An investigation into the effect of different sugars on respiration in yeast.

http://web.indstate.edu/thcme/mwking/non-glucose-sugar-metabolism.html

Glucose, fructose and galactose are all monosaccharides. Monosaccharides are the simplest form of carbohydrates and consist of one sugar. They cannot be hydrolysed into smaller carbohydrate units and they share the chemical formula of C6H12O6. All of these sugars have six carbon atoms, resulting in them being hexoses.

Sucrose

www.chemheritage.org/.../ glossary/sucrose.htm

Sucrose is a crystalline disaccharide consisting of fructose and glucose, and has the chemical formula of C12H22O11. Unlike other disaccharides, sucrose is not a reducing sugar. A reducing sugar is a sugar which has the capability of reducing various inorganic ions. The disaccharide is held together by a glycosidic bond between carbon 1 of glucose and carbon 2 of fructose. It is most commonly known as normal table sugar and is less reactive than glucose.

Maltose

 http://www.mansfield.ohio-state.edu/~sabedon/biol1025.htm#disaccharide

Maltose is a disaccharide and a reducing sugar. The chemical formula for maltose is C12H22O11. It is formed by two glucose molecules which are joined together at carbons 1 and 4 by a glycosidic bond.

Lactose

http://sci-toys.com/ingredients/lactose.html

Lactose is a disaccharide that naturally occurs in both human and cow's milk. Lactose is a reducing sugar. It is formed from glucose and galactose which are linked by a glycosidic bond between carbon one of galactose and carbon four of glucose. The molecular formula of Lactose is C12H22O11.

Sucrose, maltose and lactose are all disaccharide sugars. Disaccharides are made up of two monosaccharides which have joined together by a condensation reaction into a very short polymer. Their all share the chemical formula of C12H22O11.

Enzymes

Enzymes are biological catalysts. A catalyst speeds up a chemical reaction but remains unchanged at the end. Enzymes make it possible for chemical reactions to occur inside cells when there is no large activation energy input. They do this by lowering the activation energy required for a particular reaction. The most important part of this mechanism is the binding of the enzyme with the substrate. After binding to the enzyme, the substrate is converted to a product or products. The site on the enzyme at which the substrate binds is referred to as the active site. Each enzyme is highly specific for both the reaction and the substrate. For the yeast to be able to respire each different sugar, it needs to contain the specific enzymes for each sugar otherwise it will not be able to break the sugar down (see table below).

http://resources.ed.gov.hk/biology/english/images/virtual_lab/enzyme.gif

The enzyme complex found in yeast extract that is used to catalyze glycolysis and other reactions in alcoholic fermentation is called zymase. These enzymes break down the sugar into ethanol and carbon dioxide. The optimum temperature for the enzymes in yeast is between 20-30°C. If the temperature is above 50˚C then the enzymes will become denatured.

If the substrate concentration is increased, the rate of reaction will increase up to a point, when the rate becomes constant even if more substrate is added. When this happens, the enzyme is described as saturated with substrate.

The Collision Theory

"For a reaction to occur, particles have to collide with each other. Only a small percent result in a reaction. This is due to the energy barrier to overcome. Only particles with enough energy to overcome the barrier will react after colliding. The minimum energy that a particle must have to overcome the barrier is called the activation energy. The size of this activation energy is different for different reactions. If the frequency of collisions is increased the rate of reaction will increase. However the percent of successful collisions remains the same. An increase in the frequency of collisions can be achieved by increasing the concentration, pressure, or surface area."

Therefore, a higher concentration of yeast than sugar should result in a faster rate of respiration. More reactions should occur and more carbon dioxide should be produced. If I increase the concentration of sugar too much, the rate of respiration could decrease as the sugar particles could get in the way of each other. Heating the solution should also cause the rate of reaction to increase as it will cause the sugar and yeast particles to move around and due to the lack of space, collide more.

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Preliminary work

Before the actual experiment, I carried out some preliminary tests. These were to determine firstly, the most effective ratio of yeast: sugar and secondly, the optimum temperature to use in the experiment. This was so that in the real experiment, I would gain the most accurate results from all the different sugars and get a more detailed picture of how different sugars affect the respiration of yeast. For example, if there was too much yeast, not enough sugar or the temperature was too low, the results would be a far less accurate than if the optimum temperature and ratio were used. For all the preliminary tests, sucrose was used because it is a disaccharide that is composed of two monosaccharides, alpha glucose and fructose.

