Investigating the Effect of Temperature on the Fermentation of Yeast

Preliminary work – to plan and discover any potential flaws in the experiment

Apparatus available: Heat mat, Bunsen burner, tripod, gauze, large 400ml beaker, small 250ml beaker, retort stand, boss and clamp, bowl, delivery tube, water, thermometer 0-100oC, sealed 20ml, 10ml syringe (to collect the carbon dioxide given off from the fermentation), 20ml syringe, stopwatch, test tube and test tube rack.      5% yeast, 0.2M glucose suspension

Method:

• Set up the apparatus as shown in the diagram below.
• Fill the bowl with water
• Fill the sealed 10ml or 20ml syringe with water, to allow readings from displacement. Clamp in place.
• Fill the test tube with ?ml 5% yeast and 0.2M glucose suspension, and place it in a 400ml beaker filled to ?ml (water bath)
• Wait until the temperature of the suspension matches the desired temperature. (e.g. for 20oC make sure that the yeast is also at 20oC)
• Once at the right temperature, close the test tube with the rubber bun and place the delivery tube under the sealed syringe
•  Time for exactly ? minutes.
• After ? minutes, note the volume of gas produced, by reading off the sealed syringe.
• Repeat the experiment ? times at 20ºC, 30ºC, 40ºC, 50ºC and 60ºC, varying the temperatures by applying gentle heat from Bunsen burner.
• Record temperature and amount of gas in a table and plot results on a graph to find any anomalous results

Decisions on variables:

There are a number of variables that need to be considered in this experiment. The major ones being:

• the volume of yeast suspension
• the length of collection time
• the range of temperatures

For the range, it was obvious that I wanted as many results as possible in the fairly short time limit, but I also wanted them over a large temperature range to fully investigate the effect of temperature. During my preliminary work I realised that it would be difficult to accurately control the temperature with the apparatus available to me. I found that to maintain 5oC intervals between readings proved to be very difficult as I had to heat and cool the yeast suspension, which meant that the temperature drifted higher, or lower than required. I decided to collect results at 10ºC intervals, a big enough range to allow for experimenters error, yet close enough to allow detailed and sufficient graphic results. I would therefore test at 20ºC either side of the optimum; about 40ºC (I knew from the GCSE course that most enzymes work best at about body temperature); namely therefore at 20ºC, 30ºC, 40ºC, 50ºC and 60ºC.

The same kind of problem occurred in deciding how long to collect the carbon dioxide gas from the fermenting suspension. It had to be long enough so that there would be adequate gas for a recordable result and so that the variation of gas produced at different temperatures would be evident. However, it could not be too long, so the volume of gas collected would be no bigger than the syringe’s capacity for the predicted larger results. I experimented with different times and different volumes of yeast and considering the fact that I wanted to take fifteen readings in all (three readings for each of the five different temperatures) I decided upon 4 minutes as it gave me time to complete the experiment during a double-period.

At this point I also had to choose the size of the sealed collecting syringe. There were two sizes available to me: a 10ml syringe, with a scale of 1/2ml intervals, and a 20ml syringe, less accurate with 1ml intervals. I felt that in order to produce precise and reliable evidence I had to choose the 10ml syringe due to its accuracy. (Later in the preliminary work 10ml proved to be right as the volume of gas produced did not overfill the syringe) 

Finally, I had to decide how much of the yeast suspension to use. Knowing that I wanted to take readings every 4 minutes, I experimented with different amounts. At first I tried 10ml which did not produce enough gas to be measured in the 4 minutes. I then tried 30ml that produced a lot of gas, however it took too long to heat up and therefore did not leave enough time to take other readings. Consequently I tried 25ml of suspension and at 40oC it produced 8ml of gas. This meant that at what I expect to be the optimum temperature, the gas did not overfill the syringe.

I decided to repeat the experiments 3 times for each reading in order to maintain a high degree of accuracy and confirm anomalies.

