What Sugar And Tea Does a Kombucha Biofilm Prefer?

This project follows the Scientific Method. Review the steps before you begin.

Experimental Background

In this experiment, you will investigate how changing the substrate (tea or sweetener) for your SCOBY biofilm changes the biochemistry of the kombucha solution. In this experiment, you will change only change one substrate at a time (tea or sweetener) to create a controlled experiment and compare these changes to your black tea and granulated sugar control solution. You will track how changing each of the substrates impacts the biofilm's fermentation process and the resulting kombucha solution by taking samples of the solution and testing some of the biochemical changes (pH, temperature, and sugar content) over time. 

Experimental Set-Up

Before you start your experiment, it is important to remember that microorganisms are everywhere. They are on your skin, on surfaces, and on your equipment. Contamination of your culture can prevent the SCOBY from growing and thriving, so cleaning your equipment well and wearing gloves is very important.

1. Put gloves on before handling your equipment.
2. Clean or sterilize your equipment, including your pot, ½ gallon jars, and measuring tools with hot water and soap to remove all microbial contaminants before creating your kombucha solution.
3. Rinse very well after washing with hot water to remove any residual soap.
4. Allow equipment to dry before use. 

Experimental Protocol

To create the kombucha solution containing your substrates, you will make sweetened tea and allow it to cool before you add in the SCOBY biofilm and the kombucha starter culture that comes with the SCOBY biofilm, which also contains additional microorganisms. Repeat this protocol for each kombucha solution. For example, the protocol will create 1 of each of the following solutions: 

• Positive control kombucha (black tea + granulated sugar substrates)
• Experimental kombucha #1 (different tea substrate + granulated sugar)
• Experimental kombucha #2 (black tea + different sweetener)

The microorganisms within the biofilm will then start the complex fermentation process, interacting with the substrates to change the solution's biochemistry over time. You will take measurements throughout this process. Depending on the surrounding environment's temperature, this fermentation process can take anywhere from 1 to 4 weeks. Let's get started making a solution!

