Cleaning up Dyes from Contaminated Waters using Yeast

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

Setting Up the Gas Collection Apparatus

1. Remove the small red cap from one of the squeeze bottles.
2. Check the tip of the cap. If it is plugged, as shown in Figure 2, carefully use scissors to clip off the very top, just enough to open the cap. Do not cut off more than needed because a wider tip opening may be harder to fit into the tubing.

Image Credit: Teisha Rowland / Science Buddies

Bottle cap with point tip

Figure 2. Bottle tip that is plugged and needs to be cut open.

3. Connect the tubing to the tip opening, as shown in Figure 3. Make sure it fits tightly.

Image Credit: Svenja Lohner / Science Buddies

tube connected to the bottle opening

Figure 3. Tube connected to the bottle opening.

4. You will be collecting carbon dioxide from the yeast by displacing water trapped in an inverted graduated cylinder. Here's how to set it up:
   1. Fill your plastic tub (or bucket or dishpan) about one-third full with water.
   2. Fill the 100 mL graduated cylinder with water.
      1. If your dishpan is deep enough, fill the graduated cylinder by placing it on its side inside the dishpan. Allow any bubbles to escape by tilting the cylinder up slightly while keeping it under water. Keeping the opening of the cylinder under water, turn it upside down and attach it to the side of the dishpan with packing tape (or have your helper hold it in place).
      2. If your dishpan is not deep enough, fill the graduated cylinder completely using the faucet and cover the top tightly with plastic wrap. Quickly invert the cylinder (without spilling water) and place the opening in the dishpan, completely beneath the surface of the water. Remove the plastic wrap (under the water). Attach the cylinder to the side of the tub with packing tape (or have your helper hold it in place).
   3. The graduated cylinder should now be upside down, full of water, and with its opening under the surface of the water in the dishpan. Position the opening of the graduated cylinder so that you can place the free end of the tubing from the plastic bottle inside the graduated cylinder. Your apparatus is now ready to trap carbon dioxide from the yeast (see Figure 4).
   4. You can test your gas collection apparatus by removing the tube from the bottle top and blowing gently into the tube. The bubbles you create should be captured inside the cylinder. (You will need to reconnect the tube to the bottle and re-fill the cylinder before starting your experiment.)

Image Credit: Svenja Lohner / Science Buddies

Figure 4. Picture of the inverted graduated cylinder gas collection apparatus.

Running the Experiment

1. For each food coloring, you will want to test one bottle with active yeast and one bottle with inactive yeast. Using a permanent marker, label each of the bottles with the food coloring you will be testing, and whether it will include inactive or active yeast. If you want to test more than two food colorings, you can re-use the bottles. Make sure to rinse the bottles out thoroughly between experiments. Depending on what food colorings you are testing, your bottles could be labeled something similar to:
   1. Bottle 1: Blue, active yeast
   2. Bottle 2: Blue, inactive yeast
   3. Bottle 3: Green, active yeast
   4. Bottle 4: Green, inactive yeast
2. For each bottle, remove the lid and add 3 drops of the correct food coloring.
3. To the bottles that will be testing active yeast, add 1 teaspoon (tsp.) of sugar.
4. To all bottles, add 1/2 tsp. of dry baker's yeast.
5. Fill a pot with at least 4 cups (C) of very warm water (110°F – 115°F).
   1. Adjust the temperature of the hot water coming from the tap until it is almost too hot to hold your hands under. Use this temperature water to fill the pot. Use a thermometer to measure the actual temperature of the water and note this in your lab notebook.
6. Add 1/2 C water to your first bottle. 
7. Screw the lid onto the bottle tightly (without the tubing attached), place your thumb over the hole in the lid's tip, invert the bottle a few times (to dissolve the yeast and sugar), securely connect the tubing to the lid's tip, and then place the open end of the tubing inside your gas collecting cylinder, as shown in Figure 5. Start a stopwatch or note the starting time in your lab notebook.
   1. There should be water in the tubing as soon as it is submerged in the water. The CO₂ gas will push some water out of the tubing before the graduated cylinder starts to fill with CO₂ gas.

Image Credit: Teisha Rowland / Science Buddies

bottle connected to tubing in gas collection apparatus

Figure 5. Picture of the inverted graduated cylinder gas collection apparatus connected to the first test bottle.

