If different concentrations of caffeine including 0g, 1g, 5g, 8g are mixed into a yeast, sugar, and water solution, then the amount of carbon dioxide produced will decrease indicating less growth, as the concentration of caffeine increases, because caffeine blocks the effects of adenosine which is necessary for the cells to reproduce and increase in number.

What is Yeast?

Yeast is a type of fungi. Fungi are eukaryotic and can be single or multicellular mildews, molds, mushrooms, and yeasts, are all types of fungi. Yeast in particular is a single celled organism and commonly reproduces asexually through a process called budding. Budding takes place when a bump emerges from a parent cell, once the bump is mature it detaches itself and becomes its own cell. In nature, yeasts and fungi break down plant material and release oxygen, nitrogen, carbon, and phosphorus back into the soil. Humans regularly use yeast to make baked goods and beer. Lastly yeast is present all over the human body in small amounts, from your skin to your digestive tract.

Cellular Respiration in Yeast?

All living things including yeast produce adenosine triphosphate (ATP), it is the universal energy source for living cells. ATP produces energy to carry out energy-dependent cellular functions, including DNA and RNA synthesis, metabolic processes, and cellular division. Cellular respiration is how ATP is produced. Cellular respiration can either take place anaerobically, without oxygen, or aerobically, with oxygen. In yeast cellular respiration can be either. In the presence of oxygen, cellular respiration is when glucose is broken down, releasing energy which is captured by ATP molecules. The majority of this process takes place within the mitochondria. Water and carbon dioxide are the end result of cellular respiration.

Anaerobic respiration in yeast is known as fermentation. Fermentation is the process by which sugar is broken down by enzymes in the absence of oxygen. In the case of yeast, this enzyme is called zymase, it catalyzes the breakdown of sugar producing energy. Similar to aerobic respiration the end result is carbon dioxide but instead of water it produces ethanol. Both forms of respiration are required for growth.

How does the AMP/ATP ratio regulate yeast metabolism and growth?

The amount of adenosine monophosphate (AMP) compared to adenosine triphosphate (ATP) communicates with the cell how much energy the cell has . In yeast a protein kinase called Snf1 is activated in the presence of AMP.  When energy is low there is a high AMP to low ATP ratio, Snf1 senses this and signals the yeast to use different sources of energy instead of sugar until the environment is more stable. Without energy,  growth is put to a halt as more energy is focused on being conserved. When the cells have high energy represented by a low AMP to high ATP ratio, Snd1 is not activated allowing the cells to create proteins, fats, ribosomes, and rapid cell division.

How does Andenosine dicate Yeast's stress response?

Yeast is highly adaptable to many environments and generally performs well under stress. Within the cell, specific conditions are required to carry out cellular functions, meaning that maintaining those conditions while the outside environment changes is important. In high stress situations such as nutrient depletion, heat shock, oxidative stress, and osmotic stress, the most popular defense mechanism is burning fat as a source of energy rather than glucose. 

In yeast adenosine also dictates how it will react to stress. A receptor-like protein called G-protein coupled receptors (GPRCs) sends signals to the cell to start certain processes when they sense adenosine and its derivatives. For example when GPRCs sense a buildup of adenosine monophosphate (AMP), it indicates that the yeast is experiencing high amounts of stress due to unfavourable conditions. The GPRCs respond and signal the cells to conserve energy.

Yeast (Candida) in the human body

Yeast is naturally found in the human body including the skin, digestive system, mouth, and the vagina for anyone who has one. The yeast that is found in the body is called Candida, and is harmless at the right amounts. Healthy bacteria in the body control the balance of Candida. When the balance is off, the yeast overgrows and causes an infection called candidiasis. Depending on the location of the overgrowth it can be referred to by different names including thrush (mouth), vaginal yeast infection, and invasive candidiasis (blood).

What is caffeine?

Caffeine is a naturally occurring drug, found in coffee beans, leaves and fruit of guarana, green tea, and energy drinks, to name a few. The stimulant category is where the caffeine drug falls. Stimulants can increase the speed between the messages the brain sends to the nerves. This increased speed of messaging between the brain and the body leads to reduced drowsiness and fatigue. Negative effects of consuming caffeine can include increased dehydration, dizziness, and trembling/tremors. In some cases, caffeine can have the opposite intended effect and increase fatigue.

