The respiration of yeast in different sugar substrates

Essay, Pages 18 (4323 words)

Views

2493

Aim: The aim was to compare the respiration of yeast in different substrates of sugars, i.e. between a monosaccharide (glucose) and a disaccharide (maltose)

Theory: There are three types of Carbohydrates, monosaccharides, disaccharides, and polysaccharides. The two, which I will be looking at, are, monosaccharide (glucose) and the disaccharide (maltose)

Classification and major properties of carbohydrates

GroupPropertiesExamples

Monosaccharides general formula:(CH20)n(n = 3 to 0)Small molecules with low molecular mass; sweet tasting; crystalline; readily soluble in water.Trioses, e.g. glyceraldehyde (C3H603)Hexoses e.g. glucose, fructose (C6H12O6)

Disaccharides general formula:2[(CH2O)n] - H2OSmall molecules with low molecular mass; sweet tasting; crystalline; soluble in water, but less readily than monosaccharides.Sucrose, maltose, lactose, all with the general formula C12H22O11

From the above you can already see the differences in the properties, between the monosaccharide and the disaccharide. The disaccharides are soluble but "less readily than monosaccharides."

Monosaccharides

Monosaccharides contain carbon, hydrogen and oxygen, in the ration 1 : 2 : 1, so their general formula becomes (CH2O)n, where n an be any number between 3 and 9.

All monosaccharides also contain C=O (carbonyl) group and at least two OH (hydroxyl) groups. These two groups of atoms within the molecule are called reactive groups and play important roles in the reactions that take place within the cells.

All the sugars that occur naturally are derived from trioses. All the aldoses are formed from glyceraldehydes and all the ketones from dihydroxyacetone.

Glucose can exist in two different ring forms: one where the hydroxyl group on carbon-1 is below the ring (á-glucose) and one where the hydroxyl group is above the ring (â-glucose).

These are known as á- and â-isomers, and, because the atoms and groups are arranged differently in space, are examples of stereoisomerism. The existence of these two isomers leads to a greater variety in the formation and the properties of polymers. Starch is a polymer of á-glucose and cellulose is a polymer of â-glucose.

Disaccharides

When two monosaccharide molecules undergo a condensation reaction, a disaccharides molecule is formed and a molecule of water is removed. The bond formed between the two monosaccharide residues is a glycosidic bond. Two glucose molecules combine to form a molecule of maltose, with the removal of water.

Maltose is formed by action of amylase (enzyme) on starch during digestion in animals and during germination of seeds.

On hydrolysis, which requires water to be present, disaccharides can be split into their constituent monosaccharides. Within cells, these reactions are catalysed by specific enzymes. In the laboratory, it is possible to hydrolyse disaccharides by heating in solution with acids.

DisaccharideConstituent monosaccharidesType of glycosidic bondOccurrence and importance

MaltoseGlucose1,4Formed by action of amylase (enzyme) on starch during digestion in animals and during germination of seeds.

The above table shows the characteristics of the commonly occurring disaccharide, maltose.

Prediction: I think the respiration of yeast is affected by the size of the sugar. Therefore I think that the rate of CO2 Carbon (Dioxide) produced by the respiration of yeast will be quicker with the glucose, (monosaccharide) than, in comparison with the rate of CO2 produced with the maltose (disaccharide).

Hypothesis: Looking at the theory behind the carbohydrates involved as well as the yeast being used can justify my prediction.

The change in the respiration rate of yeast would occur due to the additional glycosidic bond in the disaccharide.

"One type of glucose is the á-glucose, which has the hydroxyl group below the ring." When compared to the maltose structure, it can be seen that the maltose can be considered to be "two á-glucose units bonded together in condensation." But when the maltose disaccharide is compared with the two separate á-glucose monosaccharides, you can observe that the "maltose has one extra bond than two á-glucose monomers: the glycosidic bond, the bond in between the two glucose subunits in maltose". Breaking this bond would slow the rate of CO2 produced in the respiration of yeast with the disaccharide in comparison to the monosaccharide.

My hypothesis can be justified by the way in which yeast digests with the chosen sugars.

"Yeast is a single-celled fungus, most in the class of Ascomycetes. The Ascomycetes yeasts include the common bread, beer, and wine-producing varieties of Saccharomyces Cereviseae." As yeast is a fungus and possesses no chlorophyll, it would be unable to photosynthesise. Therefore the yeast must obtain its carbohydrates by secreting enzymes onto the surface on which it is growing.

