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The Cold Wash Challenge: Testing How Well Enzymes Work in Laundry Detergents

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Abstract

What do you think are some of the most common stains on clothes? If you thought of grass, grease, ketchup, blood, or toothpaste stains, you are correct. Everyday activities like eating, playing outside, or doing sports can result in a lot of dirty laundry. Luckily, we have laundry detergents that can get our clothes clean again! Some of these detergents contain enzymes that are intended to boost cleaning power. In this science project, you will investigate how effective biological and non-biological laundry detergents are at different wash temperatures.

Summary

Areas of Science
Difficulty
 
Time Required
Short (2-5 days)
Prerequisites
None
Material Availability
Readily available
Cost
Low ($20 - $50)
Safety
No issues
Credits
Svenja Lohner, PhD, Science Buddies

Objective

To investigate the performance of biological versus non-biological laundry detergents at different wash temperatures.

Introduction

How many days can you go without getting a stain on your clothes? Probably not many. Our everyday activities inevitably leave marks on our clothes. Some of the most common stains come from grease, blood, coffee, ketchup, toothpaste, and grass. Luckily, there is a way to remove all of these stains: by washing your clothes. Washing dirty clothes with water alone is not enough, though. Water can only remove stains that can dissolve in water. To get rid of all the stains, including the greasy ones, we use laundry detergent. The cleaning power of detergent comes from surface active agents, or surfactants. They have complicated names, such as sodium laureth sulfate, sodium C10-16 alkylbenzenesulfonate, or C12-C15 alcohol ethoxylate. You can see these names if you look at the ingredient lists of the detergents you are using for this project.

Surfactants are unique molecules that have a water-repelling tail on one end and a water-attracting head on the other end, as shown in Figure 1. This allows them to react with water and with oil (grease), which normally doesn't mix with water.

A schematic view of a surfactant molecule.

A green oval represents the water-loving head of the molecule. A blue zig-zag line connected to it represents the water-repelling tail.


Figure 1. A surfactant molecule has a water-repelling tail and a water-attracting head.

When you wash your dirty clothes, the water-repelling tails of the surfactants stick to the grease and dirt of your stains. The water-attracting heads point outward toward the surrounding water. If many surfactant molecules attach to the same particle of grease, they form a sphere with grease in the middle, as shown in Figure 2. The trapped grease can then be easily washed away.

Schematic view of a grease particle trapped by surfactant molecules.

A brown circle in the middle represents a grease particle. The circle is surrounded by multiple surfactant molecules. The water-repelling tails of the surfactant molecules, represented by blue zig-zag lines, attach to the grease particle. Their water-loving heads, represented by green ovals, point away from the grease particle. The result is a sphere of surfactant molecules surrounding the grease particle.


Figure 2. A grease particle trapped in a sphere of many surfactant molecules.

Today, many laundry detergents contain additional ingredients to boost their cleaning power. Some of these additional ingredients are enzymes. Enzymes are biological molecules made by cells of plants and animals. They specialize in building or breaking down other molecules. The most common enzymes in laundry detergents are proteases, amylases, lipases, and mannanases. Each of these enzymes performs a specific task for removing stains. Proteases break down proteins, like those found in blood, egg, or dairy stains. Amylases break down starches in vegetable or other starchy food stains. Lipases break down fats in oil or grease stains, and mannanases break down mannans in bean gum stains. (Bean gum is extracted from the carob tree and is used as a thickening or gelling agent in lots of foods and cosmetics, such as ice cream and skin lotions.) Laundry detergents that contain enzymes are often called biodetergents or biological detergents. Watch the video below to learn more about biological detergents.

Enzymes have another advantage beyond helping to break down tough stains, and it has to do with water temperature. Heavily soiled clothes are often washed with warm or hot water to remove tough stains, but heating the water uses a lot of energy. In fact, it has been shown that about 90% of the energy for washing clothes is used to heat the washing water. Washing at lower temperatures can help save a lot of that energy. That is where enzymes come in. Enzymes in biological detergents are ideal for removing stains at lower washing temperatures. (At hot washing temperatures, enzymes can break apart and not work anymore.)

