The purpose of this investigation is to discover whether different respiratory substrates will affect the rate of respiration of yeast

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Aim

The purpose of this investigation is to discover whether different respiratory substrates will affect the rate of respiration of yeast. I will investigate this by measuring the amount of C02 evolved during anaerobic respiration. As C02 is a waste product produced during respiration, measuring the volume of c02 produced will allow me to evaluate which of the sugars are best metabolized during respiration (of yeast).

Background information

Yeast

Yeasts are a form of eukaryotic microorganisms that are used heavily in industry to be used for fermentation of alcohols and baking. Yeast digest the sugars using extracellular enzymes to break down the compounds, thus yeast are known as saprophytes, which the products will be transported into the cell of the yeast by facilitated diffusion to be respired. The yeast synthesises the enzymes required that will be specific to the substrate that they will be digesting. For example, domestic yeast is sold in as a sucrose solution, and the cell membrane of the yeast contains a high concentration of sucrose enzymes, thus the yeast adapts its digestive enzymes to its environments to digest specific sugars. Yeast can respire both through aerobic and anaerobic respiration, depending on the availability of oxygen present within the surrounding area. If oxygen is not present then fermentation occurs which converts sugars i.e. glucose into ethanol and CO2 (Glucose --> Ethanol and CO2), which is the anaerobic process. This is then respired out into surroundings. However if there is oxygen available then the sugar is fully metabolised and produces water and CO2 (glucose + oxygen --> CO2 + water), in this process energy is released for the cell to respire. In both cases of respiration CO2 is produced as a waste product, hence I have chosen it as a measure to calculate the rate of rate of respiration (based on the volume of C02 evolved). When oxygen is provided to a yeast cell then respiration follows in a series of steps, in the experiment this will only occur in small amounts as the supply of oxygen will be limited. The first of the steps of respiration is known as glycolysis.

In glycolysis the glucose/ sugar molecule is split into 2 molecules of pyruvate and thus a net gain of 2 ATP molecules takes place. The process occurs within the cytoplasm of the yeast, and occurs when 2 molecules of ATP are hydrolysed to form ADT + Pi. To get the sugar in a more reactive form 2 phosphate groups are added to it, it is then in the form known as Glyceraldehyde 3-phosphate, this has 6 carbons. This is further split into two molecules of the 3 carbon sugar, when this is split into two molecules of pyruvate an oxidation reaction takes place and electrons are transferred to NAD and thus the energy which is released from this occurrence is used to join 4 moles of ADP + Pi to form ATP. In the end of this reaction we have a net gain of 2 ATP molecules and further a reduced NAD molecule as well, the reduced NAD goes onto the electron transport chain where it is finally converted into ATP.

Enzymes

Enzymes are globular proteins, and with a tertiary structure. This complex structure forms the active site where the amino acids around the active site bind to the substrate molecule to form an enzyme substrate complex. This makes the enzyme specific for one reaction only, as other molecules won't fit into the active site. When a substrate attempts to binds to the active site, the active site changes shape and hence distorts the active site to fit around the molecule. This is referred to as the induced fit mechanism.

Temperature

Enzymes have an optimum temperature, where up to this temperature the rate increases with temperature. The rate of reaction increases because the enzyme and substrate molecules have an increase in kinetic energy hence the frequency of collisions increase. Above the optimum temperature the rate decreases the increase in heat energy breaks the hydrogen bonds holding the tertiary structure of the enzyme causing the structure of the active site to become denatured. Hence the substrate can no longer bind with the active site of the enzyme to form the enzyme substrate complex and hence the reaction is not catalysed. Hence if the respiratory enzymes (such as ATP synthase and NADH Dehydrogenase) will be denatured and respiration will stop.

pH

Enzymes have an optimum pH at which they have the highest rates of reaction. The pH affects the charge of the amino acids at the active site (as amino acids are Zwitter ions) and this causes the structure of the active site to change the substrate can no longer bind to form an enzyme substrate complex. A change in pH may disrupt the optimum pH for the extracellular digestive enzymes that are used by the yeast. Hence the substrates cannot bind to the active site and form an enzyme substrate complex, causing the rate of respiration to decrease.

