effect of temperature on the rate of respiration in yeast
Effect of temperature on the rate of respiration in yeast
My aim is to investigate the effect of temperature on the rate of respiration in yeast by using a universal indicator.
Enzymes are organic catalysts that speed up the rate of a chemical reaction without being permanently altered in the process.
- Form reversible complex with substrate.
- Not consumed in the reaction therefore they are effect in small amounts
- - (Induced fit hypothesis) react with only a single substrate.
- Many need , such as certain vitamins, to be activated.
- 2000+ enzymes per cell, different cells have different enzymes.
- Enzymes are produced by genes.
- Genetic disorders are the result of faulty enzymes.
- Operate best in optimum conditions of , , etc.
- Are controlled by feedback mechanisms.
Key and theory:
The substrates (reactants) are attracted to the enzyme molecule. They join forming an enzyme-substrate complex. The reaction occurs on an area of the enzyme molecule known as the active site producing new substrates(s) or products.
Induced fit hypothesis:
The attraction of the substrate and enzyme form an enzyme-substrate complex. It was originally referred to as the Lock and Key Enzyme Theory. The current theory suggests that the enzyme molecules are in an inactive form. To become active they must undergo a slight change in structure to more specifically accommodate the substrate(s). It is said to be "induced to fit" the substrate. Think of way your hand changes shape slight when you shake a person's hand. (Reference 1 )
Factors affecting rate of reaction:
Effect of temperature on enzyme:
As the temperature increase in an enzyme catalyzed reaction the rate of reaction also increases. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results.
The rate of reaction increases as the temperature increases. The rate of reaction increases till it reaches it its optimum level; this is when the rate of enzyme activity start to decrease and enzymes started to denatured. (Reference 2)
As the temperature increases particles start to move quickly and collide with each other with greater energy. It also increases the number of particle. It increases the number of high energy collisions which results in a reaction.
PH doesn’t just changes the change of enzyme but it can also cause denaturation. The concentration of hydrogen ions in a solution is a measure of pH, which determines how acidic or alkaline a solution is. At extreme pH conditions there is low hydrogen ions concentration. As well as that the chemical bonds between the atoms break, causes changes in the shape of the active site, which can also cause denaturation of an enzyme. This can also slow down the rate of reaction. Before that when the pH is increase it also increases the rate of reaction till the pH is reached its optimum level after that the rate of reaction started to decreases the acid or base conditions begin to disrupt some of the hydrogen bonds between loops of the protein chains. If the disruption occurs at or near the active site, the active site becomes distorted and substrate can not fit perfectly. Thus not all enzymes in the solution will be able to catalyze their reaction. With increasing or decreasing pH, more enzymes become denatured, and fewer enzymes are able to form that enzyme-substrate complex. The reaction rate continues to decrease. At some point, all the enzymes are denatured, and the reaction rate falls to zero. (Reference 3)
Enzyme concentration is directly proportional to the rate of reaction. As the enzyme concentration is increase the rate of reaction will also increase as there are a lot of active site are available for substrate to form enzyme substrate complex. However increasing enzyme concentration beyond some point it would have no effect on rate of reaction as there will be no more active sites available for substrate molecules.
The graph shows that when the concentration of enzyme is maintained constant, the reaction rate will increase as the amount of substrate is increased. However, at some point, increasing the amount of substrate does not increase the reaction rate. This is when it reaches V-max. at this point the rate of reaction stays constant. At first there is very little substrate and a lot of enzyme. An increase in the concentration of substrate means that more of the enzyme molecules can be utilized. As more enzymes become involved in reactions, the rate of reaction increases, but when it reaches V-max, all the enzymes are being involved in reactions. When this happens, some of the substrate must "wait" for enzymes to clear their active sites before the enzyme can fit with them (like a "lock and key"). (Reference 4)
Coenzymes are small proteins that join to the enzyme molecule to make it active. Like enzymes they are not permanently altered in the reactions. Many of these coenzymes are derived from vitamins and minerals that are essential for life. The absence of these cofactors can lead to vitamin and mineral deficiency diseases. An example of a coenzyme already mentioned in respiration in NAD+, which except a trios phosphate molecule to become reduced NAD+ in glycolysis. (Reference 5)
Competitive Inhibition interferes with enzyme activity by binding temporarily to the enzyme's active site. This prevents the enzyme from reacting with its normal substrate. Therefore no product made. Competitive inhibition is reversible and can be overcome by increasing the amount of substrate on which the enzyme works.
