Investigating the Effect of Ethanol Concentration on the Rate of Respiration in Yeast.
Investigating the Effect of Ethanol Concentration on the Rate of Respiration in Yeast
Respiration is the process by which the energy in food molecules is made available for an organism to do biological work. Respiration produces ATP, which is the form of energy, made by most organisms, and they use it for their survival. Yeast cell, along with all other cells respire, and I am going to investigate the effect on the rate of respiration when I add various concentrations of alcohol to it.
Scientific Theory:
There are many factors that can affect the rate of respiration in a yeast cell. In my experiment, I am going to develop this with how an alcohol affects the rate at different concentrations.
The equation for aerobic respiration is
C6H12O6 + 6O2 6CO2 + 6H2O + Energy ...
Is the standard equation before the alcohol is added. As the alcohol is the factor I am assessing, then the concentrations of the yeast solution, and how much sugar I am adding, need to be determined before I can add the different volumes of alcohol, in this case Ethanol. I am going to do this by carrying out preliminary experiments.
Glucose concentration is an important part in the reaction as this helps the reaction to get started. If too much glucose is added, then this has an osmotic effect on the yeast cell, pulling all the water out of them and preventing the from respiring. This then kills the yeast cell. Also, if not enough glucose is added then the reaction cannot begin, as it does not have enough raw materials to react with the yeast. Either way, the amount of glucose that is added has an effect on the rate of respiration, as it is the substrate for the enzymes in yeast..
Temperature can also affect how fast and how efficient the reaction is. If the yeast is heated to a temperature above 45'C, then the enzymes within the yeast are denatured. Denaturating is when the bonds holding the protein in shape are broken, and the effects it has on the enzyme are irreversible. The optimum temperature at which the yeast will work best at is approximately between 25'C and 35'C, and anything below or above this then the reaction will not occur, or the results will be in accurate. As the temperature of the reacting mixture reaches around 30'C, this is the optimum temperature, so as the temperature increases or decreases around this the molecules have more energy to move around hence more collisions. This causes the rate to increase.
Adding Ethanol can have an effect on the rate of respiration also, as the variable amounts added can act as an inhibitor to the enzymes, preventing them from working properly. So in effect, the more ethanol that is added can prevent the reaction from occurring efficiently.
Respiration releases energy in the form of ATP. In yeast, this occurs as AEROBIC respiration, and also ANAEROBIC respiration in which CO2 and Ethanol are the products formed.. To form ATP, several processes must occur for respiration to take place. The first process is Glycolysis.
Glycolysis is the first stage of respiration and takes place in the cytoplasm of a cell. In the initial phosphorylation, 2 ATP molecules are consumed. The 6C sugar phosphate breaks down to form 2, 3-carbon sugar phosphates. These are then converted to Pyruvic Acid by NAD molecules being reduced to NAD + H+. In these processes, 2 ATP molecules are formed and 2 Pyruvate molecules.
The Link Reaction is the next stage in respiration. This occurs in the mitochondria of the cell. Oxygen is required for it to take place. The Pyruvate molecules enter the Link Reaction, and the decarboxylation of the Pyruvate occurs, with CO2 being released. Dehydrogenation of the Pyruvate then occurs, with the NAD being reduced to NAD + H+. The remaining molecule is the combined with coenzyme A to form Acetyl Coenzyme A.
The Krebs Cycle (also known as the Citric acid cycle, or the Tricarboxylic acid cycle) is the next stage in Respiration. For every glucose molecule respired, the whole cycle takes place twice. So, for each turn of the Krebs Cycle, one ATP molecule is made. Each Acetyl CoA combines with an oxaloacetic acid to form a 6C citric acid. Into the Krebs Cycle, 2 Acetyl CoA, 6 NAD, 2 FAD and 2 ADP + P molecules are put into the cycle. For each complete turn, 2 oxaloacetate, 4 CO2, 6 reduced NAD, 2 reduced FAD and 2 ATP molecules are formed.
So, during respiration in a Yeast cell, 38 ATP molecules are formed.
CO2 is formed within one turn of the Krebs Cycle. This is also known as Oxidative Carboxylation. This is due to the acetyl that enters the Cycle.
Enzymes are biological catalysts that control a reaction. They can affect how fast or how slow a reaction takes place, and in respiration is no exception. There are enzymes used in respiration, for example Acetyl Coenzyme A, which is involved in the Krebs Cycle.
Enzymes adhere to a Lock and Key Theory, in which the ...
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So, during respiration in a Yeast cell, 38 ATP molecules are formed.