In the case of glucose and maltose, these enzymes are zymase and maltase. Maltose requires the specific maltase enzyme to first hydrolyse the glycosidic bond in between the glucose subunits of maltose before the zymase enzyme can break down the two á-glucose monomers from maltose. However glucose only requires the zymase enzyme to be broken down into ethanol and CO2, maltose. As the glycosidic bond needs to be hydrolysed in maltose to form two monomers of glucose, and additionally two enzymes are needed to break down maltose, is the reason why I think that the rate of CO2 produced by the respiration of yeast will be much slower with the maltose sugar (disaccharide) than with glucose sugar (monosaccharide)

When the sugars have been broken down into smaller molecules (i.e. the maltose disaccharide) by the named enzymes, the yeast will absorb the food via the mycelia surface.

The yeast can digest glucose internally as well as externally, as glucose is a relatively small molecule.

The products of the respiration of yeast are CO2 and ethanol, where the CO2 is collected in the experiment. These products occur due to the fact that yeast respires the sugars that it absorbs anaerobically. The chemical reaction for this glucose is:

C6H12O6 ---> 2C2H5OH + 2CO2

The reaction above is known as fermentation. The six-carbon sugar, glucose is broken down into two molecules of three-carbon organic acid, Pyruvic acid. As O2 (Oxygen) is absent, the Pyruvic acid is reduced to alcohol and CO2 is produced as well.

Apparatus:

·100cm3 active yeast solution (100cm3 of distilled water containing 1g of
dried yeast)

·50cm3 of sugar solution (50cm3 of water with 1g of the chosen sugar)

·250ml beaker

·Conical flask

·Glass tube and bung

·Spatulas

·10ml measuring tube

·Stirring rods

·Top-pan balance

·Water bath

·Thermometer

·Clamp stand with clamp

·Stopwatch

Variables: Some variables in my experiment need to be controlled to produce reliable results. The first variable, which I will touch upon, is the pH of the mixture. This will need to be kept constant i.e. both solutions will need to stay at a neutral pH. If this pH were altered, this would affect the ionisation of side groups in the enzymes amino acid residues and therefore affect the shape of the enzyme. A change in shape would lead to a lack of efficiency in the formation of enzyme substrate complexes. This shows that a change in pH could affect the rate of respiration and production of CO2 by the yeast cells.

Another variable is the concentration of the solutions that are to be mixed. This variable needs to be held constant through the whole experiment as a change in the concentration of sugar (substrate) or yeast (enzymes) will alter the rate of CO2 produced.

Example, if there were an increase in the concentration of yeast cells, there would be more enzymes present and thus more active sites present, therefore increasing the rate of CO2 production. This is an important reason to why the amount of yeast and sugar must be held constant. They will be controlled by using one concentration, for both the monosaccharide and disaccharide sugars (1g in 50cm3 water), and one concentration for yeast (1g of dried yeast in 100cm3 water)

A third variable that needs to be controlled is the temperature of the reaction. This will be kept constant at 40'C by placing the yeast-sugar solution in the water bath for the entirety of the reaction. 40'C is the optimum temperature for the enzymes secreted by the yeast cells, and therefore any change in temperature for glucose or maltose would alter the rate of CO2 production.

The independent variable will be the type of sugar, which is the variable I intend on changing. In the experiment, the sugar type will be altered as I am trying to see what effect a monosaccharide and disaccharide would have on the production of CO2 by yeast cells, and to compare the sets of date produced.

Fair Test: A fair test will be maintained by using the same equipment such as the same measuring cylinder to collect the CO2 to ensure a better reliability of the results, minimising any anomalous results caused.

As mentioned before, I will also make sure the temperatures are maintained to stay at 40'C. The experiment will also be considered fair if the sugars are measured out to the same concentration, so it doesn't have a bearing on the rate of CO2 produced.

Yeast Involvement: As yeast, a living organism is involved, there must be acceptable treatment of this organism.

The temperature used for this experiment will be the optimum temperature, 40'C, for the enzymes secreted by the yeast. This is to avoid denaturing if the temperature is too high. Also, if the temperature is too high, the yeast cells may die. Therefore, the optimum temperature for yeast is set for this experiment.

Avoiding Inaccuracy: The solutions will be mixed in the conical flask used to contain the reaction, to avoid any inaccuracies in the measurements, due to mixtures such as yeast sticking to the beakers after being transferred.