Non competitive inhibitors:
Competitive Inhibition interferes with enzyme activity by "binding" permanently to the enzyme therefore it permanently blocks the active site and doesn’t allow any substrate to bind with active site.
Denaturation of enzymes:
When enzymes are placed into a high temperature they start to denature. Denaturation can be defined as the loss of enough structure to render the enzyme inactive. This happens when the temperature reaches its optimum level and the rate reaction to decrease cause of denaturation of enzymes.
These denaturing reactions have standard free energies of activation of about 200 - 300 kJ mole-1 (Q10 in the range 6 - 36), which means that, above a critical temperature, there is a rapid rate of loss of activity. The actual loss of activity is the product of this rate and the duration of incubation. It may be due to covalent changes such as the deamination of asparagines residues or non-covalent changes such as the rearrangement of the protein chain. Inactivation by heat denaturation has a profound effect on the enzymes productivity. (Reference 6)
Yeasts are unicellular . The precise classification is a field that uses the characteristics of the cell. One of the more well known characteristics is the ability to ferment sugars for the production of ethanol. Yeasts multiply as single cells that divide by budding or direct division and they may grow as simple irregular filaments. These are egged shape that can only be seen by microscope. Yeast cells also digest various sugars like sucrose, fructose and glucose. The yeast's function in baking is to ferment sugars present in the flour or added to the dough. This fermentation gives off carbon dioxide and ethanol. The carbon dioxide is trapped within tiny bubbles and results in the dough expanding, or rising. Sourdough bread is not produced with baker's yeast, rather a combination of wild yeast and acid-generating bacteria. The fermentation of wine is initiated by naturally occurring yeasts present in the vineyards. Many wineries still use nature strains, however many use modern methods of strain maintenance and isolation. The bubbles in sparkling wines are trapped carbon dioxide, the result of yeast fermenting sugars in the grape juice. One yeast cell can ferment approximately its own weight of glucose per hour.
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Respiration is the release of energy from glucose or another organic chemical. Respiration most likely takes place under aerobic respiration which means efficient oxygen.
The equation for aerobic respiration:
C6H12O6 + 6O2 → 6CO2 + 6 H2O
But sometime respiration goes under an aerobically which means not enough oxygen is available to respire.
The equation to an aerobic respiration:
C6H12O6 2C3H6O3 + 2 ATP.
The chemical energy in glucose can be used to provide the energy required for growth, repair and movement. In fact most things you do require energy. Respiration is a transfer of potential chemical energy to ATP. For this to happen, respiration goes under few processes such as:
Glycolysis is the sequence of reactions that converts glucose into pyruvate with the concomitant production of a relatively small amount of ATP. Firstly glucose is phosphorylated from 2 ATP molecules to raise the energy level of the compound and reduce the activation energy barrier to the rest of the pathway. Now a compound is made which is called hexose biphosphate which is a six carbon glucose. Now this molecule split into two molecules of trios phosphate and then it’s converted into two molecules of 3 carbon pyruvate. Dehydrogenase enzymes remove hydrogen and give them to molecules of 2 NAD which gives out 4 NAD+ as a product. Where as the net production of the process is 2 molecules. However when glycolysis goes anaerobically oxygen-deficient conditions, NADH gets converted back to NAD through anaerobic mechanisms, whether homolactic or alcoholic fermentation. They can also be converted into acetyl to go under kreb cycle.
This links glycolysis to the Krebs cycle (sometimes called the citric acid cycle). Pyruvate molecules are decarboxylated (they lose a molecule of carbon dioxide) in the mitochondria. Pyruvate molecules are oxidized and converted to acetyl coenzyme A, usually abbreviated to acetyl CoA.