Preliminary experiment results:

25ml of yeast suspension

Reading taken after 4 minutes

10ml sealed syringe

(Experimental errors: ±0.5ºC, ±0.5ml)

Comments: The preliminary stage worked well, as I was able to see the general trend of the results and able to make useful changes to my experiment. Firstly, I was surprised that the reading at 50oC produced such a high volume of CO2 as I expected the enzymes to denature. However, I measured the temperature of the suspension and found that it was at 40oC. This meant that even though the water was at 50oC the temperature of the yeast was much lower and created optimum conditions for the enzymes. To avoid this problem in my real investigation I decided to wait until the yeast gets to the desired temperature before starting to collect data. Also I found that by having the same yeast suspension every time it meant that the glucose was getting more and more used up and therefore it was not a fair-test.

Prediction – including the kinetic theory

According to the Kinetic Theory, as temperature increases, so also does the rate of reaction, as the particles have more speed (kinetic energy) from heat energy. The Kinetic Theory works on another theory, the Collision Theory, which states that if two particles collide with sufficient energy (called the activation energy) bonds will be broken and a reaction will take place.

Therefore, I predict that the greater the temperature, the greater the amount of CO2 produced by fermentation, as the particles will move more quickly, therefore there will be a greater number of collisions, between substrate and enzymes, with more force, and therefore a greater rate of. In the same way I predict that at lower temperatures there will be less carbon dioxide produced, as the particles will have less energy and fewer collisions would occur. Consequently there will be a slower rate of reaction.

However, despite the above predictions, from my research and knowledge, I know that at the highest temperatures the active sites of enzymes deform, becoming denatured and unproductive. I also know that most enzymes in the human body work effectively at about 37ºC as the body constantly attempting to maintain this internal temperature by homeostasis. I can safely assume from this fact, from my preliminary work and from research and knowledge that body temperature is about the optimum temperature for enzyme activity. I think this is backed up by

As a result, I predict that there will be little products at room temperature, that there will be a constant increase, following the ‘Q10 by 2 theory’ (see background info), until about 37ºC, body temperature, and then that the enzymes will begin to denature and the amount of gas produced will decrease as the active sites can no longer bind to their specific substrate.

See prediction graph

Experimental Plan

Modification to method

I decided to get a new yeast suspension for every reading. This is because the glucose was getting more and more used up every time and consequently the accuracy and the reliability of the investigation was not at its best.

Independent variable: temperature/ oC

Dependent variable:  volume of carbon dioxide gas produced/ml

Apparatus List: 

Heat mat, Bunsen burner, tripod, gauze, large 400ml beaker, , retort stand, boss and clamp, bowl, delivery tube, water, thermometer 0-100oC, sealed 10ml syringe (to collect the gas given off from yeast), 20ml syringe, stopwatch, 3x test tube and test tube rack.

25ml yeast suspension (5% yeast, 0.2M glucose).

Method:

• Set up the apparatus as shown in the diagram below.
• Fill the bowl with water
• Fill the sealed 10ml syringe with water, to allow readings from displacement. Clamp in place.
• Fill the test tube with 25ml of 5% yeast and 0.2M glucose suspension, and place it in a 400ml beaker filled to 300ml (water bath)
• Wait until the temperature of the suspension matches the desired temperature. (e.g. for 20oC make sure that the yeast is also at 20oC)
• Once at the right temperature, close the test tube with the rubber bun and place the delivery tube under the sealed syringe
•  Time for exactly 4 minutes.
• After 4 minutes, note the volume of gas produced, by reading off the sealed syringe.
• Using a new 25ml of suspension every time, repeat the experiment 3 times at 20ºC, 30ºC, 40ºC, 50ºC and 60ºC, varying the temperatures by applying gentle heat from Bunsen burner.
• Record temperature and amount of gas in a table and plot results on a graph to find any anomalous results

Diagram:

Range: 

0-10ml of CO2, to increase precision to 0.5ml.

20ºC-60ºC, I feel that between 0-20ºC yeast will be too inactive, due to lack of energy, to receive a justifiable measurement and by 60ºC the yeast’s enzymes will have mostly denatured. This is backed up by my preliminary work. I believe that my range of temperature being tested will allow for a trend of results to become apparent and also for me to have time to repeat the experiment three times for each temperature, which could be averaged producing overall more precise and reliable data.