After I had heated the yeast and sugar to the same temperature and filled the burette with 50cm3 of water, I used a graduated pipette to put the different ratios of yeast and sugar into a boiling tube. As soon as I had done this, I put a bung in the boiling tube which was connected to a rubber tube. The rubber tube went through a beehive shelf into the burette which was placed in a water bath to prevent air entering it. This experiment was left for 10 minutes with the position of the water in the burette noted every minute.

Preliminary 1 - 30˚C – no shaking

This was to determine which ratio to use in the real experiment.

Preliminary 1 – Method

• Prepare the temperature of the water bath to 30-32˚C and place the yeast and sugar in the water to ensure they are both at the same temperature
• Fill a plastic container ¾ full with water
• Place a beehive shelf in the plastic tub
• Fill a burette with water (up to 50cm3) and whilst holding your finger over the end to avoid spilling water, place this inside the beehive shelf in the water.
• Using a clamp and clamp stand, position the burette in an upright position and tighten the clamp. Make sure it cannot move.
• Take a stretch of rubber tubing attached to a bung, and place it through the beehive shelf, and into the burette.
• Using a graduated pipette, take 9cm3 of yeast and place in a boiling tube. Keep in the water bath.
• Using a clean pipette, take 1cm3 of the sugar solution and place in the boiling tube with the yeast.
• As soon as the solution has been mixed, place the bung in the boiling tube and start the stop clock.
• Keep the solution in the water bath and record the level of water in the burette every minute for ten minutes.
• Repeat for each of the specified ratios.

Independent Variable

• Amount of Carbon Dioxide produced

Key Variables

• Ratio of yeast: sugar

Control Variables

• Sugar used – Sucrose
• Temperature of water bath (30-32˚C)
• Total amount of solution (10cm3)
• Time respiration takes place (10 minutes)

Table of results

        Time in minutes

The data shows how much the water moved down the burette (cm3) every minute.

The results for the first preliminary test were very disappointing as you can see from the above table. I concluded that this was because there was not enough yeast and sugar for respiration to occur, and that the temperature was too low for the enzymes to work efficiently.

As can also be seen from the above table, the most amount of respiration was taking place between 6:4 and 4:6. For the next experiment the amounts of yeast and sugar used were doubled to get 12:8, 10:10 and 8:12. The temperature was also increased up to 40˚C to see if it made a difference to the results. The experiment was also carried out on the ratios 15:5 and 5:15 to provide a greater range of results.

We also decided to keep the boiling tube in the water bath (to ensure that the temperature stays constant) and to constantly shake the boiling tube to cause more collisions between the yeast and the sugar.

Preliminary 2 - 40˚C - shaking

Preliminary 2 – method

• Set up experiment as in Preliminary 1 but with the temperature of the water bath 40-42˚C instead.
• After placing the bung in the boiling tube and starting the stop clock, place the boiling tube in the water bath.
• Constantly shake (not too vigorously) the boiling tube to ensure that the yeast does not separate. To make it a fair test, the same person should shake it each time.
• Record the level on the burette every minute for 10 minutes.
• Repeat for all the specified ratios.

Independent Variable

• Amount of carbon dioxide produced (cm3)

Key Variables

• Ratio of yeast: sugar

Control Variables

• Sugar used – Sucrose
• Temperature of the water bath (40-42˚C)
• Total amount of solution (20cm3)
• Time respiration takes place (10 minutes)

                              Time (minutes)

From these results, the ratio 15:5 was the most effective. This can be seen from the distance the water travelled down the burette. The results go steadily down as the ratio of yeast: sugar changes. A couple more experiments using the ratios 17:3 and 16:4 were done, to ensure that 15:5 was the best ratio to use in the actual experiment.

Neither of these ratios are as efficient as the 15:5. From all of the results using different ratios, the ratio 15:5 of yeast: sugar seems to be the best ratio for the yeast to respire to its full potential. This is because it produced the most amount of gas when it was respiring.

Preliminary 3 – No shaking

This was to determine which temperature to use in the real experiment.

During this experiment I decided to keep the boiling tube in the water bath, but not to shake it. I had planned that the boiling tube would be continually agitated, however I then identified that there was no equipment which would deliver an equal level of agitation throughout the experiment. Consequently, this would produce too great a variable so I decided not to include any agitation during Preliminary 3. For the actual experiment I will use a magnetic stirrer to ensure an even amount of agitation for each sugar.