2CH3COCOO- + 2NAD+ + 2H2O 2CH3COO- + 2NADH + 2H+ + 2CO2
The oxidised form of NAD+, is reduced to its reduced from NADH. After this it is been taken into kreb cycle and then into electron transport chain. (Reference 9)
This stage of respiration is known as kreb cycle. The Krebs cycle occurs in the matrix (fluid portion) of the mitochondrion. During this reaction a six carbon chain (citrate) is formed when coenzyme A delivers an acetyl group to oxaloacetate. Then the citrate goes under fewer changes into isocitrate then this diffuses out carbon dioxide while hydrogen is also removed by NAD to produce reduced NAD. This results in the chain of carbon by producing a a-ketoglutarate. This also diffuses carbon dioxide and produces reduced NAD. Then this converts into 4 carbon chain of succincyl coenzyme which then changes into succinate and then this releases energy compound such as FADH. This also changes into 4 carbon chain fumarate. Then this changes into malate and this releases from NAD+ to NAD+ + H+. Then this again changes into oxaloacetate and starts the cycle again. After the cycle is been round six times it releases 1 glucose molecule. (Reference 10)
Oxidative Phosphorylation and electron transport chain:
The final stage of respiration is Oxidative Phosphorylation and the electron transport chain, which are carrier molecules located inside the inner membrane of the mitochondrion. The NAD that was reduced is the starting point of the chain. The hydrogen that was removed at different stages will now come into play. This pathway functions by oxidation reactions taking place. When this happens, energy is released and ATP is synthesised. Where ATP is produced with energy captured within it, inorganic phosphate is combined with ADP. The overall process is called oxiditative phosphorylation, because of the oxidation reactions grouped with the phosphorylation.
Firstly 2 hydrogen are removed from reduced NAD and passed on to a hydrogen carrier, which reduces the carrier and oxidises the NAD. A redox reaction has taken place. After a second hydrogen carrier, the hydrogen splits in to hydrogen ions and electrons. The hydrogen ions remain in solution in the mitochondrial matrix, whilst the electrons are passed along a series of electron carries. This makes energy available which is used to convert ADP + Pi to ATP. When an electron passes from a carrier at a higher energy level to one that is lower, energy is released. Some energy is lost as heat. At the end of the chain, the hydrogen’s and electrons join with oxygen to form water and so we can see the role of oxygen.
Potentially 3 molecules of ATP can be produced from each reduced NAD molecule. However about 25% of the total energy yield of electron transfer is used to transport ADP in to the mitochondrion and ATP into the cytoplasm. Therefore each reduced NAD molecule yields the equivalent to two and a half molecule of ATP with FAD producing one and a half molecules of ATP.
The synthesis of ATP can be explained by the Chemiosmotic theory. The energy released from the electron transport chain is used to pump hydrogen ions from the matrix in to the intermembrane space. The accumulation of the hydrogen ions set up a concentration gradient, as there are more hydrogen ions in the intermembrane space relative to that in the matrix. The hydrogen ions flow down their concentration gradient in to the matrix through protein channels in the inner membrane. This protein channel is often referred to as a stalked particle that has a region called ATP synthase, which acts an enzyme. As the hydrogen ions pass through the channel their electrical potential energy is used to synthesise ATP. (Reference 11)
Methylene blue is a with : 16183. Methylene blue is widely used as a in . Solutions of this substance are blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent. The redox properties can be seen in a classical demonstration of in general chemistry, the "blue bottle" experiment. Typically, a solution is made of , methylene blue, and . Upon shaking the bottle, oxidizes methylene blue, and the solution turns blue. The dextrose will gradually reduce the methylene blue to its colorless, reduced form. Hence, when the dissolved oxygen is entirely consumed, the solution will turn colorless. Methylene blue is also used to make the reaction between and reducing sugars more visible. Methylene blue will make the yeast suspension blue and then started to get colourless or back to yeast colour depending on how high the temperature is. (Reference 12)
Alcohol fermentation starts off in glycolysis with breaking down glucose which is 6 carbon molecules into a 3 carbon molecule called pyruvic acid. This pyruvic acid is then converted to CO2, ethanol, and energy for the cell. Humans have long taken advantage of this process in making bread, beer, and wine. In these three products the same microorganism is used: the common yeast or Saccharomyces Cerevisae.
