Yeast cells use a different form of anaerobic respiration to animals. The pyruvate formed by glycolysis, is reduced to produce ethanol and carbon dioxide, instead of lactate. In this process, 2 ATP molecules are formed, releasing energy from the process.
Diagram 1 taken from http://www.chemcases.com/alcohol/
The Pyruvate is converted in Acetaldehyde, with the release of 2 carbon dioxide molecules, by the enzyme Pyruvate Decarboxylase. Acetaldehyde is then converted into ethanol by an Alcohol Dehydrogenase. The 2 H+ molecules for the ethanol are acquired from the oxidation of NADH. The 2 Hydrogen ions from the NADH are then replaced during glycolysis.
Hypothesis:
Varying the type of sugar used in the fermentation will result in some variance in the volume of ethanol produced. The larger the number of carbon atoms in each sugar ring, the larger the volume of ethanol that shall be produced.
Null Hypothesis:
Varying the types of sugar used in the fermentation process will result in no change in the volume of ethanol.
Apparatus:
Method:
- Label each 250ml flasks with the corresponding sugar it will contain and the repeat number, i.e. 1, 2 or 3.
- Place 100ml of water into each of the 250ml flasks.
- Into each of the 12 flasks, place 10g of the yeast.
- Place 10g of each sugar into their corresponding flasks.
- Agitate each flask well in a swirling motion, in order to suspend the yeast cells and sugar.
- Seal the flasks sufficiently enough to produce a seal with cotton wool that will prevent any foreign particles from contaminating mixture.
- Leave to ferment at room temperature for 1 week, without disturbing any of the flasks un-necessarily (disturbing the mixture may re-suspend the mixture and lead to an increased rate of reaction).
- Extract 50ml sample from a flask, by slowly pouring it into a measuring cylinder. Then transfer into the heating mantle and round bottomed flask apparatus.
- Place the condenser on the top of the flask and place a measuring cylinder at the end of the tube to collect the distilled ethanol. Ensure that water is flowing in the bottom of the condenser, and out of the top, this allows for better heat extraction.
- Ensure that the thermometer is inserted far enough to be just below the lip of round bottomed flask
- Turn the heating mantle on and heat at level 10 until the temperature rises to about 85°C, then turn down to heat level 3.
- Leave to heat at this temperature for about 45 minutes. Over this period of time, the measuring cylinder should be collecting the ethanol that is being distilled. Once the 45 minute is over, record the volume of ethanol collected and then dispose of the fermentation mixture in the flask, and the ethanol collected.
- In order to check if ethanol has been produced, pour a drop of tea tree oil into the measuring cylinder, if the tea tree oil emulsifies, ethanol has been produced.
Variables:
Dependent Variables:
These are the variables that shall be measured in the experiment.
In this experiment, the dependant variables are:
- The volume of ethanol produced during the fermentation process. The distillation process is the means of extracting the ethanol from the fermentation mixture. The volume of ethanol produced will be measured by the volume of ethanol collected in the measuring cylinder that is located after the condenser. The volumes collected from each sugar shall be compared to the structures of the sugars.
Independent Variables:
These are the variables that shall be changing in the experiment.
In this experiment, the independent variables are:
- The type of sugars used in the fermentation process is the only factor that shall be changed in this experiment. The sugars used have differing structures and this may have an effect on the volumes of ethanol produced during fermentation. As this is the only variable being changed, the results should be easy to link to the sugar structure.
Controlled Variables:
These are the variables that will be maintained at a constant to the best of my ability.
There are several controlled variables in this experiment. These are:
- The temperature that the fermenting mixtures are situated in. The ambient temperature in the room where the fermentation flasks are located will vary throughout the day. However, all of the flasks shall be subjected to the same temperature fluctuations throughout the fermentation process. Any variation in temperature that one flask is subjected to, that others are not, will affect the results, as increased temperatures can increase the rate of fermentation, until the yeast begin to denature at around 42°C. At this point the yeast cease to produce ethanol, and fermentation stops.
- The volumes of water in the flasks. Increasing the volume of water in the flasks will reduce the sugar solution concentration, and yeast concentration. This will result in the reaction being slowed down, affecting the volume of ethanol produced.
- The volume of sugar added to the fermentation mixture. Increasing the volume of sugar in the fermentation mixture will result in the sugar solution concentration increasing, increasing the rate of reaction. This will result in more ethanol being produced in a period of time, up until a point, at which the maximum speed of the yeast is reached.
- The volume of yeast added to the fermentation mixture. Increased volumes of yeast will result in an elevated rate of reaction, resulting in larger volumes of ethanol being produced in shorter periods of time.