Another way to make detergents more sustainable is to change how surfactants are produced. Usually, surfactants are made from petroleum, which is a nonrenewable resource. But given the limited supply of oil and its negative environmental impacts (such as greenhouse gas emissions and environmental pollution), there is a trend toward natural and more sustainable surfactants. These eco-friendly surfactants, which are also called "green surfactants" or "renewable surfactants," are made from plant-based resources, which are renewable. Sustainable surfactants have become increasingly popular, in part because they are biodegradable and non-toxic.

Detergents are a good example of how green chemistry can have a positive impact on the environment. The goal of green chemistry is to help the environment by reducing the use of harmful chemicals, preventing pollution, and saving energy and natural resources when making new products. Laundry detergents produced using the principles of green chemistry help save energy by being more efficient at lower washing temperatures and avoid the use of petroleum-based surfactants, which are associated with negative environmental impacts. The Beyond Benign resource in the Bibliography has more information on the 12 principles of green chemistry.

In this science project, you will put biological detergents to the test and compare them to laundry detergents without enzymes. You will test both types of detergents on different stains using hot and cold washing water. Do you think the non-biological detergents have caught up with biological detergents in terms of their cleaning efficiency in cold water? There is only one way to find out: Do the experiment!

Terms and Concepts

Questions

Bibliography

For help creating graphs, try this website:

  • National Center for Education Statistics, (n.d.). Create a Graph. Retrieved June 25, 2020.

Materials and Equipment

 Materials needed for the laundry detergent project.

Materials include white cotton fabric, different detergents, and different substances for creating stains.

Experimental Procedure

Preparing the Stain Swatches

  1. Use a ruler and scissors to measure and cut out 10 cm × 10 cm (4 inch × 4 inch) squares of your white fabric. You will need 24 squares for each laundry detergent you test, plus one unstained square and one extra square for each type of stain to use as a reference. For example, if you are comparing two different laundry detergents, you will need 2 × 24 = 48 squares for the detergents, plus 5 squares for the reference squares (one unstained and one for each unwashed stain type). This means that for two different detergents, you will need 53 cotton squares in total.
  2. You will do three trials for each detergent. Start by preparing 24 cotton squares for Detergent #1. On each square, use a permanent marker to write the name of Detergent #1 as well as one of the label names shown in Table 1. The labels indicate the type of stain, the temperature of the wash, and the number of the trial. (For example, "S c 1" represents starch stain, cold wash, trial 1.)
Trial Starch stain -
cold wash
Protein stain -
cold wash
Gum stain
cold wash
Fat stain -
cold wash
Starch stain -
hot wash
Protein stain -
hot wash
Gum stain -
hot wash
Fat stain -
hot wash
1 S c 1 P c 1 G c 1 F c 1 S h 1 P h 1 G h 1 F h 1
2 S c 2 P c 2 G c 2 F c 2 S h 2 P h 2 G h 2 F h 2
3 S c 3 P c 3 G c 3 F c 3 S h 3 P h 3 G h 3 F h 3
Table 1. Label names for each cotton square. One set of 24 squares needs to be prepared for each laundry detergent.
  1. Repeat the labeling process with 24 more cotton squares for Detergent #2. If you are testing more than two laundry detergents, repeat until you have 24 labeled squares for each detergent.
  2. With a permanent marker, label your reference stains. The reference squares, shown in Figure 3, will not be washed, so they do not need a detergent name. You will keep them until the end of your experiment. Label them with a letter representing the type of stain followed by an "r" for "reference," as follows:
    1. P r
    2. S r
    3. F r
    4. G r
    5. Unstained.
    Five labeled 10x10cm cotton squares, four with stains and one without.

