The Effect of Substrate on

Respiration Rate in Yeast

Aim

To determine the effect different substrates; glucose, sucrose and lactose, have on the respiration rate of yeast.

Scientific Background

Living organisms require energy from ATP to carry out many functions such as active transport. ATP is formed from joining ADP and a phosphate ion together and this process requires energy which it gets from respiration. Monosaccharides can enter at different stages of respiration but disaccharides cannot enter respiration without first being broken down into monosaccharides. Glucose is a monosaccharide and enters at the beginning of respiration but sucrose and lactose are both disaccharides and must be broken down. Sucrose is broken down into glucose and fructose and lactose is broken down into glucose and galactose. These all break down into a glucose molecule and another monosaccharide which enters lower down the process of respiration.

Although sucrose can be broken down readily by yeast, lactose can not because the enzymes to uptake and breakdown lactose are not present normally in the yeast; there is a function in the DNA of many micro organisms called the Lac Operon which is responsible for producing these enzymes when lactose is present.

A repressor prevents the RNA polymerase from producing the enzymes during protein synthesis so the enzymes are not normally present. When lactose is present, the lactose acts as an inhibitor to the repressor so then the RNA polymerase can then transcribe the enzymes and they are produced which allows lactose to be taken up and broken down.

There are two types of respiration which a living organism can undergo. In this experiment the yeast will be made to undergo anaerobic respiration. There are two types of anaerobic respiration; one pathway produces lactic acid, or lactate, and this is usual found in animals, and the other pathway produces ethanol and this is usually common to fungi like yeast. In anaerobic respiration, the pyruvate can not enter the link reaction due to the lack of oxygen, so instead it is decaroboxylated to ethanal which is then reduced to ethanol. This type of respiration is necessary to ensure that all of the yeast carries out the same type of respiration so the experiment is a fair test.

For Aerobic Respiration:

C6H12O6 + 6O2 → 6CO2 + 6H2O (+ 36 ATP)

For Anaerobic Respiration:

C6H12O6 → 2C2H5OH + 2CO2 (+ 2 ATP)

From these equations you can see that there is a significant difference in the amount of CO2 produced so it is very important that anaerobic conditions are achieved for the experiment to avoid inaccuracy caused by partial aerobic respiration.

Variables

Respiration Substrate (Controlled Variable): This will be the substrate used in respiration; glucose, sucrose or lactose, and will effect the respiration rate.

Respiration Rate (Dependant Variable): This will be determined from the amount of CO2 collected from the yeast over a certain amount of time. Rate = CO2 / Time

Constants: All other conditions should be kept constant to keep the experiment a fair test so the results are reliable.

Temperature: This will affect the rate of respiration by making the enzymes work at different rates at different temperatures. It may also denature enzymes at high temperatures. All solutions will be brought up to the same temperature of 35oC before being mixed.

Mixture Volumes: All volumes and concentrations of yeast and the substrates will be kept constant so that all of the results can be compared as they will all have taken place in the same amounts. Having different volumes and concentrations will change the rate of respiration due to there being more or less molecules that are able to react.

Yeast: It is very important to use the same batch of yeast so that its concentration is the same and it is all of the same quality otherwise there may be many factors of the makeup of the yeast, genetic or physical properties, which will affect the respiration rates.

Respiration Pathway: The yeast should only respire anaerobicaly so that each molecule of the substrate will go through the same pathway and produce the same amount of CO2. The experiment is being done in a closed environment so the yeast would eventually respire anaerobicaly which would affect the CO2 amount being produced. A period of acclimatisation will be necessary so that the yeast can use up all of the oxygen available and then when it starts respiring anaerobicaly, the timing and recording can begin.

Acclimatisation time: The yeast will be mixed with the sugar and then left for a fixed amount of time to allow for the yeast adjusting to its new conditions and to produce respiratory enzymes.

Measuring time: The time intervals of measuring the CO2 collected will be kept the same for all repeats. The total time will be 10 minutes after acclimatisation measuring in 1 minute intervals.

Hypothesis

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Acclimatisation time: The yeast will be mixed with the sugar and then left for a fixed amount of time to allow for the yeast adjusting to its new conditions and to produce respiratory enzymes.

