Grow Vegan Kombucha Leather

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

Growing the Kombucha Leather

1. You will make two kombucha recipes with different sugar concentrations. The ingredients for each recipe and their amounts are provided in Table 1.
   

   Ingredients	Recipe 1	Recipe 2
   Water (filtered or bottled)	5 cups	5  cups
   Teabags (black or green)	2	2
   Sugar	66 grams	16.5 grams
   SCOBY starter culture	1/2 cup + 1/2 pellicle	1/2 cup + 1/2 pellicle

   Table 1. Kombucha recipes.
2. Use tape and a marker to label the two pots "Recipe 1" and "Recipe 2."
3. To each pot, add five cups of water and bring to a boil.
4. Switch the oven off and add two teabags (or two teaspoons of loose-leaf tea) to each pot. If you use loose-leaf tea, use a tea infuser so you can remove the tea later. Let the tea infuse for 10–15 minutes. Then remove the teabags or tea leaves from the pots.
5. To the pot labeled Recipe 1, add 66 grams of sugar. To the pot labeled Recipe 2, add 16.5 grams of sugar. Use spoons to stir until all sugar is dissolved.
6. Place the pots back on the stove and let the sugared tea boil for about 20–30 minutes with the lid on. Boiling helps sterilize your tea to avoid the growth of any unwanted bacteria.
7. Let your tea cool to room temperature, or below 30°C. You can use a thermometer to measure its temperature if you are unsure. Make sure to sterilize the thermometer with rubbing alcohol first.
8. While the tea is cooling, label and sterilize your growth equipment.
   1. Use tape and a marker to label the two containers "Recipe 1" and "Recipe 2."
   2. Spray the insides of your growth containers with rubbing alcohol or isopropyl alcohol and rub the bottom and sides of the container with paper towels. Let the alcohol evaporate.
   3. Sterilize a 1/2 cup measuring cup the same way.
9. Clean your hands with rubbing alcohol. You don't want to risk contamination of your kombucha tea.
10. Once the tea has cooled, pour the contents of the Recipe 1 pot into the Recipe 1 growth container. Then pour the contents of the Recipe 2 pot into the Recipe 2 growth container.
11. Add the starting culture to each growth container.
    1. The starter culture consists of liquid as well as a solid pellicle. You need to transfer both to your kombucha batch.
    2. To each growth container, add 1/2 cup of the liquid kombucha starter culture. It is ok to add floating bits of solid as well.
    3. Add 1/2 of the solid SCOBY pellicle to each growth container. You can either use clean hands to separate the SCOBY pellicle or use sterilized scissors to cut the SCOBY pellicle in half. Figure 3 shows how the sugared tea with the starter culture should look.
    Image Credit: Svenja Lohner, Science Buddies / Science Buddies
    Figure 3. Growth container with sugared tea containing the kombucha starter culture, including the liquid and the solid pellicle.
12. Use pH paper to check the pH of your solutions. The starting pH of kombucha needs to be lower than 4.5 to create the optimal environment for the kombucha bacteria and yeast. During biofilm growth, the pH will drop even lower due to the acidic fermentation products. Finished kombucha has a pH between 2.5 and 3.5. You can add apple cider vinegar to your kombucha batch to adjust the pH.
13. Cover each container with cloth and secure it with a rubber band. The cloth should let air through and not touch the liquid. Alternatively, you can close the containers with plastic cling wrap. Poke some holes into the plastic wrap with a fork so air can get through.
14. Carefully place your containers in a dark place where they will not be disturbed for a couple of weeks. The growth temperature should be 16–29°C (60–85°F). The optimal temperature for growth is 27°C (85°F). Colder temperatures increase the risk of mold growth. If necessary, you can put your growth containers on a heat mat to keep them at the ideal temperature. Make sure the containers are not exposed to direct sunlight, since the SCOBY grows better in the dark. Airflow to the growth containers is also important because the SCOBY needs oxygen to grow.
15. Check your kombucha cultures every two to three days for growth. Make a data table like Table 2 for each kombucha recipe and record any observations you make. In the first week, you can expect to see initial patches of a biofilm growing on the surface of the solution. Over time the patches should grow into a solid layer of biofilm. Figure 4 shows an example of kombucha biofilm at different growth stages.
    Image Credit: Svenja Lohner, Science Buddies / Science Buddies

    In the first week, the biofilm forms patches on the surface. The second week, it covers the surface. The fourth week, the biofilm layer is thicker.