How does caffeine work?

Caffeine works to increase your alertness by blocking the effects of adenosine and increasing norepinephrine. Adenosine triphosphate (ATP) is a compound broken down by your body to release energy and adenosine as a byproduct. The brain consumes the most ATP, and it builds up in the space between cells. It is believed that when you stay awake for too long, adenosine buildup affects parts of the brain associated with alertness kicking in the body’s sleep drive (the urge to sleep). Since caffeine blocks the effects of adenosine, you feel more awake and alert, and the need to sleep is relieved.

Materials 

• Caffeine pills

• Instant quick rise yeast

• 2x 100mL graduated cylinders

• ¼ inch inside diameter vinyl tubing 5 feet long

• Thermometer

• Long shallow container

• Sharpie

• Scotch tape

• Distilled water

• Saran wrap

• ½ tsp measuring spoons

• 1 tsp

• White granulated sugar

• Pyrex glass measuring cup

• Fork

• Scissors

• Cup

• Packing tape

Procedure 1: Constructing the CO₂  Collection Apparatus

1. Gather all materials 
2. Label each 250mL squeeze bottle with the different concentrations of caffeine, 1g, 5g, 8g, as well as “control”.
3. Remove the cap of the squeeze bottle and push the 5’ vinyl tube onto the top of the 250mL squeeze bottle.
4. Fill the plastic container ⅓ full with water.
5. Fill the graduated cylinder with 100 mL of water.
6. Cut a piece of saranwap large enough to cover the opening of the graduated cylinder. Poke a small hole with a pair of scissors into the saran wrap and push one end of the 5’ vinyl tubing through. Cover the opening of the graduated cylinder with the saran wrap.
7. Place your hand over the opening of the graduated cylinder and quickly invert the cylinder into the container. Then peel away the saran wrap.
8. Add a piece of packing tape to the graduated cylinder to attach it to the container.
9. To test the apparatus remove the vinyl tube from the top of the 250mL squeeze bottle and slowly blow into it; check for water displacement. Bubbles and a decreasing water level should be visible.
10. If there is visible water displacement, steps 1-7 will need to be repeated before testing can begin.

Procedure 2: Conducting the Experiment 

1. Label each 250mL squeeze bottle with the different concentrations of caffeine, 1g, 5g, 8g, as well as “control”.
2. Microwave 250mL of distilled water for 30 second intervals until it reaches 40 degrees celsius.
3. Add 1 teaspoon of sugar to the warm water and stir until fully dissolved.
4. Once the sugar has dissolved add ½ a teaspoon of yeast to the warm water. Briefly stir the yeast mixture with a fork and pour into the “control” squeeze bottle. Repeat with the other squeeze bottles.
5. Cap the bottle tightly with the free end of the vinyl tubing. The end of the collection tube(vinyl tubing) should be under the inverted graduated cylinder in the dishpan.
6. Start a timer for 25 minutes. After 5-10 minutes the yeast should start foaming, and bubbles should be seen in the graduated cylinder. Record the time you first start seeing bubbles in the graduated cylinder.
7. Continue collecting carbon dioxide until the timer goes off.
8. Wash out the collecting tube and squeeze bottle. Re-set the inverted graduated cylinder (refer to steps 3-7 in “Procedure 1”). Repeat steps 2-6 from “Procedure 2” (1) more times with the “control” and other concentration when repeating.
9. Read the amount of grams of caffeine on the back of the pill package. Open the capsules and collect the amount of powder to have 1 gram of caffeine (or 5 and 8g when repeating).
10. Microwave 250mL of distilled water in 30 second intervals until it reaches 40 degrees celsius. Add 1 tsp. of sugar and the caffeine powder and wait until everything fully dissolves.
11. To the water, caffeine, and sugar solution, add ½ tsp of yeast. Pour the mixture into the appropriate squeeze bottle and repeat steps 5-8
12. Repeat steps 7-10 with all the other concentrations.

