volume of water displaced from the 250 ml graduated cylinder (±5%) to determine the production of CO2 gas delivered by the reaction, the stopwatch was stopped in intervals of 5 minutes during a 25 min period. 21. Then the water displaced from the graduated cylinder was recorded, by measuring the final volume in milliliters. 22. The data was collected. 23. For each of the five trials of the same glucose concentration, the same procedure was followed. After ending with one complete concentration, the same process was repeated for each of the five glucose concentrations. VIII.- Set-up of apparatus Figure 1. Glucose and yeast reaction producing CO2 Figure 2. Temperature controlled by the thermometer IX.- Safety During the development of the experiment, neither dangerous materials nor hazard reactants were used, so the need of gloves and safety goggles was not necessary. In addition, it was not used a dangerous temperature for the electric water bath. X.- Data collection The raw data obtained from the experiment is presented in the following tables. The tables contained the data of the initial volume3 of the 250 mL graduated cylinder with an 3 The initial volume is represented in the table as V0. 7 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 uncertainty of ±5 % as well as the final volume4 of water displaced by CO2 gas production, after a period of 25 min, for each of the five different concentrations of glucose (5%, 10%, 15%, 20% and 25%). Table 2. Volume of water displaced to determine CO2 (ml) production at 5% glucose concentration. Volume of water displaced by CO2 gas (ml) at 5% glucose concentration Trial 1 Trail 2 Trial 3 Trial 4 Trial 5 Time V0 (ml) Vf (ml) by V0 (ml) Vf (ml) by V0 (ml) Vf (ml) by V0 (ml) Vf (ml) by V0 (ml) (min) ±5% CO2 ±5% CO2 ±5% CO2 ±5% CO2 ±5% ±0.5 ±5% ±5% ±5% ±5% Vf (ml) by CO2 ±5% 5 13.00 26.00 83.00 97.00 15.00 25.00 90.00 102.00 16.00 29.00 10 13.00 47.00 83.00 121.00 15.00 52.00 90.00 123.00 16.00 53.00 15 13.00 64.00 83.00 141.00 15.00 72.00 90.00 143.00 16.00 72.00 20 13.00 71.00 83.00 151.00 15.00 80.00 90.00 159.00 16.00 80.00 25 13.00 81.00 83.00 161.00 15.00 92.00 90.00 164.00 16.00 89.00 Table 3. Volume of water displaced to determine CO2 (ml) production at 10% glucose concentration. Volume of water displaced by CO2 gas (ml) at 10% glucose solution Trial 1 Time Trial 2 Trial 3 Trial 4 Trial 5 VO Vf (ml) by VO Vf (ml) by VO Vf (ml) by VO Vf (ml) by VO Vf (ml) by (min) (ml) CO2 (ml) CO2 (ml) CO2 (ml) CO2 (ml) CO2 ±0.5 ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% 5 26.00 55.00 34.00 62.00 33.00 57.00 16.00 36.00 36.00 65.00 10 26.00 108.00 34.00 125.00 33.00 120.00 16.00 94.00 36.00 124.00 15 26.00 172.00 34.00 177.00 33.00 174.00 16.00 162.00 36.00 179.00 20 26.00 184.00 34.00 190.00 33.00 191.00 16.00 173.00 36.00 192.00 25 26.00 194.00 34.00 206.00 33.00 200.00 16.00 181.00 36.00 200.00 Table 4. Volume of water displaced to determine CO2 (ml) production at 15% glucose concentration. Volume of water displaced by CO2 gas (ml) at 15% glucose solution Trial 1 (ml) CO2 (ml) CO2 (ml) CO2 (ml) CO2 (ml) CO2 ±0.5 ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% VO Vf (ml) by Trial 5 (min) VO Vf (ml) by Trial 4 VO VO Vf (ml) by Trial 3 Time 4 Vf (ml) by Trial 2 VO Vf (ml) by The final volume displaced after each 5 min interval is represented in the table as V f. 8 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 5 12.00 38.00 14.00 36.00 14.00 28.00 12.00 28.00 13.00 37.00 10 12.00 85.00 14.00 78.00 14.00 70.00 12.00 74.00 13.00 75.00 15 12.00 136.00 14.00 134.00 14.00 134.00 12.00 130.00 13.00 138.00 20 12.00 186.00 14.00 200.00 14.00 187.00 12.00 184.00 13.00 189.00 25 12.00 236.00 14.00 230.00 14.00 233.00 12.00 232.00 13.00 246.00 Table 5. Volume of water displaced to determine CO2 (ml) production at 20% glucose concentration. Volume of water displaced by CO2 gas (ml) at 20% glucose solution Trial 1 Time Trial 2 VO Vf (ml) by (min) (ml) CO2 ±0.5 ±5% ±5% 5 32.00 40.00 10 32.00 15 Trial 3 Trial 4 Trial 5 VO (ml) Vf (ml) by VO Vf (ml) by VO Vf (ml) by VO Vf (ml) by ±5% CO2 (ml) CO2 (ml) CO2 (ml) CO2 ±5% ±5% ±5% ±5% ±5% ±5% ±5% 40.00 51.00 16.00 29.00 26.00 38.00 26.00 39.00 86.00 40.00 92.00 16.00 72.00 26.00 80.00 26.00 83.00 32.00 132.00 40.00 138.00 16.00 118.00 26.00 126.00 26.00 132.00 20 32.00 182.00 40.00 184.00 16.00 165.00 26.00 178.00 26.00 180.00 25 32.00 223.00 40.00 230.00 16.00 214.00 26.00 215.00 26.00 221.00 3 Table 6. Volume displaced of CO2 (cm ) production at 25% glucose concentration. Volume of water displaced by CO2 gas (ml) at 25% glucose solution Trial1 Time Trial 2 Trial 3 Trial 4 Trial 5 VO Vf (ml) by VO Vf (ml) by VO Vf (ml) by VO Vf (ml) by VO Vf (ml) by (min) (ml) CO2 (ml) CO2 (ml) CO2 (ml) CO2 (ml) CO2 ±0.5 s ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% ±5% 5 12.00 35.00 32.00 48.00 54.00 70.00 34.00 60.00 14.00 34.00 10 12.00 64.00 32.00 88.00 54.00 106.00 34.00 92.00 14.00 73.00 15 12.00 108.00 32.00 124.00 54.00 145.00 34.00 126.00 14.00 104.00 20 12.00 134.00 32.00 144.00 54.00 174.00 34.00 153.00 14.00 133.00 25 12.00 160.00 32.00 175.00 54.00 203.00 34.00 179.00 14.00 161.00 XI.