University of New England
Biddeford, ME 04005
The Relationship of Glucose and Stevia substrates on the Rate of Fermentation in Saccharomyces cerevisiae.
Fermentation is a process of anaerobic respiration, that cells can use to obtain energy when oxygen is not accessible. Fermentation serves an important function in the production of energy (ATP) when aerobic respiration is not possible. In yeast, and some bacteria, fermentation occurs by the intake and breakdown of sugar into carbon dioxide, ethanol and ATP. The sugar typically used in the process of fermentation is known as glucose. Glucose is a six-carbon simple sugar that drives many biological processes. It is also what is known to be used in the process of fermentation. Stevia is a sugar substitute used by many people every day. It is produced by a plant and contains a small amount of glucose within it. Although it is a natural source, it cannot not be broken down by the body, while glucose can be (Ashwell). Glucose is typically the driving force in fermentation, and stevia is never really used as the sugar to start it. To further understand this concept, we will study the rate of fermentation using solutions that include glucose, stevia, yeast and water.
To further explain the rate of fermentation differences we will run several test samples on varying substances: glucose, stevia and a control in order to see what substance increases the rate of fermentation most efficiently. This will be important because it will help to confirm the current understanding of glucose being the best driving factor in the rate of fermentation.
Other studies such as one conducted on the rate of fermentation by usage of glucose and sucrose supported that glucose was the most efficient in raising the rate of respiration (Berg et al. 2002). Due to this study not including a sugar replacement, we instead use stevia rather than sucrose to demonstrate the difference between the rate when using a pure glucose and a sugar substitute in the rate of fermentation. We can apply this by studying the amount of ethanol produced, a byproduct of fermentation, and use that to see how the rates of fermentation are increased by different substances.
Based on previous research, I predict that when measuring the rate of fermentation in yeast with glucose, yeast with stevia and the control yeast with water only then the solution of yeast and glucose will produce the highest amount of carbon dioxide and therefore have the highest fermentation rate.
Using a positive control of yeast and water, as well as negative controls such as glucose, stevia, yeast, an experiment was run to test the hypothesis above.
Fermentation solutions (n = 24) were placed in 125 ml Nalgene bottles with a magnetic stir bar. Glucose (n = 8), stevia (n = 8) and water (control, n = 8) were the treatments being tested. Yeast (0.4 g) was weighed ( and placed in all samples. Bottles then had 10 mL of 35 C water added to them through a pipette (. Solution in bottles were gently rocked until all yeast was dissolved. For glucose treatment, 0.1 g of glucose was added to the bottles and for stevia treatment, 0.1 g of stevia was added. All bottles were placed on stir plates with ethanol sensors placed inside. Ethanol data collection was completed through the use of logger lite, with experimental duration set at 3600 s and sampling rate = 6s/sample.
To allow complete oxygen consumption, yeast solutions were allowed to cellularly respire for 5 min. After 5 min, % ethanol (% of ethanol molecules present in gas chamber) concentrations were collected every 6 s for the duration of 60 min. After experimental trials were complete, data was analyzed using an ANOVA (α=0.05) in excel (V 2016).
The one-way ANOVA was used to determine statistical differences between % ethanol production among treatments.
The mean % of ethanol produced for the control (yeast and water) was 0.080 ± 0.045 % ethanol, for the glucose solution it was 0.429 ± 0.093 % ethanol, for the stevia solution it was 0.127 ± 0.017 % ethanol. The maximum number of % ethanol produced was 0.64 % in the glucose solution. The minimum number of % ethanol produced was 0.029 in the control. Glucose (0.429 ± 0.093) and Stevia (0.127 ± 0.017) had a higher % ethanol production than the control (0.080 ± 0.045).
A significantly statistical difference was found between solutions (ANOVA: p˂ 0.05).
Figure 1. A bar graph of the mean ethanol production in percent (±SD) of the control (dark blue), glucose (purple) and stevia (green) treatments over a 60 minute period.
To test the hypothesis that the glucose treatment had the highest rate of fermentation, I measured the ethanol production of glucose, stevia and a control over 60 minutes and found that the data supported my hypothesis. My data suggests that the glucose solution had the largest rate of fermentation, stevia had a lower rate of fermentation and the control had the lowest rate of fermentation which was to be expected. This could possibly be due to the underlying mechanism of the Crabtree effect. The Crabtree effect is the regulatory system that includes the repression of respiratory enzymes synthesized by high fermentation rates. The Crabtree effect consists of a repression in respiration by fermentation (De Deken 1965). It is likely that this influenced my data because glucose conducts the process of fermentation and since it is favored in the Crabtree effect then the rate of fermentation would be higher, which means more % ethanol is produced.
This finding was not surprising because it is like the findings of Berthels, Otero, Bauer, Thevelein, and Pretorius. They found that the fermentation rate of wine was increased when sugars like glucose and sucrose are present within the yeast (Berthels et al. 2004). This is similar to our findings in that when the glucose was present, the yeast produced a higher level of % ethanol produced meaning it has a higher rate of fermentation.
Our findings are important because it can impact the food industry. In the food industry, yeast is often used in order to make bread rise. Fermentation must occur to allow the yeast to make the bread rise and in using the information that glucose is the most affective in carrying out fermentation then it can be used to increase the rate of fermentation which in turn can raise the rate of bread production and effect the food industry greatly (Wilkin and Brainard 2012).
As a whole, our results helped to further support the concept of fermentation.
Ashwell M. Stevia, Nature’s Zero-Calorie Sustainable Sweetener: A New Player in the Fight Against Obesity. Nutrition today. 2015 May [accessed 2019 Mar 26]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890837/
J.M. Berg, J.L. Tymoczko, L. Stryer. Glycolysis is an energy-conversion pathway in many organisms. Biochemistry, W H Freeman, New York (2002)
N.J. Berthels, R.R. Cordero Otero, F.F. Bauer, J.M. Thevelein, I.S. Pretorius, Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains, FEMS Yeast Research, Volume 4, Issue 7, May 2004, Pages 683–689, https://doi.org/10.1016/j.femsyr.2004.02.005
Wilkin D, Brainard J. Fermentation. CK. 2018 Aug 6 [accessed 2019 Mar 26]. https://www.ck12.org/biology/fermentation/lesson/Fermentation-BIO/
Throughout the writing process of this lab write up, I encountered a lot of difficulty. I found that in some areas I was much stronger than I was in others. For the introduction and discussion portions of this lab, I was easily able to find sources and write those sections well. This was interesting to me, because going into it I believed it would be the two sections I thought would be the most difficult when in actuality it was not. I drafted the introduction first and went on to start my methods section and that is where you could say I fell apart. I had a difficult time with my wording of how I completed everything, I went into depth about every little detail including turning on a computer. Once I was finished with it I knew I had gone on too long and off on some sort of unneeded tangent. This resulted in me meeting up with my professor to grasp a better understanding of what exactly he needed me to do. When I went to him he helped me shorten it to about half the length I had had. It ended up being brief descriptions of what I had done with somehow including everything from the lab. I think even now, that will be something I need to continue to work on. The results portion was difficult simply because I have never had to deal with statistics and here it was being thrown my way. I worked my way through it after long hours of youtube videos but, it was eventually a success. I am glad not having to do another one of these this semester although I am sure I will see them in my future!