Modification/ Previous experiment (6):
Having carried out the previous experiments” An investigation into rates of fermentation by yeast “and “The effect of ethanol on the rate of anaerobic respiration of glucose by yeast”, we have seen that sucrose is the best respiratory substrate and the ethanol concentration of 25% is too high resulting in a reaction with infinity. In the experiment “An investigation into rates of fermentation by yeast” we investigated at which rates the different sugar-yeast mixtures, using the four sugars glucose, sucrose, maltose and lactose, move to the end of the 1mm capillary tube. The mixture containing lactose did not move, the mixture containing maltose moved only 17mm, the mixture containing glucose moved 181 mm and the mixture containing sucrose moved 352 mm. Sucrose appeared to give the best results. We suggested this could be because sucrose had to be broken down from being one glucose sugar and one fructose sugar joined by a glycosidic bond to two single monosaccharide sugars. In addition the yeast may have had a small supply of sucrase enzyme anyway start the breakdown of sucrose into its respiratory products. Glucose gave the second best results. This may have been slower as sucrose because the yeast was cold (we did not measure the temperature). If it was cold then it would have less kinetic energy and therefore less chance to collide with the glucose and respire it. Maltose gave the third best results; this could be because the yeast did not have the enzyme maltase ready to break down the maltose into two glucose molecules. Lactose did not respond to yeast at all- the mixture containing lactose did not move and no carbon dioxide was produced. One reason could be that yeast did not contain the enzyme lactase to break down lactose into glucose and galactose. As we have found out that sucrose is a better respiratory substrate than maltose, glucose and lactose we are using it in this investigation. In our other previous investigation “The effect of ethanol on the rate of anaerobic respiration of glucose by yeast” we investigated the six different ethanol concentrations: 0.00, 1.56, 3.13, 6.25, 12.50, and 25.00 on the time for the mixture containing glucose, yeast, ethanol and alkaline phenolphthalein to decolourise. We found that 6.25% is the best ethanol concentration as the decolourisation occurs fastest and that 25% of ethanol concentration does not decolourise at all. This is due to the fact that over 15% ethanol concentration the yeast will die. Moreover we found out that the method including the use of a water bath and looking at the time of decolourisation is very subjective and doesn’t give as accurate results as the use of a capillary tube. As a result we are now using a capillary tube and look at the rates of movement of the mixture containing different ethanol concentrations. Further we are now using the dilutions of ethanol of 20%, 15%, 10%, 5% and 0% to make the method easier, quicker and that we have a wide range of results.
The experiment was thought to be 90 minutes for each mixture in each capillary tube with 15- minute intervals. As our school lessons are 50 minutes only we were not able to do the experiment in this time and had to choose another one with 2-minute intervals, overall lasting 16 minutes for each mixture in the capillary tube. As a result of changing we were able to carry out the practical for each sugar twice to get more reliable results.
Detailed Prediction:
The rate of anaerobic respiration will be fastest when using the lowest ethanol concentration of 0.0% and slowest when using 20.0% of ethanol concentration. As a result we expect to see the greatest length of movement at an ethanol concentration of 0.0% and the shortest length of movement at an ethanol concentration of 20%. The most ethanol-tolerant strains of yeast can survive up to approximately 15% ethanol by volume. Concentrations below 12% don’t denature glycolytic enzymes or cause appreciable inhibition of activity. Since ethanol doesn’t accumulate within yeast cells but rapidly diffuses across the cell membrane, direct inhibition of glycolytic enzymes by intracellular ethanol is unlikely during fermentations which produce 12% ethanol or less (12). The rate of anaerobic respiration will be fastest when using 0.0% of ethanol as the yeast, containing the enzyme sucrase, splits sucrose by breaking the glycosidic bond into one glucose molecule and one fructose molecule. The glucose molecule and fructose molecule are structural isomers and give off the same products when respired. Using the ethanol concentration of 5.0%, the rate of anaerobic respiration will still be quick, but the length of movement of the mixture will be shorter than when using 0.0% ethanol concentration, because ethanol can affect the membrane permeability of yeast which slows down anaerobic respiration. The yeast plasma membrane is one of the main targets for ethanol. It has been shown that ethanol inhibits processes of mediated transport and stimulates the traffic of compounds that cross the membrane by simple diffusion. This alteration in membrane permeability may be deleterious to the cells promoting the leakage of intracellular constituents or the entry of toxic extracellular substances which disturb the composition of the cytoplasm. Protons are usually in higher concentrations in the extracellular environment and yeast cells use a powerful H⁺-pump ATPase that keeps the intracellular pH at physiological values, suitable for enzyme function and generates a proton-motive force across the plasma membrane. Ethanol increases the acidity by allowing more H⁺-ions into the cytoplasm, which will affect the pH, which will affect the enzyme activity. Most enzymes work fastest at a pH of around 7. Some however have a different optimum pH. pH is the measure of the concentration of hydrogen ions in a solution. The lower the pH the higher the hydrogen ion concentration. Hydrogen ions can interact with the R groups of amino acids, affecting the way in which they bond with each other and therefore affect their 3D arrangement. A pH which is very different from the optimum pH can cause denaturation of an enzyme (9, 10).Sucrose cannot be broken down by sucrase as fast as when using 0.0% ethanol concentration. When using an ethanol concentration of 10.0% the rate of anaerobic respiration will be even slower than when using an ethanol concentration of 5.0% because ethanol affects the membrane permeability of yeast even more and lets more hydrogen ions into the cytoplasm of yeast. The length of movement will be twice as long as the length of movement when using 20.0% of ethanol concentration. When using the ethanol concentration of 15.0% the rate of anaerobic respiration will be even slower and the length of movement will be shorter than when using an ethanol concentration of 10.0%. When using the ethanol concentration of 20.0% the rate of anaerobic respiration will be the slowest of all five ethanol concentrations and this concentration will have the shortest length of movement.
