The solution used with the highest glucose concentration will have the beads that rise to the top the most quickly. This is because the higher the glucose concentration the more chance there is of the substrate colliding with the enzyme and thus forming more enzyme substrate - complexes per second causing the yeast to produce more carbon dioxide and rising to the surface quicker.
Hypothesis
The higher the glucose concentration of the substrate the faster all the immobilized yeast reaches the surface of the substrate solution.
Plan.
The key variables for this experiment are: -
- Types of substrate
- Concentration of the substrate.
- Varying temperatures. (Keeping in mind that temperatures above 55ºC enzymes are usually denatured.)
Types of Substrate;
The type of substrate will affect the rate at which yeast respire anaerobically and produce ethanol and carbon dioxide as they may or may not be broken down by the yeast, for example, lactose solution would not be broken down by the yeast because yeast is from the Kingdom Fungi and does not contain Lactase, which is the enzyme that breaks down lactose because it is a mammalian enzyme.
If I was to use a solution of a mixture of fructose and glucose solutions rather than one of sucrose, which contains the same monosaccharides, the rate of respiration may alter as one of the stages of break down has now been eliminated. The sucrose no longer needs to be broken down into monosaccharides before they can be changed into ethanol, energy and carbon dioxide. So, I would expect the rate to be quicker if this was done. Therefore in this experiment I will be using glucose
Varying Temperatures;
Using a range of temperatures will change the activity of the yeast. It is known that 55°C is a temperature that promotes maximum respiration for the yeast in fermentation. This implies that this is the temperature at which the greatest yield of ethanol is produced in the quickest time. At any other temperature the production will be different, this is because at low temperatures the yeast have a very low kinetic energy. Increasing the temperature increase the rate increases the kinetic energy of the molecules, which causes an amplification of the speed of the particles, this in turn increases the chance of the substrate and enzyme molecules colliding with each other forming more enzyme substrate complexes per second, so the more collisions in a given period of time, the faster the rate of reaction. In this experiment I will be using a constant temperature of 40°C so that I can fully compare the effects of the other substances.
Concentration of the Substrate;
The concentration of the substrate will have an effect on the rate of anaerobic respiration in the yeast because the greater the concentration, the more chance there is of the yeast coming into contact with a substrate molecule forming more enzyme substrate complexes per second, and the more substrate there is to be converted into ethanol and carbon dioxide.
In this experiment I will be using different concentrations of glucose to observe the effects of different glucose concentrations the rate of respiration in immobilised yeast.
Method
To be able to test the rate of respiration within the yeast I will need an straightforward but accurate way of monitoring the yeast and the best approach to do this is to measure the amount of carbon dioxide produced, as a result I am going to use a method that will enable me to unmistakably see how quickly the carbon dioxide is produced. I will make “balls” of immobilised yeast; immobilised yeast are cells, which literally that have been attached to a support that takes the form of small beads. The techniques for immobilisation vary, but in this experiment I will be using is gel entrapment: the cell will be mixed with sodium alginate and distilled water to form the gel the enzyme remains "trapped" in the gel matrix. The pores will be large enough to let the substrate in, but not the cell out.
Apparatus;
- Test tubes with rubber bungs
- Glass rods – for mixing the substances
- Pipettes/ open syringes – to make balls of immobilised yeast
- Beakers
- Timers – to time the time taken for the (balls) to reach the surface
-
Thermostatically controlled water bath at 40°C – for a constant temperature
- Test-tube racks
- Gloves
- J-Cloth - for draining the immobilised yeast from the calcium chloride
- Measuring cylinders
- Weighing scale
- Labels for test tubes
Substances;
- Vaseline – to make test tubes air tight
- Calcium chloride at 2%
- 5 grams of baking yeast (Saccharomyces cerevisiae)
- Sodium Alginate at 3% (this is a nutrient which is a purified carbohydrate complex extracted from sea kelp)
- Distilled water
- Glucose solutions at 5%, 4%, 3%, 2% and at 1%
Safety measures
My investigation will be conducted safely by wearing a laboratory coat and goggles when handling chemicals and yeast. I will make sure that the bench is sterilized before I start so that the yeast is not contaminated. I will have to take care when using calcium chloride, as it irritates when is in prolonged contact with the skin so I will be using gloves when handling this substance and be gentle when handling the glassware, I will be using Vaseline so I will have to make sure that I do not handle glass when there is Vaseline on my glove as I can drop the glassware and break it.