    One square has a chocolate stain, one a ketchup stain, one an olive oil stain, and one an egg stain. The fifth cotton square has no stains. Each square is labeled with a letter representing the type of stain (P for protein, S for starch, G for gum, F for fat) and an 'r' for 'reference.' The unstained square is labeled 'unstained.'


    Figure 3. Prepared reference stains with chocolate ice cream (gum stain), ketchup (starch stain), olive oil (fat stain), and egg (protein stain).
  3. Make your protein stains.
    1. With a clean measuring spoon, take 1/4 teaspoon of your chosen protein ingredient and put it in the middle of a cotton square that you labeled with "P."
    2. With the measuring spoon, spread out the ingredient to make a circle of about 5 cm (2 inches) in diameter in the middle of the cotton square. While spreading, press the spoon lightly down on the fabric so the ingredient can get absorbed by the fabric.
    3. Repeat steps a and b with all the cotton squares you labeled with a "P."
    4. Place all the stained cotton squares on a piece of wax paper and let them dry overnight, or for at least 16 hours. Place them next to each other so the stains on one cotton square do not transfer to another cotton square. The stains will spread out into the fabric more overnight, so the next day they will look bigger.
  4. Repeat step 5 with your fat ingredient and the cotton squares labeled "F."
  5. Repeat step 5 with your bean gum ingredient and the cotton squares labeled "G."
  6. Repeat step 5 with your starch ingredient and the cotton squares labeled "S."
  7. Keep one cotton square unstained. This will be your reference that you can compare your washed cotton pieces with.

Washing the Stained Swatches

  1. Take the cotton squares labeled with Detergent #1 and F c 1, P c 1, S c 1, and G c 1. This will be your first wash trial. You will repeat the washing procedure with the trial 2 and trial 3 sets of the F c, P c, S c, and G c cotton squares. Note: If you are short in time, you can also wash all the 12 F c, P c, S c, and G c cotton squares for all trials together in one wash cycle.
  2. Take a "before wash" picture. This will be helpful for your display board.
    1. Place all 4 stained squares and the unstained cotton square next to each other on white paper. Make sure that all the pictures are taken with the same lighting, background, and camera settings so you can compare them to each other.
  3. Prepare the cold wash.
    1. Place three cups of cold tap water into your plastic container.
    2. Measure the water temperature with a thermometer. The temperature should be 20 +/- 0.5°C (or 68°F). If you need to heat the water, place it in the microwave briefly (for 15-30 seconds) and then check the temperature again. Repeat until you have the right temperature. If you need to cool the water, add an ice cube to the water, wait for it to melt, and check the temperature again. Repeat until you have the right temperature.
    3. Once the water is at the right temperature, add 1 teaspoon of your laundry detergent to the water. Swirl the water with the teaspoon to mix the detergent solution.
    4. Place the 4 stained cotton squares into the water and close the lid. Make sure the lid is sealed tightly.
    5. Set a timer for 5 minutes and start it. Then shake the container randomly in an up-and-down or side-to-side motion until the timer is done. The shaking simulates the washing cycle of a washing machine. Be careful not to spill any water. Use paper towels to clean up any spills.
    6. Once the 5 minutes are over, remove the cotton squares from the water and place them on the container lid. At this point, the stains should not transfer from one cotton square to another anymore, so you can pile the washed cotton squares on top of each other. Dispose of the washing water in the sink and rinse the container with tap water.
    7. Add 3 cups of tap water to the rinsed container and adjust the temperature to 20°C again. Place the washed cotton squares back into the water. Set a timer for 2 minutes and start the timer. Shake the container until the timer is done. This simulates the rinse cycle of a washing machine.
    8. Once the 2 minutes are over, remove the cotton squares from the water and squeeze them with your hand. This simulates the spin cycle of the washing machine, which removes excess water from the fabric.
    9. Lay out a bath towel flat on a drying rack, a table, or the floor. Place all of the washed and rinsed cotton squares stain side up and flat on the towel and let them dry completely.
  4. Repeat steps 1–3 for Detergent #1 and the cold wash two more times. For the second trial, use the F c 2, P c 2, S c 2, and G c 2 cotton squares and for the third trial use the F c 3, P c 3, S c 3, and G c 3 cotton squares. In total, you should have washed three sets of F c, P c, S c, and G c cotton squares with the same detergent and the same wash temperature. Doing at least three trials of an experiment is good practice in science and ensures that you can trust your results.
  5. Repeat steps 1–4 for Detergent #2 and for all other laundry detergents you want to test.
  6. Continue with the hot washing temperature. Follow the same procedure described in steps 1–5 with the following modifications.
    1. Use the cotton squares labeled F w, P w, S w, and G w.
    2. Adjust the water temperature for the wash and rinse cycles to 50°C +/- 0.5°C (122°F). Heat the water up in the microwave in 30 second increments until it reaches the right temperature.