- Data processing a) Calculating the production of CO2 9 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 In order to obtain the amount of carbon dioxide (CO2) gas produced from the experiment (which will be measured as volume), we must use the volume of water displaced during the 25 min period each 5 min. In the SI, the unit used to measure the volume of any substance is the cm 3. As a result, the volume of carbon dioxide (CO2) gas recorded from the experiment must be converted to cm3. The following equivalence must be used: 1 Cubic Centimeter (cm3) = 1 Milliliter Example: For 5% glucose solution initial volume: 13.00 ml = 13.00 cm3 The volume of water displaced represents the amount of CO2 gas produced by the fermentation of each of the five concentrations of glucose solution and yeast. First of all, it must be calculated the difference between the volumes of CO 2 gas displaced each 5 min interval during the 25 min period. Therefore, we must subtract the final volume of water displaced by CO2 at each 5 min interval, from the initial volume recorded by the 250 ml graduated cylinder (±5%). This procedure must be used for each of the samples collected during the experiment. In order to obtain the difference between the volume displaced each time interval and the initial volume, the following formula must be used: d ( ) Where: d = Difference = Initial volume = Volume displaced at y- time interval Example: For 5% glucose solution d ( ) d = 13.00 cm3 3 Table 7. Volume of CO2 (cm ) produced during a 25 min period at 5% glucose solution. Volume of CO2 (cm3) produced during a 25 min period at 5% glucose solution. 10 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 TIME (min) 0 5 10 15 20 25 Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 0.00 13.00 34.00 51.00 58.00 68.00 0.00 14.00 38.00 58.00 68.00 78.00 0.00 10.00 37.00 57.00 65.00 77.00 0.00 12.00 33.00 53.00 69.00 74.00 0.00 13.00 37.00 56.00 64.00 73.00 3 Table 8. Volume of CO2 (cm ) produced during a 25 min period at 10% glucose concentration. Volume of CO2 (cm3) produced during a 25 min period at 10% glucose solution. TIME (min) 0 5 10 15 20 25 Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 0.00 19.00 72.00 126.00 148.00 158.00 0.00 28.00 91.00 143.00 156.00 167.00 0.00 24.00 87.00 141.00 158.00 167.00 0.00 20.00 78.00 126.00 146.00 155.00 0.00 29.00 88.00 143.00 156.00 164.00 3 Table 9. Volume of CO2 (cm ) produced during a 25 min period at 15% glucose concentration. Volume of CO2 (cm3) produced during a 25 min period at 15% glucose solution. TIME (min) 0 5 10 15 20 25 Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 0.00 26.00 73.00 124.00 174.00 224.00 0.00 22.00 64.00 120.00 186.00 216.00 0.00 14.00 56.00 120.00 173.00 219.00 0.00 16.00 62.00 118.00 172.00 220.00 0.00 24.00 62.00 125.00 176.00 233.00 3 Table 10. Volume of C02 (cm ) produced during a 25 min period at 20% glucose concentration. Volume of CO2 (cm3) produced during a 25 min period at 20% glucose solution. TIME Trial 1 Trial 3 Trial 4 Trial 4 Trial 5 11 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 (min) 0 5 10 15 20 25 0.00 8.00 54.00 100.00 150.00 191.00 0.00 11.00 52.00 98.00 144.00 190.00 0.00 13.00 56.00 102.00 149.00 198.00 0.00 12.00 54.00 100.00 152.00 189.00 0.00 13.00 57.00 106.00 154.00 195.00 3 Table 11. Volume of CO2 (cm ) produced during a 25 min period at 25% glucose concentration. Volume of CO2 (cm3) produced during a 25 min period at 25% glucose solution. TIME (min) 0 5 10 15 20 25 Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 0.00 23.00 52.00 96.00 122.00 148.00 0.00 16.00 56.00 92.00 112.00 143.00 0.00 16.00 52.00 91.00 120.00 149.00 0.00 26.00 58.00 92.00 119.00 145.00 0.00 20.00 59.00 90.00 119.00 147.00 The following table shows the total final volume of CO2 gas displaced for each of the five different concentrations of glucose solution. 