CO2 is formed within one turn of the Krebs Cycle. This is also known as Oxidative Carboxylation. This is due to the acetyl that enters the Cycle.
Enzymes are biological catalysts that control a reaction. They can affect how fast or how slow a reaction takes place, and in respiration is no exception. There are enzymes used in respiration, for example Acetyl Coenzyme A, which is involved in the Krebs Cycle.
Enzymes adhere to a Lock and Key Theory, in which the enzyme and the substrate fit perfectly (the active site). Yet, the enzyme having an inhibitor attached to it at one part or another can stop the function and effectiveness of the enzyme. There are two types of inhibitor; Competitive Inhibitor and Non - Competitive Inhibitor. Firstly, a competitive inhibitor has a similar shape to that of the substrate, and so blocks the substrate from binding to the enzyme. A non-competitive inhibitor does not bind to the active site of the enzyme, yet binds to another part of the enzyme, which changes the shape of the active site, preventing the enzyme-substrate complex from occurring. Temperature can increase or decrease the rate at which a reaction occurs, with reference to the enzymes. An optimum temperature can be reached at which the enzyme works best at, this is usually between 35 - 45'C. on a graph, the optimum temperature is reached by a steady increasing curve, with it then levelling off and falling as it falls past its optimum and the enzymes are denatured.
Prediction:
Taking into consideration the factors that affect the rate of respiration, and the factor I am measuring, my hypothesis is that the more concentrated the ethanol becomes, when this is added to the yeast and glucose complex, this will inhibit the yeast, and so the amount of Carbon Dioxide produced will decrease. Therefore,
INCREASE IN ETHANOL CONCENTRATION
DECREASE IN VOLUME OF CO2 PRODUCED
Is my overall Prediction.
Variables:
Input Variables
o Concentration of yeast used - if there is not enough yeast added then this would cause the reaction to not start, as the activation energy is not there, as there are not enough cells, which means not enough molecules to react. If too much is added then there also may be not enough glucose to react, so the reaction does not start again, or very little CO2 is produced. The yeast that I use will need to be in a solution, so that a measurable amount of CO2 is produced. As the yeast is the source of enzymes, if this factor is increased or decreased in concentration then this will have an effect on the rate of reaction, either increasing or decreasing it, depending on the concentration.
o Temperature - an optimum temperature must be used so that the yeast can respire efficiently. A temperature above 45'C will denature the enzymes within the cell.
o Concentration of sugar used - this needs to be decided whether to add the sugar in gram by gram or to dilute it into a solution of distilled water. Recent results show that more than 6g/100ml of sugar has an osmotic effect on the yeast. It pulls all of the water from the yeast, and then kills it. If there is too much, then the concentration needs to be decreased to keep the reaction at a constant level. This is due to the fact that the sugar is the substrate, so there needs to be enough surface area to react, yet not too much so that it is wasted and the reaction is not a fair one.
As the way to measure respiration in yeast is to measure the volume of CO2 produced, then there are various ways to do this. One is to use an upturned measuring cylinder in a kidney bowl full of water. This is an efficient way to measure it, yet this only measures to an accuracy of 1ml. So it is not accurate enough for the experiment I want to do. Another way is to use a gas syringe. This also measures mainly to just 1ml, yet an upturned burette is 10x more accurate than these two methods. An upturned burette in water can measure to an accuracy of 0.1ml, which is accurate enough for my experiment.
In my experiment, there are certain factors that I need to be aware of. The first is to keep a constant temperature. This not only ensures that it is kept a fair test, yet if the temperature is allowed to go above a certain temperature, then the enzymes will be denatured. The optimum temperature that enzymes usually function well at is around 25'C to 35'C. Kinetic Theory also comes into this in that as more energy is applied to the molecules, then they will become more energetic and move around more. I need to ensure that I keep the temperature at 30'C to ensure that I get an even rate of Carbon Dioxide produced throughout my experiment. I have decided to do this in an electric water bath as this remains at a constant temperature, rather than trying to use a beaker full of water with a Bunsen burner to heat it. This is not efficient, as it cannot keep a constant temperature, therefore it is not accurate. Also this method is not safe as Ethanol can be ignited from the Bunsen burner due to it being flammable.
As I am using different concentrations of ethanol, I need to ensure that I collect various results from a variety of ethanol concentrations. I aim to use at least 5 different concentrations of ethanol, so that I can obtain a range of results.
