The effect of temperature on the survival of yeast cells

Ransford Asare

Biology Coursework

PLANNING EXERCISE

The effect of temperature on the survival of yeast cells.

The aim of my investigation is to find the lowest temperature that kills all the yeast cells in a suspension of either dried or fresh baker’s yeast.

I predict, as yeast cells contain enzymes and enzymes usually only functions best at about body temperature, which is 37oC. As the temperature increases from 40oC, the function of enzymes starts to slow as they begin to denature till it reaches about 50oC. Any temperature above this will denature the enzymes and therefore kill them. In this case, I also predict that the lowest temperature range that would kill all the yeast cells is roughly between 10oC and 60oC.

BACKGROUND INFORMATION

Yeast is a member of the fungus family and is a single-celled organism. Baker’s yeast as well as brewer’s yeast is similar in that they belong to the same species called the saccharomyces cerevisiae.

Yeast cells are sensitive to changes in temperature and therefore it affects its structure and activity. This is because its metabolism is carried out in the presence of enzymes which are active only in an optimum range of temperature. Higher ferment temperatures increase yeast activity and vice-versa. The optimum temperature range for yeast fermentation is between 32oC and 38oC. A high temperature at about 50oC destroys these enzymes and therefore kills the yeast cells. Other factors affecting yeast activity are pH and concentration of sugar.

Yeasts are affected by pH because its process during fermentation are catalyzed by enzymes which are proteins, and the tertiary structure of all proteins are affected by pH because the hydrogen bonds and ionic bonds which maintain the tertiary structure can be broken by extremes in Hydrogen and Hydroxyl ion concentration which is what pH measures. Yeast activity is constant over a pH of 4-6.

Yeast cells are also affected by concentration of sugar. This is because when there is a high concentration of sugar yeast cannot ferment because they become dehydrated due to the high osmotic pressure gradient created outside of the yeast cells by this high concentration. In this case I will make sure that I keep both the concentration of yeast and sugar the same so that the temperature is the only factor that affects them.

Fermentation is the name given only to the anaerobic respiration of yeasts .I think that as yeasts ferment sugar by respiring anaerobically, heat energy is produced because fermentation is an exothermic reaction which provides yeast with energy for its metabolism. Anaerobic respiration is the oxidation of molecules in the absence of oxygen to produce energy;

Glucose Lactic acid + Energy (ATP)

During fermentation, glucose sugar is converted to ethanol and carbon dioxide by the enzymes in the yeast;

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Glucose Lactic acid + Energy (ATP)

During fermentation, glucose sugar is converted to ethanol and carbon dioxide by the enzymes in the yeast;

C6H12O6 (aq) 2C2H5OH (aq) + 2CO2 (g)

As fermentation goes on at a constant rate, and as the temperature increases the reaction gets to a point, where the temperature is above the optimum that these yeast cells work best so fermentation will slow down as they become denatured and eventually die. In this case, it is important that brewers do not let their brewers yeast to reach too high a temperature and this can be done by using nitrogen.

Methylene blue is used as a for a number of different staining procedures. It is also used as an indicator to determine if a cell such as yeast is alive or not. The blue indicator turns colourless in the presence of active enzymes, indicating living cells but remains blue when enzymes are dead. Methylene blue can inhibit the respiration of the yeast as it picks up hydrogen ions made during the process. I will be noting this colour change in my investigation.

Based on what I have in my research, I will therefore construct an experiment that will help me to find out if my prediction was right or wrong and also come to a conclusion.

Materials;

5 cm3 10% suspension of yeast
5 cm310% solutions of sucrose or glucose (I will use glucose in my investigation as it is most commonly used in fermentation and is fermented more easily as it is a simple sugar. It is a source of energy for the yeasts).
2 drops of 1.0% methylene blue solution
Distilled water

Apparatus;

8 test tubes (includes 2 additional for control) also suggest an extra 8 to repeat experiment.
Test tube holder
Thermometer
Water bath
Stopwatch
Syringe (5cm3) I will be using this instead of a Measuring cylinder because it’s more accurate.
Dropping pipettes
Beakers (250ml)
Microscope
Slides and Cover slip would suggest Haemocytometer slides

RISK ASSESSMENT

METHOD

I will test out 5 temperatures; these will range from 10oC to 50oC with 10 degree intervals. My range ends with 60oC because it is reasonable as the optimum temperature at which yeast cells work best is at about 37oC and will denature at about 58oC, which means the indicator won’t change. This will give me a rough idea of the lowest temperature that kills the yeasts.

Measure out for each of the 5 test tubes 5cm3 of 10% suspension of yeast.

Then add 2 drops of 1.0% methylene blue solution to each of the 5 test tubes and mix thoroughly by swirling the test tube gently.

Set the water bath at first temperature which is 10oC and then put 1 of the test tube, labelled 10oC, with the mixture stated in steps 1 and 2 in it for 5 minutes.

To ensure that my result will be reliable the thermometer will remain in the water bath to monitor the temperature and also to ensure that it is kept constant.

After add 5cm3 of 10% glucose solution in the test tube with the thermometer and at the same time start the stopwatch. Time each reaction to see if there will be a colour change for 10 minutes.

If it remains blue then it means that the yeasts are dead but if it becomes colourless it indicates that the yeasts are still active.

Repeat these steps for all my other 4 test tubes using the temperatures 10, 20, 30, 40, 50 accordingly.

CONTROL

The two controls that I have established are; methylene blue with yeast and methylene blue with glucose. Each of these controls will be put in a test tube and then put in the water bath at a certain temperature along with my 5 other test tubes with a mixture of glucose, yeast and methylene blue. I will do this control for each of the temperatures in my range. This will enable me to compare the controls to my actual mixtures and figure out if there is any significance.

FAIR TEST

I will make sure that I use the same amount of 10% suspension of yeast and 10% glucose solution and same amount of drops of methylene blue solution for each of my test to ensure that is a fair test. I will also make sure that I use the same size test tube. Also my ratio of yeast suspension to glucose solution will be 1:1 because the yeast will be able to react with the glucose without too much competition. It is important also to carry out the experiment twice to see if the results are reliable and to make it a fair test.

RESULTS TABLE

Afterwards, I will observe my table and establish which of my test tubes with the highest temperature which is still blue, indicating that the yeasts are dead and then carry out the experiment further, this time, decreasing it by 1 degree at a time and then find the exact temperature that actually kills the yeast cells, when it turns colourless.

PRELIMINARY TEST

My preliminary test helped me in my investigation because it made me realise the errors in my method from my plan and gave me ideas of how to improve my investigation. Based on my preliminary investigation I found that it was difficult to have the same amount of methylene blue because it was hard to control the size of the drops therefore it makes my experiment unfair. I will therefore suggest using a dropping pipette which is more accurate. I believe it would make my results more accurate if the temperature in the water bath was measured as it can either rise or fall due to the yeasts activity. I carried out my preliminary without using a water bath to keep the temperature constant but instead a Bunsen burner. I think using this, causes a lot of errors and it’s not very accurate because the temperature might constantly rise above the temperature that you are testing or below and therefore it’s not reliable. I heated the test tubes for ten minutes. After that I took a sample of my solution and placed it on a slide. And then put a drop of methylene blue around the edge of the slide. I then viewed this slide on a light microscope and observed the colour of the yeasts. I think viewing it under a microscope to look for a colour change is also not very accurate because it will be hard to view all the yeasts in a microscope and count how many are colourless or otherwise blue. I will therefore be more accurate to use a haemocytometer, a specialised microscope slide with a grid which is used to count cells.

REFERENCES