Trial 1

Concentration: 0g

Concentration 1g

Concentration: 5g

Concentration: 8g

Trial 2

Concentration: 0g

Concentration: 1g

Concentration: 5g

Concentration: 8g

Trial 1

From the data, the test that produced the most CO₂ was “Concentration 0g test 1 (Con.0gT1)”. The tests that produced the least CO₂ were “Concentration 8g test 1 and 2 (Con.8gT1 and Con.8gT2)” and “Concentration 5g test 1 (Con.5gT1)”. “Con.0gT1” producing the highest amount of CO₂ and “Con.8gT1 and 2” producing the lowest was expected, but “Con.5gT1” also produced 0mL of CO₂ which wasn’t. 0mL of CO₂ being produced for any test is unlikely, the data appearing this way is likely due to errors in the experiment's design. The experiment allowed each test 25 minutes to produce and collect CO₂. Since the CO₂ was produced anaerobically through fermentation, as the caffeine concentration increases, 25 minutes isn’t enough time for the yeast to metabolize the total sugar. In the case of “Con.5gT1” and “Con.8gT1 and 2”, this shows that caffeine slows the rate yeast metabolizes sugar, therefore decreasing the rate CO₂ is produced. This is proved as “Con.0gT1 and T2” produced and collected 22 and 18mL of CO₂ in 25 minutes. Even then, the “Control” tests still are not the most accurate representation of the total amount of CO₂ produced. An experiment done by the University of Waterloo where 20g of white sugar and 7g of Quick-Rise yeast were mixed in 100mL of 43 degrees Celsius water, showed the solution steadily produced CO₂ for 700 minutes (11.7 hours). After 700 minutes CO₂ was produced at a slower rate for 300 minutes at 1000 minutes, 16 hours, CO₂ production evened out but the line was still on an upward trend; the graph ended at 1000 minutes. Based on the data collected from their experiment, it could be up to 490 minutes, 8 hours, of steady increased CO₂ production. After 490 minutes CO₂ production would still last for hours more at a slower rate. This number is only for the control tests, meaning the tests with caffeine would produce at a slower rate increasing the overall time. Thus proving the amount of time allotted to the tests to collect CO₂ a major flaw in this experiment. If the time had been adjusted to 24 hours the results would be different, and overall more accurate.

Another reason for the inaccuracy of the data collected potentially comes from the design of the collection apparatus. The collection tube that was connected to the squeeze bottle containing the yeast solution(s) and placed in the graduated cylinder was 5 feet in length, which was potentially too long. 5 feet is a long distance for a small amount of CO₂ to travel. When higher amounts of CO₂ are produced at a faster rate such as the “control” tests there is more pressure in the tube, moving the CO₂. During “Con.5gT2” and “Con.8gT1 and T2” it is likely the opposite occurred ; a small amount of CO₂ produced at a slow rate doesn’t create the pressure needed to push it through the 5 foot tube. In terms of “Con.5gT1” CO₂ bubbles were present at 24 minutes, this could mean they were created earlier but 24 minutes is when they successfully traveled through the tube. The solution for this would be reducing the length of the tube. When considering the length, it needs to be long enough to be in the graduated cylinder to a minimum height of 50mL, as well as, still bend and attach to the squeeze bottle. Due to these requirements a length no shorter than 1.5’ or longer than 2’ would be suitable.

Trial 2

After running the experiment again, certain test produced simmilar results as in "Trial 1", others produced vastly different results. In "Trial 2" 0g concentration, 21mL of CO₂ was collected for test 1, and 23mL for test 2. This is comparable to the CO₂ collected in the "Trial 1" 0g concentration, which was 22mL for test 1 and 18mL for test 2.

In "Trial 2", two greatley different results were produced in the 1g concentration. In test 1, 1mL of CO₂ was collected. This is inconsistent with the 8mL collected in "Trial 1" test 1 and 2, and the 9mL collected in "Trial 2" test 2. When comparing observations between the tests and the trials, a key detail takes place at 20 minutes. At 20 minutes in "Trial 1" (1g) test 1, there was no visible height increase of the yeast blooms on the bottom of the squezze bottle, but more pronounced air bubbles in the blooms was noted. Although, there was no visible growth, the bubbles indicate otherwise. This was also  observed in "Trial 1" (1g) test 2, there was no visible height increase in yeast bloom height, but bubbles started to exit the collection tube at 20 minutes. Prior to this, at 15 minutes more pronounced air bubbles were noticed in the yeast blooms. In "Trial 2" (1g) test 2, bubbles were exiting the collection tube at 20 minutes, and had been since 3 minutes prior. As well as, both the bottom and surface yeast growth continued to increase in height. In "Trial 2" (1g) test 1, at 20 minutes, there was no visible increase in height for both the surface and bottom yeast blooms. No other indicators of growth were observerd. It was not until 23 minutes when bubbles started exiting the collection tube. Throughout the obersvations one trend is apparent, at 20 minutes all the tests of the 1g concentration had signs of growth, besides "Trial 2" test 1. The other tests had been more active, and for longer periods of time, producing more CO₂ in the time alloted. Test 1 "Trial 2" showed no visible signs of growth at 20 minutes, and started having bubbles exit the collection tube at 23 minutes, allowing for only 2 minutes of collection time. The reason for the lack of yeast activity is unclear, as the procedure for this test was conducted the same, and no sources of error were made. 