Facilitated Diffusion.

Facilitated diffusion is the transport of substances across a membrane by a trans-membrane protein molecule. The transport proteins are specific for one molecule (for example a carrier protein for glucose will not diffuse a sucrose molecule). Molecules are transported down a concentration gradient, hence facilitated diffusions a passive diffusion process. This is important to respiration and my investigation because the substrates that I will be using are polar. Hence, before the yeast can start respiring the yeast first must saprophytic digest the substrate, and then absorb the products via facilitated diffusion, as the sugars are polar and cannot diffuse via lipid diffusion due to the phospholipids bi-layer.

There are two types of proteins that are used for facilitated diffusion. Channel Proteins form a water saturated channel in the membrane. This allows polar substances (usually ions) to diffuse across membranes. Carrier Proteins have a binding site for a specific solute and constantly flip between two states so that the site is alternately open to opposite sides of the membrane. The substance will bind on the side where it at a high concentration and be released where it is at a low concentration.

Independent variable

In this investigation, the independent variable is the 5 different types of sugars to be used in the experiment, Glucose, Fructose, Maltose, Lactose and Sucrose.

Dependent variable

The dependant variable will be the volume of c02 produced during yeast respiration.

Null hypothesis

There is no difference between the volumes of CO2 produced by any of these substrates when respired by yeast under the experimental conditions

Hypothesis

There will be a relationship between the substrate used and the volume of CO2 evolved by each substrate when used in respiration by the yeast.

Prediction

I predict that in this experiment that the type pf substrate used will affect the amount of C02 produced, and hence the rate of respiration. This is due to the fact that sugars are needed in order to carry out respiration. Thus the yeast will be able to carry out aerobic and anaerobic respiration. Aerobic respiration will only occur on the surface of the yeast where oxygen is available; however anaerobic respiration will dominate the respiration as the volume of 02 is limited as we go deeper into the solution. Therefore the products of respiration will be ethanol and CO2 and will yield consideberaly less ATP than aerobic respiration.

Referring to my research, I believe that although glucose and fructose are monosaccharides, they can be directly absorbed by the yeast as the yeast does not need to produce any extracellur enzymes to digest the sugars. Hence respiration will occur quicker with these two sugars. However, maltose consists of two glucose molecules, hence when the yeast manufactures maltase to digest the maltose, there will be twice as much glucose available to the yeast than just using glucose or fructose therefore there will be more glucose molecules in the same volume, so more sugars are available for respiration. Because of this it will produce a lot more CO2 in the same amount of time. However, because glucose is a polar molecule it must be transported into the cell membrane of the yeast via facilitated diffusion. If all of the carrier proteins are occupied while transporting the glucose molecule, then the amount of carrier proteins in the yeast membrane can be a limiting factor limiting the respiration.

I predict that lactose will produce the smallest volume of C02 gas because the yeast does not have the lactase gene to synthesis the lactase enzyme used for the hydrolysis of lactose enzymes lactase is not present to catalyse this reaction it will not occur. Hence if the lactose remains undigested, its monomers (glucose and galactose) cannot be used for the metabolic pathways in respiration, hence no C02 will be produced.

Apparatus

* Gas syringe

* Water bath

* Thermometer

* Boiling tube

* Measuring cylinders

* Dry yeast

* Substrates (glucose, fructose etc)

* Stop clock

* Safety goggles

* Rubber stopper

* Distilled water

* Electronic balance

* Beaker

* Universal indicator

* Acidic buffer solution

* Funnel

* Buffer Solution

Method of investigation

Steps

Accuracy

Reason for method

All apparatus that will be used to contain yeast or sugars will be cleansed using distilled water, and to set up water bath at a temperature of 40°C.

Cleaning the apparatus with distilled water will ensure that all the equipment to be used in the experiment is clean and no other substances that may have been in the equipment may interfere with my investigation, causing my results to be invalid.