Evaluating the Cleaning Performance

  1. Once all washed cotton squares are dry, assess the color of each stain.
    1. For each stain type (fat, starch, protein, and gum), you will rate the stain color on your cotton squares on a scale from 0–10. Zero means that the stain is not visible anymore; the cotton square is completely white and has the same color as the unstained reference square. Ten means that the stain hasn't changed at all; it has the same color as the stain on the unwashed reference stain square.
    2. This means the "before washing" values for each stain type (fat, starch, protein, gum) will be 10, as shown in Table 2.
    3. For your first laundry detergent, lie all the cotton squares for the first trial next to each other on white paper. Look at each of the washed cotton squares closely. Then assign a number between 0 and 10 to each washed square depending on its stain color. Record your data in a table like Table 2. Make a separate data table for each laundry detergent you test.
    4. Repeat step c to evaluate the stain colors from trials 2 and 3 for the same laundry detergent.
    5. Repeat steps c and d for the cotton squares washed with the other laundry detergent(s).
Laundry detergent:
Gum stain
  Trial 1 Trial 2 Trial 3 Average
Before wash 10 101010
Cold wash     
Hot wash      
Starch stain
Before wash 10 101010
Cold wash     
Hot wash      
Protein stain
Before wash 10 101010
Cold wash     
Hot wash      
Fat stain
Before wash 10 101010
Cold wash     
Hot wash      
Table 2. Data table in which to record your color rating results for each stain.
  1. Take an "after wash" picture of all your washed cotton squares. This will be helpful for your display board.
    1. For each laundry detergent, take a picture with all of the following cotton squares in one picture, as shown in Figure 4:
      • The four unwashed reference stain squares
      • The four cold-washed cotton squares from trial 1
      • The four hot-washed cotton squares from trial 1
      • The unstained reference square.
      Three rows of four samples, plus one unstained sample.

      The first row has the unwashed squares with prominent stains. The next two rows of squares have faded stains. One row has the hot-washed samples and one row has the cold-washed samples.


      Figure 4. An example "after wash" picture for one wash trial. The cotton squares with the unwashed reference stains are in the top row. Below are the cold-washed and hot-washed cotton squares. On the right is the unstained reference square. Note that in this picture the labels have been removed so as not to give away the results.
    2. Place all the cotton squares next to each other on white paper. Make sure that all the pictures are taken with the same lighting, background, and camera settings (it is probably best to use a white background), so you can compare them to each other.
    3. Repeat steps a and b to take separate pictures of the cotton squares from trial 2 and trial 3. In the end, you should have three pictures for each laundry detergent — one for each trial.

