An experiment to investigate the rate of anaerobic respiration of yeast in various respiratory substrates

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Adeel Ahmed                02/05/2007

Practical Method

Title

“An experiment to investigate the rate of anaerobic respiration of yeast in various respiratory substrates.”

In this experiment, the independent variable is various different respiratory substrates being used (glucose, sucrose, maltose, lactose) and the dependent variable is the rate of respiration (measured by movement of manometer fluid which moves in relation to the amount of carbon dioxide released).

Apparatus

Yeast                                        

Glucose

Sucrose

Maltose

Lactose

pH7 buffer

Top pan balance

Stopwatch

Thermometer

Manometer fluid

Capillary tube

20ml & 1ml syringe

100ml beakers

Distilled water

Water trough

Kettle

Stirring rod

Stopwatch

Spatula

Background information

My investigation will involve analysing how yeast respires in various different substrates: Glucose, Lactose, Maltose and Sucrose. All four of these respiratory substrates are carbohydrates.

Glucose

Glucose is a monosaccharide sugar, which is a 'simple sugar' that have between 3 and 10 carbon atoms per molecule. They are sweet and all soluble in H2O. It has the chemical composition C6H12O6. Glucose is a white crystalline solid but is less sweet then ordinary table sugar. Powdered dry glucose exists mainly in straight chain form. However, when glucose molecules are dissolved in water, two different ring structures are formed. See picture.        Fig 1

These ring structures are more stable in solution, so that, at equilibrium, almost all of the molecules are present as rings, with the straight chain form being a relatively short-lived intermediate. The structures of α-glucose and β-glucose differ only in the position of the ─OH and ─H groups attached to carbon atom number 1.

Lactose

Lactose is a disaccharide which is formed by condensation reactions (where water is removed) between two monosaccarides, glucose and galactose. They are joined together by a glycosidic bond. It consists of galactose and glucose molecules joined by a 1, 4 glycosidic link. 

Fig 2

Lactose is the only common sugar that is of animal origin. Other sugars, such as sucrose and fructose, can be found only in plants. In nature, the only place you can find lactose is in the milk of mammals. Lactose is the principal carbohydrate found in milk, and composes about 2 to 8 percent of milk in all mammals..

Although milk and other dairy products are the only natural sources of lactose, this sugar can also be found in many prepared foods.

Galactose is one of the two simple sugars that make up each molecule of lactose. The other simple sugar in lactose is glucose. Most birds cannot digest the lactose in milk products, because their bodies cannot make the enzyme that is necessary to break lactose down into the two simple sugars.

Sucrose

Sucrose consists of glucose and fructose joined by a 1α-2 glycoside linkage. This is a condensation reaction whereby the net result is to remove a molecule of water. The complementary process, whereby complex molecules can be split into their component parts, is called hydrolysis. Fructose is a monosaccharide that forms a five-sided ring. Together they make sucrose, which is a disaccharide made up of two monosaccharide units joined to form a single molecule.

Fig 3

Maltose

 Maltose is a crystalline disaccharide in which both monosaccharide units are glucose. It has the same empirical formula (C12H22O11) as sucrose and lactose but differs from both in structure. Maltose is produced from starch by hydrolysis in the presence of diastase, an enzyme present in malt. Maltose is hydrolyzed to glucose by maltase, an enzyme present in yeast; the glucose thus formed may be fermented by another enzyme in yeast to produce ethanol. Maltose is important in the brewing of beer. It is an easily digested food.

Fig 4


Yeast

The type of yeast being used in this experiment belongs to the branch of living organisms called Saccharomyces cerevisiae. Yeast is a fungus. Fungi are either unicellular or filamentous. All yeasts are unicellular and reproduce by budding and the parent cell buds off a daughter cell and this process is repeated indefinitely. Yeast feeds by heterotrophic nutrition because they lack chlorophyll and therefore are non-photosynthetic. They can be parasites, saprotrophs or mutulalists. Yeast consists of a chitin cell wall.

Yeast cells are microscopic, one-celled fungi made up mostly of protein which are important for their ability to ferment carbohydrates in various substances. Yeasts are widespread in nature, found in the soil and on plants.

Yeasts are well known for the making of bread and wine.

Yeast has to make energy, stored as ATP to carry out all cellular functions. To do this they can respire both aerobically when there is plenty of oxygen, but where oxygen is short, they respire anaerobically. This produces less energy, but keeps the yeast alive.

In aerobic conditions when the yeast is mixed with sugar or glucose solution, it soon starts to respire. The yeast uses sugar and oxygen dissolved in the water to produce carbon dioxide, water and energy by aerobic respiration. In anaerobic conditions when oxygen is not present, hydrogen cannot be disposed of by forming H2O. The electron transport chain stops working and no further ATP is formed by oxidative phosphorylation. Thus pyruvate is decarboxylated to ethanal, then reduced to ethanol.

