Factors that affect the respiration of immobilised yeast.
Felix Simpeh
Biology Coursework
Factors that affect the respiration of immobilised yeast
Date Completed: Thursday, 20 March 2003
To start off this experiment I feel that it is suitable to explain the main facts of this study. Below is a section explaining respiration and immobilised yeast in detail.
Like all living organisms, yeast has to make energy, stored as ATP to carry out all cellular functions. To do this they can respire aerobically when there is plenty of oxygen, or anaerobically where oxygen is short, by this, they are called partial anaerobes. This produces less energy, but keeps the yeast alive.
Pyruvic acid has to be broken down in respiration when formed by breaking down of glucose molecules, this can't be done in the same way as it is aerobically when respiring anaerobically which is how the carbon dioxide and ethanol is formed through the zymase. Here is the equation for the aerobic respiration: -
Glucose + oxygen => energy + carbon-dioxide + ethanol
Yeast can also respire without oxygen but less energy will be released. Respiration without oxygen is known as anaerobic respiration. When yeast respires anaerobically it produces alcohol. The reaction has the following equation: -
Glucose => energy + alcohol + carbon-dioxide
Cells, such as yeast are often used in industrial processes. At the end of the process the yeast is often mixed up with the product and cannot be easily separated from it. Immobilization is a method, which traps the yeast cells in a bead, which can be more easily separated from the product. The method also means that it may be possible to reuse the yeast once it has been separated from the product. This method is often used in a continuous flow system.
To observe the respiration of yeast cells, a special blue coloured dye known as Resazurin dye is used. It changes colour when the yeast cells respire. The more respiring yeast there are (or the faster yeast cells are respiring) the faster the colour changes. Likewise the rate of respiration can be measured by the amount of carbon dioxide produced.
FACTORS TABLE
The table below shows all the factors that may affect the respiration of immobilised yeast.
FACTORS
PROPORTIONALITY
WHAT HAPPENS
Temperature
The rate of reaction is directly proportional to the temperature.
Yeast works best at an optimum temperature. Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too. Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its ...
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FACTORS TABLE
The table below shows all the factors that may affect the respiration of immobilised yeast.
FACTORS
PROPORTIONALITY
WHAT HAPPENS
Temperature
The rate of reaction is directly proportional to the temperature.
Yeast works best at an optimum temperature. Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too. Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work.
Amount of beads
The number of the beads is directly proportional to the amount of carbon.
(i.e. As the number of beads are increased more carbon dioxide is produced)
At low enzyme concentration (small amount of beads) there is great competition for the active sites and the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate. Eventually, increasing the amount of beads beyond a certain point has no effect because the substrate concentration becomes the limiting factor.
Concentration
This is to do with the concentration of glucose in the solution being used. The concentration is inversely proportional to the rate of reaction. (a higher concentration means a slower reaction)
At a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low. When more substrate molecules are added, more enzyme-substrate complexes can be formed. As there are more active sites, and the rate of reaction increases. Eventually, increasing the substrate concentration yet further will have no effect. The active sites will be saturated so no more enzyme-substrate complexes can be formed.
PH
As with temperature, enzymes have an optimum pH. If the pH changes much from the optimum, the chemical nature of the amino acids can change. This may result in a change in the bonds and so the tertiary structure may break down. The active site will be disrupted and the enzyme will be denatured
PREDICTIONS
Temperature: I predict that the respiratory rate of reaction will increase as the temperature increases until the reaction reaches an optimum temperature. Above this optimum temperature, the rate of reaction will fall to zero very quickly, as the enzyme denatures.
Concentration: I predict that the yeast's respiratory rate of reaction should increase in speed as the % of the sugar increases. However this may at some point peak and therefore cease to increase in speed, one point of this investigation is to find the effect of concentration on the respiration of immobilised yeast.
Amount of Beads: I predict that as the number of yeast increases the amount of carbon dioxide will be greater in a given time.
