Yeast have to make energy, stored as ATP to carry out all cellular functions. To do this they can respire both aerobically when there is plenty of oxygen, but where oxygen is short, they respire anaerobically; by this, they are called partial anaerobes. This produces less energy, but keeps the yeast alive. Pyruvic acid has to be broken down in respiration when formed by breaking down of glucose molecules, this can’t be done in the same way as it is aerobically when respiring anaerobically which is how the carbon dioxide and ethanol is formed through the zymase. Here is the equation for anaerobic respiration:
enzymes in cytoplasm
(zymase complex)
glucose -----------------> ethanol + carbon dioxide +energy
C6H12O6 2C2H5OH CO2 210 Kj/mole
210Kj/mole in anaerobic respiration as aposed to 2890Kj/mole in aerobic respiration
There is 2ATP from each respired glucose molecule - in aerobic there is 38ATP.
Kinetic theory states that, with an increase in temperature, the rate of reactions will increase. This is due to the increase of speed of the particles, brought about by the extra energy given to them by heat. Faster particles will bring about more particle collisions and so the reaction will take lace faster. Enzymes are sensitive to temperature changes up until a certain temperature and will increase in their activity also. The reactions that take place in the enzymes will be quicker and so will create more of their products. As a general rule of thumb, it has been said that there is a doubling of the rate of reaction for every 10ºC rise this is called the ‘Q10=2’ theory. This should be evident when the concentration of the enzyme and substrate are kept the same also.
Enzymes are sensitive to temperature up untill a certain temperature where the shape of the active site is altered drastically, so much so that binding hardly ever takes place. This is called denaturisation.
Prediction
With reference to my theory, I predict that the rate and speed of respiration of glucose by yeast will increase with temperature rise up until a certain point where the enzyme used and secreted by the yeast will become denatured and cease to function, reducing the rate significantly. This is explained through Kinetic theory, yeast respiration and the nature of enzymes.
Variables
In my main experiment, I shall use the time taken for methylene blue test tube with yeast and glucose solution to turn the colour of the control as my dependant variable and the temperature as my independent variable.
Here is a list of variables that can have an affect on my experiment and also how I will control them if possible.
Temperature
Temperature of the experiment will have a great affect on the results as explained by kinetic theory. Temperature will affect the rate of yeast respiration. I shall keep the temperature of he mixture and water bath under control by using a thermometer and checking it constantly. I shall also keep swirling the thermometer to keep the heat distributed. Also, as it will take longer for the temperature inside the test tube the same as the water bath, I shall leave the apparatus for two minutes, keeping the temperature constant.
Amount of methylene blue
Methylene blue is sensitive to oxygen and will go blue with contact with oxygen and colourless with the production of NADH during glycolysis as the glucose is broken down. The amount of this would affect the accuracy of the readings as the rate of NADH production affects the methylene blue and a differing amount of methylene blue would not give fair and reliable results. I shall keep the amount of drops of methylene blue the same at each timing.
Amount of yeast
The amount of yeast is crucial, more yeast means more glucose will be respired and more products created. An imbalance will upset the results. The amount of yeast will be weighed out on an accurate top-pan balance each time.
Amount of glucose
The amount of glucose will affect the results also, as more glucose means that there are potentially more products, which would make the results accurate or the experiment fair. The glucose will be weighed out each time using an accurate top-pan balance.
Volume of water
The volume of water may have a slight affect to the results as it may cause less accuracy when distributing the heat in the test tube. The volume of water will be kept constant by using a measuring cylinder at each preparation.
Amount of shaking and acclimatisation
Acclimitisation and shaking will help to activate the yeast and prepare the solution for timing. If it is improperly mixed, acclimatised to temperature or activated, the results would not be fair and inaccurate. I will shake the test tube thoroughly each time until I can see bubbles being created well and I shall do this while it is warm to aid activation. I shall also leave this in the water bath at the required heat for two minutes, regulating the temperature with the Bunsen.