A graph to show the effect of ethanol on the rate of anaerobic respiration of sucrose (predicted)
Apparatus list (for one investigation only excludes repeats):
Table to show reasons for choice of apparatus:
Fair test:
All of these variables (including the controlled variables) will need to be controlled by the person, and to be kept the same throughout the experiment in order to keep it fair and get as accurate results as possible. The independent variable, in this experiment the ethanol concentration, has to be changed other than that all variables have to stay the same throughout the entire experiment.
Variables:
- Independent variable: different concentrations of ethanol
- Dependent variable: the distance of the sugar-yeast-ethanol mixture displaced
Table to show variables that must be controlled:
Risk assessment:
- Yeast, as it contains enzymes, can irritate membranes in the nose or eyes if inhaled. It may also cause an asthmatic attack. Yeast has a low hazard (12).
- Wear your hair tied back
- When something is spoiled it should be rinsed up immediately
- Wear goggles all the time
- Ethanol is highly flammable; when inhaled it can cause cough, headache and fatigue.
Sketch:
Method:
- Wear goggles and tie hair up.
- Collect all apparatus in front of you.
- Set up apparatus as shown in the sketch above.
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Collect 10cm3 of sucrose solution with a 10 cm3 syringe and inject it into a beaker.
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To make up the different concentrations of ethanol use the serial dilution table. Label each beaker according to their ethanol concentration. Collect 5 cm3 of 20% ethanol concentration with a 5cm3 syringe and inject it into the same beaker containing the sucrose already.
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Add 10cm3 of yeast into the beaker containing the sucrose and ethanol. Leave 1-2 minutes and stir the mixture.
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Collect 10 cm3 of the sucrose-yeast-ethanol mixture from the beaker with a 10 cm3 syringe.
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Insert the 10 cm3 syringe containing the mixture into the rubber tubing at the top of the capillary tubing.
- Gently apply pressure to the plunger of the syringe until a meniscus appears at the top of the capillary tubing.
- Mark the position of the meniscus with a marker pen and press start on the stop clock.
- At intervals of 2 minutes, over a period of 16 minutes, mark the volume and record the length of movement using the ruler. Transfer your results into a table.
- Carry out two more repeats for this ethanol concentration of 20%.
- Repeat the method for the ethanol concentrations 15%, 10%, 5%, and 0%.
- Carry out three more repeats for the ethanol concentrations 15%, 10%, 5%, and 0%.
- Transfer your results into your results table and calculate a mean for each ethanol concentration mixture.
- Highlight anomalies and exclude them from any further calculations.
- Draw a graph using your means: time against distance moved by the mixture
- Calculate 5 gradients for each ethanol concentration using the formula m=y/x
- Draw a rate graph using the average of each ethanol concentration mixture. Rate = 1/average
Serial dilution table:
Pilot
Analysis
The graph shows five best lines and the highest ethanol concentration (20%) is the steepest line as the mixture didn’t move over a long distance. The mixture containing 15% ethanol concentration is less steep and the mixture moved over a much larger distance. The 10% graph is less steep than the 15% and the 5% is less steep than the 10% graph. With containing no ethanol the 0% graph is the lowest graph of all ethanol concentrations as shown in the detailed prediction. The 0% graph moved in a very short time over a long distance, whereas the 20% ethanol concentration graph reaches its maximum of 6mm after 16 minutes.
Evaluation
The experiment went well but there are still some anomalies. They could be a result of not stirring the yeast before using it, or parallax errors. These limitations could result in anomalies which make the practical not as accurate and reliable as it could be. To attempt more accurate results we could have used more data, i.e. a longer time and repeated the experiment four times for each mixture containing a different ethanol concentration to get more accurate averages of distance displaced in a given time.
The method went well and there are no modifications to make except to ensure that the yeast has the same temperature throughout the whole investigation as once the windows were open and the biology lab was cooler compared to other days. Use a thermometer to measure the temperature of the yeast and make sure that it is room temperature. Further make sure there are no human errors made, like forgetting to start the stop watch or parallax errors, like not reading off at eye level the distance the mixture moved. Do more repeats than three as it can happen easily that the results given off in one go are different from the next results, repeat the investigation for each ethanol concentration until you get vaguely similar results and always make sure the lower the ethanol concentration in the mixture the longer the distance moved in a short time.
Bibliography:
- Advanced biology by Jones and Jones
- Advanced sciences : Biology 1
- Advanced sciences : Biology 2
- Biological sciences 1 and 2
- Advanced biology by Michael Kent
- Class notes
- Research paper: Influence of magnesium ions on heat shock and ethanol stress responses of Saccharomyces cerevisiae by Rosslyn M. Birch, Graeme M. Walker
- Research paper: The effect of ethanol on the plasma membrane permeability of spoilage yeast by Celia Quintas, Emilia Lima-Costa and Maria C. Loureiro-Dias
- Research paper: Ethanol production during Bath Fermentation with Saccharomyces cerevisiae: Changes in glycolytic enzymes and internal pH by K. M. Dombek and L. O. Ingram
- Research paper: Mechanism of ethanol inhibition of fermentation in Zymomonas mobilis by Yehia A. Osman and Lonnie O. Ingram
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Internet:
- Hazcard references from the school laboratory
References are marked as numbers in brackets, for example (3) which means number 3 in the bibliography.