Safety:

• When handling yeast and ethanol it is vital not to consume or inhale anything during the experiment.
• Make sure that the area around one’s workspace is constantly clear and tidy; make sure that chairs are placed under tables, etc so that the risk of tripping up and falling is avoided.
• Before proceeding with investigational work, make sure that apparatus is set up securely, e.g. ensuring that the beaker is placed on the centre of the gauze and therefore avoiding spillages
• When working make sure that you are not sitting down in case that if you spill anything it will not go all over you
• Place test tubes in a test tube rack to avoid spillages
• If any glass is broken it must be cleaned up immediately, in a correct fashion and, most importantly, safely to avoid any accidents.
• When the Bunsen burner flame is idle, the yellow safety flame must be on as it is easier to see, and when heating one must use the roaring purple flame.
• When conducting work around a naked flame one must be sue to keep clothing away from it, e.g. ties.

Obtaining Evidence

Observations

As the reagent produced gas in the test tube it slowly began to bubble, presumably the rising of the carbon dioxide to the surface. Larger colourless bubbles formed on the surface and resting above them was white, thick foam. As the temperature approached denaturisation point, the foam turned crisp and shrivelled. It turned into a dry solid but remained attached to the side of the test tube.

Results

Table Showing the Effect of Temperature on the Fermentation of Yeast

The instrumental errors for the equipment are:

Thermometer         - ±0.5ºC

Sealed syringe - ±0.5ml

Stop watch        - ± 1 second

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Analysis

From the graph I can see that as the temperature increases so does the amount of carbon dioxide produced until it reaches the optimum temperature, about 38ºC from the graph, and then decreases as the enzymes begin to denature.

My prediction was very accurate as there were little products at room temperature, and according to my results the optimum temperature was only about 1ºC higher than my prediction. I also correctly predicted that the enzymes would denature after 40oC and that the graph would be exponential.

Conclusion

The graph starts with little carbon dioxide production at the low temperatures. At this level there is little activity, as there is little heat and therefore energy for successful collisions. As the heat increases so does the number of collisions and the volume of CO2 produced also increases. From the graph we can see that yeast production does not occur in a linear fashion, but behaves exponentially; as the temperature rises the rate of reaction increases more rapidly. This is important as it means that yeast and other enzymes work significantly better at around the optimum temperature than at lower temperatures, as the increase of the rate of reaction is greater at each interval. This explains the great need for homeostasis.

At about 34ºC, the rate begins to slow down (again exponentially), until it peaks at about 38ºC. From this one can see that 38ºC, is the optimum temperature. This must mean that it is the temperature where the enzymes have the most energy, and therefore work the best. However on the other hand, it does not have so much energy that the hydrogen bonds begin break. In other words, 38ºC is the temperature where the enzymes have the most energy, yet does not denature.

 

From about 40-45ºC the yeast slowly begins to denaturise as some of the hydrogen bonds begin to break and some active sites begin to deform. After 45ºC, the active sites rapidly deform and most of the enzymes become denatured. This also happens exponentially (although less so), as more heat is added and more hydrogen bonds break as they have more energy from the heat, and this carries on until eventually all of the enzyme will have become fully denatured, no fermentation will take place and there will be no production of carbon dioxide.

Evaluation

Accuracy of observations / measurements

Overall, I think that the experiment went fairly well as my results were quite accurate as most of them were situated on the line of best fit. I believe that all of my decisions on variables were proven right and I did not experience any major problems with the experiment.

Anomalous results

There was only one anomalous result, at 50ºC and ringed on the graph. This occurred because I did not wait until the temperature of the yeast reached 50oC yet began collecting data when it was probably at 40oC as it produced the same result as the yeast at that temperature.

I believe that my results are reliable as I had repeated each reading three times and worked out an average. Also, my results agree with my research as I found out that the optimum temperature for fermentation is about body temperature.

Suitability of method

Overall, the method was fairly suitable however there were some problems. Once the experiment got going it was impossible to control the temperature of the yeast as the rubber bun did not allow for a thermometer to get inside. This affected the accuracy of the experiment. Also, during the timing process the yeast suspension could have become idle as I did not stir it once the time started.