Measuring time: The time intervals of measuring the CO2 collected will be kept the same for all repeats. The total time will be 10 minutes after acclimatisation measuring in 1 minute intervals.

Hypothesis

Due to the disaccharides being made of two monosaccharides, they effectively have twice the concentration after being broken down. They produce a glucose molecule and another monosaccharide which enters somewhere lower down in respiration so the respiration rates of these substrates should be much faster maybe up to twice as fast. There may be some time with these disaccharides of lower respiration rates where the disaccharides are being broken down and in the case of lactose, the enzymes required are being produced.

Prediction

The disaccharides will have a higher overall respiration rate than glucose and will have produced more CO2 after the 10 minutes for the experiment.

Safety

Mains voltage electric heating equipment is being used in close conjunction with water; care must be taken when working with this equipment. All chemicals being used are safe so no protective clothing is required. Prolonged exposure and repeated contact with yeast may cause skin and eye irritation. Care must be taken to avoid cross contamination of the chemicals which could affect the results of the experiment.

Preliminary

a) Aim
To determine suitable concentrations and volumes of yeast and the sugar solutions for use in the main experiment and to suggest improvements for the final method.

b) Apparatus

c) Method
1. Clean all equipment with de-ionised water to avoid contamination which could lead to inaccurate results. Ensure the measuring cylinder is filled to the top with water, otherwise measure the volume of gas in there before carrying out the experiment.

2. Measure out an amount of yeast and a substrate with the syringes and put them in separate test tubes in the water bath so that they can be brought up to the correct temperature.

3. When the solutions are at temperature, mix them together in the side arm test tube and start the timer for the acclimatisation period.

4. After 2 minutes, put the bung in the side arm test tube and restart the timer. After a further 2 minutes reset the timer and start recording the CO2 levels after 5 and 10 minutes. Record the CO2 amount with the volumes of yeast and substrate, the reaction time and substrate used.

5. Reset the equipment and repeat these steps with different substrate and volumes until satisfactory volumes of the yeast and substrate have been found which can be used in the main experiment.

d) Diagram

e) Results

Preliminary Analysis

The initial results show that sucrose is producing more CO2 by higher levels of respiration as predicted however there is a major problem with regards to the respiration rates with the lactose. Sucrose is producing more CO2 than glucose but the difference in CO2 levels is not large enough for it to be of any significance so many more repeats must be made at a higher level of accuracy.

A few problems did arise from the apparatus and method which can be improved upon. The inverted measuring cylinder used to measure CO2 was not very accurate and was messy and tricky to set up which slowed down the process of setting up significantly. The tube underneath the cylinder could also come away which would ruin the experiment so it would have to be redone. Water could also get into the tube which could reduce the flow of CO2 coming out of it which would cause a higher build up of pressure in the side arm test tube. The levels of CO2 recorded because of this would be less than what they should have been. This can be improved by using a gas syringe instead. This will leave a clear path through the tube from where the yeast and substrate are mixed. It is also much easier to set up and all of the connections are fixed so there is no element of leakage.

The side arm test tube also caused a problem. When adding the yeast and substrate, there was a chance that some of it could go down the arm of the test tube and clog up the rubber tube which would cause major problems in the recorded levels of CO2. Mixing the yeast and substrate during the experiment may also be required so that the true levels of respiration are recorded by aiding the diffusion of the chemicals. This would require an alternative to the side arm test tube so that the mixture will not go down the side arm when shaken. A conical flask could be used instead of the side arm test tube. A bung with a delivery tube going through it will also be used in the conical flask so that the CO2 can still be collected.

Final Apparatus

Final Method

1. Clean all equipment with de-ionised water to avoid contamination which could lead to inaccurate results. Reset the gas syringe by putting it back to 0 cm3.

2. Measure out 10cm3 of yeast and 5 cm3 of a substrate with the syringes and put them in separate test tubes in the water bath so that they can be brought up to the correct temperature.

3. When the solutions are at temperature, mix them together in the conical flask and start the timer for the acclimatisation period.

4. After 2 minutes, put the bung in the conical flask and restart the timer. After a further 2 minutes, reset the timer and gas syringe then after every minute up to and including 10 minutes, record the CO2 amount with the volumes of yeast and substrate, the reaction time and substrate used.