Lactic acid fermentation is caused by some fungi and bacteria. The most important lactic acid producing bacteria is Lactobacillus. The presence of lactic acid, produced during the lactic acid fermentation is responsible for the sour taste and for the improved microbiological stability and safety of the food. All the fermentation is done anaerobically.
This is an alcohol enzyme which plays an important part in fermentation of yeast. resulting from is converted to acetaldehyde and , and the acetaldehyde is then reduced to ethanol by an alcohol dehydrogenase called ADH1. The purpose of this latter step is the regeneration of NAD+, so that the energy-generating glycolysis can continue.
This enzyme removes carbon dioxide from pyruvate and from citrate molecules in the kreb cycle.
I predict that as the temperature increases the rate of respiration will also increases till it reaches its optimum temperature as the rate of respiration will also start to decreases as enzyme starts to denature and the rate of respiration will stop. Increasing temperature will also be broke down more quicker because active sites and the substrate molecules will gain more kinetic energy and they will start to move more quicker and also cover the larger surface area therefore they will collide with each other with greater energy and therefore there will be more successful collisions. As the temperature will increase in yeast suspension the dehydrogenase enzymes will start to remove hydrogen molecules in glycolysis and they will also remove hydrogen molecules from citrate in kreb cycle. Decarboxylase enzyme will also become active and starting to remove carbon dioxide much quicker from pyruvate and citrate in kreb cycle; where glucose will be made much more quickly as the temperature is increased.
As the temperature increases the yeast to become colourless from methylene blue will also increase as the active site and the substrate will have more kinetic energy. When the temperature is increases the rate of reaction will only increase till it reaches its optimum temperature this is where the enzyme work at there best rate but after that the enzymes will start to denatured and slow down the rate of respiration. The optimum temperature of yeast in the fermentation of sugar is 40 oC so, therefore I also predict that the optimum temperature of rate of respiration in yeast to be 40 oC. After that any higher temperature will cause chemical bonds to break and enzymes will start to denature as they will change there active site and therefore substrate will not be able to recognize its active site and no more enzyme substrate complex will be made. Decarboxylase and dehydrogenase enzyme will also denature which will affect the kreb cycle and glycolysis in respiration.
I also predict that enzyme will work very slowly or not at all below 20 oC because the enzyme will not become active and move really slowly as the molecule will move really slowly and hardly have any kinetic energy.
I also predict that the rates of respiration will double every 10 oC cause of the Q10 law which states this. This will happen till it reaches its optimum temperature and then the yeast will die.
Q10 = Rate of reaction at a temperature t + 10 c
Rate of reaction at a temperature t
The rate of reaction will double at every 10 oC in temperature. So therefore I predict that
Q10 = 2.
There are many variables which I need to control in the aspect of fair test.
- Temperature: I will be controlling temperature as I chosen to be between 20 oC - 70 oC so they are fair in order to other repetitions and as accurate as possible. The variation of temperature will increase or decrease the rate of respiration.
- Volume of methylene blue: Volume of methylene blue should be kept constant because as the volume increases it will take longer for the yeast to become colourless because there will be more hydrogen produced by the yeast which then needed to be used by methylene blue which will take longer for the yeast to get back to his colour. Therefore I will use a 1cm3 syringe and keep it as accurate as possible.
- Volume of yeast: I will be controlling the volume of yeast as this contain enzyme. By varying this will increase or decrease enzyme activity. So by increasing the volume will increase the rate of respiration. More hydrogen molecules will be picked up by methylene blue and for the yeast to become colourless from methylene blue will be much quicker. I will keep the volume of yeast constant for the whole experiment.
- The concentration of methylene blue: the concentration of methylene blue should be kept constant because it will depend on the rate of respiration as higher concentration will have more particles which then needed to be reduced by yeast which will take more time. So therefore I will keep the concentration of methylene same for the whole experiment.