    
    Figure 4. Top view of the kombucha biofilm at different stages during the growth phase.

    Every time you check on your cultures, remove the cloth from the container. Then complete the following steps:

    1. Look for any biofilm growth on the surface of the solution. Write in your data table the approximate percentage of the surface that is covered with biofilm. Once the full surface is covered with biofilm, look at the sides of the container and measure the thickness of the biofilm with a ruler. The biofilm thickness might vary along the length of the sheet. The first time you measure biofilm thickness, pick the area where the biofilm is thickest. Then measure the thickness at the same location every time. Record the measurements in your data table.
    2. Check whether bubbles are accumulating under your biofilm sheet. The bubbles are CO₂ gas produced from fermentation. They will be visible as raised areas or lighter areas under the biofilm sheet. With disinfected hands, carefully massage the bubbles to the edges of the sheet to push them out. Be careful not to push the sheet under the liquid or let too much liquid stream on top of the sheet. If the sheet sinks to the bottom of the container, it won't grow anymore, and another biofilm will form on the surface of the liquid.
    3. Look for any mold growth. If your kombucha batch gets contaminated, unwanted bacteria or fungi can grow. Sometimes it is difficult to distinguish mold from healthy kombucha. You can check this Does My Kombucha Have Mold? resource to see examples of kombucha mold and get tips on how to prevent it. If your kombucha shows mold growth, you must discard it and start a new batch.
    4. Once you are done with your kombucha observations, put the cloth back on both containers.

Harvesting and Processing the Kombucha Leather

Wait until one of your kombucha biofilms is at least half a centimeter thick, as shown in Figure 5, before harvesting both. The biofilm sheet will shrink significantly during the drying process. Thinner biofilms might not have a leather-like consistency; they may be more similar to tissue paper or printer paper.

Image Credit: Svenja Lohner, Science Buddies / Science Buddies

The lower layer is liquid kombucha tea. The top layer, which is about half a centimeter thick, is a light brown solid biofilm.

Figure 5. Side view of a kombucha biofilm mat grown for 4 weeks.

1. Once one of your biofilms reaches a thickness of 1/2 cm, harvest both biofilms. Remove the cloth from the container and, with disinfected hands, carefully take the biofilm sheet from the liquid. Note: You can use the remaining liquid as the starter culture for a new kombucha batch.
2. In the sink, rinse and wash the biofilm sheet with lukewarm water and a little bit of dish soap.
3. Place your biofilm sheet on a wooden board and measure the final thickness of the wet biofilm. Record your results in the data table.
4. Let the biofilm on the wooden board dry.
   1. It might take several days to dry at room temperature. To speed up the drying process, you can place your biofilm sheet close to a radiator or in the sun. However, you don't want to dry it at high temperatures or in the oven; otherwise, it will wrinkle.
   2. Flip the sheet every day to make sure it does not stick to the board while drying.
   3. When the sheet feels dry to the touch and it doesn't have a strong vinegar smell anymore, the drying process is complete, and your kombucha leather is ready! Dry both sheets to the same consistency. This might take longer for one sheet than the other. If you dry the sheet for too long, it will become brittle.
5. The next step is to condition your kombucha leather. Apply coconut oil to both sides of your leather and rub it in with your hands. Use enough oil so the material feels very greasy on both sides and becomes more flexible. Note: If you notice that some parts of the sheet (for example, the edges) have dried but the rest of the sheet has not, start conditioning the dried parts of the sheet. If you wait until the whole sheet is dry, the edges might already have dried out and become brittle.
6. Let the oil-coated leather sit for about 24 hours. The oil prevents the biofilm from drying out and becoming brittle.
7. Take a paper towel or clean cloth and rub the excess oil off both sides of the leather sheet. Start in one small area and work your way through both sides of the whole sheet. On each spot, you want to apply a little bit of pressure and friction to create some heat so your paper towel can absorb the excess oil.
8. Now your kombucha leather sheet is ready for testing.