Preliminary 3 – Method

• Set up experiment as in Preliminary 1 and 2 with the temperature of the water bath at 30-32˚C.
• Use the ratio of 15:5 (yeast: sugar) throughout.
• After placing the bung in the boiling tube and starting the stop clock, place the boiling tube in the water bath.
• Do not shake.
• Record the level on the burette after every minute for 10 minutes.
• Repeat for each of the specified temperatures.

Independent Variable

• Amount of carbon dioxide produced (cm3)

Key variables

• Temperature of solution

Control variables

• Sugar used – Sucrose
• Total amount of solution (20cm3)
• Time respiration takes place (10 minutes)

The results from Preliminary 3 showed that the most effective temperature was around 30˚C and the efficiency of the sugar decreased as the temperature increased. This correlates with my research on yeast (page 2) where I found that the optimum temperature for the enzymes in yeast is between 20° to 30° C

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Prediction

From my background research and my preliminary results, I have found that, to a certain extent, the more sugar, the higher the rate of respiration. When the sugar concentration is increased, the rate of reaction will increase up to a point, when the rate becomes constant even if more substrate is added. This is because the enzymes in the yeast are saturated with substrate. I have found that out of the six sugars I am using, three are monosaccharides - glucose, fructose and galactose, and three are disaccharides – sucrose, maltose and lactose. From the research I have found about the sugars, I predict the fastest rates of reaction will come from either glucose or fructose. This is because the first stage of glycolysis (see diagram, page 1) begins with glucose, which breaks down into glucose 6-phosphate, which then breaks down into fructose 6-phosphate. Fructose would miss the first two stages of glycolysis, and break down straight into fructose 6-phosphate However this is a later stage of glycolysis and the sugar which will have a faster rate of respiration will depend on which enzymes are in more abundance from the yeast, as there are specific enzymes for each monosaccharide. I predict that the third fastest rate of reaction will come from either sucrose or maltose, as sucrose breaks down into glucose and fructose, and maltose breaks down into two glucose molecules. Both glucose and fructose have fast rates of reaction but it will again depend on which enzymes are more abundant in the yeast. Sucrose and maltose will have a slower rate than fructose and glucose because it takes time to hydrolyse the disaccharide molecule into two monosaccharides. The fifth fastest sugar will be lactose, because lactose breaks down into galactose and glucose. Out of these two monosaccharides, the only one the yeast can use to respire is glucose. This is because yeast does not contain any enzymes to break down galactose, as it is generally only found in milk. The sugar with the lowest rate of reaction (if there is one at all) is galactose. It is very unlikely that there are any enzymes that can break down galactose present in the yeast.

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Variables

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Apparatus

Method

1. Prepare the temperature of the water bath to 30-32˚C and place the yeast and sugar in the water to ensure they are both at the same temperature

2. Fill a plastic container ¾ full with water

3. Place a beehive shelf in the plastic tub

4. Fill a burette with water (up to 50cm3) and whilst holding your finger over the end to avoid any spilling of water, place this inside the beehive shelf in the water.

5. Using a clamp and clamp stand, position the burette in an upright position and tighten the clamp. Make sure it cannot move.

6. Take a stretch of rubber tubing attached to a bung, and place it through the beehive shelf, and into the burette.

7. Using a graduated pipette, take 15cm3 of yeast and place in a boiling tube. Keep in the water bath.

8. Place the magnet from the stirrer into the yeast solution.

9. Using a clean pipette, take 5cm3 of the sugar solution and place in the boiling tube with the yeast and magnet.

10. As soon as the solution has been mixed, place the bung in the boiling tube and start the stop clock.

11. Keep the solution in the water bath next to the magnetic mixer and record the level of water in the burette every minute for ten minutes.

12. Repeat for each of the different sugars.

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Risk Assessment

Whilst the experiment is underway, there are various safety aspects that need to be considered.

• Take care whilst using equipment made of glass as they can easily be broken and cause cuts or wounds. If you do cut yourself, rinse the cut immediately with warm running water for 10 minutes.
• If any spillages occur, clean up immediately to prevent slipping. Also, if anything is spilt near electrical appliances, turn of the electricity and don’t operate anything electrical with wet hands.
• Safety goggles must be worn whilst measuring the yeast and sugar solutions. Some people are allergic to yeast so if it comes into contact with skin, rinse immediately.
• The experiment should take place in an area clear from unnecessary objects like coats or bags
• If any of the solutions are swallowed, rinse out mouth with water for 10 minutes.

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Bibliography

Book references

Jones, Fosbery and Taylor (2000) Biology 2, Cambridge University Press.

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