Null hypothesis:The type of sugar will have no effect on the rate of respiration of yeast. Variables

Background research

Yeast is a single celled fungus (commonly used to ferment alcohol). Yeast is a facultative anaerobe. This means that it can release energy from food through anaerobic or aerobic respiration.

Yeast will respire aerobically if oxygen is present use oxygen if it is present, If it does so; it will break down its respiratory substrate to release energy for the formation of ATP. The products of this process are carbon dioxide and water. The equation for aerobic breakdown of glucose: C6H12O6 + 6O2 6 CO2 + 6 H2O + energy

Yeast will use oxygen if it is present, and break down sugars all the way to CO2 and H2O. In the absence of oxygen, yeast will switch to an alternative pathway that does not require oxygen this will produce ethanol & CO2. The anaerobic pathway is shown below:

Fermentation is the process by which sugar is converted to alcohol using yeast, this is the anaerobic breakdown of a nutrient molecule like glucose. This process does no release all energy in the molecule but allows glycolysis to take place; the pathway of glycolysis is shown below:

During respiration, high energy carbon bonds are broken. Lower energy bonds are formed and the difference is released and used to make ATP from ADP.

Glycolysis splits glucose in to 2 molecules of 3carbon-triose phosphate, hydrogen is then released and ATP made. From this 2 molecules of pyruvate are made and in total 4 molecules of ATP. However the net gain of ATP is only 2 molecules as ATP is put in to the process at the beginning to fuel it. In the absence of oxygen glucose is broken down in to pyruvic acid but if oxygen is needed to act as the final hydrogen acceptor to break down the sugar further. Pyruvic acin then enters the next stages of respiration, which are:

The Krebs cycle – whereby hydrogen id removes from the pyruvic acid
The electron transport chain – The hydrogen is used to make ATP

The outcome of this whole process is 6ATP molecules

Metabolism and the breakdown of substances can be measured using the respiration quotient. The respiratory quotient (RQ) measures the ratio of the volume of carbon dioxide (Vc) produced by an organism to the volume of oxygen consumed (Vo). This is represented by the following equation: Vc / Vo

Enzymes – these are a major part of respiration therefore may affect the rate of respiration.

Enzymes speed up or catalyse biological reactions within the human body for example hydrolysis or condensation reactions. During a reaction the substrate will bind to the active site of an enzyme this will only occur if it is given some extra energy referred to as activation energy, in order to start the reaction. In order for a reaction to take place the substrate needs to collide with the active site, the speed at which the enzymes collide affects the rate of reaction as more collisions means a faster reaction, this can be done by heating. The core body temperature of humans is 37’C which is generally higher than hat of the surrounding environment this is the temperature at which reactions take place at a sufficient rate.

Several variables affect the rate of enzyme reaction, the table below is based on the fact that one variable is altered at a time while the others remain constant

Enzymes inhibitors

An inhibitor will have a complementary shape to that of the active site, if it binds to the enzyme it will inhibit the binding of the correct substrate and therefore inhibiting the enzymes function.

The inhibitor can bind either permanently or for a short period of time. Binding for only a short period of time means the period of inhibition is reversible, If the inhibitor binds to the active site rather than anywhere else on the enzyme becomes competitive inhibition with the correct substrate fighting for access to the active site. Permanent binding becomes non competitive as the substrate will never gain access to the active site and is therefore irreversible.

Sugars

Monosaccharides are the simplest form of carbohydrate made up of one sugar which dissolves easily in water (reducing sugars). Monosaccharides are divides in to three groups depending on their carbon atom as follows

Triose – 3C

Pentose – 5C

Hexose – 6C

The human body uses monosaccharides for two man reasons, Firstly they are used to produce energy by breaking the many hydrogen bonds present to produce ATP. Secondly they are used as building blocks for larger molecules within the human body like polysaccharides which are many monosaccharides joined and can form starch and glycogen

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Sugars

Triose – 3C

Pentose – 5C

Hexose – 6C

Disaccharides are two monosaccharides joined together during condensation (reduction of water) by a glycosidic bond as shown in the diagram below. The reverse of this process is hydrolysis (addition of water) where by the glycosidic bonds are broken, this occurs in the stomach during digestion of disaccharide sugars.

Glucose is a monosaccharide and the most common respiratory substrate in the human body, through glycolysis and other reactions it yields much of the body’s energy. It is also vital for the production of protein and lipids and its polymers form starch, cellulose and glycogen.

Fructose is one if the simplest sugars and one of the three most commonly used in the human body. Lactose is produced when sucrose is digested in the human body and is carried in the blood stream by glucose.

Galactose is found in dairy products but also synthesised by the body to form part of glycolipids and glycoproteins. Galactose is not particularly water soluble.

Maltose is the least common disaccharide; it is a reducing sugar and is forms on the partial hydrolysis of starch. It is formed by two glucose units.

Sucrose is the most common disaccharide and usually known as table sugar , it is easily digested in the stomach and absorbed in to the blood stream where it provides a rapid burst or energy. It is formed with glucose and fructose joined with a glycosidic bond.