"Trial 2" 5g concentration test 1 and 2 produced comparable results to "Trial 1" test 1 and 2. In "Trial 1" (5g) test 2, 1mL of CO₂ was collected, which is more than the other tests, but not extremley different. When looking at the observations, at 15 minutes it was noticed that the bubbles in the yeast growth were more pronounced, which was not noticed in the other tests. Still, the variation is minor.

"Trial 2" (8g) test 1, has a noticeable outlier. With all the other 8g tests having 0mL of CO₂ collected, and "Trial 2" test 1 had 8mL collected. One difference between this test ("Trial 2", 8g, test 1) and the others, is a source of human error. When mixing the solution for this test, the stirring step of the caffeine was missesd. The caffeine had been added to squeeze bottle, followed by the sugar and yeast water. This was done to ensure the full caffeine amount was in the bottle, as other tests had been overflowing when pouring the yeast, caffeine, and sugar solution. In doing so, trying to solve the issue, of not all the caffeine being in the squezze bottle, caused inconsistencies later on. As a result, the caffeine was not mixed throughout the water, and the yeast had minimal contact with it. If this had not been done, the results would likely be the same as the other 8g concentration tests. The observations  are not comparabable, as the issue started before the timed testing. 

Changes to the Experiment in the Future

The biggest changes I would make to this experiment in the furture, would be in the design of the  apparatus. As mentioned previously, the length of the collection tube should be shortened to 2 feet minimum, and no shorter than 1.5 feet. This would allow for more accuracy in the data as the CO₂ produced, would not have to travel a long distance. Second, the 100mL graduated cylinder should be replaced by one with more volume, between 250-500 mL. This would allow for a longer period of examination, without all the water being displaced. Third, changing the consturction of the apparatus, so that the collection tube could be straight, and not coiled. By elevating the container to be at the height of the squeeze bottle, the collection tube could be straight. Gas travels in a straight line until it hits something, by straightening out the collection tube, the idea is there will be less curves for the gas to bounce off of, allowing for the CO₂ to travel quicker through the tube and provide more accurate results. Lastly, as for the design of the apparatus, squeeze bottle with a slightly larger volume than 250mL should be used. This would allow for more yeast growth within the bottle before it enters the collection tube and, for all the caffeine to be present in the squeeze bottle without overflow.  One change that should be maade to the design of the experiment, would be to increase the the collection period from 25 minutes, to 24 hours. This allows the CO₂ more time to be made and collected, which would give more comparable, and accurate results. 

In conclusion, the hypothesis was incorrect.  This is because the hypothesis predicts that as caffeine concentration increases, overall CO₂ production will decrease, not the rate which is what was seen in the tests. Based on the data collected, this experiment proves that caffeine decreases the rate CO₂ is produced. In terms of the overall CO₂ collected there was not enough time for the tests with caffeine to produce CO₂.  Although, the outlier in "Trial 2" 8g concentration test 1 challenges all of these ideas, and it is unkown if the result is completley due to human error. By making the changes suggested in the analysis, more accurate and comparable data would be collected, allowing for a final, confident verdict on the hypothesis. 

Worldwide 1 in 3 people suffer from an overgrowth of Candida fungus, which includes but is not limited to thrush, vaginal candidiasis, and invasive candidiasis. Although Candida overgrowth isn’t inherently severe for healthy people it can pose a huge risk to people with compromised immune systems from cancer, HIV, diabetes, anemia, young children, and people over the age of 65. Furthermore the rise of antifungal and antibiotic resistance, means the medications prescribed to treat the infection are becoming less effective. Without proper treatment the relatively mild and easy to treat Candida overgrowth can turn into the acute condition, invasive candidiasis.