Testing the Kombucha Leather Properties

To assess how the kombucha's sugar concentration affects the material properties of your leather, you will do several tests to determine various mechanical properties of your different leather sheets. Follow the written procedure below or watch the material testing video to view each testing step.

https://www.youtube.com/watch?v=dt_eIeKJYbE

1. Qualitative description. Before you start cutting your leather sheets for testing, look at each of your sheets closely and make notes about how the material looks, feels, etc. Record your observations in a data table like Table 3.
   
   

   Sample	Qualitative description of the leather sheets
   Recipe 1 66 g sugar
   Recipe 2 16.5 g sugar

   Table 3. Data table to record the qualitative features of the different biofabrics.
2. Tensile test. In this test, you will investigate how your different leather sheets behave when they are stretched by measuring their tensile strength. Tensile strength is the load or force at failure divided by the cross-sectional area of the sample. Its unit is pascals [Pa] or megapascals [MPa].

   Equation 1:

   $$Tensile\: strength\: = \frac{Force [N]}{Cross-sectional\: area\: [m^2]}$$

   To measure the tensile strength, make a data table like Table 3 in your lab notebook and follow steps a–g to test both kombucha leather sheets.

   1. Cut three 0.5 cm × 4 cm strips of your first kombucha leather. Don't cut them from the very edges, as these tend to be thinner. Choose a section that is free of defects, such as small tears, bubbles, or ridges.
   2. Measure the thickness and width of your first strip using calipers, as shown in Figure 6. Convert your measurements to meters and record the results in your data table. Then calculate the cross-sectional area of your test sample. Record the result in your data table.
      Image Credit: Svenja Lohner, Science Buddies / Science Buddies
      Figure 6. Thickness and width measurements with calipers.
   3. Place each end of the strip into one of the clamps. The end of the sample strip should line up with the end of the metal clamp. Make sure to secure the strip tightly in the clamp.
   4. Do the tensile test. There are two ways to stretch the materials.
      1. Option A. If you have a spring scale, attach a string to one clamp, as shown in Figure 7.
         
         
         Image Credit: Svenja Lohner, Science Buddies / Science Buddies
         Figure 7. Instructions on how to attach a string to one of the compressor clamps.

         Attach the hook of the spring scale to the string loop. Hold the other clamp with one hand upright on the table. Do NOT move the clamp during the test. The test setup should look like Figure 8.

         Image Credit: Svenja Lohner, Science Buddies / Science Buddies

         A hand holds the clamp attached to one end of the sample strip. Another hand pulls on a spring scale attached to the clamp holding the other end of the sample strip.

         
         Figure 8. Tensile test setup using a spring scale.

         Then very slowly pull the spring scale straight away from the clamp so your sample gets stretched. Be careful not to bend or twist the sample. While pulling, make sure to monitor the readings on the spring scale. Pull until the sample strip breaks. Make sure that the sample broke somewhere in the middle and did not just slip off the clamp. Record the force (in newtons) that made the sample break in your data table. If your scale measures in pounds or kilograms, convert the force to newtons: 1 lb = 4.448 N, and 1 kg = 9.81 N (but only if you're in Earth's gravity!). Reset your scale to zero if you are using a trigger scale.

      2. Option B. Attach a string to both clamps as shown in Figure 9.
         Image Credit: Svenja Lohner, Science Buddies / Science Buddies
         Figure 9. A string attached to each compressor clamp holding the leather sample.

         Use the string to attach one clamp to a door handle or other sturdy object. The clamps should hang freely in the air. Use the string on the other (bottom) clamp to attach a container, like a cup or small bucket. Make sure that the container is close to the ground. This avoids spillage of its contents if your sample breaks. The test setup should look like Figure 10. Gradually add weight (e.g., metal or sand) to the container until the sample strip breaks.