- Temperature of distilling. Increasing the distilling temperature will result in more water vapour being produced, and mixing with the ethanol. This will result in there appearing to be more ethanol produced than is true. Ethanol can be diluted in water, so will not form a visible separate layer. A way of countering this is to re-distill the ethanol, to remove water impurities. This will produce more accurate results. Varying the temperature for different fermentation mixtures will result in varying quantities of water in each ethanol sample.
- The length of time that each of the 50ml samples are distilled for. In order to perform all distillations in the 2 days allocated, the length of time for distilling needed to be limited. This was limited to 45 minutes each. Increased time for some of the distillations may result in there being higher quantities of ethanol distilled, affecting the reliability of the results.
Hazards and Safety:
Under the Control Of Substances Hazardous to Health (COSHH) some chemicals in use in this experiment need to be identified and handled carefully.
Ethanol:
- Highly flammable, should be kept away from naked flames or other ignition sources at all times.
- Slightly toxic, should not be ingested, or inhaled. If any quantity of ethanol is ingested, large amounts of water should be consumed in order to dilute the ethanol. Effects of ethanol consumption include a reduction in cognitive skills. If inhaled, can have narcotic effects.
- Highly concentrated ethanol can cause permanent damage to cells if exposed to ethanol for prolonged periods of time. In order to prevent this, and ethanol that has had contact with the skin should be washed off.
- Contact with eyes can cause severe irritation, and sensitivity to light
General Lab safety:
- Any spillages should be taken care of immediately
- Any broken glassware should be disposed of appropriately and quickly to prevent injury.
- Tie hair back
- Wear goggles lab coats and gloves at all times to minimize risk of damage to eyes, hands or clothing.
- No running
- Keep all flammable materials and chemicals a reasonable distance away from sources of ignition.
- The heating mantle and flask apparatus may be hot shortly after use. It is advised that the flask remain untouched for at lest 10 minutes after the end of the heating cycle. Failure to leave to cool could result in burns and scald injuries.
Preliminary Experiment:
My preliminary experiment exposed several flaws in my original planned method. The method listed previously was adapted in specific areas, but is essentially the same as the method for my preliminary. The changes made were as follows:
- The volume of yeast used in each flask. In my original plan, i intended to use only 11g of yeast per flask. As there was no fermentation reaction visibly occurring, the volume of yeast used was increase by a factor of 10. This resulted in large a visible fermentation reaction occurring, meaning that alcohol was being produced.
- The sample size taken from the fermentation mixture was reduced from 100ml to 50 ml. This was due to the fact that 100ml took a much longer time to reach the required temperature and begin to evaporate ethanol. The 50 ml sample takes less time, so all 12 distillations could be carried out in the allotted time.
- The length of time that each distillation process takes place for. Initially, I intended to run the distillation for only 20 minutes. This was not effective as the 100ml sample was slow to heat up, and did not distill enough alcohol in the 20 minute timeframe. This lead to me increasing the length of time that distillation would occur for. When combined with the reduced volume of fermentation mixture in the heating flask, the alcohol distillation provided larger quantities of alcohol in the 40 minute timeframe.
Due to these issues, there were no repeat results gained from the preliminary work. This forced a rethink of the times and volumes involved in the actual practical. These alterations resulted in a successful experiment.
Preliminary Results:
Results:
Analysis:
There is a definite correlation between the number of carbon atoms in each hydrocarbon ring, and the volume of ethanol produced. As the volume of ethanol produced increases, the number of carbon atoms in the rings also increases, and it appears to be a direct correlation.
The V/C values are calculated by dividing the average volumes of ethanol per sugar, by the number of carbon atoms. The V/C values are all within .11ml of each other, which shows that there is a direct correlation between the volume of ethanol produced and the number of carbon atoms in the carbon rings.
In this experiment, Spearman’s rank Statistical analysis can be undertaken.
The pairs of variables need to be arranged in ascending size, in this case, the volume of ethanol produced. Then each of the results are given a rank depending on the variable. Where 2 results have an identical value, the ranks that they cover, i.e 2 and 3, are added together, and divided by the number of ranks covered, i.e 2+3/2= 2.5. this value is then given to all ranks with the same value. The paired variable, in this case Carbon Atoms, is then matched to the first Variable and their rank is ascertained in the same manner. The difference (D) is then calculated by subtracting the second rank from the first rank (Carbon Atoms Rank – Volume Rank). D is then Squared to produce DxD. All of the values in the DxD column then are added together to give the ∑DxD value.
The following equation is then used to calculate the Spearman’s rank coefficient
After using this equation, the Spearman’s rank coefficient for this experiment was 0.997. This value is very close to 1, which shows that there is a definite correlation between the number of carbon atoms in each sugar ring and the volume of ethanol produced.