3 Table 12. Final volume displaced of CO2 (cm ) production of each of glucose concentrations. Final volumes of CO2 gas (cm3) of each of glucose concentrations GLUCOSE TRIAL 1 TRIAL 2 TRIAL 3 TRIAL 4 TRIAL 5 CONCENTRATION 5% 68.00 78.00 77.00 74.00 73.00 10% 168.00 172.00 167.00 165.00 164.00 15% 20% 25% 224.00 191.00 148.00 216.00 190.00 143.00 219.00 198.00 149.00 220.00 189.00 145.00 233.00 195.00 147.00 b) Calculating mean and standard deviation 12 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 In order to analyze and to graph the data to discuss the hypothesis previously stated, the mean and the standard deviation of each of the samples must be obtained. To obtain the mean of the samples, the following formula must be used: ̄ ̄ represents the mean of the values. is all the values collected through the experiment.- represents the number of items used in the sample. Example: Taking the data collected for 5% glucose concentration at 5 min interval: ̄ ̄ cm3 The reason of using standard deviation is because in the laymen’s terms, the standard deviation represents a number which tells how far from the mean a data value is with respect to how far the other data values are from the mean. ∑ S= √ ((∑ ) ) Example: Taking the data collected for 5% glucose concentration at 5 min interval. S= √ ∑ (( ) S = 1.52 ) n x ̄ 1 2 3 4 5 13.00 14.00 10.00 12.00 13.00 62.00 169.00 196.00 100.00 144.00 169.00 778.00 2 c) Dispersion graphs 13 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 In order to analyze the data obtained by the standard deviation values of each of the five different sugar concentrations (5%, 10%, 15%, 20% and 25%), it is necessary to plot a dispersion graph. These graphs will show the correlation between each time (min) period interval and the mean volumes of CO2 gas (cm3) produced during yeast fermentation. Furthermore, the graphs will show the error bars which are helpful to appreciate the standard deviation calculated for each of the five sugar concentration in a graphical form. The error bars state how far from the mean a value is with respect to how far the other values are from the mean. In addition, the line of logarithmic regression can be appreciated from the dispersion graph. This line shows the logarithmic relationship between each mean value obtained at each 5 min. interval. From the graphs, the correlation coefficient of Pearson (R2) can be appreciated. This coefficient is necessary to determine in a mathematical way the correlation strength between the mean volumes of CO2 gas displaced. In order to have an accurate analysis form the graphs, the maximum value of 250 cm3 was chosen to be plot in the y-axis. The following tables and graphs show the standard deviation, the mean values and the regression line for each glucose concentration (5%, 10%, 15%, 20% and 25%): Table 13. Mean and standard deviation of the data collected of CO2 gas produced at 5% glucose concentration TIME (min) 0 5 10 15 20 25 TRIAL 1 Volume of CO2 TRAIL 2 Volume of CO2 TRAIL 3 Volume of CO2 TRIAL 4 Volume of CO2 TRAIL 5 Volume of CO2 (cm ) (cm ) (cm ) (cm ) (cm ) 0.00 13.00 34.00 51.00 58.00 68.00 0.00 14.00 38.00 58.00 68.00 78.00 0.00 10.00 37.00 57.00 65.00 77.00 0.00 12.00 33.00 53.00 69.00 74.00 0.00 13.00 37.00 56.00 64.00 73.00 3 3 3 3 ̄ S 0.00 12.40 35.80 55.00 64.80 74.00 0.00 1.52 2.17 2.92 4.32 3.94 3 14 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 Graph 1. Mean and standard deviation of the data collected for 5% glucose concentration GLUCOSE 5% - CO2 PRODUCTION 250 y = 38.66ln(x) - 50.84 R² = 0.99 CO2 PRODUCTION (cm3) 200 150 Glucose 5% Tendency line 100 50 0 0 5 10 15 20 25 30 TIME (min) From the previous graph it can be observed the rate of CO2 gas released in fermentation for the 5% glucose solution with yeast. The graph shows the mean values of each trial performed for the 5% glucose concentration. When using the 5% glucose concentration, the error bars show that there was a very small difference between the production of CO2 gas collected in each trial. The fermentation rate in each trial for the 5% glucose concentration did not differ significantly. The standard deviation error bars show that the difference in the values obtained for the production of CO2 gas collected in each trial was very small. This means that the results of obtained were very near between them which gives a more accurate information. Furthermore, the tendency line shows the correlation between the values obtained in each 5 min interval. As time increases, there is an increased in the production of CO 2 