         Image Credit: Svenja Lohner, Science Buddies / Science Buddies

         The compressor clamp at one end of the sample strip is attached to a hook. The compressor clamp at the other end is attached to a little bucket. A hand holds metal washers, which are added to the bucket until the strip breaks.

         
         Figure 10. Tensile test setup with a little bucket and weight.

         Again, make sure that the sample broke somewhere in the middle and did not just slip off the clamp. When the sample fails, weigh the filled container and record the weight that caused the sample to break in your data table. If needed, convert the weight to newtons first: 1 lb = 4.448 N, and 1 kg = 9.81 N (but only if you're in Earth's gravity!).

      
      
      Swipe left to see more

      Sample	Thickness [m]	Width [m]	Cross-sectional Area [m2] (Thickness × width)	Maximum applied force [N]	Tensile Strength [MPa]
      Kombucha recipe #1 with 66 g sugar
      #1
      #2
      #3
      Average
      Kombucha recipe #2 with 16.5 g sugar
      #1
      #2
      #3
      Average

      Table 4. Data table to record your data from the tensile strength test.
   5. Use Equation 1 and your data to calculate the tensile strength of your test sample. You might want to convert your result from pascals to megapascals (1 Pa = 1 × 10⁶ MPa). Record your result in your data table.
   6. Repeat the tensile test with the remaining two sample strips of the same kombucha leather.
   7. Finally, repeat steps a–f with the second kombucha leather. You want to test both kombucha leathers at least three times to make sure you can trust your results.
3. Torsion test. In this test, you will investigate how your different leather sheets behave when they are twisted. To do the torsion test, make a data table like Table 5 in your lab notebook and follow steps a–e to test both kombucha leather sheets.
   1. Cut three 0.5 cm × 4 cm strips of your first kombucha leather. Don't cut them from the very edges, as these tend to be thinner. Choose a section that is free of defects, such as small tears, bubbles, or ridges.
   2. Place each end of one strip into one of the clamps. The end of the sample strip should line up with the end of the metal clamp. Make sure to secure the strip tightly in the clamp.
   3. Hold each clamp in one of your hands with the screws both facing up, as shown in Figure 11. To twist your sample, keep one clamp still and rotate the other clamp until its screw is facing down. Now you have rotated the clamp halfway. Rotate the same clamp in the same direction so that the screw is facing up again. This makes one rotation. Continue rotating the clamp in the same direction, half a rotation at a time, until the sample breaks. Record the number of rotations until the sample breaks in your data table.
      
      
      Image Credit: Svenja Lohner, Science Buddies / Science Buddies

      In each image the right compressor clamp has been rotated by 180 more degrees.

      
      Figure 11. Torsion test setup.
   4. Repeat the torsion test with the remaining two sample strips of the same kombucha leather.
   5. Finally, repeat steps a–d with the second kombucha leather. You want to test both kombucha leathers at least three times to make sure you can trust your results.
   
   

   Trial	Kombucha recipe 1 with 66 g sugar	Kombucha recipe 2 with 16.5 g sugar
   #1
   #2
   #3
   Average

   Table 5. Data table to record the number of rotations until the sample breaks from the torsion test.
4. Flexure test. In this test, you will investigate how your different leather sheets behave when they are bent. To do the flexure test, make a data table like Table 6 in your lab notebook, and follow steps a–e to test both kombucha leather sheets.
   1. Cut three 1 cm × 4 cm strips of your first kombucha leather. Don't cut them from the very edges, as these tend to be thinner. Choose a section that is free of defects, such as small tears, bubbles, or ridges.
   2. To bend your first sample, fold it in half, as shown in Figure 12. Then slide your finger along the fold with some pressure, as if you were folding a paper airplane. Remove your finger and observe what happens. Did the sample break? Did the material spring back into its original flat state, or did it stay folded? Can you see a fold line in the material? Record your observations in your data table.
      