Plan: -
PART 1- making the immobilised yeast
Mix thoroughly 10cm3 of water with 5g of baking yeast (Saccharomyces cerevisiae) which already will have been weighed out accurately with a weighing scale
Add to that 15cm3 of 3% Sodium Alginate and mix again using a glass rod
Then I will transfer this mixture into an open syringe or a pipette so that the drops fall out slowly into a beaker, which contains 50cm3 of 2% Calcium Chloride solution this, will make the beads harden on contact.
To ensure that the beads are round I may have to adjust the height of the syringe or pipette because as they slap onto the Calcium Chloride solution their shape may become distorted, and for the maximum surface area they will have to be spherical.
I will then leave the beads in the Calcium Chloride solution so that they harden.
After this I will wash the beads and store them until the experiment.
I prepare glucose solutions at 5%, 4%, 3%, 2%, and 1% concentration for the experiments.
N.B I will put ten beads of immobilised yeast into each concentration of glucose and then repeat this three times so that there are 3 results in total for each experiment and this will allow me to have enough results to make the experiment reliable.
PART 2 – the experiment
The beads will rise, as carbon dioxide is produced because of fermentation. When yeast ferments glucose the process can be represented by the following equation: -
Oxygen (O2) + glucose (C6H12O6) Ethanol (C2H5OH) + carbon dioxide (CO2)+ Energy
- Place the beads into the labeled test tubes containing the various glucose solutions, which were in the thermostatically controlled water bath at 40º
- Insert the rubber bungs with Vaseline applied to the sides (to prevent air entering the test-tube) as soon as possible, if oxygen gets into the tubes and in contact with the yeast they will start to convert the ethanol produced into ethanoic acid.
- Start the timer to time how long it takes all the immobilised yeast to rise to the surface of the substrate solution, record the results.
Results: -
The time was recorded as all of the beads in each test tube rose to the surface and came into contact with it; adding all of the results together and then dividing by 3 worked out the average.
Looking at the table you can see that there are four underlined anomalous results in the experiment the first three could have occurred as a fault in my procedure as it was my fist experiment the first three have occurred in, I believe the that these discrepancies may have occurred as I did not apply Vaseline to the rubber anomalous result in the average results column because in the 2% concentration the results were quite varied and the experiment 1 result threw it of balance. There is only one anomalous result in the average results column. I do not think that this result really changes the trend sufficiently for me to say that there was no trend at all in the whole experiment but looking at the other two experiments I can see that there was an overall trend to show that my hypothesis was correct.
Evaluation
During my experiment there was a difficulty when trying to make the beads I changed from using an open syringe to a pipette, as it was easier for me to control the height from which I was releasing the gel with immobilized yeast.
To improve the experiment I would have used a temperature of 50°C to speed up the rate of reaction and also speed up the experiment, I would also have used bigger boiling tube so that I could have been able to see more accurately when the immobilized yeast reached the surface.
My results overall were reliable, if I did one more experiment and left the first one out my results would have been a lot more reliable than now, as you can see on the graph the trend of my average is quite deceiving as two of my experimental results show a different shape on the 2% glucose concentration my third experiment has a very large drop in the 3% which showed up in the average.
I do believe that my results are valid not taking into account the 1 pilot experiment.
If I only used the results from my 2nd and 3rd experiment this would the graph of results.
The non-anomalous results sow clearly that the higher the glucose concentration the shorter it took for the immobilized yeast to reach the surface of the glucose concentration. This shows that my hypothesis was correct.