gas. Visually, it can be observed that there is a very strong positive correlation between 15 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 the means. From the graph, it can pointed that the correlation coefficient (R 2) is 0.99, which means that as the coefficient of correlation obtained is approaching to 1, there is a very strong correlation, and there is not any outlier. The production of CO 2 gas released during fermentation was almost the same in each trial. There was not a significant difference in the rate of CO2 released in each trial for 5% glucose concentration. Table 14. Mean and the standard deviation of the data collected for the CO 2 gas produced at 10% glucose concentration TIME (min) 0 5 10 15 20 25 TRIAL 1 TRIAL 2 Volume of CO2 (cm3) Volume of CO2 (cm ) 0.00 29.00 82.00 146.00 158.00 168.00 0.00 28.00 91.00 143.00 156.00 172.00 TRAIL 3 Volume of CO2 3 TRIAL 4 TRIAL 5 Volume of CO2 Volume of CO2 (cm ) (cm ) (cm ) 0.00 24.00 87.00 141.00 158.00 167.00 0.00 20.00 78.00 146.00 157.00 165.00 0.00 29.00 88.00 143.00 156.00 164.00 3 3 ̄ S 0.00 26.00 85.20 143.80 157.00 167.20 0.00 3.94 5.17 2.17 1.00 3.11 3 Graph 2. Mean and standard deviation of the data collected for 10% glucose concentration GLUCOSE 10% - CO2 PRODUCTION 250 CO2 PRODUCTION (cm3) 200 y = 92.36ln(x) - 121.23 R² = 0.97 150 Glucose 10% Tendency line 100 50 0 0 5 10 15 20 25 30 TIME (min) 16 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 The previous graph shows the rate of fermentation process for the 10% glucose solution with yeast. It shows the mean values of each trial performed for the 10% glucose concentration in order to make a comparison between the production CO 2 released in each of the trials. From the graph it can be appreciated that the standard deviation error bars show no significant difference. The fermentation rate in each trial of 10% glucose concentration did not differ significantly. This means that the results of obtained are more accurate. Visually, the tendency line of the graph shows the correlation between the values obtained in each 5 min interval during a period of 25 min. It can be observed that there is a strong positive correlation between the means from each trial in respect to the tendency line. For 10% glucose solution there was a mean between 150 cm 3 and 200 cm3 of CO2 released. As time increases, there is an increased in the production of CO 2 gas. According to correlation coefficient (R2) given, which is 0.97; this means that as the coefficient of correlation obtained is approaching to 1, there is a very strong correlation. In addition there is not the presence of any outlier. There was not a significant difference in the rate of CO2 released in each trial for 10% glucose concentration. Table 15. Mean and the standard deviation of the data collected for the production of CO 2 at 15% glucose concentration TIME (min) TRIAL 1 TRIAL 2 TRAIL 3 TRIAL 4 TRIAL 5 Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) 0 5 10 15 20 25 0.00 26.00 73.00 124.00 174.00 224.00 0.00 22.00 64.00 120.00 186.00 216.00 0.00 14.00 56.00 120.00 173.00 219.00 0.00 16.00 62.00 118.00 172.00 220.00 0.00 24.00 62.00 125.00 176.00 233.00 ̄ S 0.00 20.40 63.40 121.40 176.20 222.40 0.00 5.18 6.15 2.97 5.67 6.58 17 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 Graph 3. Mean and standard deviation of the data collected for 15% glucose concentration GLUCOSE 15% - CO2 PRODUCTION 250 y = 124.66ln(x) - 199.23 R² = 0.9 CO2 PRODUCTION (cm3) 200 150 Glucose 15% Tendency line 100 50 0 0 5 10 15 20 25 30 TIME (min) The graph presents the rate of fermentation process for the 15% glucose solution with yeast. It shows the mean values of each trial performed for the 15% glucose concentration in relation to the production CO2 released in each trial. Visually, the graph shows that the standard deviation error bars show a very small difference between each trial. The fermentation rate in each trial of 15% glucose concentration did not differ significantly. In addition, the tendency line shows the correlation between the values obtained during a period of 25 min. There is a strong positive correlation between the means obtained from each trial and the tendency line. The 15% glucose solution shows a mean between 200 cm 3 and 250 cm3 of CO2 gas released. Moreover, the as there is a strong correlation, it can be observed that as time increases, there is an increased in the production of CO2 gas. From the graph, it can be remarked that the correlation coefficient (R2) is 0.94; the coefficient of