By cleaning my apparatus I will ensure that there are no substances that may interfere with my experiment (for example there may be a non/competitive inhibitor that may inhibit the enzymes used by yeast to digest the substrates or respiratory enzymes, thus reducing amount of C02 produced)

I will heat the water bath to 40C because I have decided to use this temperature for my experiments.

I will prepare substrate solution by adding 0.8M of sugar to 20cm3 of acidic buffer solution, and pour the solution into a boiling tube.

By using 0.8M of each substrate in solution with 20cm3 of acidic buffer solution, I will be able to maintain a constant concentration for each substrate

By maintaining the same concentration for each substrate, I will ensure that my experiment is fair and that the results are valid.

Weigh 2g of dry yeast using an electronic balance

I will use the same amount of yeast (2g) for each experiment to ensure that my experiment is a fair test.

Keeping the concentration of yeast will ensure that my test is fair, as an increase in yeast concentration will increase the amount of cells carrying out respiration hence the volume of C02 produced will increase.

I will place the 2 boiling tubes (containing substrate solution and yeast in separate tubes) into the water bath (which has been preheated to 40 °C

I will set up apparatus by connecting rubber tubing to gas syringe which I will use to measure C02 volume

After 10 minutes I will check the temperatures of the boiling tubes to ensure that both at the required temperature (40c)

This is to ensure that the temperatures in the boiling tubes are the same

When temperatures of both boiling tubes are equal at 40C, I will add the substrate to the yeast solution, as the yeast is suspended in solution. I will then measure the temperature of the boiling tubes.

I will measure the temperatures of both boiling tubes (of substrate and yeast) together so that the actual temperature when respiration begins can be recorded, and this must recording must be taken quickly

As soon as I carry out the step above, I will connect the rubber bung to the test tube which is connected to the gas syringe and start the stop clock.

Placing the rubber bung on top of the test tube will ensure that minimal amount of C02 is lost while I am connecting the bung.

I will do this so that the gas syringe can be used to measure the amount of c02 produced by yeast respiration

I will take a reading from the gas syringe at 5 minute intervals and record the volume in a table. I will continue to do this for45 minutes at 5 minute intervals for each substrate

So that the volume of C02 produced can be measured accurately.

This will enable me to accurately record the volume of c02 produced, and using a large amount of respiration per each test ensures my results are reliable. I will then be able to plot this data to calculate the rate of respiration and observe any trends in the data.

Controlled Variables

Controlled Variable

How I will control it

Why I will control it

Temperature

By placing the boiling tubes in a water bath that is heated constantly at 40C

The temperature must be controlled because the temperature will affect the rate of respiration of the yeast. If the temperature is changed, for example, too high then this may denature the enzymes used by yeast to digest substrates.

pH

I will control the pH by adding the substrate to a buffer solution

A change in pH may damage the enzymes the yeast may use to digest the substrate (such as maltase) as the pH will interact with the amino acids of the active site, and cause the active site to alter shape.

Mass of yeast

By using a constant amount of yeast for each experiment

As changing the amount of yeast will affect the volume of carbon dioxide gas evolved. This is due to the fact that more mass of yeast will cause If not controlled, then my results will be invalid.

Concentration of substrate

I will use a constant amount of substrate (0.8M) for each experiment

Changing the concentration of substrate will cause more C02 to be produced (or less if concentration is decreased) as the yeast has access to more substrates which it can digest for respiration, hence produce larger volumes of C02. Hence, if I do not keep this variable constant, my investigation will not be fair and my results will be invalid.
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Time

I will check and record the amount of C02 evolved for 5 minute intervals

Type of yeast used

Use only one type of yeast for all reactions

Different types of yeast are used for different purposes (such as yeast bred for making bread and yeast used in fermentation).

This yeast have adapted to create many enzymes regarding to the substrate that they digest most, so one type of yeast may contain many more maltase enzymes while another yeast may contain more sucrase enzymes. Hence this variable must be controlled to ensure a ...

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