- The volume of glucose: the volume of glucose is needed to control because this will increase or decrease the number of substrate. If this is increased more hydrogen will be given off and the rate of colour change will be faster. This should be controlled by it self because it is present in the yeast suspension.
- Concentration of glucose: this variable should be kept constant because increasing concentration of glucose will increase the number of substrate, thus allowing the enzymes to react quicker. Therefore the rate of respiration will increase as more substrate will be available for active sites and more enzyme substrate complex will occur.
- Batch of yeast: the same batch of yeast should be same through out the investigation whether this can affect the activity of yeast. This can affect the rate of respiration depending on how active the yeast is. The more it is the higher the rate of respiration or vice versa. Therefore I will keep the same batch of yeast for the all my experiments.
- The pH of yeast suspension: this should also be kept constant because increasing of pH will start to break chemical bonds in molecules therefore this changes the shape of active site and no more substrate molecule will form enzyme substrate complex. This will result in a denaturation of enzymes.
- Colour of end point: this should be kept same for all the experiments as I will compare this to normal yeast as this become colourless from blue.
These are the volume and indicator I have chosen to use in the experiments of respiration of yeast.
Indicator: methylene blue
Volume of indicator: 0.5 ml
Volume of yeast: 10.0 ml
Temperature: 20 - 60 oC
Choice of technique:
Why have I chosen methylene blue over TTC?
This is because TTC is more time consuming then methylene blue when the experiment reaches its end point. A colour chart would be needed if TTC is used and will take more time looking for the type of pink it has produced. It is much more harder to decide the end point, when using TTC then methylene blue because in methylene blue the blue colour present in yeast after adding methylene blue will turn back to the normal cream colour of yeast, which then can be compared to the normal yeast to see if this has reached its end point.
Why have I chosen using an indicator over measuring carbon dioxide production to test the rate of respiration in yeast?
If I would measure the carbon dioxide produce by the yeast this would make me end up with more equipment such as: gas burette, tubes etc To measure the production of carbon dioxide it will take me longer to set up an apparatus there for it can be more time consuming. A greater chance of getting anomalous results as carbon dioxide can be lost. The indicator is actually a use of dehydrogenase enzyme and measuring the production of carbon dioxide using gas burette or a gas syringe is a use of decarboxylase enzyme. Therefore I chose using a indicator which is more precise and easy to use.
Why have I chosen 0.5 ml of methylene blue and not any other volume?
This is because I think any more value volume of methylene blue will slow down the reaction as we do not have sufficient time to do the experiments; whether this will take longer to reoxidise to give back its yeast colour.
Why have I chosen temperature to be between 20-60 oC and not 10-50 oC?
This is because I think the temperature below 20 oC will hardly have any reaction because enzyme sometimes do not become active below 20 oC and if they do the reaction would be really slow because the molecules would hardly have any kind of kinetic energy for the reaction to take place. So therefore I will start my experiment from 20 oC.
List of apparatus:
- Firstly I will put on all the safety equipment on such as lab coat and goggles.
- Then I will gather all the apparatus and set them out accordingly to my experiments.
- For me to start of with 60 oC I will fill up the kettle with water and turn on the kettle to boil the water.
- I will get the test tube and the syringes.
- Then I will collect yeast in a small beaker.
- Then I will stir the yeast using a stirring rod.
- Then I will use a 10ml syringe to collect 10ml of live yeast.
- Then I will put the live yeast collected by a syringe into a test tube.
- Then I will collect hot water from a kettle into a large beaker.
- Then I will use a thermometer to check the temperature and get the temperature to 60 oC.
- Again I will stir the yeast in the test tube and then place it into a hot water for the water bath for 5 minutes and place the rubber bun immediately.
- Then I will use a 1.0ml of syringe and collect 0.5ml of methylene blue into a syringe.
- After 5 minutes I will use thermometer to check the temperature again and it its decreased then I will add some more hot water.
- When the temperature reaches 60 oC again I will take out the test tube.
- Open the rubber bun.
- Add 0.5ml of methylene blue.
- Place the rubber bun immediately.