correlation obtained is approaching to 1, so there is a very strong correlation. There was not a 18 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 significant difference in the rate of CO2 released in each trial for 10% glucose concentration. Table 16. Mean and the standard deviation of the data collected for the production of CO 2 at 20% glucose concentration TIME (min) 0 5 10 15 20 25 TRIAL 1 TRIAL 2 TRAIL 3 TRIAL 4 TRIAL 5 Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) 0.00 8.00 54.00 100.00 150.00 191.00 0.00 11.00 52.00 98.00 144.00 190.00 0.00 13.00 56.00 102.00 149.00 198.00 0.00 12.00 54.00 100.00 152.00 189.00 0.00 13.00 57.00 106.00 154.00 195.00 ̄ S 0.00 11.40 54.60 101.20 149.80 192.60 0.00 2.07 1.95 3.03 3.77 3.78 Graph 4. Mean and standard deviation of the data collected for 20% glucose concentration GLUCOSE 20% -CO2 PRODUCTION 250 y = 110.63ln(x) - 182.07 R² = 0.94 CO2 PRODUCTION (cm3) 200 150 Glucose 20% Tendency line 100 50 0 0 5 10 15 20 25 30 TIME (min) 19 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 The graph shows the rate of fermentation process for 20% glucose solution with yeast. It shows the mean values of each trial performed for the 20% glucose concentration in relation to the production CO2 released in each trial. The graph shows that from the standard deviation error bars there is a very small difference in CO2 gas production in fermentation process of each trial. The fermentation rate in each trial of 20% glucose concentration did not differ significantly. From the tendency line shows the correlation between the values obtained, meaning that there is a strong positive correlation between the means obtained from each trial. This correlation observed means that as time increases, there is an increased in the production of CO2 gas; and such increased was not very different in the rate of reaction of each of the trials that were carried out. Mathematically, the correlation coefficient obtained was 0.94; which is a value approaching to 1, so there is a very strong correlation. There was not a significant difference in the rate of CO2 released in each trial for 20% glucose concentration. Table 17. Mean and the standard deviation of the data collected for the production of CO 2 at 25% glucose concentration TIME (min) 0 5 10 15 20 25 TRIAL 1 TRIAL 2 TRAIL 3 TRIAL 4 TRIAL 5 Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) 0.00 23.00 52.00 96.00 122.00 148.00 0.00 16.00 56.00 92.00 112.00 143.00 0.00 16.00 52.00 91.00 120.00 149.00 0.00 26.00 58.00 92.00 119.00 145.00 0.00 20.00 59.00 90.00 119.00 147.00 ̄ S 0.00 20.20 55.40 92.20 118.40 146.40 0.00 4.38 3.29 2.28 3.78 2.41 20 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 Graph 5. Mean and standard deviation of the data collected for 25% glucose concentration GLUCOSE 25% - CO2 PRODUCTION CO2 PRODUCTION (cm3) 250 200 y = 77.52ln(x) - 112.48 R² = 0.97 150 Glucose 25% Tendency line 100 50 0 0 5 10 15 20 25 30 TIME (min) The previous graph represents the rate of fermentation process for 25% glucose solution concentration with yeast. It shows the mean values of each trial performed for the 25% glucose concentration in relation to the production CO 2 released in each trial. The standard deviation error bars presented in the graph show that there was no significant difference in CO2 gas production in fermentation process of each trial. The tendency line shows the correlation between the values obtained. It can be observed that there is strong positive correlation between the means obtained from each trial of 25% glucose concentration. Mathematically, the correlation coefficient was 0.971; a value approaching to the value of 1, meaning that there is a very strong correlation. There was no significant difference in the rate of CO2 released in each trial carried out for 25% glucose concentration. It can be observed that as time increases, the production of CO2 gas increases as well; and such increased was not very different in the rate of reaction of each of the trials that were carried out. 21 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 d) Calculating mean values and standard deviation for final production of CO2 of each glucose concentration. In order to compare the effect of the five different concentrations of glucose solution (5%, 10%, 15%, 20% and 25%) in the fermentation process of yeast, it is necessary to calculate and to graph the mean values of the final volume of CO 2 released for each of the five concentrations. Table 18. Values of mean and the