Spearman’s rank can also be used to accept or reject a hypothesis. In this experiment, a significance level of 5% shall be used, as this is an acceptable level for biological data. My calculated coefficient was much larger than to critical value for the 5% significance ( >0.591), therefore my hypothesis can be accepted:
Varying the type of sugar used in the fermentation will result in some variance in the volume of ethanol produced. The larger the number of carbon atoms in each sugar ring, the larger the volume of ethanol that shall be produced.
The little difference between my coefficient and the positive correlation value of 1 shows that the results have a positive correlation. The closer the calculated values are to 1, the better the evidence for a correlation. An acceptable level for correlation is said to be
The larger the number of carbon atoms, the larger number of carbon atoms that can be fixed into molecules of ethanol, hence the larger volume of ethanol as the number of carbon atoms in the rings increases. This can be supported by the results attained from both the spearman’s rank analysis, and the chart below, both of which showing a strong positive correlation.
The above chart shows the positive correlation present in this experiment. The values plotted are the averages of the volume of ethanol produced for each sugar.
The yeast cells used in this experiment must no require the entire molecule to bind to its activation site, as the values would not correlate according to the number of carbon atoms. This shows evidence that the enzymes only bind to parts of the molecule, and split that part of the molecule into fragments, ready for ethanol production. His may occur in several places on the molecule, and the intermediate products formed from the fragments.
Evaluation:
The results from the experiment were very accurate, and provided grounds for a solid conclusion, that the number of carbon atoms in a sugar ring is directly linked to the volume of ethanol hat can be produced from each sugar. The spearman’s rank correlation was chosen as it allowed me to compare the correlation between my results and the sugars from which the results were attained, and then calculate whether I should support my hypothesis or null hypothesis. In this case, I could support my hypothesis.
The results final V/C results were within .11ml of each other, which shows a degree of accuracy. However, all of the largest result was only .42ml, so the accuracy is within 26% of the largest value. The difference in each result is close enough to identify a correlation however. The V/C calculation showed that each molecule of carbon in the carbon rings allows for V/C volume of ethanol to be produced.
The volume of ethanol produced was very low, and conclusions could not be guaranteed from the differences between sugars, as he differences were so small. It is clear that lactose, a disaccharide, produced the largest volume of ethanol, due to its ability to form more molecules of ethanol than other sugars.
In future experiments, the following alterations mustbe made, in order to attain more accurate results:
- In order to attain more accurate results, the full fermentation mixture must be distilled, to ensure that all the ethanol is present in the distillation equipment, and the distillation should take place for longer. This will ensure that all of the ethanol is distilled, and total ethanol produced can be recorded instead of ethanol present in 50ml sample.
- The fermentation mixture should be heated up to a higher temperature during the fermentation. This would speed up the rate at which the ethanol is produced, and save time, by not having to run the fermentation over the period of 1 week, the suggested temperature value is around 35°C, as this is the optimum temperature for brewers yeast.
- The fermentation mixture should also be distilled for much longer, to ensure that all of the ethanol in the mixture has been removed, giving more accurate results.
- The variation in the sugars should also be increased, to provide a broader spectrum from which results could be attained. This could further compound the theory that number of carbon atoms in ring is directly linked to the volume of ethanol produced.
- More accurate measuring equipment. The measuring cylinder in which the ethanol was collected is only accurate to within 1ml and human error can often arise from misreading the volume. To overcome this, a more accurate form of measurement must be used, such as a small 5ml syringe, or 10 ml for larger volumes. The measurement levels are spaced much further apart on the syringes, so more accurate readings can be taken.
- The fermentation mixture should also undergo vacuum filtration before being placed into the distillation equipment. This will prevent any damage to the distillation equipment, and will also reduce the distillation time as there is less solid matter in the mixture.
Bibliography:
- Salters-Nuffield advanced biology A2 textbook. Very reliable source, that focuses mainly on the biological concepts of biological mechanisms. Considers ethical issues alongside the scientific issues.
- Salters Chemical ideas, Chemistry textbook. Considers the scientific aspects of chemistry, and details the processes involved. Very reliable source that provided equations and ideas about enzyme activity.
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. Reliable source that details the optimum temperature for yeast. Provides suggestions for temperatures and links to other detailed sites, none of which were used.
- Salters Chemical Storylines. Provides detailed uses of chemical processes. Stated uses for yeast and alcohol fermentation under anaerobic conditions.
- Salters-Nuffield online resources. Used to attain the method for spearman’s rank, and the critical values necessary for accepting or rejecting hypothesis. Provides techniques for multiple statistical analyses, and mathematical techniques.
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Diagram taken from this site. Provided detailed information about the processes involved in respiration, and the chemicals involved.
http://www.theartisan.net/dough_fermentation_and_temperature.htm