Lactose is a disaccharide making up a large portion of milk, it is formed by galactose and glucose.

Hypothesis:

Based on my scientific knowledge I am going to hypothesise that the monosaccharides a group will respire (breakdown) faster then the disaccharides, this is because there are fewer bonds to break and smaller subunits within each molecule. However with in the mono and disaccharide groups I am also going to predict which of the sugars will break down faster. Here is the order of reactivity I predict:

Glucose – most commonly used in the human body therefore it would breakdown relatively easy however this may not be true for yeast as yeast id not found in the human body. It also has the fewest bonds to break and is the most soluble of all the respiratory substrates I will be using. It is also used at the start of respiration therefore is a main starting point In respiration

Fructose – simplest sugar therefore would breakdown easier than the others yet with more bonds than glucose.

Galactose – is not very water soluble therefore would not respire with the ease of the previous sugars

From this order I scan predict the order of the disaccharides because of their constituent sugars:

Maltose – as it is made from two glucose molecules therefore has a very similar structure to glucose.

Sucrose – as it is made from fructose and glucose

Lactose – as it is made from glucose and galactose

Null hypothesis:

The type of sugar will have no effect on the rate of respiration of yeast.

Variables

Safety procedure

Apparatus

Procedure

The equipment will be set up as it is below:

The water bath needs to be set at the correct temperature and switched on. The bowl needs to be filled with water of any temperature as this makes no difference and the burette must be filled and turned upside down in the bowl and held in place with a clamp stand.

I will weigh out the yeast and add this to a conical flask and then add the water taking this from the water bath to ensure it is at the right temperature. This means the solution does not need to be left in the water bath for extra time to reach that of the bath. The pre weighed sugar will then be added and swirled for a few seconds. The position of the meniscus in the burette needs to be recorded. The conical flask containing my solution will then be introduced to the water bath and the rubber bung added; as this is done I will start the stopwatch. After five minutes I will remove the rubber bung and look at the burette to see how much carbon dioxide is given off by the changed position of the meniscus.

Preliminary study

The object of the preliminary study is to find out the bets possible way to carry out the experiment and minimise external factors that will affect my results. My table of variables showed me various questions I needed to answer before I can carry out my actual experiment, the questions are as follows:

How much yeast?
How much water?
How much sugar?
How long?

I attempted to answer each of these questions individually looking at the optimum result from each mini experiment.

I used the standard equipment I will use in my actual study and the same procedure, I have decided to use glucose only in my preliminary study as it the most commonly used in the human body and is a constituent of al of the disaccharides I will be using.

How much yeast?

To work this out I chose a random quantity of water, sugar and time which remained constant, I then chose varied quantities of yeast. For each different quantity of yeast I added 10ml water, 1g sugar and watched the reaction for 5 minutes. My results are as follows:

From this I can see that 1.5g yeast gave of the most CO2 therefore this must be the optimum quantity and will be carried through to my actual experiment.

How much water?

To decide this I kept my optimum yeast quantity from first experiment and kept my constant of 1g sugar and times 5 minutes. I varied my water quantity and here are the results:

This shows that 15ml water is the optimum quantity therefore I will us this is my actual experiment

How much sugar?

For this I used 15ml water, 1.5g yeast as these are the optimum amounts and times for 5 minutes, I varied the quantity of sugar I used to find an optimum quantity.

This shows me that 1g sugar is the optimum quantity which will be used in my actual study.

How long?

To do this I used the optimum quantities from the previous 3 studies and placed the solution n the water bath, I then watched the reaction until it slowed and stopped. This occurs at around 6 minutes, however I decided to time for 8 minutes in my actual study as I used glucose in this preliminary study but did not predict glucose to be the more reactive therefore I allowed time for a loner reaction to take place.

Changes to procedure resulting from the preliminary study

To make it a fair test I am going to time the swirling of the solution in the conical flask for 10 seconds to make sure everything is mixed prior to putting it in to the water bath this will mean each test is mixed the same. Also I am going to swirl the solution in the water bath for 1 minute at the beginning of the 8 minutes and for the last minute to help the respiration occur faster yet still making it a fair test. Furthermore I am going to repeat each sugar so gain an average and hopefully better identify any anomalous results. Lastly as in my background knowledge I identified the affect pH has on enzyme reaction I will record the pH of the solutions to see if this has an effect of reaction rate.