Light and atmospheric conditions
These would not have a great deal of affect on my experiment and are beyond my control. Some of the substances may be sensitive to these, but I doubt they are sensitive enough to affect the results.
Apparatus
Bunsen Burner Stopwatch Yeast Glucose Stand and Gauze
Methylene-blue
Syringe
Pipette
Boiling tubes (x2)
Beaker
Bungs
Method
In my final experiment, I shall use methylene blue in the solution. I shall time how long it takes for methylene blue to go colourless in the solution, constantly checking against a control which contains a little methylene blue for continuity. To avoid unfair contact to Oxygen in the air, I will put a layer of oil over the mixture. I shall weigh out 2g of glucose and 2g of yeast this time and 25cm3 of water to aid accuracy. My independent variable is the temperature and the dependant is the time taken to change colour of control.
I shall take readings from 20ºC to 60ºC at 10ºC intervals. I will start from 20ºC as I found out from my initial investigation that there was no respiration activity below this temperature. I shall proceed in this sequence as it is the easiest way of collecting results and will help to find out other flaws at a lower temp. Also to aid accuracy, I shall take three readings at each interval and take the mean
Summary
I found that as the temperature increased, the rate of respiration increased with it. I also found that the rate of respiration dropped of completely after a certain point, highlighting the denaturisation of the yeast’s enzymes.
Conclusion
I have found that as I increased the temperature of the yeast solution, the rate of respiration of the yeast increased to a certain point where, as the temperature rose to a certain level, (in my case about 58ºC) the rate of respiration eventually cut off. I have also found that my Q10 value is 1.43. Seeing as the most accurate value for a Q10 reaction is 2 (the rate of reaction doubling for every 10ºC rise) this makes my reaction look a bit inaccurate yet with positive signs of correlation. A Q10 reading as low as 1.43 suggests there were either faults with the method or apparatus or that the reaction was not a true Q10=2 reaction; this reaction should be a typical Q10=2 reaction, so my method or apparatus probably give the inaccuracies. I will talk further about this in strand iv to suggest reasons.
My hypothesis and prediction can be backed up with the findings; from looking at my results and graphs you can see the rise and fall of respiration, further displayed by the reaction’s Q10 reading which, although quite a lot less than 2, it still gives the presence of the reaction’s ‘sensitivity’ (through zymase) to temperature. Thus my hypothesis and prediction are shown to be present and displayed to a large extent. They are explained due to the theories of enzyme-substrate with lock and key and kinetics. Where these meet is when kinetic theory states that an increase in temperature means more particle collisions between reactants and so a faster rate of reaction; and in enzyme-substrate where the enzyme is sensitive to heat, and about a certain temperature, the active site will begin denaturing, so slowing and eventually stopping the reaction. This will give an area where the rate of respiration drops off and goes to nothing instead of a precise ‘cut-off’ point. These both apply to my experiment and were described in my planning.
My Results
To make sure that the results were as reliable as I could make them, I calculated the mean of three results at each interval when dealing with the rate and also used these to produce my log values.
I took all precautions to make the apparatus used to be reliable and give good values so I think the slight unreliability was caused by the preparation of the solution and the ‘unpredictability’ of how the reaction went that came with it. To obtain more reliable results I would want complete continuity with preparations, maybe arranging ‘sets’ of substances to create multiple solutions beforehand or preparing them but not actually activating the yeast so as to prevent any getting a ‘head start’ over the others. This would ensure that all the preparations are the same and would give continuity. I would want to be more strict and thorough with preparing solutions and mixing them up. I would want each one to be thoroughly acclimatised to the surroundings and had the same amount of methyl blue and same activating and mixing time. This would help give more reliable results throughout.
If I were to further investigate this experiment and my results, I would probably want to calculate the point where the enzymes begin to denature for respiration in yeast. I could also examine the change in rate between the intervals to determine validity and continuity, also running them through maybe more intricate calculations involving log. At this stage, I shouldn’t think there is to be much more I could do. I wouldn’t want to investigate any other v