Analyzing your Data

  1. Calculate the average color rating for each of your stains after washing. To do this, for all your laundry detergents and each of your stains listed in Table 2, add the color values from the three individual trials and then divide the result by 3. Write down the average in your data table.
  2. Graph your color rating data.
    1. For each type of stain (protein, starch, fat, gum), make a bar graph comparing the observed stain color before and after the cold or hot wash. On the x-axis, put the laundry detergent name (with or without enzyme), and on the y-axis, graph the average color value before and after the cold or hot wash.
    2. You can either make separate graphs for the cold and hot wash or use one bar graph with different colored bars indicating the different washing temperatures. The Bibliography lists an online graphing tool that can help you create graphs.
  3. Optional: Continue with steps 3–6 to do more quantitative measurements of the stain colors using a color picker tool online. You can also use any other color picker tool that is able to give you color values for saturation. If you do not want to use the color picker tool, jump to step 7.
    1. Transfer all your "after-washing" pictures to a computer and then navigate to the color picker tool website.
    2. In the "Select Image" box at the top, browse and select your first "after wash" image. Start with the first trial image of your first laundry detergent. The image should show up in the "Pick Color" box.
    3. In the "Edit and Convert Color Code" box, change the color format. From the drop-down menu, select HSL, which stands for hue, saturation, and lightness. You will compare the saturation values for each stain. Color saturation measures the intensity of a color in an image. As the saturation decreases, the color fades. As your stain will fade during washing, color saturation is a good approximation for the cleaning power of your detergent. Lower saturation values mean lighter colors.
    4. If you click on a spot on your uploaded image, the color picker tool will display a hue, saturation, and lightness value for your selected spot.
      1. Start with the four unwashed reference stains, which are equivalent to the before-wash stains. For each of the stains, sample at least five pixels by clicking on at least five spots on the stain in the image. For each pixel, write down the saturation value. Then calculate the average saturation value for each stain and record the average saturation in a data table like Table 3. You will need a separate table for each laundry detergent.
      2. Repeat step d.i for the cold-washed stains in your image.
      3. Repeat step d.i for the hot-washed stains in your image.
    5. Repeat steps b–d with your "after wash" images from trial 2 and trial 3.
    6. Repeat steps b–e for all your images taken for the other detergent(s).
Laundry detergent:
Gum stain
  Average
Saturation
Trial 1
Average
Saturation
Trial 2
Average
Saturation
Trial 3
Saturation
Average for
All Trials
Saturation
Percentage
[%]
Before wash     100
Cold wash     
Hot wash     
Starch stain
  Average
Saturation
Trial 1
Average
Saturation
Trial 2
Average
Saturation
Trial 3
Saturation
Average for
All Trials
Saturation
Percentage
[%]
Before wash     100
Cold wash     
Hot wash     
Protein stain
  Average
Saturation
Trial 1
Average
Saturation
Trial 2
Average
Saturation
Trial 3
Saturation
Average for
All Trials
Saturation
Percentage
[%]
Before wash     100
Cold wash     
Hot wash     
Fat stain
  Average
Saturation
Trial 1
Average
Saturation
Trial 2
Average
Saturation
Trial 3
Saturation
Average for
All Trials
Saturation
Percentage
[%]
Before wash     100
Cold wash     
Hot wash     
Table 3. Data table to record your stain saturations. Make a new table for each laundry detergent.
  1. Calculate the average color saturation for each of your stains after washing. To do this, for each of your stains listed in your data table 3, add the saturation values from each individual trial and then divide the result by 3. Write down the average in your data table.
  2. The next step is to compare the saturation values with each other to determine how much the stains faded for each washing condition. To do this, you will calculate the saturation percentage of the washed stain out of the saturation value of the unwashed stain.
    1. For each stain listed in your data table 3, divide the saturation average for the cold wash by the saturation average before the wash (the unwashed stain). Then multiply the result by 100 to get a percentage. For example, if the saturation value for the unwashed stain is 40, and the saturation value of the washed stain is 20, then the saturation percentage is 20 / 40 × 100, which is 50%. The stain color faded by about 50%. Repeat the calculations for each stain type and each laundry detergent. Note: The saturation percentage of the unwashed stain (which is equivalent to the before-wash stain) is 100%, as the stain hasn't been washed and thus didn't fade at all.
    2. For each stain listed in your data table 3, divide the saturation average for the hot wash by the saturation average before the wash (the unwashed stain). Then multiply the result by 100 to get a percentage. Repeat the calculations for each stain type and each detergent.
  3. Graph your color saturation data.
    1. For each type of stain (protein, starch, fat, gum), make a bar graph comparing the observed stain color saturation before and after the cold or hot wash. On the x-axis, put the laundry detergent name (with or without enzyme), and on the y-axis, graph the calculated saturation percentage before and after the cold or hot wash.
    2. You can either make a separate graph for the cold and hot wash or use one bar graph for each stain type with different colored bars indicating the different washing temperatures. The Bibliography lists an online graphing tool that can help you create graphs.
  4. Use your graphs to compare the average color values or saturation percentages for each washing temperature and each laundry detergent before and after washing. Remember, the lower the color value or saturation percentage, the more the stain has disappeared. A lower number means a higher detergent cleaning power. What conclusions can you draw from your data? Try to answer the following questions:
    1. Can you see a trend in your data based on washing temperature or type of laundry detergent?
    2. Did your stains become less saturated or even disappear after washing?
    3. How did the washing temperature affect the cleaning power of your laundry detergents?
    4. How does the cleaning power of the biological laundry detergent with enzymes compare to the non-biological laundry detergent without enzymes?
    5. Based on your data, is it better to wash your clothes at low or high temperatures?
    6. Based on your data, do you think you could save energy by using laundry detergents with enzymes?
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Variations

