How The Use of Yeast Enzymes to Glucose Substrate Has Improved Fermentation Processes

Abstract

This paper identifies the proper methods and procedures used to obtain clear data proving our hypothesis that carbon dioxide levels increase as you add a yeast enzyme to a glucose substrate. The data in the paper shows correlation; specifically that over time carbon dioxide levels rise. The causation in the paper can be deduced by the independent variable, meaning the more yeast in the mixture, the more carbon dioxide. This causation is absolute due to our precision during the process of the experiment, and it also contains sources from the Columbia University Press’s online encyclopedia, the Journal of Applied Microbiology and Biotechnology, and the Journal of the Institute of Brewing.

Introduction

“Fermentation, process by which the living cell is able to obtain energy through the breakdown of glucose and other simple sugar molecules without requiring oxygen.” (Columbia 2016) In this lab, the researchers will experiment with the levels yeast in a mixture of distilled water and glucose. With yeast being the independent variable, researchers will manipulate the amount of yeast in each mixture, while measuring the amount of carbon dioxide. Researchers will hypothesize what the data will prove, and conclude with whether their hypothesis was proved correct or disproved.

“Aristotle believed that grape juice was an infantile form of wine and that fermentation was, therefore, the maturation of the grape extract. Interest in the process of fermentation has continued through the ages, and much of modern biochemistry, especially enzyme studies, has emerged directly from the early studies on the fermentation process.” (Columbia 2016) Fermentation is also a key process in many modern industries, such that “One of the keys to obtaining better control over flavour formation may be the concentration of dissolved carbon dioxide, which has inhibitory effects on yeast growth and metabolism” (Shen 2003)

Lastly, regarding the amount of glucose. “Glucose repression is the effect conferred by high concentrations of glucose on many strains of Saccharomyces yeasts which renders various substrate utilizing systems inactive.” (Cambridge 1992)

Our hypothesis was that the more yeast enzyme [independent variable] is added, the more carbon dioxide each tube will produce. The independent variable being tested was the yeast enzyme, and the dependent variable was the carbon dioxide each tube produced, measured in centimeters.

The control group is test tube #1, which contained no yeast. There were four constant variables, those being time, the temperature of the hot water bath, the amount of glucose substrate, and the solvent of the mixture, being distilled water.

Materials

• Wax pencil
• 3 fermentation set ups (3 glass beakers and 6 large test tubes)
• 3 rubber stoppers with attached tubing
• Distilled water
• 50% corn syrup solution (original stock diluted in water by 50%)
• Yeast solution (7 grams yeast dissolved in 50ML distilled water)
• Metric Ruler
• Watch or timer
• Hot water bath (45 degrees Celsius)

Methods

1. Gather the materials for 3 fermentation set ups. (Each set up requires a glass beaker with 2 large test tubes and a rubber stopper connected to a piece of tubing.)
2. Use a wax pencil to label the 3 clean test tubes as 1, 2 and 3 then set them aside.
3. Take the other test tubes, the rubber stopper assemblies and the beakers to a hot water bath. Insert the open end of the tubing into one of the test tubes.
4. Keeping the rubber stopper dry (out of the water bath), carefully submerge the beaker, the test tube and the tubing into the hot water bath; fill the beaker and the tube with water. While under water, slip the test tube with the tubing into the beaker so that the collection tube will be upside down.
5. Bring the beaker set up out of the water bath. One end of the tubing should still be inserted inside the upside collection tube. Hold up the end of the tubing, which has the rubber stopper so the water won’t siphon or leak out.
6. Pour some water out of the beaker so the water level is at least 1 cm from the top of the beaker.
7. Assemble 2 more fermentation set up the same way before proceeding by repeating steps 3-5.
8. Add the fermentation solutions to each of the (upright) yeast fermentation test tubes according the Table 1. Be sure to add the yeast solution last.
9. Table 1: Mixture composition
10. Swirl each tube gently to mix the reactants. Place the corresponding tube in each beaker set up.
11. Put rubber stoppers into the fermentation tubes, which should force most of the water out of the tubing.
12. Wait at least one minute to allow the bubbles from the tubing to settle. There should be some air space in the base of the upside gas collection tube. If the space does not completely fill the curved bottle of the tube, wait a little longer of blow some air into the tube through another piece of tubing.
13. Check the connected tubing for kinks (bends). The carbon dioxide gas must be able to get flow into the connected tubing.
14. Mark the water level (base line) on each collection tube with a wax pencil. You will always measure the water displacement (in mm) from the base line for each time point.
15. Place ALL 3 set ups into the hot water bath. Record the water temperature.
16. At 5 minute intervals, measure the distance FROM the baseline mark to the NEW water level. Continue taking data every 5 minutes for at least 20 minutes. If the collection tube fills with the carbon dioxide gas completely before the 20 minutes then record the distance and the time the tube emptied its water. Record your data in Table 2.

Results/Data

The end results are as follows [after twenty minutes]: Tube #1 produced no carbon dioxide, Tube #2 produced 2 cm of carbon dioxide, and Tube #3 produced 10 cm carbon dioxide.

Table 2: Incubation time (minutes)

Table 3: Carbon Dioxide v. Time

CONCLUSION: The date clearly supported the hypothesis. We hypothesized that the more yeast was added, the more carbon dioxide would be produced. As seen in table 3, the higher the yeast levels, the more carbon dioxide was produced. As seen also in the data after 20 minutes, tube #3 (3ml yeast) producing almost 5 times what was produced in tube #2 with only 1ml yeast.

Given the experimental conditions tested, the only way the carbon dioxide levels could vary is due to the amount of yeast. With 3 ml glucose substrate per tube it is clear that the reaction that took place increased when more yeast was added.

There were no unexpected results, the data matched perfectly with our hypothesis, and everything went as planned. There were also no foul ups, we followed instruction precisely.

If we were to repeat the experiment, I would use consistent distilled water levels in each of the test tubes, I would also experiment with ways to measure carbon dioxide output, such as PSI, MOL, or CM3, and lastly I would increase the span of time the experiment was conducted.

Yeast fermentation is crucial in the production of alcoholic beverages, something cultures around the world have been perfecting for centuries even “Aristotle believed that grape juice was an infantile form of wine and that fermentation was therefore, the maturation of the grape extract” (Columbia 2016). Our results show just how little was known about fermentation in the old world.

Other researchers have studied not only the history of the fermentation process, (Columbia 2016), but also how to improve the performance of the yeast enzyme fermentation performance the most relevant research found is pertaining to beer flavors, where researchers hypothesized that “one of the keys to obtaining better control over flavour formation may be the concentration of dissolved carbon dioxide” (Shen 2003)