standard deviation of final production of CO 2 for each glucose concentration. Glucose Concentration TRIAL 1 TRIAL 2 TRAIL 3 TRIAL 4 TRIAL 5 Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) Volume of CO2 (cm3) 5% 10% 15% 20% 68.00 168.00 224.00 191.00 78.00 172.00 216.00 190.00 77.00 167.00 219.00 198.00 74.00 165.00 220.00 189.00 25% 148.00 143.00 149.00 145.00 ̄ S 73.00 164.00 233.00 195.00 74.00 167.20 222.40 192.60 3.94 3.11 6.58 3.78 147.00 146.40 2.41 Graph 6. Mean and standard deviation of the final amount CO2 gas released for each glucose concentration. CO2 PRODUCTION CO2 PRODUCTION (cm3) 250 222.40 200 192.60 167.20 150 5% 146.40 10% 15% 100 20% 50 74.00 25% 0 5% 10% 15% 20% 25% GLUCOSE CONCENTRATION 22 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 As it can be seen from the previous graph, the 5% glucose concentration was the concentration which produced the less amount of carbon dioxide gas (CO 2) during the experiment, with 74.00 cm3. In addition, it can be observed that there was an increased in the production of CO2 as the concentration of glucose increased until the 15% glucose concentration, which showed the maximum production of CO2. The greatest production of CO2 gas released was presented at 15% glucose concentration. However, from 15% glucose concentration it was a significant change in the production of CO2 gas for 20% and 25% glucose concentration; for such concentrations of glucose, the volume of CO2 gas released started decreasing in a significant way. From 5% to 15% glucose concentration, there was an increased in the volume of CO2 production as the concentration increased. However, as the concentration of glucose was increasing from 20% to 25%, the CO2 gas released was decreasing. In addition, from the standard deviation error bars, we can observe that for each of the different concentrations of glucose (5%, 10%, 15%, 20%, 25%) there was a relatively small difference from the data collected for the five trials carried out for each sugar concentrations. This means that the data collected of CO 2 gas released for each of the five concentrations and their respectively trials were constant during fermentation process. e) Calculating ANOVA-test. Visually, we can observe from graph 7 that the means of the final volume of CO 2 gas released from each glucose concentration are different to one another. However, in order to state whether or not the means of the final volumes of CO 2 gas released for each concentration differ to one another, it is necessary to calculate ANOVA – test5. 5 In order to carry out ANOVA – test, a normality test was previously carried out in order to ensure that the set of data chosen for ANOVA-test was well modeled by a normal distribution. 23 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 In order to perform ANOVA – test, it is necessary to group the data in a separate table. This data refers to the final volume displaced of CO2 gas after the 25 min. period for each sugar concentration. Table 19. Final volume of CO2 gas displaced for each glucose concentration after a 25 min. period. Final volume displaced of CO2 gas (cm3) after 25 min period Groups Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 5% glucose 68.00 78.00 77.00 74.00 73.00 10% glucose 168.00 172.00 167.00 165.00 164.00 15% glucose 224.00 216.00 219.00 220.00 233.00 20% glucose 191.00 190.00 198.00 189.00 195.00 25% glucose 148.00 143.00 149.00 145.00 147.00 The null hypothesis (H0) as well as the alternative hypothesis (H1) must be stated. After calculating ANOVA – test: If F < FC then H0 is accepted. Hypothesis: H0= There is no difference between the volume of CO2 gas released in each sample treated with each glucose concentration. H1= There is a difference between the volume of CO2 gas released in each sample treated with each glucose concentration. 24 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 SUMMARY Table 20. ANOVA – test summary. Groups 5% Glucose 10% Glucose 15% Glucose 20% Glucose 25% Glucose N0. of measurements 5.00 Sum Mean Varience 370.00 74.00 15.50 5.00 836.00 167.20 9.70 5.00 1112.00 222.40 43.30 5.00 963.00 192.60 14.30 5.00 732.00 146.40 5.80 ANOVA Table 21. ANOVA – test. Source of variation Between groups Within groups Sum of squares 62939.84 Freedom Degrees 4.00 Mean of squares 15734.96 354.40 20.00 17.72 Total 63294.24 24.00 F 887.98 P-value 3.31E-22 F – crit. 2.87 As F is equal to 887.98 and Fc equals to 2.87, the value of F is greater than Fc then H0 is not accepted, 887.7 >2.87. So, there is a difference between the volumes of CO 2 gas released in each sample treated with each glucose concentration. XII.