It is uncertain how much pressure, an uncontrollable variable, affects the results. The collision theory states that an increase in pressure leads to an increase in collisions and a faster rate of reaction as the particles are forced closer together. Therefore, pressure could have distorted my results, making some of the values higher than their actual value in reality.

Also, it was impossible to discover the effect the pH has on enzymes. If the suspension was acid or alkali at the start or end of the experiment it would have affected the activity of the zymase enzymes, because as stated in the background information zymase works best at a neutral pH. Another fault with the experiment is that when the experiment was started there was still air in the boiling tube, which created partial aerobic conditions, as oxygen may have been present. Also, it is uncertain what percentage of ethanol was in the suspension, remembering, as stated in the background information, that yeast becomes inactive at 10% ethanol. If the percentage increased 10% then the yeast cells would have stopped working.

I have suspicion about my results because someone else made the yeast suspension for me, a lab technician at my school. I was given the values of the ingredients used (0.2M glucose, 5% yeast) but I cannot be sure to what accuracy they carried it out or whether they thoroughly mixed the re-agent. I am also unsure how old the yeast was (which affects how well it induces fits), what isomer of glucose they used, what the rest of the re-agent was made up of or indeed if they made it up correctly at all.

Another problem was that because I used a new 25ml suspension for every reading, the glucose concentration may have varied as the suspension may have been stirred differently. However, I was proven right to renew this because had I used the same one then the glucose would have been used up more and more with every reading producing inaccurate and unreliable data.

Improvements to method

One would be to decrease the instrumental errors. Although not as applicable for the sealed syringe, one could quite easily be able to enhance the big margin of error, ±0.5ºC, for the thermometer, by replacing the alcohol thermometer with an electric one, usually with a margin for error of ±0.05ºC.

Also, as it was particularly difficult to successfully control the temperatures, and therefore significantly reduced the effectiveness of the experiment as I was unable to take averages. Therefore, another possible change to the experiment would be to use an electric water bath, accurate to the nearest to the degree, as you would be regulate the taken temperatures more easily. For example, I could take more values at around the optimum temperature, and also 35ºC and 45ºC. By investigating these temperatures I could produce a reliable and accurate line of best fit and find the optimum temperature for the fermentation of yeast. The same problem could also be solved with the use of a thermostat an electronic device used to make the temperature constant.

Another would obviously be to take more readings at different ranges. I would also increase the amount of re-agent and collection time, as by doing the latter I increase the amount of carbon dioxide collected. This means that with increased values, the percentage errors are less (±0.25ml is a smaller fraction of 40ml, for example, than 20ml).

I would have also liked to have used a buffer, which keeps the pH constant, educing the effect of it. As the yeast cells respire CO2 is produced, which is acidic. This means that the pH drops rapidly and this can cause the enzymes to denature. Using a buffer will stop the pH dropping as donates hydrogen ions into solution and therefore it fluctuates around a certain pH.

Extension work

Perhaps an alternative method could have been to measure the time taken to collect a certain amount of CO2 gas at certain temperatures. The great disadvantage of this would be the time required for the ends of the range of temperatures to create the amount of gas, especially difficult if the yeast has already denatured. Therefore, given the circumstances, especially due to the time limit, the method used was more effective.

For other experiments based on the same principle I would have used more types of yeast to see if they contain different enzymes, which might be more or less resistant to temperature. I would use a water bath, to keep the temperature constant. I would use a buffer to keep the pH constant. I would probably like to change the concentration of the yeast to see if the rate increases with the amount of glucose present.

Another good piece of further work would be to investigate the gas; one cannot be certain that it is carbon dioxide, which is being produced. For example, the glucose could be reacting to form carbon monoxide or even a non carbon-compound gas, such as hydrogen. A simple way to investigate this would be to use the carbon dioxide test: “Carbon dioxide turns limewater milky”. If this is unsuccessful other gas test could be administered

Bibliography

Biology for OCR A Bryon Dawson, Ian Honeysett.

Encarta Encyclopedia Deluxe 2000 World English
http://en.wikipedia.org/wiki/Yeast

www.bbc.co.uk/gscebitesize,