Safety is important in any experiment that is carried out. So, as I am using ethanol, I need to ensure that this is dealt with great care and kept away form any naked flame that are around, due to it being highly flammable. I also need to ensure that it is kept under a lid as it evaporates, and also does not smell too pleasant either. I also need to ensure that all hair is tied back and I wear goggles at all times.
Method:
. Make up at least 5 concentrations of chosen ethanol concentration and place into test tubes. This is done by adding a volume of ethanol and making it up with water. E.g. for a 40% solution, 4ml ethanol can be added along with 6ml of distilled water.
2. Weigh out yeast and glucose and make up solutions as required. This is done by adding required weight of yeast/sugar and adding a volume of distilled water t it.
3. Heat the yeast solution in the water bath at 30'C, so that it is ready for respiration. Whilst this is heating, set up the apparatus for collecting gas as shown in the diagram. (Appendix 5)
4. Add required amount of Yeast solution to the conical flask, and record the temperature. If needed place in the water bath to increase the temperature as a constant temperature is needed.
5. Next add required amount of sugar solution and immediately after add the required amount of ethanol (if no alcohol is added add 10ml of distilled water instead). Place bung connecting the conical flask to the apparatus. Start the timer.
6. Record how much CO2 is produced within a 1 minute time period.
7. Repeat this method about 4/5 times for each concentration until all required concentrations of alcohol have readings.
Equipment
The equipment I will need to carry out this experiment is as follows:
o Conical Flask
o Electric Water Bath
o Bung with tubing
o Burette
o Beakers
o Electronic Scales
o Kidney bowl
o Stopclock
Preliminary Experiments
To ensure that I use the best volumes and amounts of all my "ingredients" so that I can get the best results possible, I need to do some preliminary experiments. I will carry these out in the same way that I intend to carry out my experiment to ensure a fair test all the way through. I hope to achieve a suitable volume of yeast solution and sugar solution in order to achieve a good volume of Carbon Dioxide being produced.
This is the first set of results that I obtained.
Yeast Concentration
Volume used
Sugar Concentration
Volume used
Time (mins)
Volume of CO2 produced (cm3)
5g/100ml water
50ml
5g/100ml water
50ml
minute amount
I think that this produced a small amount of Carbon Dioxide due to the volume of sugar solution that I used. This caused an osmotic effect on the yeast cell, causing it to respire poorly, due to the yeast cells dying.
I then tried varying both the volumes of yeast solution and the sugar solution. These are my results:
Yeast Concentration
Volume used
Sugar Concentration
Volume used
Time (mins)
Volume of CO2 produced (cm3)
20g/100ml
30ml
5g/100ml
5ml
3.2
20g/100ml
30ml
20g/100ml
5ml
3.5
20g/100ml
30ml
20g/100ml
5ml
2
7.5
20g/100ml
0ml
20g/100ml
0ml
3.4
20g/100ml
50ml
20g/100ml
0ml
3.5
20g/100ml
50ml
20g/100ml
5ml
1.3
20g/100ml
50ml
20g/100ml
5ml
2
6.4
20g/100ml
50ml
20g/100ml
0ml
1.2
As you can see by varying the volumes (and times) this produced a variation of results. It is hard to get an optimum amount of sugar concentration to use, as if too little is used then it is not concentrated enough, and vice versa. I then decided to use a different form of sugar, sucrose to see what results I obtained.
Yeast Concentration
Volume used
Sugar Concentration
Volume used
Time (mins)
Volume of CO2 produced (cm3)
20g/100ml
50ml
20g/100ml
0ml
2
7.2
I only chose to do 1 run of this, as the Carbon Dioxide took too long to form, as the sucrose did not react with the yeast as quickly as glucose did.
I then tried to increase the sugar concentration, so the overall concentration of the sugar solution would be greater. Yet keeping the volume used fairly small, so it did not have too great an effect. These are my final set of preliminary results for Glucose;
Yeast Concentration
Volume used
Sugar Concentration
Volume used
Time (mins)
Volume of CO2 produced (cm3)
20g/100ml
50ml
40g/100ml
0ml
1.2
20g/100ml
25ml
40g/100ml
0ml
0.5
From all of these trials, I have found a suitable set of "ingredients" to use. I have highlighted these and I have decided that I will use these in my experiment as these gave the best results. Using 20g/100ml of sugar in water gives a concentrated solution and then by using 10ml of this in my experiment keeps it fairly concentrated. This then gives a reasonable amount of Carbon Dioxide being given off in 1 minute. So I have decided to use a sugar solution of 20g glucose/100ml water, and use 10ml of it. Also 20g of yeast/ 100ml of water and use 50ml of it.