1. Put on a new set of gloves.
2. Brew tea with the following steps:
   1. Boil 2 cups of bottled, filtered, or well water.
      1. Note: Filtering tap water removes the chlorine and chloramines from the water. These particular chemicals that help make our drinking water safe and antimicrobial, will inhibit the biofilm from fermenting the solution.  
   2. Steep 1 tablespoon of loose tea in a filter in the hot water for 5 minutes.
   3. Remove the tea leaves from the water.
3. Mix ½ cup of sweetener into the hot tea until it dissolves.
4. Add 5 additional cups of water to a ½ gallon jar. 
5. Add the sweetened tea to the ½ gallon jar. 
6. Take the temperature reading of the diluted sweetened tea solution in the ½ gallon jar. Wait for the solution to cool to room temperature (75℉/25℃).
   1. Note: Adding the SCOBY and starter culture before the solution is cooled will kill off the microorganisms in the biofilm and starter culture, preventing the fermentation process.
7. Once the diluted sweetened tea solution is at room temperature, add the SCOBY biofilm and the kombucha starter culture that came with the biofilm to the ½ gallon jar. 
8. Cover the jar with cloth when not taking measurements. This will allow the solution to interact with the air, permitting the process of aerobic fermentation.
9. Observe the solution and record your observations in a data table, like Table 1. Write down any changes you see to the solution, such as bubbles, color changes, clarity/turbidity, or the presence of a new SCOBY biofilm.
   1. CAUTION: Do not drink your experiment! Aseptic or sterile conditions and thorough testing are required to sell drinkable kombucha from the store. These conditions are not easily achievable at home, so do not drink your experimental kombucha!
   2. Note: The SCOBY may change locations during the fermentation process. This is expected since the air bubbles created during this process can cause the SCOBY biofilm to float around in the solution or adhere to surfaces and stay in place.  
10. Measure the pH of the solution using pH litmus paper. 
    1. Remove the cloth from the top of the ½ gallon jar.
    2. Carefully dip the bottom 1 cm of the litmus paper into the kombucha solution. 
    3. Remove the litmus paper from the solution and wait 1 minute for the solution to dry.
    4. Compare the pH litmus paper to the quantification scale (on the pH paper packaging).
    5. Record your observations in your data table, like Table 1 below. 
       1. Note: Variations in the substrate can impact the total fermentation time. When starting kombucha, the initial pH should be below 4.6. Commercially bought kombucha is typically done brewing when the pH is within the range of 2.5 - 3.0.
11. Measure the temperature of the room and each kombucha solution.
    1. Remove the cloth from the top of the ½ gallon jar to take your measurements.
    2. Follow the instructions for your thermometer. If using a physical thermometer, make sure the probe is fully below the solution surface. If using a noncontact infrared thermometer, take the surface reading of the solution.   
       1. Note: To prevent contamination, we recommend using a no-touch temperature probe. If using a physical probe, be sure to decontaminate it between each temperature reading by dipping it in isopropanol (rubbing alcohol) to disinfect it and let it air dry. 
    3. Measure the room temperature each time you measure the temperature of the solution. 
12. Measure the biochemistry of the kombucha solution using a refractometer.
    1. Test the refractometer's calibration by performing a baseline reading with the same water used to make the kombucha solutions. 
       1. Using the pipette, pipette up less than 0.5 mL of water.
       2. Flip up the cover plate of the refractometer to expose the prism assembly.
       3. Add 2-3 drops or enough volume to cover the surface with water to the top of the prism assembly.
       4. Slowly flip the cover plate onto the water to cover the prism assembly. 
          1. Note: For an accurate reading, the solution must completely cover the prism assembly surface without any bubbles. If bubbles form, pull the cover plate back up again and slowly lower it to remove them. If bubbles are still present, add more solution to prevent them from forming.
       5. Hold the refractometer horizontal to the ground on the rubber grip, and slowly move it up to your eye to see the reading through the eyepiece.
          1. Only for checking calibration:
             1. If the device was calibrated correctly, the white light line should align with the 0 at the bottom of the scale.
             2. If the device is not calibrated correctly, move the calibration screw to align the white light with the 0 line when the water is on the prism assembly. 
       6. Using the cleaning wipe included with the refractometer, wipe the liquid off the surface of the prism assembly and cover plate.
          1. Note: Using other types of wipes or fabric can scratch the prism assembly surface and impact your readings. If a wipe was not included with your refractometer, use soft materials such as eyeglass wipes. 
    2. Next, test the kombucha solution's sugar content and density. 
       1. Repeat the steps above but with a sample of the kombucha solution instead of water. To obtain more accurate readings, clean the surface of the prism assembly between readings with water. Add water to the surface of the prism assembly between each reading and wipe it off with the cloth provided with the refractometer. 
13. Place the cloth back on the kombucha solution after you are done taking each of your measurements. 
14. A solution's pH is an acidity scale representing a tenfold change in the concentration of hydrogen ions in a solution. Calculate the number of free hydrogen ions in the solution from the pH reading using the following equation:
    1. pH = -log[H+] Rearrange the equation to [H+] = 10^-pH to plug in the pH value to get the hydrogen ion concentration in moles per liter. 
       1. Example calculation: If the pH is 4. [H+] = 10^-pH = 10^-4 = 0.0001 mol/L
15. Take readings at least once a week. If possible, take additional measurements every 2-3 days in the first two weeks. Allow the fermentation process to take place over the next 1-4 weeks.

Conclusions

• How does the pH and hydrogen ion concentrations change over time? Make a graph to compare their relationship with time on the x-axis and a scale to include both pH and hydrogen ion concentration on the y-axis. Make a double-line graph to directly compare them. What is their relationship? Use the pH/acidity equation to help inform your answer.
• How does changing the substrates impact the solution's initial and final sugar content? Did the sugar ever run out based on your measurements? Why do you think that is? 
• Do the substrates impact the color of the solution? Does the color of the solution change over time? What do you think drives this change?
• How does the type of substrate impact the pH of the solution, the sugar content, the solution temperature, or the overall fermentation time? Make a graph comparing each of their relationships.
• How did the different substrates impact the biochemistry of the kombucha solution?