Time Constraints: Due to time constraints, the, cut of time, will be 1800 seconds (30 minutes) for each trial with each sugar.

Safe Test: I feel this experiment is a low risk experiment, as there are not many components, which can be deemed harmful. But, nevertheless, safety will still be a concern.

The reaction itself isn't volatile so there isn't an issue of wearing safety goggles. However, care will be needed when dealing with the equipment, such as the conical flask. As the flask is made of glass, there is a risk of breakage of glass if dropped, and wounds caused by the shards of glass. These risks can be avoided by carrying one glass object at a time, and by taking care in the usage of these glass containers.

A hazardous procedure that can be identified is carrying large containers of water. A large amount of water can cause a spillage of water onto the floor, due to the weight. This can lead to a person slipping and injuring him or herself. To minimise the risk, the water should be filled near the experiments site, so the distance you travel holding the water will be fairly small. If there is a spillage of water, this should be immediately cleaned up to prevent any accident occurring.

A substance, which can be identified as a risk, is the yeast involved. Some people may be allergic to this substance, so to minimise the risk, the person concerned should avoid skin contact. They could wear gloves and use a spatula to take out the yeast from its container. If the person allergic to the substance makes contact with the yeast, the affected body part should be washed immediately with cold water.

Method:

·Firstly, the solutions of yeast and sugar will be prepared. The two sugars used in the experiment will be glucose (monosaccharide) and maltose (disaccharide) making it easier to compare the rate of CO2 produced for both of these sugars.

·After the preparation of the two solutions, the first solution (NB1) will be placed in a conical flask. The flasks will be placed in the water bath at 40'C to let the yeast acclimatise to its optimum temperature. This is due to the fact that yeast is more efficient and occurs at a greater pace once the yeast reaches its optimum temperature.

·Once placed in the water bath, a 10ml-measuring cylinder will be filled up to the top with water, and placed upside down, with the opening below the surface of the water, ensuring no water spills out of the 10ml-measuring cylinder. The cylinder will be held in place by using a clamp stand. The conical flask containing the yeast will be mixed with the prepared sugar (NB2), and covered with the rubber bung provided. The measuring cylinder and the conical flask will be connected using the glass tube provided. The CO2 produced by the respiration of the yeast will be collected at the top of the measuring cylinder, as the CO2 will displace some of the water in the measuring tube. The amount of CO2 produced will then be recorded per unit of time. (NB3)

·The experiment will be started, and recorded every 60 seconds in case of regular reactions. The experiment will have a cut of time of 1800 seconds (30 minutes) for each trial with each sugar. Doing this experiment 4 times for each sugar will ensure preciseness and accuracy as well as proving the reliability of the results. The results will be recorded in tables and graphs for any comparisons.

NB1 - Preparation of yeast.

The yeast solution will be a mixture of 100cm3 of distilled water, which will be measured using the beaker. 1g of yeast will then be accurately measured using the top pan balance. The yeast will be placed into the beaker and the two will be mixed together using the stirring rods made available, until fully prepared.

NB2 - Preparation of sugars.

The sugar solution will be a mixture of 50cm3 of distilled water with 1g of the chosen sugar. The first sugar prepared will be the glucose. This will be taken out using the spatulas and measured on the top pan balance. When measured accurately, it will be added to the 50cm3 of water in the beaker and stirred until it is fully mixed.

The same procedure as above will be used for the making of the maltose solution.

NB3 - Unit of Time.

During the experiment, the CO2 produced will be recorded at intervals of 60 seconds (1 minute). This will be done until the 1800 seconds (30 minute) mark.

Results:

Time (in seconds) Amount of CO2 collected (in cm3)

Glucose Maltose

Conclusion: From my results, I notice my prediction was proved correct, as the results support the hypothesis I made. As shown in the graph, the average CO2 collected for glucose (monosaccharide) is quicker than the maltose (disaccharide)

The prediction can also be justified looking at the patterns and trends obtained in my results. Looking at the gradients of the two sugars you will be able to tell the rate of CO2 collected with the monosaccharide, glucose is quicker than with the disaccharide, maltose.

If the gradients from, the line of best fit, from the average of volumes of CO2 collected with glucose and maltose, are analysed we will see interesting but expected results. With glucose the gradient is 0.125 (3sf), whilst maltose is 0.0444 (3sf). Shown by the calculation above, the gradient for glucose is approximately 3 times that of the maltose, which shows that the rate of CO2 produced is approximately 3 times quicker than compared to maltose. This proves that the rate of CO2 produced is faster with glucose than with maltose.

