Anaerobic Respiration of Yeast 

Aim: To investigate the effect of temperature on anaerobic yeast respiration. 

Basic outline plan: I plan to force a solution of yeast and glucose to anaerobicly respire. I plan to measure the gas collected at allotted intervals during a set period of time, when the solution is at different temperatures. I will need equipment to accurately measure the volume of gas collected, and an indicator to show me that all no oxygen is present in my solution. I will also need to make a way to force it to anaerobicly respire by creating an air tight layer over the top of the substance. 

Prediction & Background information: 

I predict that a gas will be given off and this gas will be CO2 as we are anaerobicly respiring yeast. 

We know that Anaerobic Respiration In yeast has the following formula. 

[IMAGE]Glucose Carbon Dioxide + Ethanol + Energy 

[IMAGE]C6H12O6 2CO2 + 2C2H5OH + 210 KJ/Mole. 

I predict that the volume of gas expelled will increase with temperature. I believe this for a few reasons. The first is due to the 'Kinetic theory', this states that the extent of a molecules movement depends on its state and temperature. As temperature increases, the particles become more 'exited', and so move around and collide and react more frequently and rapidly. It is these collisions which cause the reaction. We also know that heat is a catalyst in all reactions. 

I also know that yeast is an enzyme and all enzymes have an optimal temperature at which they work best, this is around 45º for yeast. However I predict that if the temperature is raised too much, then the reaction will not occur at all. I believe this because yeast is an enzyme, and according to the, 'GCSE revision guide' and the lock and key theory, enzymes are protein molecules, and each enzyme has a different shape for a certain reactant to fit into. This is called the active sight. But protein molecules and the active sight become denatured by extreme temperature of pH. When the protein is denatured this is irreversible and the mechanism no longer works. Most enzymes become denatured around 45º, but yeast is a little stronger and so should become denatured around 50º-60º. Therefore I believe that the amount of gas collected will increase over an allotted time, until the temperature becomes to high, and then their will be no reaction taking place at all. 

Also I did some preliminary work where we used Hydrogen Carbonate indicator to detect C02 by turning yellow, and during this experiment it was made clear that more bubbles were produced in an allotted time, and the indicator turned yellow quicker with hotter temperatures. 

I think it will also be best to use the same yeast for each experiment before we make it denatured, to ensure that the concentration of glucose and yeast is constant. This will also be easier as after the oxygen has been removed for our solution, we will not have to do it again. However this could be a bad idea, because we know that anaerobic respiration in yeast produces an alcohol (ethanol) and the most common reason for which anaerobic respiration is used is to make consumable alcohol in which the Ethanol eats away at the solution and kills it. If the solution dies we could believe it became denatured because of temperature, but in fact it died through the ethanol. 

Diagrams to Explain My Prediction: 

Why I chose this plan: 

In previous experiment I have found that yeast can be a very uncooperative substance. When Diazine green is placed in it, some times it can take, 5 seconds to show that there is no oxygen present; in others it can take ten minuets. This is why I have decided to use the same yeast each time so that time is not wasted in extracting all the oxygen. 

I have previously done an experiment in which the gas produced is carried via a delivery tube, through some Hydrogen Carbonate indicator which detects CO2 by turning yellow. Although it was made obvious to us that it turned yellow quicker at higher temperature, deciding when it was yellow, and when it wasn't was up to each individual so the results would not be accurate enough to plot in a graph. That is why I have chosen an experiment where to respiration rate is easy to obtain, and human interaction is minimilised. 

I am also choosing a range of temperatures which is not very hot, as I know that if I picked a range from 60º-100º, then there would be no results from 60º - 100ºas the yeast would be denatured. Therefore, I have chosen results which I believe will give me a broad range, but will but which will show a trend. 

Apparatus: 

Conical Flask 

Delivery Tube and Bung 

Gas Syringe 

Diazine Green 

Glucose and Yeast solution 

Thermometer 

500ml beaker 

Stopwatch 

I have chosen equipment which will give me accurate, but easy results to obtain. For instance, I have chosen the gas syringe opposed to a solution of Hydrogen peroxide so that I can easily & accurately measure the volume of gas collected over an allotted time. I have chosen a stopwatch over a wrist watch as this will measure the time more accurately, and a conical flask opposed to a beaker because it is easier to obtain the gas via a bung and delivery tube from a conical flask. 