Yeasts are able to ferment, which means that they metabolise anaerobically in the presence of an organic compound, such as a sugar or an amino acid, and in the absence of an external electron acceptor. Typical examples of fermentation products are CO2 (leavening of bread), ethanol (beer and wine production) and lactic acid (formation of dairy products). Fermenting organisms gain fewer ATP molecules from each molecule of food they oxidize than aerobically respiring organisms.

Although yeast can survive during anaerobic respiration, it does not grow and multiply as it would during aerobic respiration. Anaerobic respiration releases much less energy than aerobic respiration, only 210KJ compared to 2880KJ. In anaerobic conditions most energy remains locked in the ethanol. One problem is that alcohol is poisonous in large amounts. If the concentration of alcohol gets more than 14% it kills the yeast and the fermentation stops. Thus, if the experiment was carried out in anaerobic conditions the experiment couldn't be carried out for too long.

In my opinion I believe that using Glucose as a respiratory substrate will result in the fastest rate of respiration for the yeast because Glucose is the only monosaccharide out of the four respiratory substrates therefore it will be broken down quicker as there are less bonds to break, which will consequently result in a faster rate of respiration. The other three respiratory substrates are disaccharides, consisting of two monosaccharides (one of which is glucose), so there will be more bonds to break which will take longer, resulting in a slower rate of respiration. It is difficult to tell whether yeast will use lactose as a respiratory substrate as it is known that the type of yeast used in this experiment does not have the enzymes required to metabolise Galactose, which is a constituent sugar of Lactose.

Method

  • Firstly, I will set up the apparatus as shown (see diagram).
  • Next I will create a solution consisting of 1% yeast and 2% respiratory substrate dissolved in 20 ml of pH 7 buffer (refer to 1st bullet point in justification). So, for 20 ml of pH7 buffer 1% yeast will equate to ([20/100]*1) = 0.2g of yeast. Similarly 2% respiratory substrate in 20ml pH7 buffer will equate to ([20/100]*2) = 0.4g respiratory substrate.0.2g yeast and 0.4g respiratory substrate in 20ml pH7 buffer will be used in all repeats for all sugars.
  • As I am going to conduct the experiment under anaerobic conditions (refer to 2nd bullet point in justification) I will use a 20ml syringe to draw up the solution made up of 1% yeast and 2% respiratory substrate and remove any excess oxygen in the syringe by pushing the solution to the very end of the syringe. This will generate almost completely anaerobic conditions for the yeast to respire in.
  • For the purposes of this experiment I will need to keep all the possible confounding variables constant (except for the independent variable). This includes keeping such variables as temperature, time, and pH constant. To keep the temperature constant, I will use a kettle and tap water in order to keep the temperature constant at 30 degrees Celsius (3rd bullet point in justification). To check the temperature remains at 30 degrees Celsius I will use a thermometer to constantly make sure the temperature remains constant and if the temperature drops below 30 degrees Celsius, I will add hot water accordingly to increase the temperature.
  • As mentioned earlier, in order to keep the pH constant I will be dissolving the yeast and respiratory substrate in a solution of pH7 buffer.
  • Before I begin to measure the rate of respiration I will allow the apparatus to equilibrate for approximately 5 minutes. This is to allow internal and external pressure to balance and allow for any expansion. It will also allow the yeast to reach a respiratory rate typical for 30 degrees Celsius.
  • I will allow the yeast to respire in the sugar solution for a maximum of 12 minutes (refer to 4th bullet point in justification) with three minutes intervals at minutes 0, 3, 6, 9, 12 to record my results.
  • In order to generate results from the experiment, I will note down the distance moved along the capillary tube by the manometer fluid from a starting point at each of the 3 minute time intervals. Thus the data collected will be quantitative data (refer to 5th bullet point in justification). The distance moved by the manometer fluid will correspond to the amount of carbon dioxide respired by the yeast in each of the sugar solutions during anaerobic respiration.
  • I will repeat the experiment with each respiratory substrate 10 times to ensure that I have a large enough sample to make fairly accurate and reliable conclusions from. Although 30 sets of repeats for each sugar would provide me with more accurate results from which to generate statistics and relevant conclusions from, I feel that this would be too time consuming and would not be a realistic objective given the time constraints.  