5. Reset the equipment and repeat the experiment with the same substrate and different substrates until at least 5 repeats for each substrate have been done.

Final Diagram

Results

Analysis

The table shows the CO2 collected at certain time intervals during the experiment. It is opposite to what is predicted which was both sucrose and lactose producing more CO2 than glucose. Lactose did not produce any CO2 from respiration after the acclimatisation period which would suggest it had an inability to break down lactose. This suggests that either the Lac Operon was not present in this sample of yeast or that there was not enough time given for the Lac Operon to operate.

The prediction suggested that glucose would have a higher initial rate of respiration due to the fact that the yeast does not have to break it down before respiring it. This could also suggest that not enough time was given for the experiment and that sucrose may have produced a much higher amount of CO2 than glucose if left for longer. The shape of the graph shows that the gradient of the line of best fit is increasing which shows the rate of respiration is increasing. This shows that it was only the beginning of the reaction as the rate of respiration was increasing. This could be because the substrate is being absorbed into the cell increasing the concentration in the yeast cells.

The Mann Whitney-U Test can determine if these is a significant difference between the results. Two hypotheses are chosen for if the results have a significant difference or not.

Null Hypothesis: There is no difference of rate of respiration for yeast with different substrates.

Alternative Hypotheses: This is a difference of rate of respiration for yeast with different substrates.

For this test I will ignore lactose for the fact that there was an obvious significant difference which will be evaluated separately.

R = 35.5 (Sum of ranks for lesser substrate)

n1 = n2 = No. of results for substrate. = 6

U = n1n2 + n1(n1 + 1) / 2 – R

U = 6*6 + 6(6+1)/2 – 35.5 = 21.5

U’ = n1n2 – U

U’ = 36 – 21.5 = 14.5

U’ < U therefore U’ is used.

Ucrit for 6 + 6 set of results = 5

U’ > Ucrit

14.5 > 5 therefore the values are not significant enough so accept Null Hypothesis.

These set of results are not significant for P > 0.05 so there is no difference of rate of respiration for yeast with different substrates.

Evaluation

It is obvious that this experiment was not carried out perfectly and there are many improvements that could be made. First of all, there is a major anomaly with a set of results for sucrose which is highlighted on the table of results. The levels of CO2 for this particular set of results were much lower than the others which suggests that there was a problem with that particular experiment. This could have been caused by a blockage in the rubber tube or in the air syringe, or possibly an error in the mixture of the yeast and substrate used. This set of results was rejected and not included in the averages taken.

There was also a major flaw in the fact that the yeast could not respire lactose. This could have been due to many factors such as the absence of the Lac Operon or there was not enough time given for the Lac Operon to work. An improvement to this would be to leave the experiment to run for a much longer time and to try to use a different sample or species of yeast which may have the Lac Operon present.

Due to the fact that the results for sucrose where much lower than predicted, there could have been a problem with the method. This could be that the time for the experiment was not long enough. This shape of the graph suggests this as the gradient was getting steeper and not starting to reduce and level off which it will do as it gets to its maximum respiration rate and then runs out of substrate to respire. Extending the time of the experiment would be a major improvement and may show the results with a much higher significance than what was found in the Mann Whitney-U test.

There are many improvements that could be made to the equipment and method which are listed below.

There were also problems with the methods and techniques which could also be improved. Here is a list of these problems and possible improvements.

Six repeats were taken for each substrate so that anomalies could be found and removed. This made the results more reliable and suggested that there was an obvious trend to the results as shown by increasing amounts of CO2. The preliminary showed a trend line which was of a similar shape to the results found however it could have been extended to show a longer time allowed for the reaction which would have shown a more thorough prediction.

Bibliography

Respiration Information

Advanced Sciences – Biology 1 - Mary Jones, Richard Fosbery, Dennis Taylor.

Lac Operon, Respiration and ADP/ATP Information

Advanced Sciences – Biology 2 - Mary Jones, Jennifer Gregory.

Lac Operon Diagram and Information

Sugar Diagrams and Information

Mann Whitney-U Test Information

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