How Temperature Effect On Anaerobic Respiration In Yeast?

Enzymes are the tools of life. They do almost everything in a cell. Virtually every one of life's chemical reactions is driven by some enzyme. Enzymes are organic catalysts that work like a starter on a car. With a small input of energy from the starter, a car can run on its own energy all day. They don't get not used up or changed when they "jumpstart" another molecule. They can perform the same task over and over working very fast.

They can carry out their task thousands of times per second. Each enzyme has its own unique structural shape that allows it to bind to or "unlock" certain other molecules called substrates. When the correct "lock" and "key" come together, a chemical bond is formed.

Lock and Key Theory

The lock and key structure is a theory on why enzymes catalyse reactions. This theory states that all enzymes and substrates (the object which the enzyme act on) have specified structures (active site) and chemical properties.

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The substrate fits into the enzyme's active site, and they react. The substrate is broken down, and then the enzyme can act on the next substrate. This can be done as quickly as 32 million particles per second. This means that, like only a certain and correctly sized and shaped key can fit into a certain lock, only a certain substrate (key) can fit into a certain active site (or key hole) in the enzyme(lock). This is displayed clearly in the diagram below.

Kinetic Theory

This is a theory that suggests as temperature increases, the molecules move faster, due to increased kinetic energy.

This causes the enzyme and substrate molecules to meet more often meaning the rate at which the product is formed will increase. However, as the temperature continues to increase the hydrogen and ionic bonds, which hold the enzyme in shape, break and the active site will no longer accommodate the substrate. When this happens we say the enzyme has denatured, this cannot be reversed.

Preliminary Experiment

Aim: To establish a basic relationship between temperature and respiration rate in yeast.

Before completing the final experiment I decided to do a preliminary experiment in order to get some basic results and in order to get a good understanding of what I was doing and to get used to the apparatus I would be using.

Preliminary method: I took a test tube consisting of yeast and water, added two small drops of diazine green and also poured liquid paraffin in the test tube to work as a seal. I then put a rubber bung with a plastic pipe in the top of the test tube; I then submerged the test tube in water of various temperatures, with one end of the tube in the yeast solution and one end in a large beaker full of water. I waited for the yeast solution to turn pink due to the diazine green and then, using a stopwatch, counted the number of bubbles produced each minute by the C02 being released by anaerobic respiration in the yeast solution.

This preliminary experiment proved to be relatively unsuccessful. The results were very vague and inaccurate. I found that the temperature was inconsistent, as I wasn't adding cold or hot water when necessary. Also I didn't allow time for the yeast solution to equilibrate, which may well have added to the inaccurateness of the results. Bubbles also are a very vague measurement, in my final experiment I will use a gay syringe and measure the volume of CO2 produced. I took all of these matters into account when carrying out the final experiment in order to make it as accurate and efficient as possible.

Prediction: In the final experiment I will see the yeast respire anaerobically as in the preliminary experiment. I know this because the gas given out is CO2, this is the waste gas given out during anaerobic respiration.

Below are the word and symbol equations for anaerobic respiration.

glucose ethanol + carbon dioxide + energy

C6H12O6 2C2H5OH + 2CO2

In my final experiment I expect to see an optimum temperature of approximately 45�C. This, I think, will be when the enzymes and the substrates will have the most kinetic energy thus forming the most products. Although I only went up to 40�C in my preliminary experiment I believe that the enzymes will be able to work at a slightly higher temperature. I expect them to denature at 60-70�C.

Final Experiment

Aim: To measure the rate of anaerobic respiration in yeast at different temperatures using a gas syringe.

Method:

1. Using a beaker I set up a water bath at 30C.
2. I then poured 20cm of the yeast solution into a small conical flask and added 2 drops of diazine green.
3. I then added enough liquid paraffin to create a layer on top of the yeast.
4. I submerged the conical flask in the water bath at 30C.
5. I then waited for the indicator to turn pink.
6. Once Pink I put a rubber bung with a tube connected to a gas syringe in the top of the conical flask.
7. Once I had put on the rubber bung, I started the stopwatch and took a reading of the volume of CO2 produced every 5 minutes for 15 minutes.
8. Using the same yeast I repeated this experiment at 40C, 45C, 50C and 60C.
9. I recorded my results in a table with the average volume of gas at each temperature.