- Conclusion As it could be observed in the information presented in the previous tables and graphs, the hypothesis previously stated was not correct. At the moment of analyzing the raw data, it could be noticed that the production of carbon dioxide gas by the fermentation of glucose was increasing while the glucose concentration was increasing. However, this increase in CO2 gas production was present only when using 5%, 10% and 15% glucose concentrations; but when fermenting yeast at 20% and 25% glucose concentrations, the production of carbon dioxide gas started to decrease considerably. 25 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 When using 5% glucose concentration, there was a mean of 74.00 cm 3 of carbon dioxide (CO2) gas production. This concentration of glucose sugar was the one producing the less volume of CO2 gas released. Furthermore, analyzing graph 1 with each of the values collected from the five trials of sample concentration; it could be observed by means of the standard deviation error bars that there was a very small difference between the production of CO2 gas collected for each trial, giving a more accurate information. In addition, the R2 coefficient, 0.99, approached to 1, there was a very strong correlation, and there were not any outlier. So the production of CO 2 gas released during fermentation was almost the same in each trial. For the 10% glucose concentration, it could be observed a significant difference between the mean of CO2 of 5% glucose concentration, releasing a mean volume of 167.20 cm3. The amount of such gas increased at a 10% concentration. In addition, from graph 2 it could be appreciated that the standard deviation error bars show no significant difference. The fermentation rate in each trial of 10% glucose concentration did not differ significantly. With a correlation coefficient (R2) of 0.97, there was a very strong positive correlation. Analyzing the data provided by the samples of 15 % glucose concentration, it could be seen that at this concentration, there was a greater production of carbon dioxide with a mean of 226.40 cm3 released. At this concentration, glucose fermented yeast at a higher rate. Furthermore, it was showed in graph 3, by means of the standard deviation, that there was a small difference on the amount of CO2 released between the data values collected from each trial. Moreover, the correlation coefficient was 0.94; giving a very strong positive correlation. There was not a significant difference in the rate of CO 2 released in each trial for 10% glucose concentration. However, the refutation of the hypothesis stated is presented when analyzing the data from the 20% glucose concentration. At this concentration, the volume of CO2 released decreased in a significant way. For this concentration was expected a greater volume released of such gas; however, it happens the other way around, with a decrease in the released of CO2. Furthermore, there is present the greater difference in the volume produce of CO2 of each of the trials of the sample, with a mean of 192.70 cm 3. 26 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 When analyzing the results of the 25% glucose concentration, it was a greater decreased on the production of CO2. For this concentration, and based on the hypothesis, it was expected the major production of CO2; however, there was a greater decreased on CO2 with reference to the 20% concentration. In addition to these results, the standard deviation error bars presented in the graph 5 showed that there was no significant difference in CO2 gas production in fermentation process of each individual trial. Mathematically, the correlation coefficient was 0.97, meaning that there was a very strong positive correlation. There was no significant difference in the rate of CO2 released in each trial carried out for 25% glucose concentration. As it was shown in the data collected from the experiment the effect of increasing the concentration of glucose with respect to the volume of CO2 released was not as it was expected from the hypothesis stated. At the beginning, as the concentration from 5% increased to a 10% glucose concentration; however as the concentration increased from 15% to 25%, there was a decreased in the volume of carbon dioxide gas released. According to Damon, McGonegal, and Ward, (2009) a reason for the decreased on carbon dioxide volume released from yeast respiration, it