The rate of CO2 produced with a monosaccharide is quicker than it is with a disaccharide. This can be explained by the fact that the maltose (disaccharide) has an additional glycosidic bond compared to two á-glucose molecules. Breaking of this glycosidic bond, found between the two maltose molecules, is the reason the rate of CO2 produced by respiration of yeast with disaccharide is slow, in comparison to that of a monosaccharide.

As yeast uses an extra enzyme maltase to break the glycosidic bond, this further justifies and supports my hypothesis. Maltose (disaccharide) has an additional glycosidic bond that needs to be hydrolysed, so that á-glucose can be formed, and the reaction proceeds with the glucose molecules being broken down into ethanol and CO2 by zymase. This is a longer procedure when compared to glucose (monosaccharide) has, glucose is broken down by the enzyme zymase into ethanol and CO2 where it doesn't have an extra bond that needs to be broken. As the disaccharide needs an additional bond to be broken with two enzymes, this is the reason, why the respiration of the yeast is quicker for the monosaccharide, glucose when compared to the disaccharide, maltose.

When looking upon the graphs, the volume of CO2 produced, were fairly linear, with a strong positive correlation. This is normal of an enzyme-catalysed reaction when variables such as pH, temperature, sugar substrate amount and the concentrations of the solutions, are kept constant. This is due to the fact, as there are no external factors affecting the function of the enzymes and the respiration of yeast, the reaction will proceed at a steady rate, indicated by the linear nature of the data shown in the graphs plotted.

The gradient being high in glucose and low in comparison to Maltose can be explained by the structural differences in the sugar substrates. As the disaccharides (Maltose) have an extra glycosidic bond compared to the two separate á-glucose molecules.

As stated in my hypothesis it is "the breaking of this additional bond in between the two maltose subunits of á-glucose that reduce the rate of CO2 produced when yeast reacts with a disaccharide. In addition, yeast must also use a second enzyme called maltase to break the glycosidic bond in between the two-disaccharide monomers. This means that the monosaccharide (glucose) requires only zymase to be broken down into ethanol and CO2 whilst the disaccharide (maltose) requires both zymase and maltase. Maltase is needed to first hydrolyse the glycosidic bond so that the two á-glucose are formed before the continuation of its reaction in a similar manner to monosaccharides by breaking down the two á-glucose molecules into ethanol and CO2 using the enzyme zymase. This would cause the slower reaction in disaccharide while respiring, as two enzymes must be employed as well as the disaccharides containing the extra glycosidic bond. Justification of this is shown in the graph, as the volume of CO2 obtained is less maltose when compared to glucose.

After thorough examining on the results table, I don't think were any prolific anomalies. However, the results were not exactly identical proving that the rate was quicker in one test when compared to another test. For example, when looking at glucoses first test, the last result was 2.5cm3 of CO2. This was 0.1cm3 slower. Although this is not very significant, in an ideal test, the three results would have been the same.

Nevertheless, despite this small difference, I think the experiment justified my results greatly.

Evaluation: Overall, I believe the plan produced worked well when put to practice. The controlling of variables, like the temperature remaining at the optimum, was controlled well, and the procedure conducted was the best possible with the equipment provided. I also think the safety aspect of the plan was handled excellently with very low risk. I think this was the reason the results were as accurate and reliable as they could be.

The results obtained can be seen to be very accurate. This can be justified by the positive correlations in the graphs of both sugars. This is further evidence that in this enzyme catalysed fermentation reaction, the variables were held constant precisely, due to the steady rate of CO2 produced in the reactions. With strong and reliable results, the conclusion obtained will be strengthened, showing that there is a faster rate of CO2 production when yeast respires a monosaccharide sugar, than a disaccharide sugar.

Even though strong reliable results were produced, I think this could have been strengthened for more accurate results. Anomalies, despite being small and minor, could be attributed to time constraints. Biology lessons are 1 hour long, meaning the yeast had to be prepared a day before the experiment. This meant the yeast had to be stored in places with low temperature or high temperatures. This could be due to inadequate storage. With the yeast being prepared early, that meant the solution was not fresh, probably causing a few enzymes to denature.