8. Within 5–10 minutes, if the yeast is alive and active, the yeast solution should start foaming, and you should see bubbles collecting in the graduated cylinder. The bubbles are CO₂ gas produced by active yeast. Note the time when you first start seeing bubbles in your lab notebook.
9. Decide how long to collect CO₂ gas. Somewhere around 15-30 minutes is probably good, but you may need to adjust for your particular conditions. Be sure the graduated cylinder does not overfill with gas, as it will no longer capture and measure all of the gas. Use the same amount of time for all of your tests.
10. When the time is up, note how much CO₂ gas was collected by observing how much water was displaced from the graduated cylinder. Carefully tilt the cylinder to be vertical, without losing any of the gas, to most easily measure the amount of trapped gas.
11. Disconnect the bottle from the tubing and set it aside. You will be observing it over the next 3 days.
12. Re-fill your gas collection cylinder and repeat steps 4 to 11 with another bottle. If the water in the pot has cooled to below the recommended temperature range, refill it with new hot water, as described in step 4. 
    1. Which conditions generated CO₂? Which conditions did not? Why do you think this is?
13. Note: For each food coloring you want to test, you will want to test it a total of 3 times (with both active and inactive yeast). Because the kit only comes with 4 bottles, after you are done measuring the CO₂, you can carefully pour each bottle's contents into a different, separate bottle or jar, leaving the lid loosely attached in case more gas is produced. Thoroughly rinse any bottles you need to reuse. Alternatively, you can wait for 3 days, until you are done performing observations with the samples in the first 4 bottles, and then rinse and reuse the bottles.  

Analyzing Changes in Dye Over Time

1. Observe the liquid in the bottles each day. Record observations in your lab notebook.
   1. Are any of the bottles changing color? If so, how?
   2. Do any of the bottles with the same food coloring look different if they had active yeast (conditions with sugar included) compared to inactive yeast (no sugar added)? If you see differences, why do you think this is?
   3. How do the bottles change 1 and 2 days after starting the experiment?
2. When it has been 3 days since you started your experiment, analyze how the colors in the liquids have changed using serial dilutions. If any of the dye in any condition has been broken down, the liquid in that bottle will look lighter in color. For example, if you tested dark blue dye, and the blue dye has been broken down by the yeast, the liquid may look light blue in color. You can learn more about serial dilutions by watching this video on How to Make a Serial Dilution. Perform the serial dilution analysis as follows:
   
   1. Label six baby food jars (as shown in Figure 6) as follows: 100%, 50%, 25%, 12.5%, 6.25%, or 3.13%. 

Image Credit: Teisha Rowland / Science Buddies

small glass jars labeled with percentages

Figure 6. Six baby food jars labeled 100%, 50%, 25%, 12.5%, 6.25%, and 3.13%.

3. Fill all jars, except for the one labeled 100%, with 1/4 C water.
4. Fill the 100% jar with 1/2 C water and 3 drops of the food coloring you want to analyze first. (This is the same as the original solution you prepared, so it will be your 100% solution.) 
5. Measure out 1/4 C from the 100% jar and pour it into the 50% jar. 
6. Measure out 1/4 C from the 50% jar and pour it into the 25% jar.
7. Measure out 1/4 C from the 25% jar and pour it into the 12.5% jar.
8. Measure out 1/4 C from the 12.5% jar and pour it into the 6.25% jar.
9. Measure out 1/4 C from the 6.25% jar and pour it into the 3.13% jar.
10. Your serial dilution series is now complete and should look similar to the jars shown in Figure 7.

Image Credit: Teisha Rowland / Science Buddies

jars filled with blue liquid

Figure 7. Six baby food jars with blue liquid in a serial dilution.

11. Take the first bottle you want to analyze and carefully pour off some of the liquid into a clean baby food jar. When pouring from the bottle, stop before you disturb the yeast at the bottom of the bottle.
12. Hold the jar with your sample next to your serial dilution jars and try to match the darkness or lightness of your sample as closely as you can to the serial dilution jars.
    1. Try the best you can to find the closest matches in color. It may be that your sample is in between two of the serial dilution jars. For example, if your sample looks slightly darker in color than the 12.5% jar but slightly lighter than the 25% jar, then you could consider it to be between 12.5% and 25%. 
    2. It is possible that the sample has changed color and does not easily match any of the serial dilution jars.
    3. Record your observations in your lab notebook.
13. Repeat steps 11 and 12 with your remaining bottles.
14. Repeat the Running the Experiment and Analyzing Changes in Dye Over Time sections of the procedure twice more. This means you should test each food coloring (with both active and inactive yeast) a total of 3 times. Scientists always repeat their experiments to make sure their findings are true and reproducible.
    1. Thoroughly rinse any bottles you need to reuse them. 

Analyzing Your Data

1. What do your results tell you about whether live, active yeast is able to break down or reduce the amount of dye in a water sample?
   1. Was the yeast only active when sugar was included in the bottle? Remember that when yeast are alive and active, they produce CO₂ gas. 
   2. How do the results of the bottles (testing the same food coloring) with and without sugar compare to each other? How did the colors in each pair of bottles look one, two, and three days after starting the experiment?
   3. By about what percentage could the yeast decrease the amount of dye in the water for the different food colorings tested?
   4. Were all dyes affected the same way by the yeast? Why or why not?
   5. Can you think of ways that the yeast could be used to more efficiently break down or reduce the amount of dye in a water sample? How might you design an experiment to determine whether the water is safe (or safer) for aquatic organisms after the yeast breaks down the dye in the water? You can also check out the Variations section, below, for additional ideas.