Results:

Analysis:

From looking at the graph from these results you can clearly see that as the temperature rises from 30C to 50C, the rate of reaction and the volume of gas produced through anaerobic respiration also rise. This is due to the Kinetic Theory. As the yeasts temperature rises, the molecules gain more kinetic energy and begin to move at much greater speeds. This causes the enzymes to collide with the substrates much more frequently, meaning many more keys will find there locks, which in turn causes more products to be formed. This is the reason for more CO2 being produced as the temperature increases, as the respiring at a much higher rate.

However once we pass 60C the reaction rate drops drastically. This is due to enzymes denaturing. Enzymes have optimum temperatures. This is where they work at the most effective rate. As we can see these enzymes optimum temperature is approximately 50C. However if you exceed that temperature by too much, the enzymes will die and denature. This stops them from working and they are then useless. This explains the drastic plummet in the graph after 50C. The enzymes have denatured and are no longer working.

My prediction forecasted this result as I saw this occur in the preliminary bubble counting experiment. At 60C there were very few bubbles produced which also suggests that the enzymes had denatured as in this experiment. My preliminary experiment also helped me predict a fairly good optimum temperature of approximately 45C.

Evaluation:

I believe that my data is relatively reliable for a number of reasons. I have achieved enough results to get the pattern that I expected to see. I used 5 different temperatures and repeated each one 3 times. This gives me 15 pieces of data to work with which is enough for me to achieve the pattern I wanted.

Also I used a precision piece of equipment in the gas syringe. This is an extremely accurate piece of apparatus and has given me very accurate results. I also could have used a burette, but I feel a gas syringe is just as effective if not more so.

I tried my best to control the temperature by using a thermometer to get the water bath at the desired heat. I added hot and cold water whenever necessary in order to keep it at the temperature. This is not the most accurate way of keeping the water bath at one temperature, however it is fairly accurate and was one of few ways available to me.

I also gave time for equilibration at each temperature to ensure the results were accurate for that temperature and the enzymes had grown used to the newly adjusted heat.

The majority of my readings are close to the average any my graph looks relatively similar to that of the textbook.

However I do not like my results at 45C. They don't seem to fit in with the rest of the results. The average volume of gas produced was less than at 40C. This cannot be due to denaturing as the enzymes did not denature at 50C. Therefore due to the Kinetic theory the reaction rate should be higher at 45c than at 40c. However it is not. I believe that this is most likely due to the fact that I made a mistake when collecting my results for 45. It is most likely that I let the temperature drop and forgot to top it up with hot water. This would make sense as my results for 45�C look more like results for approximately 35C. Due to this fact I circled it on the graph as an anomaly and decided not to use it in the line of best fit.

I came across one other anomaly at 50C. My first two readings were as I expected and came out fine. However my final reading at 15 minutes was far lower than the other two. Only 17 cm compared with 42cm and 33cm. I am certain that this is due to the enzymes denaturing. I must have added too much hot water to the water bath, causing the temperature to rise above 50C and denature the enzymes causing them to work much less effectively. You can see this result as the red circled one in my results table. I decided not to use it in the average for 50C as it was an anomaly that would have drastically changed the average and shape of my graph.

There is not a great deal I could have done to improve my experiment. I could have been a slightly more careful when adding hot or cold water, as this seemed to be the cause of my two anomaly's. I also could have used a burette instead of a gas syringe in order to get more accurate readings. Also it may have been useful to get results at more temperatures and also take more repeats in order to make the averages more accurate. This would also make it much far easier to identify any anomalies.

Improved Experiment Diagram:

Despite the various ways in which I could improve my experiment, I still can depend on my results. I got the data I set out to get and achieved my aim. Even though I got two anomalies' I managed to spot them and exclude them from the experiment, giving me sufficient, good quality data that allowed me to draw a firm conclusion.