Method

Set up equipment as above
weigh out yeast, sugar and measure water
add yeast, water and sugar to a clean conical flask and swirl for 10 seconds
record position of meniscus in burette
add solution to water bath, put in bung and start timer
swirl solution in water bath for 1 minute
wait and observe for 6 minutes
Swirl again for last minute
remove from water bath and record new position of meniscus
calculate CO2 given off using this method

Initial volume- final volume = final result

Test pH
Repeat again for same sugar
repeat for all other sugars
repeat again with no sugar as a control group
Repeat finally with no yeast as another control group

Results

Glucose – PH 5.3

Fructose – PH 4.58

Sucrose – PH 4.81

Maltose – 4.83

Lactose – 5.67

Galactose – 5.14

The two control groups did not respire at all show therefore have not been included in the table below

Discussion

Explanation of findings

My hypothesis predicted that glucose would give off the most carbon dioxide in the given time due to the ease with which it is broken down. However my findings show that this is not the case. Glucose in fact gave off less CO2 than 3 of the other sugars, which were fructose, sucrose &maltose. Looking at the graph for glucose I can see that both experiments were quite consistent as both lines of the graph show a steady increase at the same rate with only a slight variation on the end amount of CO2 produced. However despite the apparent consistency it did not prove they hypothesis to be correct.

In fact Fructose respired the most on average. However there was a largely significant difference between the volume of CO2 collected in the individual experiments. One of the experiments showed a slow steady reaction where as the other showed a much faster reaction not at all in keeping with the rates of any of the other reactions and contradicting my hypothesis.

Galactose also disproved my hypothesis, as it did not fall where I predicted it would in sequence.

Sucrose gave off much more CO2than originally thought reacting at a faster rate than other sugars however it did show a difference between both tests.

Maltose also showed a significant difference between the two tests performed and like the other sugars one reacted as expected and another did not once again disproving my hypothesis.

Lastly lactose, predicted to give off the least CO2 still came near the bottom with a small difference between the experiments and a steadily increasing reaction, which falls more or less in to what I predicted for it.

The experiments were also different for two different reasons, one is the rates of reactions varies i.e. how steep the gradient of the graphs is and the second is the total volume of CO2 produced. These do not necessarily correlate with each other as lactose had a steeper gradient than sucrose so it would be thought it also gave of the most CO2 where as it did not.

Here is my predicted order against the order in my results:

Glucose Fructose

Fructose Sucrose

Galactose Maltose

Maltose Glucose

Sucrose Lactose

Lactose Galactose

These shows that not even one of my predictions were correct and all of the monosaccharides didn’t even respire before the disaccharides. Fructose may have given off the most because it is the simplest sugar but as maltose is made up of two

Glucose molecules there is no way that is should be above glucose in order.

Whilst analyzing the results I came across another factor they seemed to be consistent with rate of reaction. I recorded the pH of each experiment to see if this was a factor in rated of reaction in this case and found that with increasing reactivity there was a decreasing pH. The difference in pH was not large but consistent as from fructose to galactose the pH increased. However this is a correlation relationship showing that there is a relationship between pH and reaction rate it does not show that one affected the other, to prove this further research would be needed.

Limitations and modifications

During my experiment a few limitations were encountered. Many of the limitations came about by the experiment being conducted in a classroom setting with other around doing the same study or similar to myself. Firstly the scales used were very sensitive which although is a good thing, the other students in the room walking past affected the reading while I was weighing my yeast and sugar samples. To correct this in future I would make time when there is no one else in the room to weigh out all of my samples, which would perhaps give me a higher level of accuracy. Furthermore I had to share a water bath due to limited supplies, like myself the student I shared with was swirling her sample throughout her experiment. However we were not doing this at the same time so when she was swirling hers it was disturbing the water causing mine to move about, this may have caused in increase reaction of my solution and therefore meant the my experiment was not a fair test.

Working together with a person you share water bath with may modify this as then both students can make theirs a fair test.

A final limitation was encountered when it was time for me to swirl my experiments, the speed and enthusiasm with which I did this may have varied. A minute can be a long time to perform a repetitive potentially tiring task and it is human nature to slow down once they begin to get bored or tired, this would have affect the rate of reaction. A modification for this may me short bursts of movement at set intervals during the experiment to make it a fairer test.

There is also a question of the accuracy of measuring the CO2 given off by hand, as the measuring cylinder has to be filled up and read correctly then the volume of CO2 needs to be correctly read which leaves room for further human error. This may be decreased if another person verified the readings and may make the results more accurate.

Finally the experiment was only repeated once therefore if two contradictory readings were taken there is no way to know which id correct and which in fact is an anomalous result. A modification for this would be to repeat the experiment twice in the hope of gaining at least two sets of results the same.

If these changes were implemented it would be appropriate for a statistical test such as standard deviation to be used to work out how spread out the results are and therefore how accurate the experiment it. However in the case of this test the results clearly do not match that of the hypothesis and standard deviation would be pointless. If the experiment was repeated and appeared to be more accurate then standard deviation would be a useful test to carry out.

Implications & suggestions for further research

The limitations of this experiment and the poor results mean that this study is not very significant at all. It showed an unpredicted reaction for all sugars and a variety of anomalous results. Further research performed in this area would first have to include a more accurate modification of this experiment with the changes I suggested in limitations and modifications. Also I would suggest testing each sugar 3 time this would give a better idea of what result is correct. In this study on many occasions I was faced with 2 different results and no way of knowing which one is correct, a third result may back up on eof the first two.

After completing this I would research further in to the affect of pH on the respiration of yeast as my study showed it is a factor but does not show in which way.

Bibliography