      
      Image Credit: Svenja Lohner, Science Buddies / Science Buddies
      Figure 12. Folding the sample strip in half.
   3. Repeat the flexure test with the remaining two sample strips of the same kombucha leather.
   4. Finally, repeat steps a–c with the second kombucha leather. You want to test both kombucha leathers at least three times to make sure you can trust your results.
   
   
   Swipe left to see more

   Trial	Did the sample break?	Does the material spring back into its original flat state?	Do you see the fold line?
   Kombucha recipe 1 with 66 g sugar
   #1	yes no	yes no	yes no
   #2	yes no	yes no	yes no
   #3	yes no	yes no	yes no
   Totals	__ yes  __ no	__ yes  __ no	__ yes  __ no
   Kombucha recipe 2 with 16.5 g sugar
   #1	yes no	yes no	yes no
   #2	yes no	yes no	yes no
   #3	yes no	yes no	yes no
   Totals	__ yes  __ no	__ yes  __ no	__ yes  __ no

   Table 6. Data table to record your data from the flexure test.
5. Sewing test. In this test, you will investigate whether your leather can be sewn easily, which is crucial for most textile applications. To do the sewing test, make a data table like Table 7 in your lab notebook and follow steps a–e to test both kombucha leather sheets.
   1. Cut two 3 cm × 3 cm square pieces of your first kombucha leather. Don't cut them from the very edges, as these tend to be thinner. Choose a section that is free of defects, such as small tears, bubbles, or ridges.
   2. Place both leather squares on top of each other.
   3. Use a sewing needle and sewing thread and try to sew the leather squares together. Make a single straight seam along the edges of the square using a backstitch. If you don't know how to backstitch, you can review this backstitch tutorial (Method 1).
   4. Repeat steps a–c with the second kombucha leather.
   5. How easy or difficult is it to sew the materials? Do the materials break? Record your observations in your data table. Then rank both materials from easiest to sew (1) to hardest to sew (2) in your data table.

Sample	Can you sew your leather without breaking it?	Rank from easiest to sew (1) to hardest to sew (2)
Kombucha recipe 1 with 66 g sugar	yes no
Kombucha recipe 2 with 16.5 g sugar	yes no

Table 7. Data table to record your data from the sewing test.

Analyzing your Data

In this section, you will analyze your data and come up with a conclusion about how the sugar concentration affects the properties of your bacteria-grown kombucha leather.

1. Biofilm growth
   1. Use the data from data table 2 to make a line graph showing the thickness of the SCOBY biofilm sheet over time. Put the time in days on the x-axis and the biofilm thickness in milimeters on the y-axis.
2. Tensile strength data
   1. For data table 3, calculate the average tensile strength for each sugar concentration. Record the result in your data table.
   2. Make a bar graph of your data. Put the sugar concentration on the x-axis and the average tensile strength on the y-axis.
3. Torsion test data
   1. For data table 4, calculate the average number of rotations that made the sample break for each sugar concentration.
   2. Make a bar graph of your data. Put the sugar concentration on the x-axis and the average number of rotations that made the sample break on the y-axis.
4. Flexure data
   1. Review your data table 5 and for each question count the total number of yes and no answers for each sample. Record the results in your data table.
5. Look at your data tables (2–6), graphs, and observations and try to draw conclusions from your results about which kombucha recipe makes the better leather replacement. Take the following questions into consideration.
   1. How did the different sugar concentrations affect the SCOBY biofilm growth?
   2. What similarities or differences do you see between the two processed leather sheets?
   3. How did the amount of sugar change the material properties of the different leathers?
   4. Based on your results, what function does sugar have in your kombucha recipe?
   5. Can you explain your results?
   6. Which of the tests you conducted do you think are most relevant for a leather alternative material?
   7. Is there a possibility that other factors could have affected your test results? If yes, which ones?
6. Based on your results, do you think kombucha leather is a good leather replacement?
7. Do you think the kombucha leather you made in this project is more or less sustainable than conventional leather? Use the 12 principles of green chemistry to answer this question.