METHOD
Using my information on respiration of enzymes, it is clear that one of the products of the reaction is carbon dioxide. Therefore to measure the rate of reaction, I could measure the rate at which carbon dioxide is produced. For this experiment I will need:
* Beads; as the source of yeast
* Glucose solution
* A water bath in which I can heat both enzymes and substrate
* Thermometers to ensure both liquids are at the correct temperature
* Measuring cylinders in order to measure the amount of carbon dioxide produced.
* A timer to enable me to work out the rate at which carbon dioxide is produced
* A basin of water
* Conical flask in which the reaction will take place
* Delivery tube
The First stage is manufacturing the immobilised yeast (the beads) and glucose solution.
Apparatus
* Sodium alginate
* 1.4% calcium chloride
* Distilled water
* 250ml beaker
* 10ml syringe
* Tea strainer.
* Glucose
* Distilled Water
* Glass Rod and ruler [15 centimetres]
Making the Beads
. Mix thoroughly 10cm3 of water with 5g of yeast (Saccharomyces cerevisiae), which already will have been weighed out accurately with a weighing scale
2. Add to that 15cm3 of 3% Sodium Alginate and mix again using a glass rod
3. 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.
4. 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.
5. I will then leave the beads in the Calcium Chloride solution so that they harden.
6. After this I will wash the beads and store them until the experiment.
7. I prepare glucose solutions at 5%, 4%, 3%, 2%, and 1% concentration for the experiments.
Test for Temperature:
To test my prediction I will heat the beads and glucose to a given temperature and allow them to react in the conical flask, starting the timer at the beginning of the reaction. The carbon dioxide given off will pass through the delivery tube and bubble up into the measuring cylinder, which will be set full of water in a basin. I will allow the reaction to continue for a set period of time before using the measuring cylinder to measure the amount of oxygen produced.
FIG. 1.1
The results will then be recorded in a table, and then graphed after the experiment has been conducted at a satisfactory amount of temperatures.
My preliminary experiment suggests that suitable quantities to use would be 20cm3 glucose and 10 beads, timed over a period of two minute during the reaction. I am now able to perform the experiment at a range of temperatures between 0 oC and 80 oC. If the rate of reaction is still above zero beyond 80 oC, I will continue the experiment for higher temperatures. My research suggests however that most enzymes become denatured before 80 oC.
[I shall increase the temperature by 10oC after every test]
Test for Amount of Beads:
I predicted that as the number of beads increases the amount of carbon dioxide would be greater in a given time.
To test this I will mix beads and glucose to a given number of beads and allow them to react in the conical flask, starting the timer at the beginning of the reaction. The carbon dioxide given off will pass through the delivery tube and bubble up into the measuring cylinder, which will be set full of water in a basin.
I will allow the reaction to continue for a set period of time [i.e. two minutes] before using the measuring cylinder to measure the amount of oxygen produced.
My preliminary experiment suggests that, the suitable increasing interval is 20 beads, so I will increase the number of beads by 20 after every test.
Test for Concentration
This is the most complex experiment.
Before starting the test I will prepare about ten solutions of glucose using the method above with each haven a concentration 10M greater than the previous (i.e. from 10M,20M,30M and so on).
To test my prediction that the yeast's respiratory rate of reaction should increase in speed as the % of the sugar increases I will carry the following method.
I will mix the beads with different concentrations of glucose and allow them to react in the conical flask, starting the timer at the beginning of the reaction. The carbon dioxide given off will pass through the delivery tube and bubble up into the measuring cylinder, which will be set full of water in a basin.
I will allow the reaction to continue for two minutes before using the measuring cylinder to measure the amount of oxygen produced.
Like the other tests I will set up my apparatus us in Fig 1.1
RESULTS
[In 2minutes,with glucose solution at conc.:]
Temperature
Amount of Carbon dioxide (cm3)
Test 1
Test 2
Test 3
0
20
30
40
50
70
80
[In 2minutes,at room temperature]
Concentration
Amount of Carbon dioxide (cm3)
Test 1
Test 2
Test 3
[In 2minutes,at room temperature and glucose conc. 5M]
Number of beads
Amount of Carbon dioxide (cm3)
Test 1
Test 2
Test 3
20
40
60
80
00
20