could be because when increasing the concentration of a substance, there is an increased of molecular collisions; however, as yeast contains certain types of enzymes, such enzymes have a certain limit to which they can work at a maximum rate. As a result, if we continue increasing the concentration of substrate, there will be a point in which the enzymes will be working as far as possible until they cannot work efficiently, so the rate of reaction starts decreasing. Another reason for the decreased of the volume of CO 2 released as the concentration of glucose was increasing is because as the experiment was carried out within a closed system, the production of carbon dioxide gas as the concentration of glucose was increasing, it could have killed the yeast inside the flask. 27 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 XIII.- Evaluation Along the experiment it was possible to obtain reliable data when following cautiously the method previously designed step by step with the objective of avoiding any possible problem in terms of the control of variables. In addition, a set of five trials were performed for each sugar concentration (5%, 10%, 15%, 20% and 25%) in order to gather current and reliable data for the analysis. However, there will be always problems that can arise when performing the experiment which can be improved for future repetitions of the experiment. The data gathered from the production of carbon dioxide (CO2) by means of the final water displacement of the 250 ml graduated cylinder (±5 %), it can be 80% accurate due to the lost of CO2 present when mixing the substances and at the moment of closing the flasks by means of the rubber stopper; a reason why this happened is because when mixing the glucose solution with yeast, the reaction was very fast and there is a loss in carbon dioxide gas from the closed system to the environment. However, the fermentation process was carried out in a way in which it was able to obtain quantitative data with respect to the volume of carbon dioxide gas released. In addition, when performing this experiment a problem aroused related with temperature. As it was pointed out, the optimum temperature to carry out the fermentation reaction was 37°C; however, the room temperature form the laboratory affected the optimum temperature set on the electric water bath. Even though precautions were taken in terms of maintaining the optimum temperature as the use of the incubator and the use of the electric bath, the control of the temperature was difficult to maintain due to the opening of the electric water bath. Besides that, another possible weakness of the experiment was the period of time considered for the collection of data during the fermentation process. As the data was collected at 5 min intervals during a 25 min period due to the laboratory conditions, the data gathered was limited to a fraction of the process. As a result, this can be considered as a limitation for the overall analysis since it can probably affect the accuracy of the data for the approval of the hypothesis. 28 “The Effect of Different Concentrations of Glucose in the Anaerobic Respiration by Yeast / Mayo 2012 XIV.- Improvements An improvement for the experiment can be the fact of increasing the period of time for fermentation. As the data was collected during a 25 min period, the data collected corresponds only to a fraction of the complete fermentation process. It would be best to record the data until the fermentation process has ended, in order to have more accurate information about the rate of CO2 released. Another improvement in order to enhance the experiment could be to have a better control over the room temperature from the laboratory where the experiment was performed. Even though some precautions were taken with respect to maintain the optimum temperature of 37°C as the use of the incubator and the electric water bath, the opening of the water bath was a limitation because the 125 ml flasks (±5 %) in which the fermentation process was taking place were exposed to the room temperature from the laboratory. XV.- Bibliography Clark, Jim. (2002). The effect of concentration on reaction rates. Web. 24 Oct 2011. <http://www.chemguide.co.uk/physical/basicrates/concentration.html Damon, Alan, McGonegal, Randy, Tosto, Patricia, & Ward, William. (2009). Higher level Biology developed specifically for the IB diploma. Pearson Education, Inc. Miller, K. R., & Levine, Joseph S. (2006). Biology. Boston, Massachusetts: Pearson Education, Inc. 29