The few anomalies may have resulted due to the reactions not beginning as soon as they were placed in the water baths. This was because the solution was still acclimatising to the environment and conditions before starting. (Ie the 40'C temperature of the water bath)

Also with a lack of time, results weren't widely ranged. The test for each sugar was conducted up to a limit of 40 minutes, due to arriving to class, getting ready, and time involved for packing up. If more time was available, more trials could have been done, and with more trials, trends produced could have been analysed. But saying this, I was very surprised with the results obtained in the limited time. The results were adequate enough for this write up and as I have stated before, I do think this experiment went really well. In an ideal experiment it would have been great to have at least 5 or 6 repeated trials for more reliable results. Although realistically, I do realise how limited time was, and despite the time constraints, I think the experiment was good enough.

Another factor was the schools resources. There was a shortage of equipment such as conical flasks. The lack of equipment meant it was difficult to make use of the equipment provided in the scarce time allocated. For instance, repeats for glucose could not be carried out at the same time, due to the shortage.

Air being trapped in the measuring cylinder when filling it up with water, was a difficulty I experienced during the experiment. This could and probably is due to the lack of quality apparatus. The clamps provided weren't top notch. They were wobbly, but had to be used to hold up the cylinder. Shaking of the clamp could have lead to air being trapped. This however is a minor irregularity, and I don't think it would have a huge significance on the conclusion and the numbers of tests were enough to make the data reliable.

Except from human errors, a difficulty experienced was the water was being trapped in the glass tube above the waterline. This meant the CO2 needed to build up a high enough pressure to push the water out and move into the cylinder. This could have caused a delay in the production of CO2. The delay in the reaction may also have been the build up of pressure to dislodge the water in the measuring cylinder and replace it with CO2 and this means that there was a gas trapped in the delivery tube for a certain period of time. However, this might not have contributed to the delay, as the yeast may have needed to acclimatise to the conditions before the experiment fully began.

I do feel minor anomalies didn't have a huge affect on the conclusions drawn from the experiment. This is due to repeats or number of trials, being done accurately ensure the reliability and precision of the data. The conclusion could have been strengthened by the improvement in equipment as well as additional time. This could have allowed for fresh yeast preparation and if the limited time for the test were extended, this would have provided more reliable results. Anomalies would have been reduced in this way and this would have also solidified the reliability of the results as I stated before. I do believe, however, that the practical and therefore conclusion could have been improved and strengthened by improvements to the equipment. Improvements such more apparatus provided, like conical flasks, to aid in repeats tests. Also, time could have been extended to get a wider ranger of results. This would help in having a better trend to analyse data from.

I think if there was an alternative method for measuring the rate of respiration with different sugars, this could improve the precision and accuracy of the experiment. If there was equipment to record the weight lost from the yeast sugar solution, I think the accuracy would be greater. For example we could have used equipment like a top-pan balances which was sensitive to the changes in weight. I think the weight lost is equivalent and would be more accurate than timing the CO2 produced. This would erase human errors, such as reading of the measuring cylinders, and the results produced would be more precise.

My final point is by preventing the yeast-sugar solution to respire aerobically by placing lubricate over the reacting solution would prevent the rate of respiration from fluctuating, as it would be respiring both aerobically as well as anaerobically. This would have made it easier to compare the different results for different sugar substrates (prevention of external factors). The idea of using such equipment like a top-pan would be impractical as it would be difficult to maintain a constant temperature outside the water bath. If there weren't a limitation of time, then a proposal of testing two types of monosaccharides (like Glucose and Fructose) as well as two disaccharides (like Maltose and Lactose) would have been handy for bigger comparisons between the two structures.

This would help to form a better relationship between sugar substrates and the rates of respiration with yeast as well as confirming with detail as they could be directly compared (maltose with glucose, fructose with lactose) and evidence would be stronger that monosaccharides respire quicker than disaccharides due to the extra glycosidic bond in them. This would provide clear and sound evidence on the relationship between the respiration rates with specific monosaccharides and disaccharides

Despite this, I once again feel the experiment went really well and the plan used for the practical was well done.

Bibliography

BOOK TITLE AUTHORS

Nelson Advanced Science - John Adds, Erica Larkcom

Molecules and Cells (AS textbook) and Ruth Miller

Encyclopaedia Britannica

Microsoft Encarta Encyclopaedia 1999

INTERNET ADDRESSUSED FOR

http://www.google.comYeast.

http://www.37.comMonosaccharides