Method: The big beaker was filled up half way with tap water and using the thermometer the temperature was measured, the first temperature is 25°C so if it is too cold, hot water from the kettle, or even the hot tap should be added if it is too hot add ice to cool the water down. When the temperature is right our pre prepared solution of Glucose and Yeast should have around 4-5 drops of Diazine green added to it and sealed with a layer of liquid paraffin. The flask should be corked and shook, and then the solution should be allowed to settle. The flask should be placed into the prepared water and allowed to equilibrate to the temperature for 5 minutes, making sure the temperature of the water bath is still 25°C, if after 5 minutes the solution hasn't changed to a pink colour allow it to stand in the water bath until it does change to a pink colour. This will show you there is no oxygen present in the yeast solution so anaerobic respiration can take place. Now the water bath with the flask still in it should be placed near the gas syringe, ready to place the delivery tube's cork over the top of the flask. The gas syringe should be set to 0 and the stop watch reset. The delivery tube should be placed with its cork over the flasks neck so that all gas will go into the gas syringe and not into the surrounding air. When the cork is air tight the stop watch should be started and the results recorded. The experiment should be repeated three times for this temperature, remembering to set the gas syringe back to 0 every time, then the other temperatures should be done in the same way. 

Analysing Evidence and Drawing Conclusions 

As temperature increased, so did the amount of gas given off in a certain time up to 50º. 

My first graph proves that as predicted, the amount of gas collected rises with the temperature (1), then after the temperature reaches its maximum, or optimum temperature (2), it plummets quickly as it has become denatured (3). 

The second graph proves that a constant rate of gas production was achieved with the equilibrated yeast, despite the different temperatures, as each one shows a straight line, and the only difference is the gas produced per minute. 

The rise then sudden plummet happened for a few reasons as I will explain. The first reason is due to the 'Kinetic theory', this states that the extent of a molecules movement depends on its state and temperature. As temperature increases, the particles become more 'exited', and so move around and collide and react more frequently and rapidly. It is these collisions which cause the reaction. We also know that heat is a catalyst in all reactions. 

I also know that yeast is an enzyme and all enzymes have an optimal temperature at which they work best, this is around 45º for yeast. However I know that if the temperature is raised too much, then the reaction will not occur at all. I believe this because yeast is an enzyme, and according to the, 'GCSE revision guide' and the lock and key theory, enzymes are protein molecules, and each enzyme has a different shape for a certain reactant to fit into. This is called the active sight. But protein molecules and the active sight become denatured by extreme temperature of pH. When the protein is denatured this is irreversible and the mechanism no longer works. Most enzymes become denatured around 45º, but yeast is a little stronger and so should become denatured around 50º-60º. 

I also found that the experiment tied very closely with my prelim., in my prelim as temperature was raised more bubbles were produced in an allotted time, and it turned Hydrogen Carbonate indicator yellow quicker, which in effect is exactly the same as what happened in my real experiment, except we collected the gas instead of counting the bubbles for increased accuracy, and we did not feel the need to test the gas. 

I think my conclusion is valid and matches the whole range of experiments as it seems to follow a trend and the rules, and seems to fit in n a graph. 

Evaluation 

I thought the results obtained were very accurate and conclusive. They seem consistent as the show straight lines on graph two, which means a constant rate as it should be and a steady rise then a sudden plummet in graph 1 as it suggests there should be. However, on my repeats there are some noticeable differences between attempt one and three, with two, for the volume of gas given off per minute. At 35ºC and 40ºC the volume of gas collected seems to be consistently higher then the other two, and even in 40ºC goes above the results for 45ºC, which is not what I would have expected. The explanation I can offer for this is that there must have been some change in the temperature, and I must have not stirred it consistently, which makes that set of data in accurate. I thought all of my other results generally fitted the overall trend well however the differences between the gas given off each minute per temperature seems to be a little random, even if it does follow the general trend. 

I though the experiment allowed the prediction to be tested very well as it proved the points that I wanted it to, and very concussively as well. I thought my method allowed the results to be sufficiently accurate although we did run the risk of killing the yeast by its own ethanol which could disguise itself as denaturing. 

I think my results were reliable enough to support the conclusion especially due to the fact that there are no obvious abnormalities. There are a few results hat do not fit the overall trend such as the cumulative amount of gas expelled as shown on graph 1 does not quite fit into a curved line but still however shows a general trend. 

If I was to test the same variable again I think I would have pre boiled my glucose and water solution with a paraffin layer already coating it and then add a yeast tablet, so we would not have to wait for the oxygen to disperse. And I would also use an electric water bath to heat the water each time, with an electric thermometer to achieve more accurate results. If I could make the water bath very accurate I would test every degree for a 10º period between 40º and 50º to discover exactly when the optimal point for this yeast enzyme is.