  • The focus of this project was to compare biological laundry detergents with enzymes to non-biological laundry detergents without enzymes. Most of these detergents contain surfactants made from petroleum. As mentioned in the Introduction, there is another type of detergent that contains sustainable surfactants made from plants. Repeat the experiment with a green or eco-friendly laundry detergent with plant-based surfactants. How effective are these detergents?
  • Whereas in this project, you simulated a wash cycle by submerging your stained cotton squares in water and shaking them, the everyday washing in many households happens in a washing machine. How do your results change if you repeat the projects, but instead of hand-washing, you machine-wash your stained cotton squares? Make sure to use a low-load setting and switch between the cold and hot.
  • Your results should tell you how the cleaning effectiveness of your laundry detergents compares between a hot and cold washing temperature. If you get comparable cleaning effectiveness with a cold wash, you can save a lot of energy, as mentioned in the Introduction. Try to find out how much heating energy you can actually save when washing your clothes at 20°C instead of 50°C. For your calculation, assume that a standard, not high-efficiency washing machine uses about 20 gallons of water per load. What would be your energy savings in kilowatt-hours (kWh), and how much money could you save per load?
  • Enzymes are great at removing stains at low washing temperatures. However, as enzymes are biological molecules, they break apart under certain conditions. For example, enzymes can become inactive when the pH or the temperature is too low or too high. How do the pH values of biological and non-biological detergents compare? Would your results change if you were to heat up your biological detergent to 60°C before using it for washing?
  • Green or sustainable laundry detergents produced with the concept of green chemistry in mind are becoming more and more popular. Being mindful of energy usage and the exploitation of natural resources is just a subset of the 12 principles of green chemistry. The use of less-toxic chemicals and the biodegradability of products are other factors that matter. Can you design an experiment that could evaluate the toxicity or the biodegradability of laundry detergents?

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General citation information is provided here. Be sure to check the formatting, including capitalization, for the method you are using and update your citation, as needed.

MLA Style

Lohner, Svenja. "The Cold Wash Challenge: Testing How Well Enzymes Work in Laundry Detergents." Science Buddies, 22 Mar. 2023, https://www.sciencebuddies.org/science-fair-projects/project-ideas/GreenChem_p009/green-chemistry/laundy-detergent-enzymes. Accessed 24 Feb. 2024.

APA Style

Lohner, S. (2023, March 22). The Cold Wash Challenge: Testing How Well Enzymes Work in Laundry Detergents. Retrieved from https://www.sciencebuddies.org/science-fair-projects/project-ideas/GreenChem_p009/green-chemistry/laundy-detergent-enzymes


Last edit date: 2023-03-22
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