- Shake the test tube for 3 seconds.
- Then place the test tube back into the beaker containing 60 oC of hot water.
- And immediately start the stop watch.
- I will repeat each temperature twice and do 5 different temperatures.
To carry out my experiments I will do 20 oC, 30 oC, 40 oC, 50 oC, 60 oC as my temperatures and I will do 2 repetitions for each of them.
Precision and reliability:
I will make sure that I am very precise while I am measuring volume of yeast and volume of methylene blue that there are no air bubbles presences in the syringe. If this happens this will increase or decrease the enzyme activity by increase or decreasing the volume of yeast. This can have an affect on rate of respiration. Also if the volume is not same in each repetition this will not show a fair test and shoes inconsistency and inaccurate, which can lead to unreliable results. Therefore I will use precise syringes 10ml syringe for 10ml of live yeast and 1.0ml syringe for 0.5ml of methylene blue. Using precise instrument will help me increase the precision of my results.
When I am reading my thermometer to check the temperature I will come down on scale with an eye level to the thermometer because changes in temperature and not reading it accurately can affect rate of respiration which can lead to unreliable results.
The starting and the end point of respiration of yeast will also depend on time of starting and ending the clock. Therefore I will keep the clock close to me so I start and end immediately so the yeast doesn’t get more respire. It will make my results more accurate.
I will use digital clock rather than an analogue one because this will increase precision as digital one is significantly better. It is harder to read off the seconds in analogue one. By using a digital watch this will make my results more reliable.
I will use clean test tube so there is any extra yeast on the sides of the test tube which then can be mixed with the live and gets contamination which will give me unreliable results. So I will use clean and dry test tubes because there also can be a contamination with water.
I will continuously be using stirring rod and keep mixing the yeast before my experiments because by not mixing the yeast so the solution does not go to the bottom and when I collect it with the syringe I get the equal amount. This will increase accuracy of my results.
I have used test tubes other then boiling tubes because boiling tubes have larger surface area and the colour change is slightly slower because the reaction doesn’t heat up as quicker due to larger surface area than in test tube where they have slightly smaller surface area.
Reference 1: Biology book 1
Reference 4: biology book 1
Reference 6: (
Reference 9: http://www.chemsoc.org/networks/learnnet/cfb/respiration.htm
Reference 10: Biology book 2
Reference 11: Biology book 2
Table showing time taken for the yeast suspension to reoxidise from methylene blue blue colour.
To work out rate I used formulae: 1000/time
To work out Q10 I will use its formulae for it which is:
Q10 = Rate of reaction at a temperature t + 10 c
Rate of reaction at a temperature t
In my investigation I have looked at how different temperature affect on rate of respiration in yeast by using a redox indicator such as methylene blue which converts yeast colour into blue and then it is reoxidise back into the normal yeast colour by dehydrogenase enzymes. They usually produce hydrogen and break down glucose molecules.
I think that my prediction is partially right as I predicted that the as the temperature increases the rate of respiration will also increase and that what happens in my experiments and as shown in my background information. This is due to more and more enzyme substrate complex and induced fit hypothesis reactions has taken place cause of collision theory of molecule where they gain more kinetic energy and collide with each other with greater power and more successful collision takes place due to the increase in temperature. But as shown in my background information that as the temperature exceed over 37 oC the rate of reaction will start to decrease and enzymes will start to denatured but this doesn’t happen in my experiment where I predicted that 40 will be the optimum temperature but that doesn’t happen; my rate of respiration keep increases. My results shows that at 60 oC there was the greater respiration taken place this may be a cause of where dehydrogenase enzymes become highly reactive and break down glucose much more quicker and produce a lot of hydrogen atoms which allows glycolysis to continue which uses ATP and NAD molecule and gives a net production of two extra energy molecules. Also there may be reason that experiments were taken place in a cool environment which can also have an affect of optimum temperature of yeast. As I have seen during my experiments that the rate of respiration is getting faster and faster and there is no signs of denaturation of enzyme whether this lets me know that the optimum temperature of yeast respiring may be over 60 oC where the stop watch was stopped before 25 seconds.