I then decided to try my yeast and sugar solution and add the alcohol, as I would do in my experiment. I tried this by using 4 different concentrations of alcohol, 0%, 100%, 50% and 10%. These are my results: in order of 0%, 100%, 50%, and 10% Ethanol concentration.
Yeast Concentration
Volume used
Sugar Concentration
Volume used
Time (mins)
Volume of CO2 produced (cm3)
% Of Ethanol solution
20g/100ml
50ml
20g/100ml
0ml
8.2
0%
20g/100ml
50ml
20g/100ml
0ml
3.9
00%
20g/100ml
50ml
20g/100ml
0ml
7.8
50%
20g/100ml
50ml
20g/100ml
0ml
20.4
0%
As I expected, with low concentrations of Ethanol, this gives a high volume of Carbon Dioxide being produced, yet as the ethanol concentration increases, the volume of Carbon dioxide given off decreases.
I will now carry out my actual experiment and record my results in a table and plot a graph of the averages.
My table is shown in Appendix 2, with the graph as shown below.
Analysis
I have now obtained my results, as shown in appendix 2 and I have plotted a graph of the averages from my results. On both my graph and table of results, there is a clear significance in how the rate of respiration reacts in relation to the concentration of ethanol added. It shows that as the concentration of ethanol increases, the volumes of Carbon dioxide produced decreases. Yet, this occurs after a concentration of 50/60% of ethanol is added. Up until this amount, it slightly increases. This could be due to a number of factors, Yeast concentration, sugar concentration, freshness of yeast, how well the sugar dissolved in the solution. Then, up to 100% ethanol concentration, it then decreases again. As there was a maximum at 50/60%, then this could be that the enzymes were denatured at the concentrations up to and after 50/60%. Another reason for the variation, could be that all of the substrate has reacted, therefore there is no more substrate to react. Also, as the ethanol acts as an inhibitor, it prevents the yeast from respiring. As the ethanol becomes more and more concentrated, this causes more of the yeast's active site to become inhibited. And so this gave low volumes of Carbon Dioxide being produced. On some of my results, particularly at 50%, the yeast respired more initially, and slowed down towards the 1-minute mark. I found that the other concentrations done the opposite to this and reacted more at the end of the 1-minute time period.
Evaluation
Overall I am quite pleased and satisfied with how my experiment went. I achieved a good set of results that were varied in the way I had hoped for and mainly supported my predictions. I had many positive aspects of both my experiment and my results. My experiment worked very well in that everything went the way it should have and that I achieved what I wanted - bubbles of Carbon Dioxide. I felt that the equipment I had chosen was well suited to my experiment. The pieces I chose were accurate and gave a clear reading of what I wanted. I chose the burette as it was clear as to what the readings shown, and is accurate to 0.1ml. using a burette made it a lot easier to read the digits as it could be set at eye level to read. The results I obtained fitted with my predictions in certain ways, yet not all ways. As I repeated each experiment three times, each of these was quite close to each other, yet overall the general trend was not as I had hoped. I think that this was mainly due to the fact that the yeast I used was not fresh every day I carried out my experiment. Due to the amount of results I had to take, and the number of people carrying out the experiments, the availability of fresh yeast was not always there. Time was also a large limiting factor in my experiment. I found it hard to get all of the concentrations I had hoped for as to set up and carry out the experiment took time, as well as cleaning and washing apparatus and making up fresh ingredients each time. So, I was only able to achieve the concentrations that I have in my results. Space within my working area also prevented me from carrying out my experiment as accurately as I should. So my results could be more accurate.
The results I obtained anyway I think support my hypothesis well, yet not extremely well. I predicted that as the ethanol concentration increased then the volume of Carbon Dioxide produced would decrease. This occurred yet not as consistently as I would have hoped it to. I think that this is due to the limiting factors in my experiment that I have just discussed.
If I were to carry out this experiment again then the major element I would change would be the time scale. I would allow myself a reasonable time period, yet enough to ensure that fresh yeast was used every day, and maybe have a one-day limit for this. Also I would probably change how I dissolved my sugar in the water. The way that I done it was accurate, yet not extremely accurate as not all the sugar dissolved. I could possible heat the solution to allow the sugar to dissolve, then cool to room temperature to ensure a fair test. Also, I could extend my preliminary experiments more and vary the amount of sugar I used more. I also think that I could use a variety of alcohols, for example propanol or butanol, and see whether they have a different effect on the yeast, or that they change the way it respires. I think that that would be all I would change as overall my experiment and the equipment was well used.
Biology Coursework A2
Nikki Jagait 13T