Invasive candidiasis is a major fungal infection that can become life-threatening without the proper diagnosis and treatment. It develops when Candida enters the bloodstream, this is called candidemia, and spreads to other parts of the body such as the bones, central nervous system, liver, and heart. Candidemia is one of the most common healthcare-associated infections, putting people receiving treatment at hospitals for other conditions at risk. Among hospital patients, premature babies, illegal drug users/abusers, people with vein catheters, people of colour, and anyone with a weakened immune system are at risk of Candidemia. Symptoms of invasive candidiasis include abdominal pain, chills or fevers, low blood pressure, muscle aches, skin rashes, and weakness or fatigue. Depending on the location symptoms can vary. For example, invasive candidiasis in the eye can produce symptoms such as blurred vision and sensitivity to light. These symptoms make the condition difficult to diagnose, as the majority of people developing the condition are recovering or simultaneously have illnesses that present the same symptoms. In 2015 the mortality rate for invasive candidiasis was 30-60%, per the National Health Institute.

One issue that could be worsened by invasive candidiasis is leaky gut syndrome. Leaky gut syndrome is a hypothetical condition, as there is still debate and not enough information to determine whether it is a symptom or its own condition. Everyone's gut is semi permeable and designed to absorb water and nutrients from consumed food. Leaky gut syndrome is the result of increased permeability or hyperpermeability, resulting in the gut absorbing more than water and nutrients. Because it causes hyperpermeability it is easily susceptible to bacteria and fungi in the bloodstream such as invasive candidiasis. Without treatment invasive candidiasis will spread everywhere including the gut, but leaky gut syndrome could increase the rate Candida is absorbed and spread. 

Caffeine could be used as a topical treatment to treat Candida. Caffeine molecules are small enough to penetrate the skin. Within five minutes caffeine can penetrate the epidermis layer of the skin. For caffeine to be used as a topical treatment for Candida, the concentration would need to be higher than what was used in this test. A 2022 study conducted by the Scientific Research Department at Imam Abdulrahman Bin Faisal University, found that caffeine inhibited the growth of Candida biofilms on dentures after 24-48 hours. They found the most effective concentration to be 60 mg/mL. Although caffeine has not been used as a topical antifungal for skin Candida, their experiment confirmed its antifungal properties, and this experiment confirmed that it slows the rate of growth. Between both experiments, caffeine could be an antifungal treatment or additive in the future.

Caffeine is also significantly cheaper than current prescription antifungals. Worldwide it is estimated that 3.8 million people died due to fungal infections, in 2023. This is more than how many people died (630,000) from AIDS related illnesses, in 2023. If caffeine ever developed into its own topical antifungal treatment, it could make antifungal treatment more accessible to impoverished communities. As an additive, it could allow for less of the main active ingredient to be used, also lowering the costs.

• When pouring from the graduated cylinder into the Pyrex cup some water splashed out, since an unspecified amount came out it could not be replaced (Concentration 0g test 2 "Trial 1").
• When opening one of the caffeine capsules it exploded and some of the powder could not be collected (5g caffeine collection).
• When pouring the 5g solutions into the squeeze bottles from the Pyrex cup, there was a multitude of caffeine that had to be scraped out, so the entire 5 grams was not in the squeeze bottle.
• During the 5g concentration test 2 ("Trial 1"), there was some leaking that occurred from the top of the squeeze bottle for about 5 minutes.
• When carrying the squeeze bottle for concentration 8g test 1 ("Trial 1") some solution spilled out (approximately 5mL) as it was very full.

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I acknowledge my gratitude towards my science teacher, and science fair cordinator Ms. Burkell. Her help, feedback, and dedication has been invaluable. I also thank Mr. Bykovskikh and his fiance, aswell as Ms. Burkell for helping me pivot when challenges arose in designing my experiment. I also thank Dr. Rose for his feedback, and mentorship. Lastly, I thank my family for asking questions, providing suggestions and encouragement. All the support I have received has made my project better, aswell as made me a better scientist.