Justification

  • As I mentioned in my method, I will generate a solution consisting of 20ml pH7 buffer, 1% yeast (0.2g) and 2% sugar (0.4g). It is important that all variables, except the independent variable, are kept constant. Therefore, in order to keep the pH constant I must use a buffer which will ensure that pH is the same in all repeats of the experiment. I have decided to use pH7 buffer because the solution will be neutral. It is known that yeast enzymes work at their optimum speed at a neutral pH, i.e. pH7, so using a pH7 buffer solution would ensure the solution will be neutral at the start of the experiment. Most enzymes work in a narrow pH range. pH changes outside this range alter ionic charges of acidic and basic groups of amino acids. The tertiary structure and active sit will therefore be altered, so the substrate cannot bind. Extremes of pH can cause denaturation due to acid hydrolysis of the enzyme, making the enzyme inactive; therefore slowing the rate at which respiratory substrate can be broken down, slowing the rate of respiration. Respiration may completely stop due to the denaturation of all the enzymes associated with breaking down respiratory substrate. I have used a higher proportion of respiratory substrate in comparison to yeast because in order for the yeast to respire at an optimum rate throughout the experiment there must be excess substrate present. The enzymes will only work at their maximum speed if there is enough substrate present. I feel that using 0.2g yeast and 0.4g of respiratory substrate will be sufficient for the yeast to respire at its maximum for the period of 12 minutes.
  • I have decided to carry out my experiment under anaerobic conditions. Although I mentioned earlier that yeast releases much less energy under anaerobic conditions than aerobic conditions, I feel that it would be very difficult to carry out the experiment accurately under aerobic conditions as it would be very difficult to keep oxygen concentrations constant. Therefore anaerobic conditions will be used as I feel this would provide me with more accurate and reliable results from which to analyse my data from and draw relevant conclusions. However, it must be taken into account, that by using anaerobic conditions ethanol is produced and remains locked inside the solution. One problem is that ethanol is poisonous in large amounts. If the concentration of ethanol gets more than 14% it kills the yeast and respiration stops. Thus, if the experiment was carried out in anaerobic conditions the experiment couldn't be carried out for too long.
  • I will keep the temperature constant at 30 degrees Celsius throughout the experiment. I have decided to use a temperature of 30 degrees Celsius because I have found that the optimum temperature of yeast under anaerobic conditions (in fermentation) is approximately 30-40 degrees Celsius. I decided to use a temperature of 30 degrees as I feel this temperature will be easiest to maintain and keep constant over quite a long period of time as room temperature is closest to 30 degrees than 40 degrees. To do this I will use a water trough with water at 30 degrees filled to the top, with the syringe containing the yeast and sugar solution completely immersed in water. It is essential that the syringe is completely immersed in the water as all parts of the solution must be at the same temperature so that temperature will not affect the experimental results and thus cannot act as a confounding variable. Heating the solution to higher temperatures will mean the substrate molecules will gain more kinetic energy, therefore they will move around faster, increasing the likelihood of successful collisions between enzyme and substrate, and formation of enzyme-substrate complexes. Therefore more enzyme-substrate complexes will be formed and so the respiratory substrates will be broken down at a faster rate and the rate of respiration will increase. Respiration needs to occur at a fast enough rate in order for me to efficiently measure calculate the rate of respiration and compare between different substrates.
  • I will record the rate of respiration of the yeast for a maximum of 12 minutes because soon after this time the ethanol concentration in the solution will become too great and will kill the yeast off, thus slowing down the rate of respiration. Therefore to eliminate the threat of this confounding variable, the experiment will only commence for 12 minutes, with readings taken at every 3 minute intervals (0, 3, 6, 9 and 12 minutes) to generate a large enough sample of data to analyse and draw conclusions from.
  • I will take readings at every 3 minute intervals to produce a set of quantitative data to analyse from. I will use quantitative data rather than qualitative data because quantitative data is easier to collect, analyse, and draw conclusions from. Using quantitative results will allow me to use Statistics more effectively and efficiently when analysing my results than qualitative results which will be very difficult to analyse. I feel using Statistics to analyse my data will be a very effective method in the interpretation of my results. The type of Statistics I will be using to analyse and compare the rate of respiration between the different respiratory substrates will be the t-test and standard error and 95% confidence limits test. The t-test compares two sets of data and analyses whether the differences between the sets of data are due to chance alone, or whether they are statistically significant, in which case there must be a biological explanation for the results. A null hypothesis is used which states there will be no significant difference between the sets of data. The standard error and 95% confidence limits test looks at the mean, standard deviation and the standard error for each set of data (each substrate) and determines the spread of the data. The 95% confidence limits can then be worked out using these calculations. Confidence Limits = Mean + or – (1.96 * Standard Error). If the 95% confidence limits do not overlap, there is a 95% chance the four means are different, therefore we would reject the null hypothesis as there will be a significant difference between the volumes of CO2 produced by the yeast in the different respiratory substrates.
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Results and Statistical Analysis

To interpret these results, I will use the Standard Error and 95% confidence limits test. This test involves working out the mean, standard deviation and standard error for each of the four sets of data above and applying the 95% confidence limits to each set of data. A graph is drawn to illustrate the data calculated and if the 95% confidence limits do not overlap, there is a 95% chance ...

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