# Temperature experiments with the cerevisiae population

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## Testing Hypothesis 1:

Comparing the different temperatures that the S. cerevisiae population left to grow, it can be seen based on both the cell concentration and the graph that below 30oC the population tends to grow more as the temperature increases; the yeast population almost doubles when temperature increases from 5oC to 15oC and almost triples when temperature increases from 15oC to 30oC. Above 30oC the growth of the population is highly decreased; yeast population becomes almost 3.5 times less when temperature increases from 30oC to 50oC and when temperature increases from 50oC to 60oC the population decreases very slightly. As a result, the highest S. cerevisiae population growth is observed at 30oC. Consequently this should be the optimum temperature. Moreover below the optimum point, as temperature increases the population increases more from its initial value than it does at temperatures above the optimum point. Also, it can be seen that the standard deviation varies between different temperatures. The standard deviation in 15oC (7146.4 cells/mL) is higher than 5oC (4682.8 cells/mL). The standard deviation at 60oC (5449.4 cells/mL) and 50oC (5180.7 cells/mL) is very close. The standard deviation at 30oC (24235.5) is the highest of all the other temperatures. Overall the hypothesis 1 is confirmed.

Testing Hypothesis 2:

Evaluating the yeast population growth at the different pH levels, it can be seen that the increase of population above and below the value of pH 6 is almost the same. The fact that at pH 6 it is observed the highest population growth implies that this is the optimum pH level. The lowest growth is observed at pH 3 and pH 8. In these specific pH levels the growth is slightly higher at pH 8 (population increases approximately 1.7 times) than it is at pH 3 (population increases approximately 1.3 times). The growth is higher in pH 8 as it is closer to the optimum pH. At pH 4 the increase in population is almost the same as it is at pH 8. Both pH 4 and pH 8 differ by 2 pH levels from the optimum level but the yeast population at pH 4 increases approximately 1.982 times where at pH 8 the population increases 1.7 times. This shows that S. cerevisiae operates better at acidic conditions. The standard deviation at pH 3 (4621.4 cells/mL) is lower of that at pH 8 (5855.4 cells/mL) and pH 4 (9470.8 cells/mL). Moreover the standard deviation of pH 4 is higher than it is at pH 8. The hishest deviation is observed at pH 6 (14029.9). Overall hypothesis 2 is confirmed.

Testing Hypothesis 3:

Analysing the growth of S. cerevisiae at different glucose concentrations and for 24 hours of fermentation, the results obtained show that in the absence of glucose (0 mL glucose) from the culture the yeast population didn't increase at all. The only increase that was observed from its initial population was 1.091.1 times, meaning that this 0.1 increase may have occurred due to the capacity of energy within the yeast cells. At 1mL glucose concentration it was observed sufficient growth. The yeast population almost doubled from its initial value (increased approximately by 1.8 times). In higher glucose concentration (2mL glucose) the yeast cells population respond greater and as a result a higher population growth was observed. The initial population increased 3.9 times, meaning that almost quadrupled. In even higher glucose concentrations (3 mL glucose) the population increased highly again but not enough so to be able to say that at 24 hours of fermentation S. cerevisiae requires more energy to reach the maximum replication capacity. The population increased 3.954.00 times, almost the same of that of 2mL concentration. Moreover, based on the graph plotted for glucose concentrations, it can be seen that after 2mL of 2% glucose concentration the yeast population reaches plateau without any further increase. So the limiting growth glucose concentration is at 2mL. The standard deviation at 0mL (5642.6 cells/mL) is lower than it is at 1mL (6812.5 cells/mL). At 2mL the standard deviation (8855.0 cells/mL) is the highest of all. The standard deviation at 3mL (5780.2 cells/mL) is lowest than it is at 2mL. Overall the hypothesis 3 is confirmed.

Testing hypotheses 1, 2, 3:

Based on the three different hypotheses it can be clearly seen that in conditions in which there is highest growth of S. cerevisiae cells there is highest standard deviation. At optimum conditions where it is observed the highest growth, there is also the highest standard deviation. In conditions where there is little growth there is also small standard deviation and increases when conditions improve. In hypothesis 3, where the standard deviation is smaller in 3mL (5780.2 cells/mL) than in 2mL (8855.0 cells/mL) besides the fact that was almost equal high growth, shows that in 3mL there was more sufficient glucose and hence more cells performed good and as a result there was more equal distribution. A possible explanation for the diversion of the rest of the factors could be that: Since the overall growth conditions were not ideal in all nutrients, some cells performed better and some worse. This is more obvious when the yeast grows at the optimum pH, Temperature and Glucose. The standard deviation increases at the optimum conditions compared to the other pH, temperatures and glucose concentrations where it is smaller. This shows that growth is variable even though these factors are optimum because other nutrients are needed for consistent growth.

## 4.3 Weaknesses and Improvements

Weakness

Improvement

In the populations of yeasts cells that were counted in the microscope, there were both alive and dead cells

or denaturated cells.

A dye such as methylene blue could be used to determine in each counting the live and the dead or inactive cells. The cells which would remain colourless would indicate enzyme activity and the dead or denaturated cells would be turned into blue.

Methylene blue should be used only after the fermentation has finished because it inhibits the yeast cells by consuming the hydrogen ions that are produced during respiration.

The test tubes, where the yeast cultures were left for fermentation, were slightly closed on the top with cotton in order to prevent the entrance of other microorganisms. This cotton plug prevented the easy flow of fresh air inside the test tube. This limited the availability of oxygen supply that the yeasts required in order to grow aerobically.

The test tubes can be placed to ferment aerobically in a closed container such as BioFlo 3000. This kind of bio processing systems provide a wide range of options that enables the researcher to adjust a standard air flow which includes different options of certain proportions oxygengasair which can respond to oxygen-demanding yeasts.

There was absence of some basic element sources in every yeast culture that are necessary for better fermentation conditions such nitrogen and phosphorus sources. This leadsto relatively low cell growth comparing to the growth that could be achieved without the absence of such elements.

Bacto-peptone can be used as an organic nitrogen source. Yeast extract makes available many bio nutrients required for the fermentation of yeast cells. It also provides essential water soluble vitamins, amino acids, peptides and carbohydrates.

## Chapter 5: Conclusion

After the examination of S. cerevisiae cells at all of different growth factors of temperature, pH, substrate (glucose) concentration, it can be finally stated that the cells of this yeast species show higher growth at the following conditions:

Temperature: 30oC

pH level: pH6

Substrate concetration: Glucose concentration of 2mL

Also through this research was deduced that as fermentation conditions improve the cell growth increases and standard deviation increases as well. Furthermore, at optimum conditions the standard deviation is the highest as a result of variation between cells.