I have also predicted that there Q10 value will be = 2 whether this seems to be correct as shown in my table where Q10 values are shown. This is due to the 4 steps of respiration (glycolysis, kreb cycle, link reaction, oxidative phophorylation) which have taken rightly so.
I have quoted the optimum temperature of enzyme wrongly, but I can still say that most of my prediction was right as the temperature increase and the rate of respiration increases and the value Q10 = 2. As you can see that in my table at 50 oC the Q10 value is 2.04 which are very close to 2. Therefore this prediction of mine is correct.
I think this was the suitable way of investigating the rate of respiration in yeast other than collecting the gas such as CO2, which can give me a lot of anomalous results such as gas lost in evaporation. By using an indicator was a much more suitable way of collecting the rate of respiration and suggesting the end point of the experiments.
I have identified some anomalous results such as for the 20 temperature that the difference between repetition 1 and repetition 2 is 43 seconds which means that it has taken longer for the second repetition. This also brings the average much higher. I think the anomalous result is the first repetition this can be a caused of which is discussed under the next heading.
Limitation / improvement:
These are the limitation which can be a caused of anomalous results in the order where the most significant at the top.
Limitation 1: Controlling the temperature of the experiment and the water bath. Temperature was varying most of the time in my experiment where it was hard to control because of the room temperature which causes the temperature to increase and decrease and I was adding hot water and ice to bring the temperature down or to increase.
Improvement: To make this improvement next time I will use thermostatically controlled water bath. This can maintained the temperature through out the experiments and increase accuracy of my experiments and reduced anomalous results.
Limitation 2: Deciding the end of the experiments. To decide the end point of my experiments I have used another test tube containing live yeast without any contamination to match in the eye level with the one containing methylene blue which has been reoxidised.
Improvement: This is a human error which can not be improved by any equipment. Although I had live yeast in another test tube for me to determine the end point but still I cannot tell if all methylene blue is oxidised or not. For me to improve this limitation I can rather wait a bit longer next time and for methylene blue to be fully oxidised. This will make my results reliable.
Limitation 3: Air bubble in the syringes while I am measuring the volume of methylene blue and volume of yeast. The air bubble can cause the volume of yeast and methylene blue to increase and decrease which can result in rate of respiration to increase or decrease. If this increases or decreases by ±0.5ml which can cause can error of 5 – 10 %.
Improvement: I will make sure that I squired out if there is any air bubble present then collect the methylene blue or yeast again slowly and with the open nozzle. This will make my results much more accurate and reliable.
Limitation 4: Stopping and starting the stop watch at right time. This limitation is also cause by a human error. This suggests that the actual starting time of the experiments may be a bit earlier or a bit late or vice versa. If this increases or decreases by ±0.5ml which can cause can error of 5 – 10 %.
Improvement: To make this improvement I will make sure that after adding methylene blue I start the stop watch straight away and as I see the end point I stop the clock straight away. This will increase accuracy in my results.
My graph doesn’t show the results I expected but it doesn’t mean that there not reliable. My results has certainly affected by few limitation such as not controlling the temperature and deciding the end point. Other then few errors I think my results are much more reliable. Errors which I think affected the reliability were controlling the temperature which was not really maintained; as I said before that it increases or decreases by the room temperature where then I have to use a thermometer to add hot water or ice. This could be easily done if we had the right equipments such as thermostatically water bath which maintains it self and increase reliability. I think my result was more precise as I did two repetitions and most of them were closer to each other than 20 which has a difference of 43 seconds but this does not affect the accuracy of my results. I think my results was very accurate as I didn’t allow a lot of air bubbles in my syringes and keeping the temperature of experiments as closest as possible.
To draw conclusions I say that my results are valid although my experiments was not really reliable as I predicted that the optimum temperature would be around 40 which doesn’t happen this is because of errors and limitation which have affected my results and making it unreliable. Nevertheless I think that my result are precise and as accurate as they could be but they are unreliable and they are quite valid to some extend as my prediction partially supported my conclusions as I said that the temperature will increase the rate of respiration in yeast.
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